Microsoft Word - 2016ejtm26(1) 37-60 SpPMD Abstracts Epub x PUBMED.doc European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   37 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Muscle  Decline  in  Aging  and  Neuromuscular  Disorders  -­‐  Mechanisms  and  Countermeasures   Terme  Euganee,  Padova  (Italy),  April  13  -­‐  16,  2016   Hotel  Augustus,  Viale  Stazione  150  -­‐  35136  Montegrotto  Terme  (Padova),  Italy  &  Villa  dei  Vescovi,  Luvigliano  di  Torreglia  (Padova),  Italy   Phone  +39  049  793  200  -­‐  Fax  +39  049  793518  -­‐  http://www.hotelaugustus.com/english/pages/hotel_augustus.php  -­‐  E-­‐mail:  info@hotelaugustus.com   Organizers:  Ugo  Carraro,  Helmut  Kern,  Christiaan  Leeuwenburgh,  Werner  Lindenthaler,  Francesco  Piccione,  Carlo  Reggiani,  Marco  Sandri ---------------------------------------------------------------------------------------------------------------------------------------------------------------- ABSTRACTS     WEDNESDAY  April  13,  2016   Hotel  Augustus,  Viale  Stazione  150,  Montegrotto  Terme  (Padova),  Italy   Exercise  and  FES  in  premature  and  late  aging:       Tutorial  and  get-­‐together,  H  Kern,  U  Carraro,  Chairs   Functional  Electrical  Stimulation  (FES)  of  Aging  Muscle   Helmut  Kern   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and   Physical   Rehabilitation,  Department  of  Physical  Medicine  and  Rehabilitation,   Wilhelminenspital  Wien,  Austria  Italy   E-­‐mail:  wil.pys.kern-­‐forschung@wienkav.at     Physical   exercise   is   known   to   have   beneficial   effects   on   muscle   trophism   and   force   production   modulating   signaling   pathways   involved   in   fiber   type   plasticity,   muscle   growth   and   mitochondria   respiratory   efficiency.1   It   has   been   shown   that   the   decrease   of   muscle  mass  and  strength  observed  in  aging  is  linked  to  intracellular   and   extracellular   abnormalities,   that   is,   sarcoplasmic   reticulum-­‐to-­‐ mitochondria   malfunctions   and   extracellular   matrix   metabolism,   respectively.2   Lifelong,   high-­‐level   physical   activity   delays   the   medium  and  long  term  effects  of  aging.3  Furthermore,  when  healthy   seniors   are   exposed   to   regular   neuromuscular   Functional   Electrical   Stimulation   (FES)   training   for  a  period  of  9  weeks  outcomes  are  an   increase   in   muscle   strength   and   muscle   fiber   size   and,   most   importantly,  an  increase  of  fast  fibers,  the  more  powerful  of  skeletal   muscle  motor   units.4   Electron  microscopy   analysis   of   aging  muscle   show   remodeling   of  mitochondrial   apparatus   as   a   consequence   of   fusion   phenomena   that   are   consistent   with   adaptation   to   physical   exercise.5  Altogether   these   results   indicate   that   the  FES-­‐dependent   beneficial   effects   on   muscle   force   and   mass   are   associated   with   changes   in   mitochondrial-­‐   and   sarcoplasmic   reticulum-­‐related   proteins   involved   in   Ca2+   homeostasis,   providing   new   targets   to   develop  therapeutic  strategies  to  promote  healthy  aging.   1. Egan   B,   Zierath   JR.   Exercise   metabolism   and   the   molecular   regulation   of   skeletal   muscle   adaptation.   Cell   Metabolism.   2013;17:162-­‐84.     2. Konopka   AR,   Sreekumaran   Nair   K.   Mitochondrial   and   skeletal   muscle   health   with   advancing   age.   Molecular   and   Cellular   Endocrinology  2013;379:19-­‐29.   3. Zampieri   S,   Pietrangelo   L,   Loefler   S,   et   al.   Lifelong   Physical   Exercise   Delays   Age-­‐Associated   Skeletal   Muscle   Decline.   J   Gerontol   A   Biol   Sci   Med   Sci   2015;70:163-­‐73.   doi:   10.1093/gerona/glu006.  Epub  2014  Feb  18.   4. Kern   H,   Barberi   L,   Löfler   S.   et   al.   Electrical   stimulation   counteracts   muscle   decline   in   seniors.   Front   Aging   Neurosci.   2014;  6:189.   5. Pietrangelo   L,   D'Incecco   A,   Ainbinder   A,   Michelucci   A,   Kern   H,   Dirksen   RT,   Boncompagni   S,.Protasi   F.   Age-­‐dependent   uncoupling  of  mitochondria   from  Ca2+   release  units   in   skeletal   muscle.  Oncotarget.  2015;34:35358-­‐71.   *****     In-­‐Bed  Gym  and  FES:  Fighting  muscle  weakness  by  take-­‐ home  strategies   Ugo  Carraro,  Andrea  Marcante,  Alfonc  Baba,  Francesco  Piccione   IRCCS  Fondazione  San  Camillo  Hospital,  Venice,  Italy   E-­‐mail:  ugo.carraro@ospedalesancamillo.net   All   permanent   or   progressive   muscle   contractility   impairments   (including   age-­‐related   muscle   power   decline)   need   permanent   managements.   Beside   eventual   pharmacology   therapy,   a   home-­‐ based   physical   exercise   approach   is   helpful,   in   particular   for   bed-­‐ rested   or   bed-­‐ridden   patients.1   Awaiting   development   of   implantable   devices   for  muscle   stimulation,   i.e.,   of   electroceuticals   as   effective   as   pace-­‐makers   for   cardiac   arrhythmias   or   cochlear   implants  for  hearing  loss,  education  of  hospitalized  patients  to  take-­‐ home   physical   exercise   managements   is   an   effective   low   cost   alternative.2  Frail  elderly  due  to  advanced  age  or  associated  diseases   are  often  hospitalized  for   long  periods  of  time.  There,  their  already   modest  amount  of  daily  physical  activity  is  reduced,  contributing  to   limit   their   independence   up   to   force   them   to   the   bed.   Inspired   by   the   proven   capability   to   recover   skeletal   muscle   contractility   and   strength   by   home-­‐based   Functional   Electrical   Stimulation   (h-­‐bFES)   even  in  the  worse  cases  of  neuromuscular  traumatic  injuries,3-­‐5  but,   mainly   guided   by   common   sense,   we   suggest   a   short   (15-­‐20   minutes)  daily  sequence  of  fifteen  easy  volitional  physical  exercises   that   are   performed   in   bed   (In-­‐Bed   Gym).   If   sedentary   borderline   persons  challenge,  but  not  stress,  them-­‐self,  in  a  few  days  in  hospital   In-­‐Bed   Gym   may   increase   muscle   strength,   fatigue   resistance   and   independence   in  daily   life   activities.   In   surgical   units   this  will   grant   standing  of  patients  soon  after  operation,  a  mandatory  measure  to   prevent   thromboembolism   risk.   In-­‐Bed  Gym   helps   also   to  mitigate   the  bad  mood  that  accompanies  mobility   limitations,  strengthening   patients’   confidence   in   recovering   partial   or   total   independence6   Continued   regularly,   In-­‐Bed   Gym   may   help   to   maintain   the   independence   of   frail   older   people   and   to   reduce   the   risk   of   the   possible   serious   consequences   of   accidental   falls.   In-­‐Bed  Gym  may   also  mitigate   eventual   arterial   hypertension,   a  major   risk   factor   in   elderly  people.7   1. Albert   SM,   King   J,   Boudreau   R,   Prasad   T,   Lin   CJ,   Newman   AB.   Primary   prevention   of   falls:   effectiveness   of   a   statewide   program.   Am   J   Public   Health.   2014   May;104(5):e77-­‐84.   doi:   10.2105/AJPH.2013.301829.  Epub  2014  Mar  13.   2. Gillespie  LD1,  Robertson  MC,  Gillespie  WJ,  Sherrington  C,  Gates   S,   Clemson   LM,   Lamb   SE.   Interventions   for   preventing   falls   in   older   people   living   in   the   community.   Cochrane   Database   Syst   Rev.   2012   Sep   12;9:CD007146.   doi:   10.1002/14651858.CD007146.pub3.   3. Kern   H,   Carraro   U,   Adami   N,   et   al.   Home-­‐based   functional   electrical   stimulation   rescues   permanently   denervated  muscles   in  paraplegic  patients  with  complete  lower  motor  neuron  lesion.   Neurorehabil   Neural   Repair   2010;24:709-­‐21.   doi:   10.1177/1545968310366129.  Epub  2010  May  11.   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   38 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   4. Carraro  U,  Kern  H,  Gava  P,  et  al.  Biology  of  muscle  atrophy  and   of   its   recovery  by  FES   in  aging  and  mobility   impairments:   roots   and   by-­‐products.   Eur   J   Transl   Myol   –   Basic   Appl   Myol   2015;25:211-­‐30.   5. Kern   H,   Barberi   L,   Löfler   S,   et   al.   Electrical   stimulation   counteracts   muscle   decline   in   seniors.   Front   Aging   Neurosci.   2014   Jul   24;6:189.   doi:   10.3389/fnagi.2014.00189.   eCollection   2014.   6. Carneiro   LS,   Fonseca   AM,   Serrão   P,   et   al.   Impact   of   physical   exercise   on   catechol-­‐O-­‐methyltransferase   activity   in   depressive   patients:  A  preliminary  communication.  J  Affect  Disord.  2016  Jan   1;193:117-­‐122.  doi:  10.1016/j.jad.2015.12.035.   7. Börjesson  M,  Onerup  A,   Lundqvist   S,  Dahlöf  B.   Physical   activity   and   exercise   lower   blood   pressure   in   individuals   with   hypertension:  narrative  review  of  27  RCTs.  Br  J  Sports  Med  2016   Jan   19.   pii:   bjsports-­‐2015-­‐095786.   doi:   10.1136/bjsports-­‐2015-­‐ 095786.  [Epub  ahead  of  print].   *****   THURSDAY  April  14,  2016   Hotel  Augustus,  Viale  Stazione  150,  Montegrotto  Terme  (Padova),  Italy   Workshop  “Circadian  rhythms  in  skeletal  muscle”     C  Leeuwenburg,  S  Schiaffino,  Chairs     Introduction  by  S  Schiaffino,  Padova,  Italy   Circadian  rhythms  and  the  molecular  clock  in  skeletal  muscle   Karyn  A.  Esser   University  of  Florida,  Gainesville,  FL,  US   E-­‐mail:  "Esser,Karyn"  kaesser@ufl.edu     Disruption   of   circadian   rhythms   in   humans   and   rodents   has   implicated  a  fundamental  role  for  circadian  rhythms  in  aging  and  the   development   of   many   chronic   diseases   including   diabetes,   cardiovascular  disease,  depression  and  cancer.  The  molecular  clock   mechanism   underlies   circadian   rhythms   and   is   defined   by   a   transcription-­‐translation  feedback  loop  with  Bmal1  encoding  a  core   molecular   clock   transcription   factor.   Germline   Bmal1   knockout   (Bmal1   KO)   mice   have   a   shortened   lifespan,   show   features   of   advanced   aging   and   exhibit   significant   weakness   with   decreased   maximum   specific   tension   at   the   whole   muscle   and   single   fiber   levels.1  We   tested   the   role   of   the  molecular   clock   in   adult   skeletal   muscle   by   generating   mice   that   allow   for   the   inducible   skeletal   muscle-­‐specific  deletion  of  Bmal1   (iMSBmal1),2  Here  we   show   that   disruption   of   the   molecular   clock,   specifically   in   adult   skeletal   muscle   is  associated  with  a  muscle  phenotype   including   reductions   in   specific   tension,   increased   oxidative   fiber   type,   and   increased   muscle   fibrosis   similar   to   that   seen   in   the   Bmal1   KO   mouse.     Remarkably,   the  phenotype  observed   in   the   iMSBmal1-­‐/-­‐  mice  was   not  limited  to  changes  in  muscle.    Similar  to  the  germline  Bmal1  KO   mice,   we   observed   significant   bone   and   cartilage   changes   throughout   the   body   suggesting   a   role   for   the   skeletal   muscle   molecular  clock   in  both  the  skeletal  muscle  niche  and  the  systemic   milieu.3  This  emerging  area  of  circadian  rhythms  and  the  molecular   clock  in  skeletal  muscle  holds  potential  to  provide  significant  insight   into  intrinsic  mechanisms  of  the  maintenance  of  muscle  quality  and   function   as   well   as   identifies   a   novel   crosstalk   between   skeletal   muscle,  cartilage  and  bone.   1. Andrews   JL,   Zhang   X,   McCarthy   JJ,   et   al.   CLOCK   and   BMAL1   regulate   MyoD   and   are   necessary   for   maintenance   of   skeletal   muscle   phenotype   and   function.   Proc   Natl   Acad   Sci   U   S   A   2010;107:19090-­‐5.   doi:   10.1073/pnas.1014523107.   Epub   2010   Oct  18.   2. Hodge   BA,   Wen   Y,   Riley   LA,   et   al.   The   endogenous   molecular   clock   orchestrates   the   temporal   separation   of   substrate   metabolism   in   skeletal   muscle.   Skelet   Muscle   2015;5:17.   doi:   10.1186/s13395-­‐015-­‐0039-­‐5.  eCollection  2015.   3. Schroder  EA,  Harfmann  BD,  Zhang  X,  et  al.  Intrinsic  muscle  clock   is  necessary  for  musculoskeletal  health.  J  Physiol  2015;593:5387-­‐ 404.  doi:  10.1113/JP271436.  Epub  2015  Nov  23.   *****   Intrinsic  and  extrinsic  control  of  circadian  gene  expression  in   skeletal  muscle   Kenneth  A.  Dyar  (1,6),  Stefano  Ciciliot  (1),  Guidantonio  Malagoli   Tagliazucchi  (2),  Giorgia  Pallafacchina  (1,4),  Jana  Tothova  (1),  Carla   Argentini  (1),  Lisa  Agatea  (1),Reimar  Abraham  (1),  Miika   Ahdesmäki  (5),  Mattia  Forcato(2),  Silvio  Bicciato  (2),  Stefano   Schiaffino  (1,4),  Bert  Blaauw  (1,3)   (1)  Venetian  Institute  of  Molecular  Medicine  (VIMM),  Padova,  Italy;   (2)   Center   for   Genome   Research,   Department   of   Life   Sciences,   University   of   Modena   and   Reggio   Emilia,   Modena,   Italy;   (3)   Department  of  Biomedical   Sciences,  University  of  Padova,   Italy;   (4)   Institute  of  Neurosciences,  Consiglio  Nazionale  delle  Ricerche  (CNR),   Padova;   (5)   Institute   for   Medical   Informatics,   Statistics   and   Epidemiology,   University   of   Leipzig,   Germany,   (6)   Present   address:   Molecular   Endocrinology,   Institute   for   Diabetes   and   Obesity,   Helmholtz  Zentrum  München,  Germany   E-­‐mail:  kenneth.dyar@gmail.com   Physical   activity   and   circadian   rhythms   are   well-­‐established   determinants   of   human   health   and   disease,   but   the   relationship   between   muscle   activity   and   the   circadian   regulation   of   muscle   genes   is   a   relatively   new   area   of   research.   We   compared   the   circadian   transcriptomes   of   two   mouse   hind-­‐limb   muscles   with   vastly   different   circadian   activity   patterns,   the   continuously   active   slow   soleus   and   the   sporadically   active   fast   tibialis   anterior,   in   the   presence  or   absence  of   a   functional   skeletal  muscle   clock   (skeletal   muscle-­‐specific  *Bmal1*  KO).  In  addition,  we  compared  the  effect  of   denervation   on   muscle   circadian   gene   expression.   We   found   that   different  skeletal  muscles  exhibit  major  differences  in  their  circadian   transcriptomes,  yet  clock  gene  oscillations  were  essentially  identical   in   fast   and   slow   muscles.   Furthermore,   denervation   caused   relatively  minor  changes  in  circadian  expression  of  most  clock  genes,   yet   major   differences   in   expression   level,   phase   and   amplitude   of   many   muscle   circadian   genes.   Our   studies   suggest   that   a   major   physiological   role   of   the   skeletal   muscle   clock   is   to   prepare   the   muscle  for  the  transition  from  the  light/inactive/fasting  phase  to  the   dark/active/feeding  phase,  in  anticipation  of  periodic  fluctuations  in   fuel  supply  and  demand.   1. Dyar  KA,  Ciciliot  S,  Wright  LE,  et  al.  Muscle  insulin  sensitivity  and   glucose  metabolism  are  controlled  by  the  intrinsic  muscle  clock.   Mol   Metab   2014;3:857.   doi:   10.1016/j.molmet.2014.09.002.   eCollection  2014  Dec.   2. Dyar  KA,  Ciciliot  S,  Tagliazucchi  GM,  et  al.  The  calcineurin-­‐NFAT   pathway   controls   activity-­‐dependent   circadian   gene   expression   in   slow   skeletal   muscle.   2015;4:823-­‐33.   doi:   10.1016/j.molmet.2015.09.004.  eCollection  2015.   3. McCarthy  JJ,  Andrews  JL,  McDearmon,  et  al.  Identification  of  the   circadian  transcriptome   in  adult  mouse  skeletal  muscle.  Physiol   Genomics  2007;3:86-­‐95.  Epub  2007  Jun  5.   *****   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   39 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy       Workshop  “Diaphragm,  structure  and  function”   C  Leeuwenburgh  and  C  Reggiani,  Chairs   Bench  to  bedside  research  on  critical  illness  myopathy  (CIM)   and   ventilator   induced   diaphragm   muscle   dysfunction   (VIDD):  Underlying  mechanisms  and  intervention  strategies   Lars  Larsson   Department  of  Physiology  and  Pharmacology,  Department  of  Clinical   Neuroscience,   Clinical   Neurophysiology   section,   Karolinska   Institutet,  Stockholm,  Sweden   E-­‐mail:  lars.larsson@ki.se     Muscle  wasting   in   intensive  care  unit  (ICU)  patients  may  be  related   to   the   primary   disease,   but   there   is   heterogeneity   of   underlying   disease   and   pharmacological   treatment   among   patients   exhibiting   similar  outcomes.  Thus,  it  is  highly  likely  that  a  common  component   of   ICU   treatment   per   se   is   directly   involved   in   the   progressive   impairment  of  muscle  function  and  muscle  wasting  during  long-­‐term   ICU   treatment.   The   specific   mechanisms   underlying   the   muscle   wasting   and   impaired   muscle   function   associated   with   the   ICU   intervention  are  poorly  understood  in  the  clinical  setting.  1  This  is  in   part  due  to  heterogeneity  in  pharmacological  treatment,  underlying   disease,   clinical   history   etc.   There   is,   accordingly,   compelling   need   for  experimental  animal  models  closely  mimicking  the  ICU  condition,   including   long-­‐term   exposure   to   mechanical   ventilation   and   immobilization   (lack  of  weight  bearing  and  activation  of  contractile   proteins,   i.e.,   “mechanical   silencing”).   In   this   project,   the   muscle   dysfunction,  which   by   far   exceeds   the   loss   in  muscle  mass   in   limb   and   respiratory   muscles   in   patients   with   CIM2,3   and   VIDD,4   have   been   investigated   in  detail  at   the  cellular  and  molecular   levels   in  a   rodent   experimental   ICU   model,   allowing   detailed   studies   in   an   immobilized  and  a  mechanically  ventilated  rat  for  long  durations  (up   to  weeks-­‐months).    The  long-­‐term  scientific  goals  of  the  research  are   to:   (a)  define  the  causative  agents,   (b)  develop  sensitive,  quick  and   accurate   diagnostic   tools   and   monitoring   devices,   and   (c)   develop   efficient   intervention   strategies.  This  project,  which   focuses  on   the   mechanisms  underlying   the   severely   impaired   limb  and   respiratory   muscle   function   (CIM   and   VIDD)   in   response   to   long-­‐term   mechanical   ventilation   and   immobilization   and   the   introduction   of   specific   intervention  strategies,  constitutes  a  significant  component   of  the  attempt  to  achieve  these  long-­‐term  goals.   1.   Friedrich   O,   Reid   MB,   Van   den   Berghe   G,   Vanhorebeek   I,   Hermans   G,   Rich   MM,   Larsson   L..   The   Sick   and   the   Weak:   Neuropathies/Myopathies   in   the   Critically   Ill.   Physiol   Rev     2015;95:1025-­‐109.   2.   Larsson  L,   Li  X,  Edstrom  L,  et  al.  Acute  quadriplegia  and   loss  of   muscle   myosin   in   patients   treated   with   nondepolarizing   neuromuscular  blocking  agents  and  corticosteroids:  mechanisms   at   the   cellular   and  molecular   levels   [see   comments].   Crit   Care   Med  2000;28:34-­‐45.   3.   Ochala   J,   Gustafson   AM,   Diez   ML,   et   al.   Preferential   skeletal   muscle   myosin   loss   in   response   to   mechanical   silencing   in   a   novel   rat   intensive   care   unit   model:   underlying   mechanisms.   J   Physiol  2011;589:2007-­‐26.   4.   Corpeno  R,  Dworkin  B,  Cacciani  N,  et  al.  Time  course  analysis  of   mechanical   ventilation-­‐induced   diaphragm   contractile   muscle   dysfunction   in   the   rat.   Journal   of   Physiology-­‐London   2014;592:3859-­‐80.   *****       A  finite  element  approach  to  diaphragm  mechanics:   multiscale  modeling  from  fiber  to  whole  muscle   Lorenzo  Marcucci  (1,2),  Piero  Pavan  (2,3),  Luana  Toniolo  (1),  Lina   Cancellara  (1),  Arturo  Natali  (2,3),  Carlo  Reggiani  (1,2)   (1)   Department   of   Biomedical   Sciences,   University   of   Padova;   (2)   Center   for  Mechanics  of  Biological  Materials,  University  of  Padova;   (3)  Department  of  Industrial  Engineering,  University  of  Padova,  Italy   E-­‐mail:  carlo.reggiani@unipd.it   The   diaphragm   is   the   most   important   inspiratory   muscle.   It   has   a   thin,   dome-­‐shaped   structure,   and   separates   the   thoracic   and   abdominal   cavities,   mechanically   interacting   with   the   surrounding   organs   during   its   contractile   function.   Moreover,   its   muscle   fibers   have   a   complicated   geometry,   connecting   to   several   ribs,   lumbar   vertebral   bodies   and   to   the   central   tendon.   All   these   peculiar   features   make   it   a   very   challenging   organ   to   be   modeled   and   analyzed  by  means  of  finite  element  methods.  We  present  our  work   aimed  at  creating  a  finite  element  model  of  the  human  diaphragm.   A   computational   model   based   on   a   realistic,   potentially   patient   specific,   geometry,   may   help   in   the   understanding   of   diaphragm   related  pathologies,   such  as  chronic  obstructive  pulmonary  disease   (COPD),  mechanical   ventilation   (MV)   induced   diaphragm   inactivity,   amyotrophic   lateral   sclerosis   (ALS)   or   even   for   respiratory   tumor   motion   tracking   to   reduce   radiation   treatment   side  effects.  On   the   other   side,   several   diaphragm   pathologies   are   related   to   relevant   modifications   of   the   single   fiber   properties,   such   as   contractile   weakness,   atrophy,   or   reduced   cross   sectional   area.   Moreover   different  fiber  isoforms  differently  affect  the  whole  muscle  behavior.   In   the   design   of   our   model,   we   adopted   a   bottom-­‐up   approach,   trying  to  explain  the  whole  muscle  behavior  starting  from  the  single   fiber   characterization.   Therefore,  we   first   developed  a  model   for   a   single  fiber  based  on  a  three  elements  Hill's  model  which   is  able  to   reproduce   the   classic   protocols   for   the   force   velocity   curve   (isometric   contraction   followed   by   an   isotonic   contraction   at   different   external   forces)   as   well   as   the   slack-­‐test   protocols   (isometric   contraction   followed   by   a   rapid   shortening   imposed   to   the   fiber.  We   then   characterized   the  parameters   for   both   fast   and   slow  human  skeletal  fibers,  based  on  the  original  experimental  data   obtained  in  our  lab,  taking  into  account  the  effects  of  temperatures   as   single   fiber   mechanics   is   generally   studied   at   low   temperature   (12-­‐20°C).   The   characteristic  width  of   the  elements   in   the  mesh  of   the   diaphragm   is   several   tens   of   microns,   which  means   that   each   element   likely   include   several   slow   and   fast   fibers.   Then   a   characterization  of  the  mixed  fiber  bundles  is  needed.  We  therefore   characterized  a  mesh  describing  a  bundle  of  fast  and  slow  fibers  to   reproduce   the   mixing   effect   on   the   force   velocity   curve.   The   transversal   connections   linking   each   other   adjacent   fibers   were   taken   into   consideration.   The  mesh   for   the   bundles  was   based   on   histological  cross  sections  of  diaphragm,  which  can  be  found  in  the   literature   [1].   Finally,   we   included   our   results   in   a   3-­‐D   mesh   representing  the  human  diaphragm.  The  bottom-­‐up  approach  based   on  the  real  behavior  of  single  bundles  of  fibers,  is  particularly  useful   to  predict  how  different  pathologies  at  the  fiber  level  can  influence   the  whole  muscle  performance.   1. Hooijman   PE,   Beishuizen   A,   Witt   CC,   et   al.   Diaphragm   muscle   fiber  weakness  and  ubiquitin-­‐proteasome  activation   in  critically   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   40 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   ill   patients.   Am   J   Respir   Crit   Care  Med   2015;191:1126-­‐38.   doi:   10.1164/rccm.201412-­‐2214OC.   *****   Plasticity  of  mouse  diaphragm:  aging  and  response  to   endurance  training   Silvia  Quartesan  (1),  Lina  Cancellara  (1),  Sara  di  Siena  (2),  Cinzia   Calabrese  (2),  Fabio  Naro  (2)  Carlo  Reggiani  (1)   (1)   University   of   Padova,   Department   of   Biomedical   Science;   (2)   University   of   Roma   "La   Sapienza",   Department   of   Anatomy,   Histology,  Forensic  Medicine  and  Orthopedics,  Italy   E-­‐mail:  carlo.reggiani@unipd.it   All   skeletal   muscles   undergo   an   age-­‐dependent   loss   of   mass   and   functional  deterioration.  As   follow-­‐up  of   the   functional   impairment   of   lower   limb   muscles,   the   elderly   experience   limitations   in   locomotion   and   eventually   lose   the   possibility   of   independent   life.   Accordingly,   an   impairment   in   respiratory   function   is   the   expected   effect  of  the  age-­‐dependent  decline  of  respiratory  muscles.  In  spite   of  its  essential  role  in  breathing  the  age-­‐dependent  modifications  of   diaphragm   muscle   (DIAm),   which   is   the   main   inspiratory   muscle,   have  received  less  attention  than  limb  muscles  (Greising  et  al  2013,   Elliott   et   al   2015).   In   this   study   we   aimed   to   investigate   the   age-­‐ related  changes  of  the  mouse  diaphragm  and  the  possible  impact  of   moderate   endurance   training.   The   respiratory   function   in   mice   is   very   different   compared   to   humans.   Breathing   in  mice   as   in   other   small   mammals   is   characterized   by   very   high   frequency   (400-­‐ 500/min)   and   low   tidal   volume   (<0.5  ml/breath).   Thus,   the  murine   diaphragm   is   a   fast   muscle,   characterized   by   aerobic   metabolic   activity,   which   allows   it   to   avoid   fatigue.   Murine   diaphragm   is   particularly   susceptible   to   damage   when   dystrophin   or   other   proteins   linked   to   dystrophin   are   absent   (Stedman   et   al   1991,   Randazzo   et   al   2013,   Giacomello   et   al   2015).     In   spite   of   those   significant   differences,   murine   muscle   can   be   considered   a   useful   model   to   study  human  diaphragm.  Three  groups  of  healthy   female   CD1  mice  were  studied:  young  (Y,4  months),  old  (O,  20  months),  and   oldest-­‐old   (OO,   32   months).   In   each   group,   two   subgroups   were   formed:  sedentary  and  trained.  Training  protocol  included  moderate   intensity   running   on   treadmill   (13   cm/sec,   for   30   minutes)   5   days/week  for  8  weeks.  After  mice  sacrifice,  DIAm  was  dissected  and   immediately   stored   at   -­‐80°C.   Proteins  were   analysed  with  8%  SDS-­‐ PAGE   (Talmadge   and   Roy,   1993)   to   separate   myosin   heavy   chain   (MyHC)   isoforms  and  with  10%  SDS,   followed  by  Western  Blot  and   staining  with  antibody  specific  to  PLIN5,  TOM20  and  Actin.  RNA  was   prepared   for   Real   Time   PCR.   The   analysis   of   MyHC   isoforms   expression   revealed  a  predominance   (80-­‐90%)  of   fast   intermediate   isoforms  (2A  and  2X)  with  minor  components  of  slow/1  and  fast  2B   isoforms.  Aging  of  sedentary  mice  was  accompanied  by  an  increase   of  slow  MyHC  (from  4%  at  4  mo  to  9%  at  32  mo),  a  decrease  in  fast   intermediate   2A   and   2X   (from   91%   at   4   mo   to   85%   at   32   mo),   a   minor  variation  of  fast  2B  MyHC  (from  4%  to  6%).  Training  induced  a   very   small   fast-­‐to-­‐slow   shift   in  4  months  mice  and,   surprisingly,   an   opposite,   slow-­‐to-­‐fast   transition   in   32   mo   old   mice.   The   mitochondrial   density   evaluated   by   the   TOM20/actin   ratio   was   virtually   unchanged   in   the   sedentary   mice   of   the   3   age   groups.   Training  caused  opposite  effects  in  young  and  old  mice:  in  the  young   the   mitochondrial   density   increased   two   folds   in   trained   mice   compared   to   sedentary   controls,  whereas   in   the  old  mice,   training   caused   a   significant   reduction   in   mitochondrial   density.   The   presence  of  adipocytes  in  the  muscles  was  evaluated  using  PLIN5  as   a  marker.  The  concentration  of  PLIN5  showed  an  increase  with  age   (approximately   2   folds)   in   both   sedentary   and   trained   mice.   In   conclusion,   our   results   show   that   DIAm   undergoes   an   age-­‐ dependent   fast-­‐to-­‐slow   fibre   type   transition   as  most   of  muscles   of   the   mouse.   This   is   accompanied   by   a   preserved   mitochondrial   density.   Training   can   revert   the   age-­‐dependent   change   in   myosin   isoform   expression   but   is   not   able   to   induce   in   the   elderly   the   mitochondrial  biogenesis  which  can  be  observed  in  the  young   1. Elliott   JE,  Greising  SM,  Mantilla  CB,  Sieck  GC.  Functional   impact   of   sarcopenia   in   respiratory   muscles.   Respir   Physiol   Neurobiol   2015  Oct  20.  pii:  S1569-­‐9048   2. Giacomello   E,   Quarta   M,   Paolini   C,   et   al.   Deletion   of   small   ankyrin   1   (sAnk1)   isoforms   results   in   structural   and   functional   alterations   in   aging   skeletal   muscle   fibers.   Am   J   Physiol   Cell   Physiol  2015;308:C123-­‐38.     3. Greising   SM,   Mantilla   CB,   Gorman   BA,   Ermilov   LG,   Sieck   GC.   Diaphragm   muscle   sarcopenia   in   aging   mice.   Exp   Gerontol.   2013;48:881-­‐7.   4. Randazzo  D,  Giacomello  E,  Lorenzini  S,  et  al.  Obscurin  is  required   for  ankyrinB-­‐dependent  dystrophin  localization  and  sarcolemma   integrity.  J  Cell  Biol  2013;200:523-­‐36.     5. Stedman   HH,   Sweeney   HL,   Shrager   JB,   et   al.   The   mdx   mouse   diaphragm   reproduces   the   degenerative   changes   of   Duchenne   muscular  dystrophy.  Nature  1991;352(6335):536-­‐9.   6. Talmadge  RJ,  Roy  RR.   Electrophoretic   separation  of   rat   skeletal   muscle   myosin   heavy-­‐chain   isoforms.   J   Appl   Physiol   (1985).   1993;75:2337-­‐40.   *****   7-­‐day  denervation-­‐atrophy  is  also  absent  in  emi-­‐diaphragm   of  oldest  rats   Elena  Germinario  (1),  Barbara  Ravara  (1,2),  Valerio  Gobbo  (3),  Ugo   Carraro  (4),  Daniela  Danieli   (1)   Interdepartmental   Research   Center   of   Myology   (CIR-­‐Myo),   Department  of  Biomedical   Sciences,  University  of  Padova,   Italy;   (2)   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and   Physical   Rehabilitation,   Vienna,   Austria;   (3)   Institute   of   Neuroscience,   Consiglio   Nazionale   delle   Ricerche,   Padova   (Italy);   (4)   IRRCS   Fondazione  Ospedale  San  Camillo,  Venezia,  Italy   E-­‐mail:  daniela.danieli@unipd.it   Denervated   left   emi-­‐diaphragm   of   adult   rats   is   well   known   to   paradoxically   increase   in   weight   during   the   first   ten   days   after   denervation,   then   it   atrophy   as   any   other   denervated   muscle.1   Increased  activity  of  eIF-­‐2   initiation   factor  accounts  at   least   in  part   for  the  enhancement  of  protein  synthesis  in  2-­‐day  denervated  emi-­‐ diaphragm   of   adult   rats.2   Nothing   is   known   about   the   behavior   of   the   denervated   emi-­‐diaphragm   in   oldest   (30-­‐month)   rats.   We   compared   in   two   groups   of   four   animals   (3-­‐month   adults   vs   30-­‐ month   oldest   male   rats)   the   effect   on   functional   and   structural   properties  of  7-­‐day  denervation  in  the  diaphragm  and  in  denervated   EDL  and  Soleus  leg  muscles.  At  30-­‐month  the  body  weight  of  the  rats   is   high   significantly   heavier   than   that   of   the   3-­‐month   animals   (gr   289+/-­‐44  vs  401+/-­‐38,  mean+/-­‐SD,  p<  0.001).  Despite  the  increase  in   body  weight  the  weight  of  the  innervated  EDL,  and  SOL  muscles  are   lower   in   the   oldest   rats.   Equal   or   slightly   lower   are   also   sizes   of   muscle   fibers   in   30-­‐month   innervated   EDL,   SOL   and   diaphragm.   Contrastingly,   the  response  to  7-­‐day  denervation   is  muscle  related:   EDL  and  SOL  leg  muscles  show  a  15%  decrease  in  muscle  fiber  size,   while   the   muscle   fiber   of   denervated   left   emi-­‐diaphragm   are   indistinguishable   from   contralateral   innervated  muscle   in   3-­‐month   and  30-­‐month  rats.  Of  note  is  the  fact,  that  in  the  7-­‐day  denervated   left  emi-­‐diaphragm  of   the  oldest   rats   (30-­‐month)   slow-­‐type  muscle   fiber   present   a   5.7%   highly   significantly   increase   in   fiber   size,   and   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   41 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   that   in   two  out   of   the   four   rats   total  muscle   fiber   size   analyzed   in   semi-­‐thin   section   after   fixation   for   electron   microscopy   present   highly   significant   increase   (+   14.5   %)   when   compared   with   the   contralateral  innervated  emi-­‐diaphragm.  On  the  other  hand,  muscle   fibers  of  7-­‐day  denervated  EDL  and  SOL  of   the  same  rats  show  the   expected  decrease  in  size  (-­‐18.7  %). In  conclusion,  both  young  and   old   diaphragms   appear   to   be   unresponsive   to   7-­‐day   denervation   atrophy.   1.   Carraro  U,  Morale  D,  Mussini  I,  Lucke  S,  Cantini  M,  Betto  R,   Catani  C,  Dalla  Libera  L,  Danieli  Betto  D,  Noventa  D.  Chronic   denervation  of  rat  hemidiaphragm:  maintenance  of  fiber   heterogeneity  with  associated  increasing  uniformity  of  myosin   isoforms.  J  Cell  Biol  1985;100:161-­‐74.   2.   Carraro  U,  Catani  C.  eIF-­‐2  initiation  factor  activity  in   postribosomal  supernatant  of  hypertrophying  rat  diaphragm.   FEBS  Lett  1980;110:173-­‐6   3.   Ljubicic  V,  Joseph  AM,  Adhihetty  PJ,  Huang  JH,  Saleem  A,   Uguccioni  G,  Hood  DA.  Molecular  basis  for  an  attenuated   mitochondrial  adaptive  plasticity  in  aged  skeletal  muscle.  Aging   (Albany  NY).  2009;1:818–30.  Published  online  2009  Sep  12.     *****   Workshop  “Neuromuscular  adaptations  to   exercise  and  ES  in  aging”                                       Lee  Sweeney,  Marco  Sandri,  Chairs   CIR-­‐Myo  -­‐  Lecture  1   Autophagy  and  the  control  of  neuromuscular  junction   Marco  Sandri,  Padova,  Italy   Francesca  LoVerso  (1),  Silvia  Carnio  (1),  Rüdiger  Rudolf  (5),  Marco   Sandri  (1,2,3,4)   (1)   Venetian   Institute   of   Molecular   Medicine,   Padova,   Italy;   (2)   Department  of  Biomedical   Sciences,  University  of  Padova,   Italy;   (3)   Institute   of   Neuroscience,   Consiglio   Nazionale   delle   Ricerche,   Padova,   Italy.   (4)   Department   of   Medicine,   McGill   University,   Montreal,  Canada;  (5)  Institut  für  Toxikologie  und  Genetik,  Karlsruhe   Institute  of  Technology.   E-­‐mail:  marco.sandri@unipd.it     Neuromuscular  Junction  (NMJ)   is  the  synapse  that  connects  motor-­‐ neuron   with   skeletal   muscle   and   its   stability   is   critical   for   muscle   contraction.   Inherited   or   acquired   conditions   that   perturb   the   components   of   this   unit   lead   to   weakness,   denervation   and,   ultimately,  paralysis.1,2  The  signals  that  control  such  complex/critical   structure   in   adulthood   are   largely   unknown.   Here  we   show   that   a   retrograde  signal  is  released  from  adult  skeletal  muscles  to  maintain   a   stable   and   functional   NMJ.   An   efficient   autophagy   system   is   required   for  a   functional  NMJ  because   it   controls  MuSK  clustering3   Impairment   of   autophagy   or   inhibition   of   MuSK   leads   to   NMJ   dismantle  and  denervation.  These   results   identify  MuSK-­‐autophagy   axis  as  a  retrograde  signal  that  is  required  for  NMJ  maintenance  and   function  in  adulthood.   1.   Jang  YC,  Van  Remmen  H.  2011.  Age-­‐associated  alterations  of  the   neuromuscular  junction.  Exp  Gerontol  46:193-­‐198.   2.   Rudolf   R,   Khan   MM,   Labeit   S,   Deschenes   MR.   2014.   Degeneration  of   neuromuscular   junction   in   age   and  dystrophy.   Front  Aging  Neurosci  6:99.   3.   Carnio  S,  LoVerso  F,  Baraibar  MA,  Longa  E,  Khan  MM,  Maffei  M,   Reischl  M,   Canepari  M,   Loefler   S,   Kern  H,   Blaauw  B,   Friguet   B,   Bottinelli  R,  Rudolf  R,  Sandri  M.  2014.  Autophagy  impairment  in   muscle   induces   neuromuscular   junction   degeneration   and   precocious  aging.  Cell  Rep  8:1509-­‐1521.   *****   Autophagic  flux  of  CHRN  is  regulated  by  RAB5-­‐GTPase  and   T145  phosphorylation  level  of  SH3GLB1  at  mouse   neuromuscular  junction  in  vivo   Franziska  Wild  (1,2,3),  Muzamil  Majid  Khan  (1,2,3),  Rüdiger  Rudolf   (1,2,3)   (1)   Interdisciplinary   Center   for   Neuroscience,   University   of   Heidelberg,   Germany;   (2)   Institute   of   Molecular   and   Cell   Biology,   Mannheim  University   of  Applied   Science,  Germany;   (3)   Institute  of   Toxicology   and   Genetics,   Karlsruhe   Institute   of   Technology,   Germany   E-­‐mail:  r.rudolf@hs-­‐mannheim.de     Endolysosomal   carriers   containing   nicotinic   acetylcholine   receptors   (CHRN)   are   either   recycled   (1)   or   degraded   via   autophagy   during   atrophic  conditions  (2,  5).  However,  regulatory  processes  underlying   their   processing   have   remained   elusive.   We   have   recently   shown   that   internalized   CHRN   are   accompanied   by   TRIM63   (MuRF1)   and   SH3GLB1   (3,   4).   Here  we   further   dissected   the   role   of   SH3GLB1   in   regulating   the   autophagic   flux   of   CHRN.   Upon   sciatic   denervation,   we   found   a   tight   regulation   of   SH3GLB1/phosphorylated-­‐T145   (p-­‐ T145)   SH3GLB1   protein   amounts.   While   p-­‐T145   SH3GLB1   levels   remained   constant   upon   induction   of   muscle   atrophy,   the   non-­‐ phosphorylated  SH3GLB1  protein  was   increased.  Overexpression  of   a   T145  phosphomimetic  mutant   (T145E)   of   SH3GLB1   slowed  down   the   processing   of   CHRN   endolysosomes   while   as   T145   phosphodeficient  mutant   (T145A)   of   SH3GLB1   strongly   augmented   it.   The   slow   processing   of   CHRN   endolysosomes   brought   about   by   T145E   was   rescued   upon   co-­‐expression   of   the   early   endosomal   orchestrator  RAB5,  suggesting  a  role  of  SH3GLB1  in  regulating  CHRN   endocytic   trafficking   at   steps   upstream   of   autophagosome   formation.   1.   Bruneau   E,   Sutter   D,   Hume   RI,   Akaaboune  M.   Identification   of   nicotinic   acetylcholine   receptor   recycling   and   its   role   in   maintaining   receptor   density   at   the   neuromuscular   junction   in   vivo.  J  Neurosci  2005;25:9949–59.   2.     Carnio   S,   LoVerso   F,   Baraibar   MA,   Loet   al.   Autophagy   Impairment   in   Muscle   Induces   Neuromuscular   Junction   Degeneration  and  Precocious  Aging.  Cell  Rep  2014;8:1509–21.   3.     Khan  MM,   Strack   S,  Wild   F,   et   al.   Role   of   autophagy,   SQSTM1,   SH3GLB1,  and  TRIM63  in  the  turnover  of  nicotinic  acetylcholine   receptors.  Autophagy  2014;10:123–36,.   4.     Rudolf  R,  Bogomolovas   J,  Strack  S,  et  al.  Regulation  of  nicotinic   acetylcholine   receptor   turnover   by   MuRF1   connects   muscle   activity   to   endo/lysosomal   and   atrophy   pathways.   Age   (Dordr)   2013;35:1663–74,.   5.     Rudolf   R,   Khan  MM,   Labeit   S,   Deschenes  MR.   Degeneration   of   Neuromuscular   Junction   in   Age   and   Dystrophy.   Front   Aging   Neurosci  2014;6:99.   *****   CK2-­‐dependent  phosphorylation  in  skeletal  muscle   regulates  neuromuscular  junction  stability  and   mitochondrial  homeostasis   Said  Hashemolhosseini   Institut   für   Biochemie,   Friedrich-­‐Alexander   Universität   Erlangen-­‐ Nürnberg,  Erlangen,  Germany   E-­‐mail:  said.hashemolhosseini@fau.de   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   42 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Protein   kinase   CK2,   a   pleiotropic   serine/threonine   kinase,   plays   an   important  role  in  many  different  biological  processes  inside  of  cells.1   Conditional  muscle-­‐specific  CK2  mutant  mice   lack  grip  strength  and   show  muscle   fatigability.2  We   identified   the   role   of   CK2   in   skeletal   muscle  cells  as  a   regulator  of  neuromuscular   junction  maintenance   by   phosphorylation   of   different   protein   members   of   the   postsynaptic   apparatus.2,3   Moreover,   CK2   is   involved   in   ensuring   proper  mitochondrial  homeostasis   in  skeletal  muscle  fibers  by  fine-­‐ tuning  mitochondrial  protein   import  through  the  translocase  of  the   mitochondrial   outer   membrane.   In   absence   of   CK2-­‐dependent   phosphorylation   of   a   mitochondrial   outer   membrane   translocase   protein,   muscle   fibers   undergo   accelerated   mitophagy,   as   demonstrated  by  an  up-­‐regulated  PINK/Parkin/p62  pathway.   1.   Cozza   G,   Pinna   LA,  Moro,   S.   Kinase   Ck2   inhibition:   An   update.   Current  medicinal  chemistry.  2012;20:671-­‐93.   2.   Cheusova   T,   Khan   MA,   Schubert   SW,   et   al.   Casein   kinase   2-­‐ dependent   serine   phosphorylation   of   MuSK   regulates   acetylcholine   receptor   aggregation   at   the   neuromuscular   junction.  Genes  Dev  2006;20:1800-­‐16.   3.   Herrmann  D,  Straubinger  M,  Hashemolhosseini  S.  Protein  Kinase   CK2  Interacts  at  the  Neuromuscular  Synapse  with  Rapsyn,  Rac1,   14-­‐3-­‐3gamma,   and   Dok-­‐7   Proteins   and   Phosphorylates   the   Latter  Two.  J  Biol  Chem  2015;290:22370-­‐84.   *****   Spatial  distribution  of  neuromuscular  junction  components.   Pretzels  are  more  complex  than  we  have  anticipated   Tomasz  Prószyński   Laboratory   of   Synaptogenesis,   Department   of   Cell   Biology,   Nencki   Institute  of  Experimental  Biology,  Warsaw,  Poland   E-­‐mail:  t.proszynski@nencki.gov.pl     Mammalian   neuromuscular   junctions   (NMJs)   undergo   a   postnatal   topological   transformation   from  a  simple  oval  plaque  to  a  complex   branch-­‐shaped   structure   often   called   a   “pretzel”.   Although   abnormalities   in   NMJ   maturation   and/or   maintenance   are   frequently  observed  in  neuromuscular  disorders,  such  as  congenital   myasthenic   syndromes   (CMSs),   the   mechanisms   that   govern   synaptic   developmental   remodeling   are   poorly   understood.   It   was   reported  that  myotubes,  when  cultured  aneurally  on  laminin-­‐coated   surfaces,   form   complex   postsynaptic   machinery,   which   resembles   that   at   the   NMJ.   Interestingly,   these   assemblies   of   postsynaptic   machinery   undergo   similar   stages   in   developmental   remodeling   from   “plaques”   to   “pretzels”   as   those   formed   in   vivo.   We   have   recently   demonstrated   that   podosomes,   actin-­‐rich   adhesive   organelles,   promote   the   remodeling   process   in   cultured  myotubes   and   showed   a   key   role   of   one  podosome   component,   Amotl2.  We   now   provide   evidence   that   several   other   known   podosome-­‐ associated  proteins  are  present  at   the  NMJ   in  vivo  and  are   located   to   the   sites   of   synaptic   remodeling.   Additionally,   we   identified   proteins  that  interact  with  Amotl2  in  muscle  cells.  We  show  that  two   of   them:   Rassf8   and   Homer1,   together   with   other   podosome   components,  are  concentrated  at  postsynaptic  areas  of  NMJs  in  the   indentations   between   the   AChR-­‐rich   branches.  Our   results   provide   further   support   for   the   hypothesis   that   podosome-­‐like   organelles   are   involved   in   synapse   remodeling   and   that   Rassf8   and   Homer1   may  regulate  this  process.   This   research   was   supported   by   grants   2012/05/E/NZ3/00487,   2013/09/B/NZ3/03524   and   2014/13/B/NZ3/00909   from   the   National  Science  Centre  (NCN).   1.   Proszynski   TJ,   Gingras   J,   Valdez   G,   Krzewski   K,   Sanes   JR.   Podosomes   are   present   in   a   postsynaptic   apparatus   and   participate   in   its   maturation.   Proc   Natl   Acad   Sci   U   S   A   2009;106:18373-­‐8.   2.     Proszynski  TJ,  Sanes   JR,  Amotl2   interacts  with  LL5β,   localizes   to   podosomes  and  regulates  postsynaptic  differentiation  in  muscle.   J  Cell  Sci.  2013;126(Pt  10):2225-­‐35.   3.   Bernadzki   KM1,   Rojek   KO,   Prószyński   TJ.   Podosomes   in  muscle   cells   and   their   role   in   the   remodeling   of   neuromuscular   postsynaptic  machinery.  Eur  J  Cell  Biol  2014;93:478-­‐85.   4.   Kummer   TT,   Misgeld   T,   Lichtman   JW,   Sanes   JR.   Nerve-­‐ independent   formation  of  a   topologically   complex  postsynaptic   apparatus.  J  Cell  Biol  2004;164:1077-­‐87.   *****   Mitochondrial  Alarmins  are  key  intercellular  signals  in  the   degeneration  &  regeneration  of  the  neuromuscular  junction   Michela  Rigoni  (1),  Elisa  Duregotti  (1),  Samuele  Negro  (1),  Michele   Scorzeto  (1),  Irene  Zornetta  (1),  Bryan  C.  Dickinson  (2),  Christopher   J.  Chang  (2),  Cesare  Montecucco  (1)   (1)  Department  of  Biomedical   Sciences,  University  of  Padova,   Italy;   (2)   Department   of   Chemistry   and   Molecular   and   Cell   Biology,   University  of  California,  Berkeley,  USA   E-­‐mail:  cesare.montecucco@gmail.com     The  neuromuscular  junction  is  one  of  the  few  human  tissues  capable   of   complete   regeneration   after  major   damages.  We   have   set   up   a   reliable  model   of   acute   degeneration   of   the  motor   axon   terminals   followed   by   complete   recovery   of   function.2   We   have   found   that   alarmins   released   by   mitochondria   of   the   degenerating   nerve   terminal  are  key  factor  that  act  on  the  perisynaptic  cells  and  muscle   fibre.2   These   cells   are   activated   and   release   signals   that   act   retrogradely  on  the  nerve  terminal   inducing   its  regeneration.  Some   of   these   signals   are   currently   being   investigated   by   imaging   and   transcriptomics  methods.   1.   Sanes   M   &   Lichtman   JW.   Development   of   the   vertebrate   neuromuscular  junction.  Annu  Rev  Neurosci  1999;22:389-­‐442.   2.   Duregotti   E,   Negro   S,   Scorzeto   M,   Zornetta   I,   Dickinson   BC,   Chang   CJ,   Montecucco   C,   Rigoni   M.   Mitochondrial   alarmins   released   by   degenerating   motor   axon   terminals   activate   perisynaptic   Schwann   cells.   Proc   Natl   Acad   Sci   U   S   A   2015:112:497-­‐505.   *****     Introduction  by  Gerta  Vrbova,  London,  Uk     Comparison  of  electrical  stimulation  to  exercise,       Gerta  Vrbova,  Dept  of  Life  Sciences  UCL,  London,  UK       E-­‐mail:  g.vrbova@ucl.ac.uk   Two   fundamental   differences   exist   between   voluntary   muscle   contractions   and   those   induced   by   electrical   stimulation   .   During   voluntary  movements  motor  units  are  activated  asynchronously  and   a  strict  hierarchical  order  of  recruitment  is  always  maintained  where   the  smallest  motor  units  are  activated  first  followed  by  contractions   of   larger  units.  Thus  during  voluntary  movement   the   largest  motor   units  are   least  active  and  are  used  only  during  maximal  effort.  This   order  of  recruitment   is  cancelled  when  electrical  stimulation  of  the   muscle   is   used;   indeed   due   to   the   biophysical   properties   of   the   axons   that   innervate   the   muscle   the   largest   motor   units   are   activated  preferentially   and   therefore   the  parts   of   the  muscle   that   are   usually   used   rarely   are   active   most   frequently.   Thus   during   electrical   stimulation   it   is   the   motor   units   that   are   normally   least   active   that   experience   the   biggest   increase   in   their   use   and   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   43 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   consequently   the   biggest   change   in   their   characteristic   properties.   Thus   electrical   stimulation   by   bypassing   the   hierarchical   order   of   recruitment,   indeed  by  reversing   it,   is  able   to  activate   those  motor   units   and   muscle   fibres   that   can   only   be   activated   during   most   strenuous  exercise.  It  can  therefore  exploit  the  adaptive  potential  of   muscle   more   efficiently   then   exercise   and   maintain   much   higher   levels   of   activity   over   time   then   exercise.   This   enhanced   activity   is   restricted   to   specific   target   muscles   and   is   unlikely   to   have   unwanted  systemic  effects.  Finally,  high  amounts  of  activity  can  be   imposed   on   a   muscle   from   the   beginning,   since   the   CNS,   cardiovascular   and   other   systems   will   not   interfere   or   limit   the   amount  of  activity   carried  out  by   the  muscle,   as   is   the   case  during   exercise   (see   for   review   Pette   and   Vrbová,   1999).1   On   the   other   hand,   there   are   several   functions   that   electrical   stimulation   of   individual  muscle  groups  cannot  accomplish  and  that  are  unique  to   exercise   induced   activity.   During   exercise-­‐induced   activity   coordinated  movement   is   carried  out  and   it   is   therefore   likely   that   the   individual’s   skills   in   carrying   out   movement   of   this   kind   will   improve.   Thus,   while   exercise   can   improve   coordination,   electrical   stimulation   is   unlikely   to   do   so.   In   addition   the   flexibility   of   joints   and  lengthening  of  muscles  can  be  improved  by  exercise  but  not  by   electrical  stimulation.  Particular  exercise  regimes  such  as  Pilates  and   yoga  are  particularly  effective  in  achieving  these  goals.  Improvement   of   the   cardiovascular   system   is   also   easier   achieved   by   exercise.   Nevertheless,   it   can   be   argued   that   having   muscles   that   are   less   fatiguable   than   usual,   an   advantage   that   is   readily   achieved   by   electrical   stimulation,   enables   the   individual   to   exercise   more   efficiently   and   achieve   all   the   goals   regarding   fitness  more   readily   and  in  a  shorter  time.   1.   Pette   D,   Vrbová   G.   What   does   chronic   electrical   stimulation   teach   us   about   muscle   plasticity?   Muscle   Nerve.   1999;22:666-­‐ 77.  Review.   CIR-­‐Myo  -­‐  Lecture  2   Difference  in  muscular  adaptation  due  to  regular   exercise  or  through  electrical  stimulation   Christiaan  Leeuwenburgh     University  of  Florida,  Gainesville,  FL,  US   E-­‐mail:  "Leeuwenburgh,Christiaan"  cleeuwen@ufl.edu     Regular   exercise   and   neuromuscular   electrical   stimulation   (NMES)   have   been   used   in   a   variety   of   settings   for   different   populations.   Various   modes   of   exercise   (eccentric,   concentric,   resistance   and   aerobic)  and  NMES  training  regimes  (localization,  intensity,  duration,   frequency)   exist   for   healthy   subjects   and   athletes,   patients   in   a   variety  of  rehabilitation  and  preventive  settings,  either  partially  or  in   totally   immobilized   subjects.   Both   standard   exercise   interventions   and   NMES   have   been   shown   to   be   effective   in   preventing   the   decrease   in  muscle  strength,  muscle  mass  and  the   loss   in  oxidative   capacity   of   skeletal   muscles   following   multiple   types   of   surgical   (orthopedic)   procedures.     However,   it   is   still   not   entirely   clear   whether   biological   adaptations   are   similar   and   the   duration   differences   in   their   adaptation   duration.  We   will   discuss   potential   biological  differences  in  adaption  at  the  neuromuscular  junction  and   potential   differences   in   bioenergetics   adaptation.   Future   research   needs   to   determine   potential  molecular   differences   and   beneficial   post  adaptation  differences.   1.   Ahn  B,  Beaver  T,  Martin  T,  Hess  P,  Brumback  BA,  Ahmed  S,  Smith   BK,   Leeuwenburgh   C,   Martin   AD,   Ferreira   LF   Phrenic   nerve   stimulation   increases   human   diaphragm   fiber   force   after   cardiothoracic   surgery..   Am   J   Respir   Crit   Care   Med   2014;42:e152-­‐6.  doi:  10.1097/CCM.0b013e3182a63fdf.   2.   Martin  AD,  Joseph  AM,  Beaver  TM,  Smith  BK,  Martin  TD,  Berg  K,   Hess   PJ,   Deoghare   HV,   Leeuwenburgh   C.   Effect   of   intermittent   phrenic   nerve   stimulation   during   cardiothoracic   surgery   on   mitochondrial   respiration   in   the   human   diaphragm.   Crit   Care   Med  2014;42:e152-­‐6.  doi:  10.1097/CCM.0b013e3182a63fdf.   3.   Powers   SK,   Ji   LL,   Leeuwenburgh   C.   Exercise   training-­‐induced   alterations  in  skeletal  muscle  antioxidant  capacity:  a  brief  review   Med  Sci  Sports  Exerc. 1999;31:987-­‐97.   4.   Joseph  AM,  Adhihetty  PJ,  Leeuwenburgh  C.  Beneficial  effects  of   exercise  on  age-­‐related  mitochondrial  dysfunction  and  oxidative   stress   in   skeletal   muscle.   J   Physiol.   2015. Oct   27.   doi:   10.1113/JP270659.  [Epub  ahead  of  print]   *****   FRIDAY  April  15,  2016   Villa  dei  Vescovi,  Luvigliano  (Padova),  Italy     Introduction  by  Carlo  Reggiani,  Padova,  Italy     CIR-­‐Myo  –  Lecture  3     Therapeutic  Targets  for  Muscular  Dystrophy     Lee  Sweeney,  University  of  Florida,  Gainesville,  US   H.  Lee  Sweeney,  Margaret  M.  Sleeper,  Sean  C.  Forbes,  Ai  Shima,   Glenn  A.  Walter,  and  David  W.  Hammers   Myology  Institute,  University  of  Florida,  Gainesville,  FL,  US   E-­‐mail:  “Lee  Sweeney"  lsweeney@mail.med.upenn.edu     Duchenne  muscular  dystrophy  (DMD)  is  caused  by  loss  of  the  force   transmitting  and  membrane  complex  organizing  protein,  dystrophin,   and  is  characterized  by  progressive  muscle  deterioration  with  failed   regeneration   and   replacement   with   a   fatty-­‐fibrous   matrix.1   Dystrophin  replacement   therapies   to  date  have  shown  only   limited   ability   to  slow  the  disease  process,  and  thus   therapeutics   targeting   other  aspects  of  the  disease,  which  can  be  used  in  combination,  are   needed.   One   potential   target   is   nuclear   factor   κB   (NFkB,   which   is   upregulated  in  the  DMD  muscles.2  We  examined  a  novel  class  of  NF-­‐ kB  inhibitors  in  mdx  mouse  and  golden  retriever  muscular  dystrophy   (GRMD)  dog  models  of  DMD.  These  orally  bioavailable   compounds   improved   the   phenotype   of   voluntarily   run  mdx  mice,   in   terms   of   amount   of   activity,   muscle   mass   and   function,   inflammation,   and   fibrosis.   Surprisingly,   the   muscles   were   also   more   resistant   to   contraction-­‐induced   damage,   which   we   demonstrated   was   significant   increases   in   dysferlin,   a   protein   required   for  membrane   damage   repair.   We   also   evaluated   the   cardiac   impact   of   a   phosphodiesterase   5   (PDE5)   inhibitor,   tadalafil,   which   has   been   shown  to   improve  blood   flow   in  exercising  skeletal  muscles   in  mdx   mice  and  human  DMD  patients.3  Cardiomyopathy  is  a  leading  cause   of  mortality  among  DMD  patients  and  is  well  modeled  by  the  golden   retriever   muscular   dystrophy   (GRMD)   dog   model   of   DMD.   Prophylactic   use   of   the   PDE5   inhibitor,   tadalafil,   improved   GRMD   histopathological   features   of   the   hearts,   decreased   levels   of   the   pathogenic   cation   channel   TRPC6,   increased   phosphorylation   of   TRPC6,   decreased  m-­‐calpain   levels   and   indicators   of   calpain   target   proteolysis,   and   elevated   levels   of   the   dystrophin   ortholog,   utrophin.   The   progressive   loss   of   cardiac   function  was   significantly   slowed  by   these  effects.  These  data  demonstrate   that  prophylactic   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   44 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   use   of   tadalafil   delays   the   onset   of   dystrophic   cardiomyopathy,   which   is   likely   attributed   to   modulation   of   TRPC6   levels   and   permeability  and   inhibition  of  protease  content  and  activity,  which   results   in   higher   levels   of   the   protective   protein,   utrophin.   Thus   PDE5   inhibition   and   NF-­‐kB   inhibition   are   potential   therapeutics   to   consider  in  developing  a  combinatorial  approach  to  the  treatment  of   DMD.   1.   Forbes   SC,  Willcocks   RJ,   Triplett  WT   et   al.  Magnetic   resonance   imaging   and   spectroscopy   assessment   of   lower   extremity   skeletal  muscles   in   boys  with  Duchenne  muscular   dystrophy:   a   multicenter  cross  sectional  study.  PLoS  One  2014;9:e106435.   2.   Acharyya   S,   Villalta   SA,   Bakkar   N,   et   al.   Interplay   of   IKK/NF-­‐ kappaB   signaling   in   macrophages   and   myofibers   promotes   muscle   degeneration   in   Duchenne   muscular   dystrophy.   J   Clin   Invest  2007;117,889-­‐901.   3.   Nelson   MD,   Rader   F,   Tang   X,   et   al.   PDE5   inhibition   alleviates   functional   muscle   ischemia   in   boys   with   Duchenne   muscular   dystrophy.  Neurology.  2014;82:2085-­‐91.   *****   10.00    Mobility  impairments  in  aging  and  myopathies,  I   Christiaan  Leeuwenburg,  Ugo  Carraro  Chairs     Handling  growth  and  glucose  through  muscle  IGF-­‐I   Elisabeth  Barton   University  of  Florida,  Gainesville,  FL,  US   E-­‐mail:  “Barton,Elisabeth  R"  erbarton@ufl.edu     IGF-­‐I  and  insulin  are  intrinsically  connected  through  their  actions  on   the   IGF-­‐I   and   insulin   receptors   to   regulate   blood   glucose   (1).   Reduced  circulating  IGF-­‐I  can  be  compensated  by  heightened  insulin,   but   chronically   elevated   insulin   can   lead   to   insulin   resistance   and   ultimately   diabetes   .   Further,   increased   circulating   or   local  muscle   IGF-­‐I  may  enhance  glucose  uptake.   If   IGF-­‐I   from  muscle  and   liver   is   equivalent,   then   loss   of   muscle   IGF-­‐I   should   result   in   a   similar   pathologic   diabetic   state   (2).   By   extension,   if   muscle   IGF-­‐I   is   elevated,   it   may   serve   a   protective   role   in   glucose   homeostasis,   either   through   increased  muscle  mass   providing   a   greater   glucose   sink  (3),  or  through  increased  hybrid  receptor  activation  by  IGF-­‐I  (4).   To   address   the   impact   that   these   factors   have   on  metabolism,  we   elevated   IGF-­‐I   by   local   AAV-­‐IGF-­‐I   injections   into   both   hindlimbs   of   adult  male  mice  (5),  and  reduced  muscle  IGF-­‐I  through  an  inducible   muscle   specific   deletion   of   Igf1.    Mice  were   subjected   to   tests   for   body   composition,   glucose   uptake,   and   energy   expenditure   compared   to   age-­‐matched   controls.   It   was   not   surprising   that   the   hindlimb   injections   boosting   IGF-­‐I   levels   only   in   a   small   group   of   muscles  did  not  alter   the  whole  animal  body  composition.  Further,   when   mice   were   subjected   to   treadmill   running   for   60   minutes,   there   were   no   significant   changes   in   blood   levels   of   glucose   or   lactate  pre-­‐  or  post-­‐exercise.  While   increased  muscle  mass  did  not   appear  to  alter  basal  glucose  uptake,  increased  IGF-­‐I  content  altered   contraction   induced   glucose   uptake   in  muscle  when   normalized   to   mass.   To  understand   the   consequences  of   diminished  muscle   IGF-­‐I   production,  we  generated  mice  with   inducible  muscle  specific   IGF-­‐I   deletion,  with   induction   in  adult  mice.   In  mice  with  muscle  specific   deletion   of   Igf1,     glucose   levels   following   uphill   treadmill   running   increased   by   10%,   in   contrast   to   controls   where   blood   glucose   decreased  by  ~40%,   supporting   that   glucose   clearance   is  mediated   in   part   through   muscle   IGF-­‐I.     Further,   a   marked   impairment   of   glucose   clearance   occurred   following   a   simple   glucose   tolerance   test.   Based   on   these   results,  we   assert   that   the   IGF-­‐I   produced   by   the  muscle  has  an  endocrine  function,  and  like  IGF-­‐I  produced  by  the   liver,   modulation   of   muscle   levels   of   IGF-­‐I   will   lead   to   changes   in   glucose  homeostasis.   1.   Clemmons  DR.  Metabolic   actions   of   insulin-­‐like   growth   factor-­‐I   in  normal  physiology  and  diabetes.  Endocrinol  Metab  Clin  North   Am  2012;41:425-­‐43.   2.   Yakar   S,   Liu   JL,   Fernandez   AM,   et   al.   Liver-­‐specific   igf-­‐1   gene   deletion   leads   to   muscle   insulin   insensitivity.   Diabetes   2001;50:1110-­‐8.   3.   Musarò   A,   McCullagh   K,   Paul   A,   Houghton   L,   Dobrowolny   G,   Molinaro  M,  Barton  ER,  Sweeney  HL,  Rosenthal  N.  Localized  Igf-­‐1   transgene  expression   sustains  hypertrophy  and   regeneration   in   senescent  skeletal  muscle.  Nat  Genet  2001;27:195-­‐201.   4.   Entingh-­‐Pearsall  A,  Kahn  CR.  Differential  roles  of  the  insulin  and   insulin-­‐like   growth   factor-­‐I   (IGF-­‐I)   receptors   in   response   to   insulin  and  IGF-­‐I.  J  Biol  Chem  2004;279:38016-­‐24.   5.   Barton   ER.   Viral   expression   of   insulin-­‐like   growth   factor-­‐I   isoforms  promotes  different  responses  in  skeletal  muscle.  J  Appl   Physiol  2006;100:1778-­‐84.   *****   Therapeutic  approach  through  liposomes  carrying   antioxidant  in  skeletal  muscle   Rosa  Mancinelli  (1),  Simone  Guarnieri  (1),  Alessio  Rotini  (1),  Viviana   Moresi  (2),  Cecilia  Bombelli  (3),  Simona  Sennato  (4),  Stefania  Fulle  (1)   (1)   Dept.   of   Neuroscience,   Imaging   and   Clinical   Sciences,   “G.   d’Annunzio”   University,   Chieti-­‐Pescara;   (2)   Dept.   of   Anatomy,   Histology,   Forensic   Medicine   and   Orthopedics,   “La   Sapienza”   University,   Rome;   (3)  Dept.   of   Chemistry,   “La   Sapienza”  University,   Rome;  (4)  Dept.  of  Physics,  “La  Sapienza”  University,  Rome,  Italy   E-­‐mail:  "Rosa  Mancinelli"  r.mancinelli@unich.it     Sarcopenia   is   the   age-­‐related   loss   of   muscle   mass,   strength   and   function  leading  to  loss  of  muscle  power,  which  in  the  end  results  in   frailty   and   disability.   At   molecular   level,   sarcopenia   is   a   complex   condition   characterized   by   insufficient   antioxidant   defense   mechanism,   increased   oxidative   stress   and   altered   function   of   respiratory  chain  (Fulle  S.,  2005).   It  has  been  hypothesized  that  the   accumulation   of   oxidative   stress   is   also   related   to   an   impaired   regeneration   cooperating   to   the   atrophic   state   that   characterizes   muscle  ageing  (Beccafico  S.,  2007).  To  the  purpose,  we  investigated   the   myogenic   process   in   satellite   cells,   the   skeletal   muscle   stem   cells,   as   myoblasts   and   myotubes   collected   by   human   Vastus   Lateralis  skeletal  muscle  of  young  and  old  subjects  through  needle-­‐ biopsies  (Pietrangelo  T.,  2011).  NBT  and  H2DCF-­‐DA  assays  were  used   to  measure   O2•-­‐   and   ROS   production,   respectively.   Data   revealed   that   oxidant   species   are   more   concentrated   in   elderly   myoblasts   compared  to  young  ones.    To  evaluate  if  mitochondria  are  affected   by   ROS   using   JC-­‐1   assay   we   found   that   in   elderly   myoblasts   mitochondrial  transmembrane  potential  decreases  much  more  than   in  young  ones  probably  due  to  their   lower  endogenous  antioxidant   abilities.  Furthermore,  MitoSOX™  Red  reagent  was  used  to  measure   directly   O2•-­‐   in  mitochondria.  We   found   that   in   elderly  myoblasts   O2•-­‐  production  is  increased  respect  to  young  ones  and  the  result  is   worsened   in   myotubes.   Gene   expression   analysis   revealed   that   genes  involved  in  atrophic  and  ubiquitin-­‐proteasome  pathways  were   upregulation   together   with   the   dysregulation   of   the   proliferative   one   suggesting   an   alteration   at   gene   expression   level   in   elderly   myoblasts   vs   young   ones   (Pietrangelo   T.,   2009).   In   an   attempt   to   ameliorate   muscle   regeneration   in   elderly   mitochondrion-­‐specific   liposomes   carrying   antioxidant   were   synthetized.   The   toxicity   of   liposomes  were   tested   on   human   satellite   cells   and   C2C12   cells,   a   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   45 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   murine   skeletal   muscle   cell   line.   Preliminary   results   demonstrated   that  liposomes  made  using  DPPC  97.5%/BOLA  2.5%  gave  the  lowest   toxicity  at  24-­‐48-­‐72  hours.  Overall,  if  we  need  more  data  and  further   analysis,   up   to   day   our   data   suggest   that   oxidative   stress   impairs   muscle  regeneration  in  elderly  subjects.   1.   Fulle   S, Di   Donna   S,   Puglielli   C,   Pietrangelo   T.   Age-­‐dependent   imbalance   of   the   antioxidative   system   in   human   satellite   cells.   Exp  Gerontol  2005;40:189-­‐97.   2.   Beccafico  S,  Puglielli  C,  Pietrangelo  T,  Bellomo  R,  Fanò  G,  Fulle  S.   2007,   Age-­‐dependent   effects   on   functional   aspects   in   human   satellite  cells.  Ann  N  Y  Acad  Sci.  1100:345-­‐52.   3.   Pietrangelo  T,  D'Amelio  L,  Doria  C,  Mancinelli  R,  Fulle  S,  Fanò  G.   2011,  Tiny  percutaneous  needle  biopsy:  An  efficient  method  for   studying   cellular   and   molecular   aspects   of   skeletal   muscle   in   humans.  Int  J  Mol  Med  27:361-­‐67.  doi:  10.3892/ijmm.2010.582.   4.   Pietrangelo  T,  Puglielli  C,  Mancinelli  R,  et  al.  Molecular  basis  of   the   myogenic   profile   of   aged   human   skeletal   muscle   satellite   cells  during  differentiation.  Exp  Gerontol  2009;44:523-­‐31.   *****   Molecular  determinants  of  proteins  localization  to  triadic   junction   Enea  Liguori,  Daniela  Rossi,  Vincenzo  Sorrentino   (1)   Molecular   Medicine   Section,   Department   of   Molecular   and   Developmental  Medicine,  University  of  Siena,  Italy   E-­‐mail:  vincenzo.sorrentino@unisi.it     The   sarcoplasmic   reticulum   (SR)   is   organized   in   longitudinal   and   junctional   SR   (j-­‐SR).   In   skeletal  muscle,   this   latter   domain   together   with   the   T-­‐tubules   form   specific   junctions   called   triads,   where   proteins   regulating   the   excitation-­‐contraction   coupling   mechanism   assemble.   Junctophilins   (JPs)   are  directly   involved   in   the   formation   and  maintenance  of  triads.  Basically,  they  are  anchored  to  the  SR  via   their   C-­‐terminal   transmembrane   domain   (TMD),   while   their   N-­‐ terminus   contains   eight   MORN   motifs,   which   associate   with   the   phospholipids  of   the  T-­‐tubules.  Nevertheless,  how   JPs  are   targeted   to   triads   is   not   known.   The   roles   of   the   N-­‐terminal   and   the   C-­‐ terminal  regions  of  JP1  in  this  process  were  investigated.  Expression   in  primary  myotubes  and/or  muscle   fibers  of   JP1  deletion  mutants   lacking  the  TMD  resulted  in  protein  distribution  at  both  the  surface   sarcolemma   and   the   T-­‐tubules,   confirming   that   MORN   motifs   are   involved   in   JP1   interaction   with   the   sarcolemma,   but   are   not   sufficient   to   restrict   its   localization   at   the   T-­‐tubules.   On   the   other   hand,  progressive  deletion  of  the  eight  MORN  motifs  or  even  of  the   entire   cytosolic   region,   did   no   affect   JP1   localization   at   triads,   indicating   that   the   presence   of   the   TMD   is   sufficient   for   JP1   localization   at   the   j-­‐SR.   FRAP   analysis   performed   on   a   GFP-­‐TMD   fusion  protein  expressed  in  myotubes  indicated  that  this  protein  has   a   high   mobility,   suggesting   the   absence   of   strong   protein-­‐protein   interactions  occurring  at  the   j-­‐SR.  Further  work   is  needed  to  better   understand   the   molecular   mechanisms   driving   TMD-­‐mediated   JP1   localization  at  triads.   Barone  V,  Randazzo  D,  Del  Re  V,  Sorrentino  V,  Rossi  D.  Organization   of   junctional   sarcoplasmic   reticulum   proteins   in   skeletal   muscle   fibers.  J  Muscle  Res  Cell  Motil  2015  Sep  15.  [Epub  ahead  of  print]   Rossi  D,   Bencini   C,  Maritati  M,   et   al.  Distinct   regions  of   triadin   are   required  for  targeting  and  retention  at  the  junctional  domain  of  the   sarcoplasmic  reticulum.  Biochem  J  2014;458:407-­‐17.   Golini  L,  Chouabe  C,  Berthier  C,  et  al.   Junctophilin  1  and  2  proteins   interact   with   the   L-­‐type   Ca2+   channel   dihydropyridine   receptors   (DHPRs)  in  skeletal  muscle.  J  Biol  Chem  2011;286:43717-­‐25.   *****         CASQ1  mutations  in  human  skeletal  muscle  diseases   Valeria  del  Re,  Valentina  Polverino,  Alessandra  Gamberucci,   Virginia  Barone,  Vincenzo  Sorrentino   Molecular   Medicine   Section,   Department   of   Molecular   and   Developmental  Medicine,  University  of  Siena,  Italy   E-­‐mail:  vincenzo.sorrentino@unisi.it     Physical   Calsequestrin   (CASQ)   is   the   major   protein   of   the   sarcoplasmic  reticulum  of  striated  muscle  that  binds  Ca2+  with  high   capacity   and   moderate   affinity.   CASQ   exist   as   a   monomer   and   polymers,  depending  on  Ca2+  concentration.  CASQ  switches  from  an   unfolded   state   to   a   folded   monomer   when   the   ionic   strength   increases   allowing   the   formation   of   front-­‐to-­‐front   first   and   then   back-­‐to-­‐back   interactions   in   higher   Ca2+   concentrations.   Recently   we  reported  one  mutation  in  the  skeletal  CASQ1  gene  in  a  group  of   patients  with  a  vacuolar  myopathy  characterized  by  the  presence  of   inclusions   containing   CASQ1   and   other   SR   proteins.   The   CASQ1   mutation   (CASQ1D244G)   affects   one   of   the   high-­‐affinity   Ca2+-­‐ binding  sites  of  the  protein  and  alters  the  kinetics  of  Ca2+  release  in   muscle   fibers   from   patients.   Expression   of   the   CASQ1D244G   in   myotubes  and  in  mouse  fibers  causes  the  appearance  of  SR  vacuoles   containing   aggregates   of   the  mutant   CASQ1  protein   that   resemble   those   observed   in   patients.   Studies   of   Ca2+   release   showed   an   increase   in   Ca2+   storage   in   CASQ1WT   COS-­‐7   transfected   cells   whereas  no  increase  was  observed  in  CASQ1D244G.  Moreover  both   CASQ1WT   and   CASQ1D244G   were   expressed   in   bacteria,   purified   and   analysed   for   their   ability   to   polymerize   at   increasing   Ca2+   concentrations.  The  results  obtained  indicate  that  the  CASQ1D244G   protein   polymerizes   at   lower   Ca2+   levels   and   more   rapidly   than   CASQ1WT.   These   results   suggest   that   the   CASQ1D244G   mutation   interferes   with   the   correct   process   of   Ca2+-­‐dependent   protein   polymerization  causing  altered  intracellular  calcium  storage  and  the   formation  of  protein  aggregates.   Rossi   D,   Vezzani   B,   Galli   L,   et   al.   A   mutation   in   the   CASQ1   gene   causes   a   vacuolar   myopathy   with   accumulation   of   sarcoplasmic   reticulum  protein  aggregates.  Hum  Mutat  2014;35:1163-­‐70.   Lewis  KM,  Ronish  LA,  Ríos  E,  Kang  C.  Characterization  of  Two  Human   Skeletal   Calsequestrin   Mutants   Implicated   in   Malignant   Hyperthermia   and   Vacuolar   Aggregate   Myopathy   J   Biol   Chem   2015;290:28665-­‐74.   Wang   L,   Zhang   L,   Li   S,   et   al.   Retrograde   regulation   of   STIM1-­‐Orai1   interaction  and  store-­‐operated  Ca2+  entry  by  calsequestrin.  Sci  Rep   2015;5:11349.   *****   Calcium  Entry  Units  (CRUs):  discovery  of  intracellular   junctions  that  promote  assembly  of  STIM1  and  Orai1  in   skeletal  muscle   Simona  Boncompagni   CeSI   -­‐   Center   for   Research   on   Ageing   &   DNICS   -­‐   Dept.   of   Neuroscience,  Imaging,  and  Clinical  Sciences,  Univ.  G.  d'Annunzio  of   Chieti,   Italy   &   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and  Physical  Rehabilitation,  Wien,  Austria   E-­‐mail:  s.boncompagni@phobos.unich.it     European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   46 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Store-­‐operated   Ca2+   entry   (SOCE),   also   referred   to   as   capacitative   Ca2+   entry,   plays   an   important   role   in   intracellular   Ca2+   regulation.   SOCE   is   a  ubiquitous  Ca2+  entry  mechanism,   first  described   in  non-­‐ excitable   cells,   that   is   triggered   by   depletion   of   intracellular   Ca2+   stores   (endoplasmic/sarcoplasmic   reticulum,   respectively   ER   and   SR).   SOCE   is   coordinated   by   the   interaction   of   stromal   interaction   molecule  1  (STIM1),  which  acts  as  the  Ca2+  sensor  in  the  ER  lumen,1   and   Orai1,   the   Ca2+-­‐permeable   channel   in   the   plasma  membrane   (PM).2  Specific  Gap  of  Knowledge.  SOCE  is  also  well-­‐documented  in   skeletal   muscle,3   where   it   limits   muscle   fatigue   during   repetitive   fatiguing   stimulation.4   Several   studies   suggest   that   STIM1-­‐Orai1   coupling  occurs  within  the  pre-­‐formed  SR-­‐TT  junctions  of  the  triad,5   also   known   as   Ca2+   release   units   (CRUs),   the   sites   of   excitation-­‐ contraction  (EC)  coupling.  However,  the  precise  subcellular  location   of  STIM1-­‐Orai1  SOCE  complexes  in  skeletal  muscle  has  not  yet  been   unequivocally   identified.   Recent   breakthroughs.   Here   we   show   by   electron  microscopy  (EM)  that  prolonged  muscle  activity  drives  the   formation  of  previously  unidentified  intracellular  junctions  between   the  SR  and  extensions  of  the  TTs  membrane.  The  activity-­‐dependent   formation   of   these   unique   SR-­‐TT   junctions   reflects   a   striking   and   unexpected  remodeling  of  the  existing  sarcotubular  system  at  the  I-­‐ band   of   the   sarcomere.   Using   immunohistochemistry   and   immunogold   labeling   for   EM  we   demonstrate   that   these   junctions   contain  the  molecular  machinery  known  to  mediate  SOCE:  STIM1  in   the  SR  and  Orai1  channels,  which  move  into  the  I  band  as  a  result  of   the  elongation  of   existing   TTs.   Thus,   these  newly   formed   junctions   are  referred  to  as  Ca2+  Entry  Units  (CEUs),  the  first  new,  molecularly-­‐ defined  subcellular  structure  in  skeletal  muscle  in  over  30  years.  We   propose   that   CEUs:   a)   play   a   fundamental   role   in   coordinating   STIM1-­‐Orai1   coupling   in   muscle,   b)   represent     the   structural   framework   for  SOCE  providing  an   ideal  Ca2+  entry  pathway   to   refill   SR  stores,  and  c)  plays  a  key  role  during  repetitive  stimulation.   1. Roos  J,  DiGregorio  PJ,  Yeromin  AV,  et  al.  STIM1,  an  essential  and   conserved  component  of  store-­‐operated  Ca2+  channel  function.   J  Cell  Biol  2005;169:  435-­‐45.   2.   Feske  S,  Gwack  Y,  Prakriya  M,  Srikanth  S,  Puppel  SH,  Tanasa  B,   Hogan  PG,  Lewis  RS,  Daly  M,  Rao  A.  A  mutation  in  Orai1  causes   immune  deficiency  by  abrogating  CRAC  channel  function.  Nature   2006;441,179-­‐85.   3.   Kurebayashi  N,  Ogawa  Y.  Depletion  of  Ca2+  in  the  sarcoplasmic   reticulum   stimulates   Ca2+   entry   into   mouse   skeletal   muscle   fibres.  J  Physiol  2001;533,185-­‐99.   4.   Zhao  X,  Yoshida  M,  Brotto  L,  Takeshima  H,  Weisleder  N,  Hirata  Y,   Nosek  TM,  Ma   J,  Brotto  M.  Enhanced   resistance   to   fatigue  and   altered   calcium   handling   properties   of   sarcalumenin   knockout   mice.  Physiol  Genomics  2005;23,72-­‐8.   5.   Launikonis   BS,   Rios   E.   Store-­‐operated   Ca2+   entry   during   intracellular   Ca2+   release   in   mammalian   skeletal   muscle.   J   Physiol  2007;583,81-­‐97.   *****   CIR-­‐Myo  -­‐  Lecture  4   Deep  Brain  Stimulation  in  Parkinson  and  dystonia   Angelo  Antonini   Parkinson   and   Movement   Disorders   Unit,   IRCCS   Hospital   San   Camillo,  Venice  &  1st  Neurology  Clinic,  University  Hospital  of  Padua,   Italy   E-­‐mail:  "Angelo  Antonini"  angelo3000@yahoo.com     Deep   brain   stimulation   (DBS)   of   the   subthalamic   nucleus   (STN)   or   globus   pallidus   internus   (GPi)   is   now   an   established,   safe   and   effective   treatment   option   for   Parkinson’s   disease   (PD),   used   by   more   than   125,000   patients   worldwide.   Solid   scientific   evidence   indicates  that  it  can  significantly  alleviate  motor  disability,  levodopa-­‐ induced  complications  and  improve  a  patient’s  overall  quality  of  life.   The   indications   for   DBS   have   expanded   in   recent   years,   including   earlier   application   in   Parkinson   patients,   use   in   other   motor   disorders   such   as   dystonia   and   essential   tremor,   and   the   potential   for   use   in   intractable   epilepsy   and   psychiatric   disorders,   for   example.   In   Parkinson   patients   DBS   produces   a   marked   improvement  in  motor  fluctuations  and  dyskinesias  even  if  evidence   suggests   that   the   reduction   in   motor   disability   is   greater   with   medications   than   with   STN-­‐DBS.   Benefits   associated   with   DBS   persists  for  many  years,  although  disability  still  progresses,  reflecting   degeneration  in  non-­‐dopaminergic  sites.   It   is  noteworthy,  however,   that  the  use  of  DBS  is  limited  not  only  by  several  restrictive  exclusion   criteria,   but   also   by   the   comparatively   high   risk   of   severe   complications,   such   as   neuropsychiatric   morbidity,   intracranial   bleeding   (which   can   occur   in   2-­‐8%   of   patients   undergoing   stereotactic  neurosurgery),  and  in  some  cases  increased  mortality.   1.   Dafsari  HS,  Reddy  P,  Herchenbach  C,  et  al  Beneficial  Effects  of   Bilateral  Subthalamic  Stimulation  on  Non-­‐Motor  Symptoms  in   Parkinson's  Disease.  Brain  Stimul  2016;9:78-­‐85.   2.   Volkmann  J,  Albanese  A,  Antonini  A,  et  al.  Selecting  deep  brain   stimulation  or  infusion  therapies  in  advanced  Parkinson's   disease:  an  evidence-­‐based  review.  J  Neurol  2013;260:2701-­‐14.   3.   Siri  C,  Duerr  S,  Canesi  M,  et  al.  A  cross-­‐sectional  multicenter   study  of  cognitive  and  behavioural  features  in  multiple  system   atrophy  patients  of  the  parkinsonian  and  cerebellar  type.  J   Neural  Transm  2013;120:613-­‐8.   4.   Antonini  A,  Pilleri  M,  Padoan  A,  et  al.  Successful  subthalamic   stimulation  in  genetic  Parkinson's  disease  caused  by  duplication   of  the  α-­‐synuclein  gene.  J  Neurol  2012;259:165-­‐7.   5.   Antonini  A,  Isaias  IU,  Rodolfi  G,  et  al.  A  5-­‐year  prospective   assessment  of  advanced  Parkinson  disease  patients  treated  with   subcutaneous  apomorphine  infusion  or  deep  brain  stimulation.  J   Neurol  2011;258:579-­‐85.   5   Cilia  R,  Marotta  G,  Landi  A,  et  al.  Cerebral  activity  modulation  by   extradural  motor  cortex  stimulation  in  Parkinson's  disease:  a   perfusion  SPECT  study.  Eur  J  Neurol  2008;15:22-­‐8.   *****   CIR-­‐Myo  -­‐  Lecture  5   Link  between  malignant  hyperthermia  (MH)  and   environmental  heat  stroke  (EHS):  just  a  medical  hypothesis?   Feliciano  Protasi   CeSI   -­‐   Center   for   Research   on   Ageing   &   DNICS   -­‐   Dept.   of   Neuroscience,  Imaging,  and  Clinical  Sciences,  Univ.  G.  d'Annunzio  of   Chieti,  Italy   E-­‐mail:  feliciano.protasi@unich.it     EC   coupling   in  muscle   links   the   transverse(T)-­‐tubule   depolarization   to   release   of   Ca2+   from   the   sarcoplasmic   reticulum   (SR).1-­‐2   These   membranes   communicate   in   specialized   structures,   i.e.   calcium   release   units   (CRUs),   thanks   to   a   cross-­‐talk   between   voltage-­‐ dependent   Ca2+   channels   CaV1.1   (or   dihydropyridine   receptors,   DHPRs)   in   the   T-­‐tubule   and   Ca2+   release   channels,   or   ryanodine   receptors   type-­‐1   (RYR1),   in   the   SR.3-­‐5   Mutations   in   the   gene   encoding   for   RYR1),   the   SR   Ca2+   release   channel,   underlie   debilitating,   life-­‐threatening  muscle   disorders   such   as   central   core   disease   (CCD)   and   malignant   hyperthermia   (MH).   To   date,   MH   is   only   seen   as   a   clinical   syndrome   in   which   genetically   predisposed   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   47 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   individuals   respond   to   volatile   anesthetics   in   the   operating   room   with  potentially   lethal  episodes  characterized  by  elevations   in  body   temperature   and   rhabdomyolysis   of   skeletal   muscle   fibers.   However,   virtually   identical   over-­‐heating   episodes   have   been   reported   in   individuals   also   after   exposure   to   environmental   heat,   physical  exertion,  or  even  during  febrile  illness.  The  life-­‐threatening   nature  of  EHS  underscore  the  critical  need  for  a  deeper  mechanistic   understanding  of  these  syndromes  and  for  the  development  of  new   and  effective  treatments.  Specific  Gaps  of  Knowledge.  A)  Mutations   in  RYR1  have  been  found  in  many,  but  not  all,  MH  cases  suggesting   the  potential  involvement  of  additional  genes  in  the  pathogenesis  of   this   syndrome.   B)   The   relationship   between   classic   MH   and   over-­‐ heating   episodes   triggered   by   different   stressors   (heat,   exertion,   fever,  etc.)  is  not  yet  widely  recognized.  C)  The  cascade  of  molecular   mechanisms   that   from   SR   Ca2+   leak   leads   to   rhabdomyolysis   of   muscle   fibers   are   still   unclear   and   needs   to   be   fully   elucidated.   Recent   breakthroughs.   In   the   last   years,   thanks   to   the   support   of   Telethon   (GGP08153   and   GGP13213),   we   have   moved   significant   steps  forward.  We  have  demonstrated  in  animal  models  that:  A)  MH   episodes  can  result  not  only  from  mutations  in  RYR1,  but  also  from   mutations  in  proteins  that  interact  with  RYR1  (such  as  Calsequestrin-­‐ 1,   CASQ1);   B)   the   mechanisms   underlying   hyperthermic   episodes   triggered   by   anesthetics   and   by   heat   and   exertion   are   virtually   identical,   suggesting   that   these   syndromes   could   be   possibly   treated/prevented   using   similar   treatments;   C)   during   lethal   MH/EHS   crises  Ca2+   leak   from   intracellular   stores   results   in   a   feed-­‐ forward  mechanism  mediated  by  excessive  production  of  oxidative   species  of  oxygen  and  nitrogen  (ROS  and  RNS),  which  eventually  will   lead   to   depletion   of   the   SR   and   to   massive   activation   of   Store   Operated  Ca2+  Entry  (SOCE).   1.  Schneider   MF.   Control   of   calcium   release   in   functioning   skeletal   muscle  fibers.  Annu  Rev  Physiol  1994;56:463-­‐84.   2.  Franzini-­‐Armstrong   C,   Protasi   F.   Ryanodine   receptors   of   striated   muscles:  a  complex  channel  capable  of  multiple  interactions.  Physiol   Rev  1997;77:699-­‐729.   3.  Maclennan   DH,   Zvaritch   E.   Mechanistic   models   for   muscle   diseases   and   disorders   originating   in   the   sarcoplasmic   reticulum.   Biochim   Biophys  Acta  2011;1813:948-­‐964.   4.  Jungbluth  H.  Central  Core  Disease.  Orphanet  J  Rare  Dis  2007;2:25-­‐30.   5.  Denborough  M.  Malignant  hyperthermia.  Lancet  1998;352:1131-­‐6.   *****   Workshop    “Mobility  in  elderly”:   Molecular  approaches  –  H  Kern,  A  Musarò,  Chairs     Insights  into  the  oxidative  stress-­‐mediated  sarcopenia  and   dismantlement  of  NMJ   Antonio  Musarò   Institute   Pasteur   Cenci   Bolognetti,   Unit   of   Histology   and   Medical   Embryology,  IIM,  Sapienza  University  of  Rome,  Italy   E-­‐mail:  antonio.musaro@uniroma1.it     A   crucial   system   severely   affected   in   different   pathological   conditions   is   the   antioxidative   defense,   leading   to   accumulation   of   ROS.  The  discovery   that   the  anti-­‐oxidant   status  decreases  with  age   and  it  is  affected  in  several  pathological  conditions,  such  as  disuses,   chronic   fatigue   syndrome,   cancer,   muscular   dystrophy   and   amyotrophic   lateral   sclerosis   (ALS),  has  placed  oxidative  stress  as  a   central  mechanism   in   the  pathogenesis  of   these  diseases.  A  critical   aspect  underlying  the  mechanisms  of  age-­‐related  muscle  loss  is  the   reduction   in   the   number   of   nerve   terminals,   fragmentation   of   the   neuromuscular   junctions   (NMJs),   and   a   decrease   in   neurotransmitter   release.   However,   considering   that   skeletal   muscles   in   one   of   the   tissues   that   generate   considerable   ROS,   an   important   issue   to   address   is   whether   a   selective   alteration   of   oxidative   stress   balance   in   skeletal   muscle   is   sufficient   to   induce   these   alterations.   Preliminary   results   demonstrate   that   muscle   specific   accumulation   of   oxidative   stress   induces   mitochondria   dysfunction/alterations   and   triggers   NMJs   dismantlement,   associated   with   higher   rate   of   Acetilcholine   Receptor   (AchR)   turnover   and   morphological   alterations   of   the   pre-­‐synaptic   neuromuscular   endplate.  We   also   defined   the   potential   molecular   mechanisms   that  mediate   the   toxic   effects   of   oxidative   stress   and   NMJs  dismantlement.   1. Musarò  A,  Fulle  S,  Fanò  G.  Oxidative  stress  and  muscle   homeostasis.  Curr  Opin  Clin  Nutr  Metab  Care  2010;13:236-­‐42.     2.   Jang  YC1,  Van  Remmen  H.  Age-­‐associated  alterations  of  the   neuromuscular  junction.  Exp  Gerontol  2011;46:193-­‐8.   *****   Muscle  trophism  and  mitochondria  dynamics  in  ageing   human  skeletal  muscle  trained  with  Electrical  Stimulation   Sandra  Zampieri  (1,2),  Cristina  Mammucari  (1),  Marco  Sandri  (1,  3),   Vanina  Romanello  (3),  Antonio  Musarò  (4),  Laura  Barberi  (4),   Feliciano  Protasi  (5),  Laura  Pietrangelo  (5),  Aurora  Fusella  (5),   Stefan  Loefler  (2),  Jan  Cevka  (6),  Nejc  Sarbon  (7),  Rosario  Rizzuto   (1),  Ugo  Carraro  (8),  Helmut  Kern  (2)   (1)   Department   of   Biomedical   Sciences,   Padova,   Italy;   (2)   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and   Physical   Rehabilitation,   Vienna,   Austria;   (3)   Venetian   Institute   of  Molecular   Medicine,   Dulbecco   Telethon   Institute;   (4)   DAHFMO-­‐Unit   of   Histology   and   Medical   Embryology,   IIM,   Sapienza   University   of   Rome,   Italy;   (5)   CeSI   -­‐   Center   for   Research   on   Aging   &   DNICS,   Department   of   Neuroscience,   Imaging   and   Clinical   Sciences,   University   G.   d'Annunzio,   Chieti,   Italy;   (6)   Faculty   of   Physical   Education   and   Sport,   Comenius   University,   Bratislava,   Slovakia;   (7)   University   of   Primorska,   Science   and  Research  Centre,   Institute   for   Kinesilogical   Research,   Koper,   Slovenia;   (8)   IRCCS   Fondazione   Ospedale  San  Camillo,  Venezia,  Italy   E-­‐mail:  sanzamp@unipd.it   Physical   exercise   is   known   to   have   beneficial   effects   on   muscle   trophism   and   force   production   modulating   signaling   pathways   involved  in  fiber  type  and  muscle  growth  also  via   intracellular  Ca2+   and   inducing   specific  mitochondrial   adaptations.   Several   evidences   both   in   vitro   and   in   vivo,   have   demonstrated   that   during   muscle   contraction,   Ca2+   concentration   in   the   mitochondrial   matrix   is   increased.   Importantly,   alterations   of   mitochondrial   Ca2+   homeostasis   controlled   by   mitochondrial   calcium   uniporter   (MCU)   has  been  recently  shown  to  regulate  muscle  mass  in  vivo  in  mice.  In   skeletal   muscle,   mitochondria   exist   as   dynamic   network   that   is   continuously  remodeled  through  fusion  and  fission  phenomena  that   are   important   for   maintenance   of   functional   mitochondria.   In   particular,   fission   occurs   upon   the   recruitment   of   dynamin-­‐related   protein   1   (DRP1),  while   fusion   is   controlled   by  mitofusins   (MFN)   1   and   2   and   by   optic   atrophy   1   (OPA1).   OPA1   also   regulates   mitochondrial  adaptations  to  bioenergetic  conditions  at  the  level  of   inner   membrane   ultrastructure   and   remodeling   of   mitochondrial   cristae,   controlling   muscle   atrophy   and   mitochondria   respiratory   efficiency.  It  has  been  shown  that  the  decrease  of  muscle  mass  and   strength   observed   in   age-­‐related   Sarcopenia   is   linked   to   abnormalities   of  mitochondrial  morphology,   number   and   function.   In   the   present   study   we   intended   to   investigate   the   effects   of   9   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   48 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   weeks  strength  training  by  neuromuscular  electrical  stimulation  (ES)   in   comparison   to   voluntary   leg   press   (LP)   in   sedentary   70yrs   old   subjects   on   mitochondria   dynamics,   MCU   and   mitochondria   respiratory   chain   enzymes   modulation.   Our   results   show   that   ES   mediated  muscle  structural  and  functional  improvements  are  linked   to   signaling   pathways   related   to   muscle   mass   regulation   and   enhanced   MCU,   and   COX   IV   respiratory   chain   enzyme.   Electron   Microscopy   ultrastructural   analyses   showed   remodelling   of   mitochondrial   apparatus   as   a   consequence   of   fusion   phenomena   that   is   consistent   with   adaptation   to   physical   exercise   and   with   increased   OPA1   protein   expression   levels   as   documented   by   WB   analyses.  LP  training  showed  moderate  effects  both  at  structural  and   functional  level,  with  no  impact  on  mitochondrial  dynamics,  that  are   consistent   with   a   milder   protocol   of   training   in   comparison   to   neuromuscular   ES.     Altogether   these   results   indicate   that   the   ES-­‐ dependent   beneficial   effects   on   muscle   mass   and   force   are   associated  with   changes   in  mitochondrial-­‐related  proteins   involved   in   Ca2+   homeostasis   and   mitochondrial   shape,   providing   new   targets   to   develop   therapeutic   strategies   counteracting   Sarcopenia   and  promoting  healthy  ageing.   1.   Rudolf   R,   Mongillo   M,   Magalhaes   PJ,   Pozzan   T.   In   vivo   monitoring  of  Ca(2+)  uptake  into  mitochondria  of  mouse  skeletal   muscle  during  contraction.  J  Cell  Biol  2004;166:527–36.   2.   Zampieri  S,  Pietrangelo  L,   Loefler  S,  Fruhmann  H,  Vogelauer  M,   Burggraf   S,   Pond   A,   Grim-­‐Stieger   M,   Cvecka   J,   Sedliak   M,   Tirpáková  V,  Mayr  W,  Sarabon  N,  Rossini  K,  Barberi   L,  De  Rossi   M,  Romanello  V,  Boncompagni  S,  Musarò  A,  Sandri  M,  Protasi  F,   Carraro   U,   Kern   H.   Lifelong   Physical   Exercise   Delays   Age-­‐ Associated  Skeletal  Muscle  Decline.  J  Gerontol  A  Biol  Sci  Med  Sci   2015;70:163-­‐73.   3.   Kern  H,  Barberi  L,  Löfler  S,  Sbardella  S,  Burggraf  S,  Fruhmann  H,   Carraro  U,  Mosole   S,   Sarabon  N,  Vogelauer  M,  Mayr  W,   Krenn   M,   Cvecka   J,   Romanello   V,   Pietrangelo   L,   Protasi   F,   Sandri   M,   Zampieri   S,   Musaro   A.   Electrical   stimulation   (ES)   counteracts   muscle  decline  in  seniors.  Frontiers  in  Aging  Neuroscience,  2014;   6:189.   4.   Konopka   AR,   Sreekumaran   Nair   K.   Mitochondrial   and   skeletal   muscle   health   with   advancing   age.   Mol   Cell   Endocrinol   2013;379:19–29.   5.   Mammucari  C,  Gherardi  G,  Zamparo  I,  Raffaello  A,  Boncompagni   S,   Chemello   F,   Cagnin   S,   Braga   A,   Zanin   S,   Pallafacchina   G,   Zentilin  L,  Sandri  M,  De  Stefani  D,  Protasi  F,  Lanfranchi  G,  Rizzuto   R.  The  Mitochondrial  Calcium  Uniporter  Controls  Skeletal  Muscle   Trophism  In  Vivo.  Cell  Reports  2015;10:1269–1279.   6.   Pietrangelo   L.,   D'Incecco   A,   Ainbinder   A,  Michelucci   A,   Kern   H,   Dirksen   RT,   Boncompagni   S,   Protasi   F.   Age-­‐dependent   uncoupling   of   mitochondria   from   Ca2+   release   units   in   skeletal   muscle.  Oncotarget  2015;34:35358-­‐71.   *****   Ectopic  expression  of  Merg1a  in  skeletal  muscle  of  mice   modulates  NFκB  activity   Sohaib  Hameed  (1),  Kevin  Bradley  (1),  Luke  Anderson  (1),  Chase   Latour  (1),  Nicole  Dethrow  (1),  Emi  Hayashi  Park  (1),  Mariam   Hashmi  (1),  Sandra  Zampieri  (2,3),  Ugo  Carraro  (4),  Amber  Pond  (1)   #Southern  Illinois  University  School  of  Medicine,  Carbondale,  IL,  US;   (2)   Department   of   Biomedical   Sciences,   Padova,   Italy;   (3)   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and   Physical   Rehabilitation,  Vienna,  Austria;   (4)   IRCCS  Fondazione  Ospedale   San   Camillo,  Venezia,  Italy   E-­‐mail:  "Amber  Pond"  apond@siumed.edu     Skeletal   muscle   atrophy   is   a   debilitating   loss   of   muscle   mass   (resulting  from  decreased  myofiber  size)  and  strength  that  normally   occurs   during   aging,   muscle   disuse   /   inactivity,   neuropathies   /   myopathies  and  with  other  pathological  diseases.    The  ether-­‐a-­‐gogo   related   gene   (ERG1a)   is   a   K+   channel   that   is   up-­‐regulated   in   atrophying   gastrocnemius   muscles   of   both   unweighted   and   cachectic   mice.1   Ectopic   expression   of   mouse   erg1a   (Merg1a)   in   mouse   muscle   increases   ubiquitin   proteasome   activity   by   up-­‐ regulation  of  at   least  one  known  E3  ligase,  MuRF1.2  Because  Murf1   expression   is   up-­‐regulated   by   increased   NFκB   activity3,   we   hypothesized   that   ectopic   expression   of   Merg1a   would   increase   NFκB  activity  and  lead  to  increased  Murf1.  To  test  this,  we  electro-­‐ transferred   plasmid   encoding   an   NFκB   firefly   luciferase   (FFL)   reporter  and  a  second  plasmid  encoding  Renilla  luciferase  (Ren)  into   mouse  gastrocnemius  muscles.    We  added  a  Merg1a  plasmid  in  the   left   leg   while   adding   a   control   plasmid   in   the   right.   We   then   harvested   muscle   on   days   0-­‐7   and   assayed   for   both   FFL   and   Ren   activities  and  used  the  FFL  to  Ren  ratio  as  a  measure  of  NFκB  activity   (to   correct   for   differences   in   transfection   efficiency).   Surprisingly,   the  muscles   expressing  Merg1a   showed   a   decreased  NFκB   activity   when  compared   to   the  controls.     Thus  we  hypothesized   that   there   may   be   a   factor   present   in   a   physiological   model   of   atrophy   that   would   cause   MERG1a   to   modulate   NFκB   activity   differently.     Because   sciatic   nerve   transection   does   not   produce   an   increase   in   Merg1a  expression,  but  it  increases  NFκB  activity3,  we  repeated  our   electro-­‐transfer   with   mice   and   then   denervated   both   legs   18-­‐24   hours   later.  We   harvested  muscles   at   days   0,   2,   4,   7   and   10   post-­‐ denervation.   The   Merg1a   treated   muscle   still   experienced   a   decrease  in  NFκB  activity.  Thus,  we  conclude  that  ectopic  expression   of   Merg1a   modulates   NFκB   activity   in   both   innervated   and   denervated   skeletal   muscle.   Future   studies   will   include   efforts   to   determine  if  this  finding  is  truly  physiological  and,  if  so,  then  by  what   mechanism  does  MERG1a  affect  NFκB  activity.   1.   Wang   X,   Hockerman   GH,   Green   HW,   Babbs   CF,   Mohammad   SI,   Gerrard  D,  Latour  MA,  London  B,  Hannon  KM,  Pond  AL.  Merg1a  K+   channel   induces  skeletal  muscle  atrophy  by  activating  the  ubiquitin   proteasome  pathway.  FASEB  J  2006;20:1531-­‐3.   2.Pond   AL;   Nedele   C,   Wang   W-­‐H,   Wang   X,   Walther   C,   Jaeger   C,   Bradley   KS,   Du   H,   Fujita   N,   Hockerman   GH,   Hannon   KM.   The   MERG1a  channel  modulates  skeletal  muscle  MuRF1,  but  not  MAFbx,   expression.  Muscle  &  Nerve.  2013;49:378-­‐88.     3.Mittal  A,  Bhatnagar   S,   Kumar  A,   Lach-­‐Trifilieff   E,  Wauters   S,   Li  H,   Makonchuk   DY,   Glass   DJ,   Kumar   A.   The   TWAEK-­‐Fn14   system   is   a   critical   regulator  of  denervation-­‐induced  skeletal  muscle  atrophy   in   mice.  J  Cell  Biol  2010;188:833-­‐49.   *****   Molecular  adaptation  of  MHC  and  Ca2+-­‐handling  proteins  of   human  skeletal  muscle  to  aging  and  FES:  an  in  situ  study   Simone  Mosole  (1,2),  Sandra  Zampieri  (1,2),  Sandra  Furlan(3),   Barbara  Ravara  (1,2);  Hanna  Fruhmann  (2),  Stefan  Löfler  (2),   MIchael  Vogelauer  (4),  Helmut  Kern  (2,4),  Ugo  Carraro  (5),  Pompeo   Volpe  (1),  Alessandra  Nori  (1)   (1)   Laboratory   of   Translational   Myology   of   the   Interdepartmental   Research   Center   of   Myology,   Department   of   Biomedical   Science,   University   of   Padova;   2)   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation  and  Physical  Rehabilitation,  Vienna,  Austria;  (3)  Institute   of  Neuroscience  Consiglio  Nazionale  delle  Ricerche,  Padova,  Italy;  (4)   Institute  of  Physical  Medicine  and  Rehabilitation,Wilhelminenspital,   Vienna,   Austria;   (5)   IRRCS   Fondazione   Ospedale   San   Camillo,   Venezia,  Italy   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   49 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   E-­‐mail:  simone.mosole@studenti.unipd.it   Physical   activity   plays   an   important   role   in   preventing   chronic   disease  and  muscle  degeneration  in  adults  and  the  elderly  persons.1   Age-­‐related  changes   in  skeletal  muscle   innervation,   independent  of   patent   peripheral   neuropathies,   are   known   to   contribute   to   the   decline   in   quality   of   life   often   reported   in   older   population.2   The   changes   and   the   mechanism(s)   by   which   they   occur   are   not   well   understood.   We   had   the   opportunity   to   examine   the   effects   of   lifelong   high-­‐level   physical   activity   comparing   cohorts   of   young   adults   and   septuagenarians   either   sedentary   or   recreational   sportsmen,   collecting  what   is,   in   our   opinion,   strong   evidence   that   aging  atrophy  is,  at  least  in  part,  a  result  of  progressive  denervation   that  can  be  counter-­‐acted  by   lifelong  high-­‐level  exercise.3  We  used   immunolabeling  methods   to   analyze   the   fiber   type   composition   of   muscle  biopsies.  Our  main   results  demonstrate   that  biopsies   from:   1.   young   men   seldom   contain   denervated   (0.2±0.5   %),   or   transforming  muscle  fibers  (0.5±0.6  %);  2.  sedentary  seniors  contain   both   denervated   (2.6±1.9   %),   coexpressing   myofibers   (1.8±1.7   %)   and  a  few  reinnervated  clustered  myofibers  of  the  fast  type  (3.0±4.7   %);   and   3.   senior   sportsmen   present   with   a   larger   percentage   of   healthy,   slow   myofibers   (up   to   68.5±14.1   %,)   that   appear   mainly   clustered   in   slow   fiber-­‐type  groupings   (7.9±7.4  %).   4,5  Data  analysis   reveals   that   there   was   no   difference   between   the   athletic   and   sedentary   senior   groups   in   terms   of   their   very   low   percentages   of   muscle   fibers   co-­‐expressing   fast   and   slow   MHCs   (0.6±0.6   %),   suggesting   that   lifelong   exercise   does   not   induce   motor   unit   transformation.  On  the  other  hand  the  recreational  sportsmen  had   both   considerably   higher   percentages   of   slow-­‐type   myofibers   and   greater   numbers   of   slow   fiber-­‐type   groupings,   providing   sound   evidence  that  lifelong  cycles  of  denervation/reinnervation  occurred.   It  appears   that   lifelong  exercise  protects  muscle   function  by  saving   otherwise   lost   muscle   fibers   through   reinnervation   by   different,   mainly  slow,  motor  axons.6   On   the   other   hand,   volitional   exercise   is   not   always   feasible   or   people  are  reluctant  to  do  it  and  other  strategies  should  be  applied   such  as  Functional  Electrical  Stimulation  (FES).7  This  study  shows  the   effects   in   situ   of   FES   in   human   Vastus   Lateralis   (VL)   muscle   of   sedentary   elderly   people,   in   particular   on   the   key   process   of   Ca2+   uptake   and   release   and   related   control   mechanisms   that   are   essential   in   muscle   adaptation.   Through   immunofluorescence   analysis  of  muscle  cryosections,  a  huge   increment  of  NFAT  positive   nuclei   was   found   after   treatment   (from   3%   to   60%);  moreover   an   increment   of   P-­‐CamkII   was   observed   by   western   blotting   analysis.   These   findings   indicate   that   FES   activate   the   CaM-­‐dependent   phosphatase   signaling   (known   to   be   involved   in  muscle   plasticity).   Muscle  total  homogenates  were  obtained  from  biopsies  performed   before  and  after  completing  a  nine  weeks  FES  treatment  on  a  group   of   volunteers   and   Calsequestrin   (CASQ),   SERCA,   Sarcalumenin,   protein   expression   was   determined   by   Western   blot.   After   FES   significant   increase   of   SERCA2   and   Sarcalumenin   and   decrease   of   CASQ1   were   observed.   Immunofluorescence   analysis   were   also   performed   to   localize   in   situ   MHCII/SERCA2   co-­‐expressing   muscle   fibers,  an  interesting  tool  to  identify  subpopulation  of  muscle  fibers   involved   in  muscle  adaptation.  The  overall   results   indicate   that   the   applied   FES   protocol,   simulating   a   motoneuron   slow-­‐type   firing   pattern,   potentiates   Ca2+   uptake   and   storage   in   skeletal   muscle   fibers   validating   at   molecular   level   the   FES   strategy   as   a   safe   and   effective  rehabilitation  strategy  in  elderly  persons.   1. Faulkner   JA,   Larkin   LM,   Claflin   DR,   Brooks   SV.   Age-­‐related   changes   in   the   structure   and   function   of   skeletal  muscles.   Clin   Exp  Pharmacol  Physiol  2007;34:1091–6.   2. Mitchell  WK,  Williams  J,  Atherton  P,  Larvin  M,  Lund  J,  Narici  M.   Sarcopenia,   dynapenia,   and   the   impact   of   advancing   age   on   human  skeletal  muscle  size  and  strength;  a  quantitative  review.   Front  Physiol  2012  Jul  11;3:260.  doi:  10.3389/fphys.2012.00260.   eCollection  2012.   3. Crane   JD,   Macneil   LG,   Tarnopolsky   MA.   Long-­‐term   aerobic   exercise   is   associated  with   greater  muscle   strength   throughout   the   life   span.   J   Gerontol   A   Biol   Sci   Med   Sci   2013;68:631–638.   doi:10.1093/gerona/gls237.   4. Mosole  S,  Rossini  K,  Kern  H,  et  al.  Significant   increase  of  vastus   lateralis   reinnervation   in   70-­‐year   sportsmen   with   a   lifelong   history  of  high-­‐level  exercise.  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol   2013;23:117-­‐22.   5. Mosole  S,  Carraro  U,  Kern  H,  et  al.  Long-­‐term  high-­‐level  exercise   promotes   muscle   reinnervation   with   age.   J   Neuropathol   Exp   Neurol  2014;73:284-­‐94.  doi:  10.1097/NEN.0000000000  000032.   6. Zampieri   S,   Pietrangelo   L,   Loefler   S,   et   al.   Lifelong   Physical   Exercise   Delays   Age-­‐Associated   Skeletal   Muscle   Decline.   J   Gerontol   A   Biol   Sci   Med   Sci   2015;70:163-­‐73.   doi:   10.1093/gerona/glu006.  Epub  2014  Feb  18.   7. Kern   H,   Barberi   L,   Löfler   S.   et   al.   Electrical   stimulation   counteracts   muscle   decline   in   seniors.   Front   Aging   Neurosci.   2014;  6:189.   *****   Tat-­‐MyoD  fused  proteins,  together  with  C2C12  conditioned   medium,  are  able  to  induce  equine  adult  mesenchymal  stem   cells  towards  the  myogenic  fate   Chiara  Gomiero  (1),  Tiziana    Martinello  T(1),  Alessandro  Negro  (2),   OhadTopel  (3),  Roberta  Sacchetto  (1),  Marco  Patruno  (1)   (1)   Department   of   Comparative   Biomedicine   and   Food   Science,   University  of  Padova,   Italy;   (2)  Department  of  Biomedical   Sciences,   University   of   Padova,   Italy;   (3)   VTH   -­‐Koret   School   of   Veterinary   Medicine,  The  Hebrew  University  of  Jerusalem,  Israel   E-­‐mail:  marco.pat@unipd.it   The  Tat  protein  is  able  to  translocate  through  the  plasma  membrane   and   when   it   is   fused   with   other   peptides   may   act   as   a   protein   transduction   system.   This   ability   appears   particularly   interesting   to   induce   tissue-­‐specific   differentiation   when   the   Tat   protein   is   associated  to  transcription  factors.  In  the  present  work  the  potential   of   the   complex   Tat-­‐MyoD   in   inducing   equine   peripheral   blood   mesenchymal   stromal   cells   (PB-­‐MSCs)   towards   the   myogenic   fate,   was   evaluated.   Results   showed   that   the   internalization   process   of   Tat-­‐MyoD  needs  the  absence  of  serum  and  the  nuclear   localization   of   the   fused   complex   is   observed   after   15   hours   of   incubation.   However,   the   supplement   of   Tat-­‐MyoD   only   was   not   sufficient   to   induce  myogenesis  and,  therefore,  in  order  to  achieve  the  myogenic   differentiation   of   PB-­‐MSCs,   conditioned   medium   was   added.   The   latter   was   obtained   coculturing   PB-­‐MSCs   with   C2C12   without   a   direct   contact.   These   results   suggest   that   TAT-­‐mediated   protein   transduction   system,   if   supported   by   conditioned   medium,   might   represents   a   useful   methodology   to   induce   myoblasts   differentiation.   Gao   Y,   Connell   JP,   Wadhwa   L,   Ruano   R,   Jacot   JG.   Amniotic   Fluid-­‐ Derived   Stem   Cells   Demonstrated   Cardiogenic   Potential   in   Indirect   Co-­‐culture   with   Human   Cardiac   Cells.   An   Biomed   Eng   42:2490-­‐500   Martinello   T,   Bronzini   I,   Maccatrozzo   L,   et   al.   Canine   adipose-­‐ derived-­‐mesenchymal  stem  cells  do  not  lose  stem  features  after   a  long-­‐term  cryopreservation.  Res  Vet  Sci.  2011;91,18–24.   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   50 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Hidema   S,   Tonomura   Y,   Date   S,   Nishimori   K.   Effects   of   protein   transduction   with   intact   myogenic   transcription   factors   tagged   with   HIV-­‐1   Tat-­‐PTD   (T-­‐PTD)   on   myogenic   differentiation   of   mouse  primary  cells.  J  Biosci  Bioeng    2012;113,5–11.   Dietz  GPH,  Bähr  M.  Delivery  of  bioactive  molecules  into  the  cell:  the   Trojan  horse  approach.  Mol  Cell  Neurosci  2004;27:85–131.   *****       Mobility  impairments  in  aging  and  myopathies   Stefano  Masiero,  Francesco  Piccione,  Chairs   Decline  of  power  developed  by  skeletal  muscles  based  on   world  records  of  Master  athletes  from  30  to  110  years   Paolo  Gava  (1),  Ugo  Carraro  (2)   (1)   Laboratory   of   Translational   Myology   of   the   Interdepartmental   Research   Center   of   Myology,   Department   of   Biomedical   Science,   University  of  Padova;   (2) IRRCS  Fondazione  Ospedale  San  Camillo,   Venezia,  Italy   E-­‐mail:  “Paolo  Gava”  Paologavastra@alice.it     Both   strength   and   power   developed   by   human   skeletal   muscles   decline   with   increasing   age.1,2   The   athletic   world   records   of   the   Master   athletes   of   age   ranging   from   35   years   to   100   years   are   an   excellent  proof  of  such  decline  in  every  track  and  field  competition.   The  world   record   performances   of   running,   jumping   and   throwing   events   can   be   transformed   into   dimensionless   parameters   proportional   to   the  power  developed   in   the   trials.   Such  parameter   ranges  from  1  for  the  Senior  world  record  (i.e.  the  maximum  human   performance)   to   lower  values   for   the  Master  athletes  of   increasing   age  down   to   0   for   a   null   performance.3,4  With   this   procedure   the   declines   of   the   power   parameter   with   increasing   age   can   be   analysed  and  compared:   the  trend-­‐lines    start   to  decline  very  close   to  the  age  of  30  years  and  arrive  to  0  around  the  age  of  110  years   for   every   athletic   discipline   (running,   jumping   and   throwing).5   The   comparison   of   the   various   trend-­‐lines   show   significantly   different   rates   of   decline.   Each   declining   trend-­‐line   reaches   a   “critical”   threshold   at   different   ages   for   the   running,   jumping   and   throwing   activities.   Such   thresholds   indicate   different   age   limits   for  most   of   everyday   tasks:  walking,   climbing   stairs  and   lifting  weights  above  a   table.  The  decline  of   the  Master  world   records,   transformed   into  a   dimensionless   power   parameter   declining   from   1   toward   0   with   increasing  age  is  the  decline  of  the  power  developed  by  each  one  of   us,   starting   from  1   in  our   youthful   age  and  declining   toward   lower   values  with  increasing  age.  There  are  no  reason,  for  each  one  of  us,   to   decline   differently   from   the   world   record-­‐men,   provided   that   each   of   us   remains   in   stable   fitness   condition   without   disabling   pathologies.   1.   Hill   AV.   The  physiological   basis   of   athletic   records.   Sci  Monthly   1925;21:  409  -­‐  28.   2.   Mitchell  W  K,  Williams  J,  Atherton  P.  Sarcopenia,  dyapenia,  and   the  impact  of  advancing  age  on  human  skeletal  muscle  size  and   strength;  a  quantitative  review.  Frontiers  in  Physiology  20123:  1-­‐ 18.   3.   Gava   P,   Kern  H,   Carraro  U.   Age-­‐associated   power   decline   from   running,   jumping,   and   throwing   male   Masters   world   records.   Exp   Aging   Res   2015;41:115-­‐35.   doi:   10.1080/0361073X.2015.   1001648.   4.   Baker   AB,   Tang,   YQ,   Turner  MJ.   Percentage   decline   in  masters   superathlete  track  and  field  performance  with  aging.  Exp.  Aging   Res  2003;29,  47-­‐65.     5.   Runge   M,   Rittweger   J,   Russo   CR,   Schiessl   H,   Felsenberg   D.   Is   muscle  power  output  a  key   factor   in   the  age-­‐related  decline   in   physical   performance?   A   comparison   of   muscle   cross   section   chair-­‐rising   test   and   jumping  power.  Clin  Physiol   Funct   Imaging   2004;24:335-­‐40.   *****       Electrical  Stimulation  in  neuromuscular  impairments:  an   educational  case  report   Andrea  Marcante  (1),  Paolo  Gargiulo  (2),  Ugo  Carraro  (1),   Francesco  Piccione  (1)   1   IRCCS   Fondazione   Ospedale   San   Camillo,   Venezia   Lido,   Italy;   (2)   Institute   for   Biomedical   and   Neural   Engineering/   Biomedical   Technology   Centre   Reykjavik   University   &   Landspitali   Reykjavik,   Iceland   E-­‐mail:  andrea.marcante@ospedalesancamillo.net   We  present   a   case   report   of   atypical   amyotrophic  neuralgia  of   the   suprascapular   nerve   with   isolated   denervation   atrophy   of   rotator   cuff  muscles  and  related  biomechanics  impairments  of  the  shoulder.   The  patient,  lamenting  yearly-­‐long  unsatisfactory  results  of  standard   clinical  physiotherapy,  after  a  baseline  re-­‐evaluation  at  our  hospital,   have   been   treated   for   the   last   seven   months   with   an   additional   personalized   home-­‐based   Electrical   Stimulation   protocol   using   triangular  currents  for  denervated  muscles.  At  the  end  of  the  follow   up   we   observed   clinical,   radiological   (False-­‐color   CT)1   and   neurophysiological   (needle-­‐EMG)2   improvements.   The   case   report   provides   the   opportunity   to   discuss   a   rehabilitative   pathway   for   diagnostics   and   rehabilitation   of   patients   suffering   of   peripheral   denervation,  a  condition  that  is  still  a  challenge  for  clinicians.   1.   Carraro   U,   Edmunds   KJ,   Gargiulo   P   3D   false   color   computed   tomography   for   diagnosis   and   follow-­‐up   of   permanent   denervated   human   muscles   submitted   to   home-­‐based   Functional   Electrical   Stimulation.   Eur   J   Transl  Myol   -­‐  Basic  Appl   Myol  2015;25:129-­‐40.   2.   Pond  A,  Marcante  A,  Zanato  R,  Martino  L,  Stramare  R,  Vindigni   V,  Zampieri  S,  Hofer  Ch,  Kern  H,  Masiero  S,  Piccione  F.  History,   mechanisms  and   clinical   value  of   fibrillation  analyses   in  muscle   denervation  and  reinnervation  by  Single  Fiber  Electromyography   and   Dynamic   Echomyography.   Eur   J   Transl   Myol   -­‐   Basic   Appl   Myol  2014;24:41-­‐54.   *****   Gut  inflammation  and  chronic  low  back  pain:   physiopathology   Sergio  Veneziani,  Christian  Testa,  Claudio  Carlo  Castelli   Ortopedia   e   Traumatologia   e   Dipartimento   di   Emergenza-­‐Urgenza,   Azienda  Ospedaliera  “Papa  Giovanni  XXIII”,  Bergamo,  Italy   E-­‐mail:  sveneziani@hpg23.it   The  Chronic  low  back  pain  (CLBP)  is  a  disabling  condition  affecting  a   majority   of   people   of   the  western   countries.   It   also   deeply   affects   the   quality   of   life   as   it   is   often   linked   to   multidimensional   disturbances   such   as   poor   sleep,   mood   disorders,   chronic   fatigue   and  joint  pain.  There  is  no  condition  with  higher  social  and  economic   expenses.  It  has  been  reported  that  only  a  minority  of  patients  with   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   51 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   gut   inflammation   suffers   from   intestinal   symptoms.   In   a   previous   paper   it   was   proposed   that   gastrointestinal   disturbances,   beyond   mechanical  issues,  could  be  overlooked  in  the  management  of  these   patients.  Dietary  changes  were  successful   in  the  positive  resolution   of   the  described  clinical  case.  Here  we   further   test   this  hypothesis.   We   measured   on   5   subjects   specific   parameters   related   to   gastrointestinal  and  digestive  physiology  that  have  been  associated   with   metabolic   and   immuno-­‐related   pathological   conditions.   Inflammation   in   the   gut   can   lead   to   altered   mucosa   permeability.   The  entrance   in   the  blood   stream  of  abnormal  molecules  activates   the  immune  system  in  a  cascade  of  events  affecting  remote  systems   and   possibly   the   integrity   of   structures   like   the   neuromuscular   junction  or  the  pathways  of  energy  production.  Conditions  that  are   currently   managed   by   orthopaedists,   reumatologists   and   neurologists   could   benefit   from   a   screening   of   the   gastrointestinal   functionality.   1.   Ferreira   PH,   Ferreira  ML,  Maher   CG,   Refshauge   K,   Herbert   RD,   Hodges   PW.   Changes   in   recruitment   of   transversus   abdominis   correlate   with   disability   in   people   with   chronic   low   back   pain.   British  Journal  of  Sports  Medicine  2009  May  26:bjsports61515.   2.   Holmberg  S,  Thelin  A,  Stiernstrom  E,  Svardsudd  K.  Low  back  pain   comorbidity   among   male   farmers   and   rural   referents:   a   population-­‐based   study.   Annals   of   Agricultural   and   Environmental  Medicine  2005;12:261-­‐8.   3.   Masuko   K,   Nakamura   H.   Functional   somatic   syndrome:   how   it   could   be   relevant   to   rheumatologists.   Modern   Rheumatology   2007;17:179-­‐84.   4.   Hadjivassiliou   M,   Sanders   DS,   Grünewald   RA,   et   al   Gluten   sensitivity:   from   gut   to   brain.   The   Lancet   Neurology   2010;   9:   318-­‐30.  doi:  http://dx.doi.org/10.1016/S1474-­‐4422(09)70290-­‐X.   5.   Dal   Pont   E,   D'Incà   R,   Caruso   A,   Sturniolo   GC.   Non-­‐invasive   investigation   in   patients   with   inflammatory   joint   disease”   J   Gastroenterol  2009;20:2463-­‐8.   6.   Veneziani  S,  Christian  Doria  Ch,  Falciati  L,  Castelli  CC,  Fanò-­‐Illic  G.   Return  to  competition  in  a  chronic  low  back  pain  runner:  beyond   a  therapeutic  exercise  approach,  a  case  report.  Eur  J  Trans  Myol   -­‐  Basic  Appl  Myol  2014;24:203-­‐7.   *****   Novel  insights  into  skeletal  muscle  function  by   mechanomyography  in  health  and  disease   Fabio  Esposito   Department  of  Biomedical  Sciences  for  Health,  Università  degli  Studi   di  Milano,  Milan,  Italy   E-­‐mail:  fabio.esposito@unimi.it     In  the  last  decades,  a  growing  body  of  literature  focused  on  the  use   of  mechanomyography   (MMG)   as   a  means   to   study   non-­‐invasively   skeletal  muscle  mechanical  activity.  MMG  signal  is  detectable  at  the   skin  surface  during  the  dimensional  changes  of  active  muscle  fibres   that   generate   pressure   waves   due   to   voluntary   or   evoked   contractions.1,2   A   novel   application   is   the   use   of   an   electromyographic   (EMG),   MMG,   and   force   (F)   signals   combined   approach  as  a  tool  to  partition  the  electrochemical  and  mechanical   events   underpinning   the   electromechanical   delay   during   muscle   contraction   (EMD)   and   relaxation   (R-­‐EMD).3,4   This   approach   has   been  utilized  to  evaluate  the  changes  in  the  electrochemical  and  the   mechanical   components   of   EMD   and   R-­‐EMD   under   several   physiological   conditions   (local   fatigue,   muscle   temperature   manipulation   and   muscle-­‐tendon   unit   stretching).   Under   all   these   circumstances,   the   approach   presented   a   high   reliability   and   sensitivity.  Myotonic   dystrophy   type   1,   the  most   frequent   form   of   inherited   muscular   dystrophy5   involves   a   broad   spectrum   of   systemic   complications.   The   main   features   at   the   skeletal   muscle   level  are  muscle  weakness  and  grip  and  percussion  myotonia.  Distal   muscles  are  generally  more  compromised  than  the  proximal  ones.  In   clinical   settings,   muscle   weakness   and   myotonia   are   usually   determined  on  patients  with  DM1  qualitatively  or  semiquantitatively   by  the  Medical  Research  Council  scale,  by  dynamometry,  and/or  by   physician’s   handgrip   evaluation.   Hence,   a   valid,   non-­‐invasive,   and   reliable   tool   to   assess   the   degree   of   muscle   dysfunction   in   DM1   could  be  of  great   interest   for  clinical   trials   involving  new  therapies.   Therefore,  the  aims  of  the  study  were:  (i)  to  assess  the  reliability  and   sensitivity   of   the   measurement   of   the   electromechanical   delay   components  during  both  contraction  and  relaxation  in  patients  with   DM1;   and   (ii)   to   evaluate   and   discuss   possible   differences   in   delay   components’  duration  between  patients  with  DM1  and  healthy,  age-­‐ matched   controls   (HC).   EMD   and   R-­‐EMD   electrochemical   and   mechanical   components   duration   and   reliability   of   the   measurements  were  investigated  during  skeletal  muscle  contraction   and   relaxation   in   a   group   of   patients   with   DM1   (n   =   13)   and   in   healthy  controls  (n  =  13).  EMG,  MMG,  and  F  were  recorded  from  the   tibialis  anterior  (distal  muscle)  and  vastus  lateralis  (proximal  muscle)   muscles   during   maximum   voluntary   and   electrically-­‐evoked   isometric   contractions.   The   electrochemical   and   mechanical   components   of   the   electromechanical   delay   during   muscle   contraction   and   relaxation   were   calculated   off-­‐line.   Maximum   strength  was  significantly  lower  in  DM1  than  in  controls  under  both   experimental   conditions.   All   electrochemical   and   mechanical   components   were   significantly   longer   in   DM1   in   both   muscles.   Measurement   reliability   was   very   high   in   both   DM1   and   controls.   The   high   reliability   of   the   measurements   and   the   differences   between  DM1   patients   and   controls   suggest   that   the   EMG,  MMG,   and   force   combined   approach   could   be   utilized   as   a   valid   tool   to   assess  the  level  of  neuromuscular  dysfunction  in  this  pathology,  and   to   follow   the   efficacy   of   pharmacological   or   nonpharmacological   interventions.   1.   Cè   E,   Rampichini   S,   Esposito   F.   Novel   insights   into   skeletal   muscle  function  by  mechanomyography:  from  the  laboratory  to   the  field.  Sport  Sciences  for  Health  2015.  in  press.   2.   Orizio   C,   Gobbo  M,   Diemont   B,   Esposito   F,   Veicsteinas   A. The   surface  mechanomyogram  as  a  tool  to  describe  the  influence  of   fatigue   on   biceps   brachii   motor   unit   activation   strategy.   Historical   basis   and   novel   evidence.   Eur   J   Appl   Physiol   2003;90:326-­‐36.  Epub  2003  Aug  16.   3.   Rampichini   S,  Cè  E,   Limonta  E,   Esposito   F.   Effects  of   fatigue  on   the   electromechanical   delay   components   in   gastrocnemius   medialis   muscle.   Eur   J   Appl   Physiol   2014;114:639-­‐51.   doi:   10.1007/s00421-­‐013-­‐2790-­‐9.  Epub  2013  Dec  21.   4.   Cè  E,  Rampichini  S,  Limonta  E,  Esposito  F.  Fatigue  effects  on  the   electromechanical   delay   components   during   the   relaxation   phase   after   isometric   contraction.   Acta   Physiol   (Oxf)   2014;211:82-­‐96.  doi:  10.1111/apha.12212.  Epub  2014  Jan  2..   5.   Meola   G.   Clinical   aspects,   molecular   pathomechanisms   and   management  of  myotonic  dystrophies.  Acta  Myol.  2013;32:154-­‐ 65.  Review.     *****   CIR-­‐myo  -­‐  Lecture  6     Muscle  and  bone  assessed  by  Color  CT,  gait   analysis  and  EMG   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   52 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Kyle  J.  Edmunds(1),  Iris  Árnadóttir  (1),  Magnus  K.  Gíslason  (1),  Ugo   Carraro  (2),  Paolo  Gargiulo  (1,3)   (1)  Institute  for  Biomedical  and  Neural  Engineering,  Reykjavík   University,  Reykjavík,  Iceland;  (2)  IRRCS  Fondazione  Ospedale  San   Camillo,  Venezia,  Italy;  (3)  Department  of  Rehabilitation,  Landspítali,   Reykjavík,  Iceland   E-­‐mail:  paologar@landspitali.is     The  optimum  metric  for  assessing  changes  in  skeletal  muscle  quality   remains   debated.   Identifying   a   novel   quantitative   method   for   muscle  assessment  in  this  regard  would  allow  for  the  generalizability   of  such  studies  to  clinical  practice  and  therefore  aid  in  the  indication   of   compensatory   targets   for   clinical   intervention.1-­‐3  While   there   is   much   extant   literature   reporting   the   use   of   average   HU   values   to   investigate  muscle  quality  and   its  utility  as  a  comorbidity   index,  no   studies  have  yet  to  utilize  the  entire  radiodensitometric  distribution.   The   increasing   prevalence   of   sarcopenic   and   cachexic   muscular   degeneration   necessitates   the   establishment   of   a   robust   quantitative   muscle   assessment   methodology.4   Herein,   we   hypothesize   that   rigorously   quantifying   entire  HU  distributions   can   elicit  much  more   information   regarding  muscle  quality   than  extant   methods   that,   to   date,   only   utilize   average  HU   attenuation   values.   This  study  reports  the  development  and  use  of  this  method,  wherein   we   assess   upper   leg   muscle   quality   utilizing   nonlinear   trimodal   regression   analysis   with   radiodensitometric   distributions   from   computed   tomography   (CT)   scans   of   a   healthy   young   adult,   a   healthy   elderly   subject,   and   an   SCI   patient   with   complete   lower   motor   neuron   denervation.   Results   from   this   assessment   highlight   the  utility  of  utilizing  entire  HU  attenuation  value  distributions  and   identify   novel   parameters   from   these   analyses   that   could   provide   further   insight   into   how   muscular   degradation   can   be   optimally   quantified.   1.   Baumgartner  RN,  Koehler   KM,  Gallagher  D,   et   al.   Epidemiology   of   sarcopenia   among   the   elderly   in   New   Mexico.   American   Journal  of  Epidemiology  1998;147:755–63.   2.   Janssen   I,   Heymsfield   SB,   Ross   R.   Low   relative   skeletal   muscle   mass  (sarcopenia)  in  older  persons  is  associated  with  functional   impairment   and   physical   disability.   Journal   of   the   American   Geriatrics  Society,  2002;50:889–96.     3.   Newman  AB,  Kupelian  V,  Visser  Met  al.   Sarcopenia:  Alternative   Definitions   and   Associations   with   Lower   Extremity   Function.   J   Am   Geriatr   Soc   2003;51,1602–9.   doi:10.1046/j.1532-­‐ 5415.2003.51534.x   4.   Carraro   U,   Edmunds   KJ,   Gargiulo   P.   3D   False   Color   Computed   Tomography   for   Diagnosis   and   Follow-­‐up   of   Permanently   Denervated   Human   Femoral   Muscles   Submitted   to   Functional   Electrical   Stimulation.   Eur   J   Transl   Myol   2015;25:5133.   doi:   10.4081/ejtm.2015.5133.  eCollection  2015  Mar  11.  Review.   5.   Carraro  U,  Kern  H,  Gava  P,  Hofer  C,  Loefler  S,  Gargiulo  P,  Mosole   S,  Zampieri  S,  Gobbo  V,  Ravara  B,  Piccione  F,  Marcante  A,  Baba   A,  Schils  S,  Pond  A,  Gava  F.  Biology  of  Muscle  Atrophy  and  of  its   Recovery  by  FES   in  Aging  and  Mobility   Impairments:  Roots  and   By-­‐Products.   Eur   J   Transl   Myol   2015;25:221-­‐30.   doi:   10.4081/ejtm.2015.5272.  eCollection  2015  Aug  24.  Review.   *****   SATURDAY  April  16,  2016   Hotel  Augustus,  Viale  Stazione  150,  Montegrotto  Terme  (Padova),  Italy      CIR-­‐Myo-­‐Lecture  7     The  RISE  Project:  roots,  outcomes,  byproducts   Helmut  Kern,  Vienna,  Austria   Ludwig  Boltzmann  Institute  of  Electrical  Stimulation  and  Physical   Rehabilitation,  and  Dept.  of  Physical  Medicine  and  Rehabilitation,   Wilhelminenspital  Wien,  Austria   E-­‐mail:  helmut.kern@wienkav.at   During   the   last   decade   we   contributed   to   rehabilitation   in   aging   studying  effects  of  physical  exercise  induced  by  Functional  Electrical   Stimulation   (FES)   in   the   special   case   of   Spinal   Cord   Injury   patients   affected   by   complete   injury   of   the   Conus   Cauda,   a   syndrome   in   which   the   denervated   leg  muscles   are   fully   disconnected   from   the   nervous   system.   Denervated   human   muscles   become   unexcitable   with  commercial  electrical   stimulators  and  undergo  ultra   structural   disorganization  within  a  few  months  from  SCI,  while  severe  atrophy   with  nuclear  clumping  and  fibro-­‐fatty  degeneration  appear  within  3   and  6  years,  respectively.1-­‐4  To  counteract  these  progressive  changes   a  novel  therapy  concept  for  paraplegic  patients  with  complete  lower   motor  neuron  denervation  of  the  lower  extremity  was  developed  in   Vienna:   home-­‐based   functional   electrical   stimulation   of   long-­‐term   denervated  muscles  (h-­‐b  FES).  New  electrodes  and  a  safe  stimulator   for   h-­‐b   FES   have   been   designed   to   reverse   severe   atrophy   by   delivering   high-­‐intensity   (up   to   2,4   J)   and   long-­‐   duration   impulses   (up   to   150   ms)   able   to   elicit   contractions   of   denervated   skeletal   muscle   fibers   in   absence   of   nerves.5,6   Specific   clinical   assessments   and   trainings   were   developed   at   the   Wilhelminenspital   Wien,   Austria,7  based  on  sound  evidence  from  animal  experiments.8  Main   results  of  the  clinical  study  on  patients  which  completed  the  2-­‐year   h-­‐b  FES  training  were:  1.  significant  increase  of  muscle  mass  and  of   myofiber   size,   with   striking   improvements   of   the   ultra-­‐   structural   organization;   2.   recovery   of   tetanic   contractility   with   significant   increase   in   muscle   force   output   during   electrical   stimulation;   3.   capacity   to   perform   FES-­‐assisted   stand-­‐up   and   stepping-­‐in-­‐place   exercise.9-­‐12     The   study   demonstrated   that   h-­‐b   FES   of   permanent   denervated   muscle  is  an  effective  home  therapy  that  results  in  rescue  of  muscle   mass,   function  and  perfusion.  Additional  benefits  are   improved   leg   cosmetic  appearance  and  enhanced  cushioning  effect  for  seating.   1.   Rossini   K,   Zanin   ME,   Carraro   U.   To   stage   and   quantify   regenerative   myogenesis   in   human   long-­‐term     permanent   denervated  muscle.  Basic  Appl  Myol  2002;  12:  277–286.   2.   Kern  H,  Boncompagni   S,  Rossini   K,  Mayr  W,   Fanò  G,   Zanin  ME,   Podhorska-­‐Okolow   M,   Protasi   F,   Carraro   U.   Long-­‐term   denervation   in  humans  causes  degeneration  of  both  contractile   and   excitation-­‐contraction   coupling   apparatus   that   can   be   reversed   by   functional   electrical   stimulation   (FES).   A   role   for   myofiber  regeneration?  J  Neuropath  Exp  Neurol  2004;  63:  919– 931.   3.   Boncompagni  S,  Kern  H,  Rossini  K,  Hofer  C,  Mayr  W,  Carraro  U,   Protasi   F.   Structural   differentiation   of   skeletal   muscle   fibers   in   the  absence  of  innervation  in  humans.  Proc  Natl  Acad  Sci  U  S  A.   2007;  104:  19339–  19344.   4.   Kern   H,   Carraro   U,   Biral   D,   Adami   N,   Zampieri   S.   Severely   atrophic  muscle  fibers  with  nuclear  clumps  survive  many  years   in   permanently   denervated   human   muscle.   The   Open   Pathology  Journal  2009;  3:  106-­‐110.   5.   Mayr  W,  Bijak  M,  Rafolt  D,  Sauermann  S,  Unger  E,  Lanmüller  H.   Basic   design   and   construction   of   the   Vienna   FES   implants:   existing   solutions   and   prospects   for   new   generations   of   implants.  Med  Eng  Phys  2001;  23:  53–60.   6.   Hofer   C,  Mayr  W,   Stöhr   H,   Unger   E,   Kern   H.   A   stimulator   for   functional  activation  of  denervated  muscles.  Artif  Organs  2002;   26:  276–279.   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   53 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   7.   Kern  H,  Hofer  C,  Mayr    W,    Carraro    U.    European    Project  RISE:   Partners,   protocols,   demography.   Basic   Appl  Myol/   European   Journal  of    Translational  Myology  2009;  19:  211-­‐216.   8.   Squecco   R,   Carraro  U,   Kern     H,     Pond     A,     Adami     N,   Biral   D,   Vindigni   V,   Boncompagni   S,   Pietrangelo   T,   Bosco   G,   Fanò   G,   Marini  M,  Abruzzo  PM,  Germinario  E,  Danieli-­‐Betto  D,  Protasi   F,   Francini   F,   Zampieri   S.   Despite   lost   contractility,   a   sub-­‐ population   of     rat   muscle   fibers   maintains   an   assessable   excitation-­‐  contraction  coupling  mechanism  after  long-­‐standing   denervation.  J  Neuropath  Exp  Neurol  2009;68:1256-­‐68.   9.   Kern  H,  Carraro  U,  Adami  N,  Hofer  C,   Loefler  S,  Vogelauer  M,   Mayr   W,   Rupp   R,   Zampieri   S.   One   year   of   home-­‐based   Functional  Electrical  Stimulation  (FES)  in  complete  lower  motor   neuron  paraplegia:  Recovery  of  tetanic  contractility  drives  the   structural   improvements   of   denervated   muscle.   Neurol   Res   2010;  32:  5-­‐12.   10.  Kern   H,   Carraro   U,   Adami   N,   Biral   D   Hofer     C,     Forstner   C,   Mödlin  M,   Vogelauer  M,   Boncompagni   S,   Paolini   C,  Mayr  W,   Protasi   F,   Zampieri   S.   Home-­‐based   Functional   Electrical   Stimulation   (h-­‐b   FES)   recovers   permanently   denervated   muscles   in   paraplegic   patients   with   complete   lower   motor   neuron  lesion.  Neurorehab  Neur  Rep  2010;  24:  709-­‐721.   11.  Gargiulo   P,   Reynisson   PJ,   Helgason   B,   Kern   H,   Mayr   W,   Ingvarsson   P,   Helgason   T,   Carraro   U.   Muscle,   tendons,   and   bone:   structural   changes   during   denervation   and   FES   treatment.  Neurol  Res.  2011;  33:  750-­‐758.   12.  Boncompagni   S.   Severe   muscle   atrophy   due   to   spinal   cord   injury   can   be   reversed   in   complete   absence   of   peripheral   nerves.  European  Journal  Translational  Myology  -­‐  Basic  Applied   Myology  2012;  22:  161-­‐200.   *****    Workshop  Functional  Rejuvenation  in  Aging   Werner  Lindenthaler,  Winfried  Mayr,  Chairs   The  effects  of  chronic  electrical  muscle  stimulation  (EMS)   following  nerve  injury  on  time  to  reinnervation  and   appropriate  reinnervation  of  muscular  targets   Mike  P  Willand  (1)  ,  Joseph  Catapano  (1),  Jennifer  J  Zhang  (1),   Hermann  Lanmueller  (2),  Ewald  Unger  (2),  Martin  Schmoll  (2,5),   Jon  Cheetham  (3),  Jonathan  Norm  Ducharme  (3),  Marta  Cercone   (3),  David  Zealear  (4),  Yike  Li  (4),  Jonathan  C.  Jarvis  (5),  Gregory   Borschel  (1),  Tessa  Gordon  (1)   (1)   Division   of   Plastic   and   Reconstructive   Surgery,   Department   of   Surgery,   The   Hospital   for   Sick   Children,   Toronto,   ON,   Canada;   (2)   Center   for   Medical   Physics   and   Biomedical   Engineering,   Medical   University   Vienna,   Vienna,   Austria;   (3)   College   of   Veterinary   Medicine,   Cornell   University,   Ithaca,   NY,   USA;   (4)   Department   of   Otolaryngology,  Vanderbilt  University,  Nashville,  TN,  USA;  (5)  School   of   Sports   and   Exercise   Sciences,   Liverpool   John  Moores  University,   Liverpool,  UK.   E-­‐mail:  mike.willand@gmail.com     Functional   recovery   after   peripheral   nerve   injury   is   reduced   when   axon  growth  is  misdirected  to  reinnervate  muscles  other  than  their   original   targets.1-­‐3  Here  we  review  the  effects  of  chronic  electrical   muscle   stimulation   (EMS)   following   peripheral   nerve   injury   in   rat,   canine,   and   equine  models   of   peripheral   nerve   injury.   Specifically,   we   examine   whether   EMS   accelerates   reinnervation   of   muscular   targets  and   if   these   targets  are  appropriately   reinnervated  by   their   original   axons   following   nerve   injury.   Methods:   In   the   Sprague   Dawley   rat,   the   lateral   gastrocnemius   nerve   was   transected   and   immediately   repaired.   The   soleus   muscle   was   implanted   with   electrodes   and   connected   to   a   mini   stimulator   implanted   intra-­‐ abdominally.   Muscles   were   stimulated   daily   using   a   12-­‐hour   day   time  pattern  of  a  10  second  burst  of  20  Hz  once  per  hour  followed   by  a  12-­‐hour  night  time  pattern  of  20  Hz  (10  seconds  on,  20  seconds   off).   This   stimulation   pattern   was   delivered   for   2   months.   Appropriate   reinnervation  of   the  soleus  muscle  was  assessed  using   retrograde   labeling   of   the   soleus   nerve.   Functional   recovery   was   assessed   by  measuring   isometric   soleus  muscle   forces.   In   the   dog,   the   recurrent   laryngeal   nerve   was   transected   bilaterally   and   immediately   repaired.   Electrodes   were   implanted   to   stimulate   the   posterior   cricoid   arytenoid   (PCA)  muscles   bilaterally.  Muscles  were   stimulated   continuously   using   either   a   10   or   40   Hz   pulse   train   for   ninety   days.   Appropriate   reinnervation   was   measured   using   electromyography  methods.  Functional  recovery  was  assessed  using   a   treadmill   exercise   test   and   vocal   fold   movement   during   hypercapnia.   In   the   horse,   the   right   recurrent   laryngeal   nerve  was   injured   using   a   stainless   steel   probe   pre-­‐chilled   in   liquid   nitrogen   and   placed   on   the   nerve   for   two   minutes.   Electrodes   were   implanted  into  the  right  PCA  muscle  and  connected  to  an  implanted   stimulator.   Muscles   were   stimulated   for   one   hour   once   every   12   hours  at  22  Hz   (3.5  seconds  on,  6.5  seconds  off)   for  twenty  weeks.   Functional  recovery  was  assessed  using  a  treadmill  exercise  test  and   examining   arytenoid   abduction   and   tracheal   inspiratory   pressure   endoscopically.   Results:   Retrograde   labeling   in   the   rat,   demonstrated   that   EMS   of   the   soleus   muscle   had   no   effect   on   directing  the  original  soleus  neurons  back  to  reinnervate  the  muscle   following   nerve   injury   and   repair.   Muscle   twitch   forces   were   significantly   greater,   however,   tetanic   forces   were   not   different   whether   the  muscle  was  stimulated  or  not.   In   the  dog,  progressive   addition  of  samples  to  the  study  showed  that  exercise  tolerance  and   glottal   area   following   hypercapnia   was   maximal   in   dogs   that   received  PCA   stimulation  at  10  Hz.4  EMG  measurements   in  3  dogs   suggest   that   PCA  muscles   stimulated   at   10   Hz   were   preferentially   reinnervated  by  their  original  motoneurons  whereas  those  muscles   stimulated   at   40   Hz   or   were   unstimulated   had   random   reinnervation.   Horses   that   had   the   PCA   muscle   stimulated   had   improved   function   as   demonstrated   by   lower   negative   tracheal   inspiratory   pressures.   These   improvements   occurred   sooner   after   injury  and  at  lower  exercise  intensities  than  horses  that  did  not  have   the  PCA  muscle   stimulated.  However,   functional   recovery   returned   to   near   baseline   in   all   horses   suggesting   that   the   original   nerve   injury  was  not  severe  enough.  Conclusions:  Despite  stimulating  the   soleus   muscle   in   rats   using   a   pattern   resembling   natural   activity   before  and  during  the  time  of  reinnervation,  EMS  did  not  encourage   the   original   motoneurons   that   were   connected   to   the   stimulated   muscle  to  return.  However,  in  the  dog,  10  Hz  stimulation  promoted   selective  reinnervation.  One  limitation  in  the  dog  study  is  the  small   sample   size   which   needs   to   be   expanded   to   provide   adequate   statistical   power.   In   the   horse,   EMS   enhanced   the   speed   of   functional  recovery  despite  a  nerve  injury  that  was  not  as  severe  as   one   in   both   dogs   and   rats.   Nevertheless,   in   all   animal   models   stimulation   did   not   negatively   impact   functional   recovery   with   muscle  forces   in  the  rat  being  higher  with  stimulation  and  dynamic   airway  measurements  being  enhanced  in  both  dogs  and  horses  that   had  their  muscles  stimulated  following  nerve  injury.   1.   Willand   MP,   Nguyen   MA,   Borschel   GH,   Gordon   T.   Electrical   stimulation   to   promote   peripheral   nerve   regeneration.   Neurorehabil   Neural   Repair   2015   Sep   10.   pii:   1545968315604399.  [Epub  ahead  of  print]  Review.   2.   Willand   MP,   Chiang   CD,   Zhang   JJ,   Kemp   SW,   Borschel   GH,   Gordon   T.   Daily   electrical   muscle   stimulation   enhances   functional   recovery   following   nerve   transection   and   repair   in   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   54 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   rats.   Neurorehabil   Neural   Repair   2015;29:690-­‐700.   doi:   10.1177/1545968314562117.  Epub  2014  Dec  11.   3.   Willand  MP.  Electrical   stimulation  enhances   reinnervation  after   nerve  injury.  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2015;25:243-­‐8.   4.   Zealear   DL,   Mainthia   R,   Li   Y,   Kunibe   I,   Katada   A,   Billante   C,   Nomura  K.  Stimulation  of  denervated  muscle  promotes  selective   reinnervation,   prevents   synkinesis,   and   restores   function.   The   Laryngoscope  124,  E180–E187  (2014).   *****   Establishing  a  hypertrophy  model  in  rats  using  ‘SpillOver’   stimulation  to  cause  co-­‐contraction   Jonathan  C.  Jarvis  (1),  Martin  Schmoll  (1,2),  Manfred  Bijak  (2),   Michael  Haller  (2),  Ewald  Unger  (2),  Hazel  Sutherland  (3),  Hermann   Lanmueller  (2)   (1)   School   of   Sport   and   Exercise   Sciences,   Liverpool   John   Moores   University,   Liverpool,   UK;   (2)   Center   of   Medical   Physics   and   Biomedical   Engineering,   Medical   University   of   Vienna,   Austria;   (3)   Department   of   Musculoskeletal   Biology,   Institute   of   Ageing   and   Chronic   Disease,   University   of   Liverpool,   UK   Research   Institute   for   Sport   and   Exercise   Sciences,   Liverpool   John   Moores   University,   Liverpool,  UK.   E-­‐mail:  "Jarvis,  Jonathan"  J.C.Jarvis@ljmu.ac.uk     The   cellular   mechanisms   underpinning   the   maintenance,   gain   and   loss   of   muscle   mass   are   of   great   interest   at   present,   given   the   popularity   of   bodybuilding,   the   potential   for   increased   muscle   metabolism   to   reduce   the   damage   caused   by   diabetes   and   insulin   resistance,  and  the  key  role  of  muscle   function  associated  with  the   decline  in  mobility  and  well-­‐being  associated  with  ageing.  There  are   many  model  systems   in  which  to  make  experimental   investigations   of  muscle  hypertrophy   in   rodents.1-­‐9   It   is   generally   considered   that   increased  average  force  generation  (loading)  is   important,  although   hypertrophy   can   be   achieved   in   some   muscles   by   pharmaceutical   agonism   of   the   androgen   receptor   family,   or   by   genetic   manipulation   of,   for   example,   the   response   to   IGF.   Models   have   included   removal1,2,3   or   denervation   of   agonists   to   generate   constant   overload,   various   training   modalities   such   as   squats,3     lifting,4,5   or   jumping   for   a   food   reward,   and   treadmill   or   ladder   climbing   exercise,6   sometimes   with   added   weight7   to   increase   the   muscular   effort.   We   have   designed   a   hypertrophy   model   using   programmed   exercise   by   stimulating   agonists   and   antagonists   simultaneously.  In  the  rat  hind  limb,  the  dorsiflexor  muscles  that  lift   the   foot   are   supplied  by   the   common  peroneal  nerve  whereas   the   plantarflexor   muscles   are   supplied   by   the   tibial   nerve.   The   plantarflexors  are   the   larger  and  stronger  group  so   it   is  possible   to   generate  loaded  contractions  of  the  dorsiflexors  by  activating  them   fully   at   the   same   time   as   a   partial   activation   of   the   plantarflexors.   We   have   achieved   this   with   a   single   channel   implant   by   careful   positioning  of   the  electrodes  with   the   cathode  under   the   common   peroneal  nerve  and  the  anode  near  to  the  tibial  nerve  as  it  runs  on   the  proximal  posterior  surface  of  the  gastrocnemius  muscle.  The  key   to   success   in   this   model   is   the   ability   to   adjust   remotely   the   stimulating   current   and   to   choose   a   stimulation   pattern   that   generates   high   force   contractions  with  minimal   disturbance   to   the   subject.  Using  the  new  miniVStim  device  developed  between  Vienna   and  Liverpool  we  are  able  to  programme  an  ‘adaptation’  pattern  so   that  the  subject  is  accustomed  to  the  sensation  of  muscle  activation   at   a   low   level   before   the   loaded   contractions   are   made.   With   stimulation   in   one   session   per   day   of   5   sets   of   10   repetitions   at   100Hz   (2s   ON   2s   OFF)   and   2.5   minutes   between   sets,   we   have   achieved   hypertrophy   of   the   tibialis   anterior   muscle   giving   an   increase   in  wet  weight  of  between  11,5  and  13,7%   in  5  rats  over  4   weeks.  We  will  use   this  model   to   investigate   further   the  sensitivity   to  hypertrophy  of  the  various  fibre  types  and  the  cellular  pathways   that  are  activated  in  this  response.   1.   Goldberg  AL,  Etlinger  JD,  Goldspink  DF,  Jablecki  C.  Mechanism  of   work-­‐induced   hypertrophy   of   skeletal   muscle.   Med   Sci   Sports   1975;7:185–98.   2.   Ianuzzo   CD,   Gollnick   PD,   Armstrong   RB.   Compensatory   adaptations   of   skeletal   muscle   fiber   types   to   a   long-­‐term   functional  overload.  Life  Sciences  1976;19:1517–23.   3.   Gollnick   PD,   Timson   BF,   Moore   RL,   Riedy   M.   Muscular   enlargement  and  number  of   fibers   in  skeletal  muscles  of   rats.   J   Appl  Physiol  Respir  Environ  Exerc  Physiol  1981;50:936–43.   4.   Tamaki   TE,   Uchiyama   SH,   Nakano   SH.   A   weight-­‐lifting   exercise   model  for  inducing  hypertrophy  in  the  hindlimb  muscles  of  rats.   Med  Sci  Sports  Exerc  1992;24:  881–8.   5.   Wong  TS,  Booth  FW.  Skeletal  muscle  enlargement  with  weight-­‐ lifting  exercise  by  rats.  J  Appl  Physiol  1988;65:  950–4.   6.   Duncan  ND,  Williams  DA,   Lynch  GS.   Adaptations   in   rat   skeletal   muscle   following   long-­‐term   resistance   exercise   training.   Eur   J   Appl  Physiol  Occup  Physiol  1998;77:372–8.   7.   Hornberger  TA,  Farrar  RP.  Physiological  hypertrophy  of  the  FHL   muscle   following   8  weeks   of   progressive   resistance   exercise   in   the  rat.  Can  J  Appl  Physiol    2004;29:16–31.    8.   Adams   GR,   Cheng   DC,   Haddad   F,   Baldwin   KM.   Skeletal   muscle   hypertrophy   in   response   to   isometric,   lengthening,   and   shortening   training  bouts  of  equivalent  duration.   J  Appl  Physiol   2004;96:1613–8.   9.   Garma  T,  Kobayashi  C,  Haddad  F,  Adams  GR,  BodellPW,  Baldwin   KM.  Similar  acute  molecular  responses  to  equivalent  volumes  of   isometric,  lengthening,  or  shortening  mode  resistance  exercise.  J   Appl  Physiol,  2007;102:135–43.   *****   Investigating  Energy  Efficiency  for  different  Pulse  shapes   and  electrode  arrangements  to  activate  motor  neurones   Martin  Schmoll  (1,2),  Jonathan  C.  Jarvis  (1)   (1)   School   of   Sport   and   Exercise   Sciences,   Liverpool   John   Moores   University,   Liverpool,   UK;   (2)   Center   of   Medical   Physics   and   Biomedical  Engineering,  Medical  University  of  Vienna,  Austria.   E-­‐mail:  martin.schmoll@meduniwien.ac.at     One   of   the   main   determinants   of   the   size   of   neural   implantable   pulse   generators   is   the   size   of   the   battery.   The   challenge   for   engineers   is   to   design   devices   that   are   small   in   volume   whilst   fulfilling   their   stimulation   task   as   long   as   possible.   Efficient   stimulation  methods  are  crucial  for  their  success.    Wongsarnpigoon   pointed   out   three   different   types   of   efficiency   relating   to   nerve   activation.1  A  “charge-­‐efficient”  stimulation  has  the  positive  effect  of   reducing   tissue   damage.   As   the   battery   size   is   proportional   to   the   maximal   instantaneous   power   required,   “power-­‐efficient”   stimulation  could  reduce  battery-­‐size  and  therefore  the  overall  size   of  an  implant.  An  “energy-­‐  efficient”  stimulation  on  the  other  hand,   increases   the   battery   lifetime.   We   have   compared   different   waveforms   according   to   their   “energy-­‐efficiency”.   Six   different   waveforms   have   been   investigated   (rectangular   monophasic,   rectangular   biphasic,   rectangular   biphasic   with   interphase   gap,   gaussian   biphasic,   exponential   biphasic,   asymmetric   rectangular   biphasic   with   interphase   gap)   to   clarify   some   of   the   potentially   useful   efficiencies   noted   in   other   studies.2-­‐7   Another   interesting   aspect   that   has   been   investigated   in   our   latest   experiments,   is   a   comparison  between  monopolar  and  bipolar  stimulation,  and  some   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   55 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   investigation   of   the   transition   between   the   monopolar   and   the   bipolar   configuration   in   terms  of   stimulation  efficiency.   For  bipolar   stimulation,   two   stainless-­‐steel   loop   electrodes  were   placed   under   the   common   peroneal   nerve   of   rats   under   buprenorphine/isofluorane  anaesthesia.   In  the  monopolar  situation   the   anodal   electrode   was   a   hypodermic   needle   placed   under   the   skin  more  than  50mm  away  from  the  cathode.  The  cathode  was  the   same  in  both  cases.  The  isometric  force  produced  at  optimal  length   by   the   extensor-­‐digitorum-­‐longus   muscle   was   measured   using   a   load-­‐cell.   Our   results   showed   a   noticeable   difference   between   monopolar  and  bipolar  stimulation.  Monopolar  stimulation  showed   generally   higher   energy   levels   than   bipolar   stimulation   to   activate   the   motor   neurones   of   the   common   peroneal   neve.   While   the   introduction   of   an   interphase-­‐gap   increased   the   threshold   current,   where  the  force  was  10%  of  the  control-­‐force,  in  the  bipolar  case,  a   reduction   was   observed   in   the   monopolar   situation.   In   previous   bipolar  experiments  we  found  an  increased  energy  requirement  for   threshold   activation   with   asymmetric   waveforms   while   the   monopolar  stimulation  again  showed  a  reduction.  The  results  show   similar   effects   as   described   in   literature   when   using   monopolar   stimulation.   Nevertheless   we   achieved   different   results   for   the   bipolar   stimulation   regime.   It   is   therefore   important   to   bear   these   differences   in   mind,   when   designing   electrodes   and   patterns   of   stimulation  to  improve  stimulation  efficiency.   1.   Wongsarnpigoon  A,  Woock  JP,  Grill  WM.  Efficiency  Analysis  of   Waveform  Shape  for  Electrical  Excitation  of  Nerve  Fibers.  IEEE   Trans  Neural  Syst  Rehabil  Eng  2010;18:319–28.   2.   Shepherd  RK,  Javel  E.  Electrical  stimulation  of  the  auditory   nerve:  II.  Effect  of  stimulus  waveshape  on  single  fibre  response   properties.  Hearing  Research  1999;130:171–88.   3.   McKay    CM,  Henshall  KR.  The  perceptual  effects  of  interphase   gap  duration  in  cochlear  implant  stimulation.  Hearing  Research   2003;181:94–9.   4.   Carlyon  RP,  van  Wieringen  A,  Deeks  JM,  Long  CJ,  Lyzenga  J,   Wouters  J.  Effect  of  inter-­‐phase  gap  on  the  sensitivity  of   cochlear  implant  users  to  electrical  stimulation,”  Hearing   Research  2005;205:210–24.   5.   Prado-­‐Guitierrez  P,  Fewster  LM,  Heasman  JM,  McKay  CM,   Shepherd  RK.  Effect  of  interphase  gap  and  pulse  duration  on   electrically  evoked  potentials  is  correlated  with  auditory  nerve   survival.  Hear  Res  2006;215:  47–55.   6.   Weitz  AC,  Behrend  MR,  Ahuja  AK,  Christopher  P,  Wei  J,  Wuyyuru   V,  Patel  U,  Greenberg  RJ,  Humayun  MS,  Chow  RH,  Weiland  JD.   Interphase  gap  as  a  means  to  reduce  electrical  stimulation   thresholds  for  epiretinal  prostheses,”  J  Neural  Eng  2014;11:   016007.   7.   Macherey  O,  van  Wieringen  A,  Carlyon  RP,  Deeks  JM,  Wouters  J.   Asymmetric  Pulses  in  Cochlear  Implants:  Effects  of  Pulse  Shape,   Polarity,  and  Rate,”  J  Assoc  Res  Otolaryngol,  vol.  2006;7:253–66.   *****   MiniVStim18B:  A  highly  configurable  battery  powered  FES   implant  for  long-­‐term  implantation  in  small  animals   Manfred  Bijak  (1),  Ewald  Unger  (1),  Michael  Haller  (1),  Martin   Schmoll  (1,2),  Jonathan  C.  Jarvis  (2),  Hermann  Lanmüller  (1)   Center   of   Medical   Physics   and   Biomedical   Engineering,   Medical   University  of  Vienna,  Austria.   (2)  Liverpool   John  Moores  University,   UK     E-­‐mail:  manfred.bijak@meduniwien.ac.at     According   to   PubMed   roughly   10%   of   the   annually   added   publications   in   the   Life   Sciences   describe   findings   obtained   from   animal  models.  Since  half  of  these  studies  are  done  in  mice  and  rats   it   can   be   assumed   that   there   is   a   need   for   implantable   electrical   stimulators   which   are   flexible,   reliable   and   small   enough   (~1   cm3)   that  implantation  is  possible  in  mice.  It  is  important  that  animals  do   not  have  to  be  isolated  during  stimulation  periods  and  that  they  can   run   freely.   MiniVStim   12A   is   a   battery   powered   implantable   electrical   stimulator   able   to   deliver   constant   current   monophasic,   rectangular  pulses  up  to  2mA  and  1ms  pulse  width  (@1kOhm).  It   is   easy   to   use   because   the   required   stimulation   pattern   is   preprogramed  during  manufacturing.  On,  off  or  different  stimulation   patterns   can   be   cyclically   activated   with   a   strong   magnet,   also   through  the  skin.  This  implant  has  an  outer  diameter  of  15  mm  and   a   volume   of   1.2   cm3.   MiniVStim   12B   has   the   same   mechanical   dimensions   but   can   be   fully   programed   via   a   transcutaneous   bidirectional   data   link.   Both   types   of   implants   are   already   successfully   used   in   studies.1   The   latest   generation   of   implants   is   represented   by   the   new  MiniVStim18B.   It   is   slightly   larger   (22mm   outer  diameter,  5.3  cm3)   than   its  predecessors  but  offers  an  8   fold   longer   battery   life.   Additionally,   it   can   deliver   biphasic   constant   current   pulses   and  extends   the   stimulation  parameter   range  up   to   8mA  at  a  maximum  output  voltage  of  10V  and  with  a  pulse  width  of   5ms   (@1kOhm)   for   monophasic   and   2x5ms   for   biphasic   pulses.   Lifetime   is   strongly   dependent   on   the   chosen   stimulation   pattern.   For  example,  monophasic  stimulation  with  a  duty  cycle  of  20%  (20%   on,  80%  off  time)  and  2mA,  100Hz,  250µs  pulse  width,  1kOhm  load,   leads   to   a   battery   lifetime   of   300   days   and  when   stimulating  with   8mA   life   time   comes   to   70   days.   Under   the   same   circumstances   except   choosing   stimulation   frequency   of   10Hz   a   lifetime   of   1000   days  and  450  days  could  be  expected.  The  very  low  standby  current   consumption   (<8µA)   helps   to   increases   the   battery   life   time   proportionally   when   stimulation   is   intermittently   applied,   like   in   most  applications.   If   there   is  no   stimulation  at  all,   the   ‘shelf   life’   is   nearly  4  years.   Qin  W,  Sun  L,  Cao  J,  Peng  Y,  Collier  L,  Wu  Y,  Creasey  G,  Li  J,  Qin  Y,   Jarvis  JC,  Bauman  WA,  Zaidi  M,  Cardozo  C.  The  central  nervous   system  (CNS)-­‐independent  anti-­‐bone-­‐resorptive  activity  of  muscle   contraction  and  the  underlying  molecular  and  cellular  signatures.  J   Biol  Chem  2013:288:13511–21.   *****   Chronic  electrical  stimulation  in  rodents:  Results  of  a   feasibility  study   Michael  Karbiener   ENT  University  Hospital  Graz,  Department  of  Phoniatrics,  Medical   University  of  Graz,  Austria   E-­‐mail:  michael.karbiener@medunigraz.at     *****   Chronic  neurostimulation  for  treatment  of  age  related   laryngeal  muscular  atrophy   Marcus  Gugatschka  (1),  Michael  Karbiener(  1),  Justin  Perkins  (2),   Claus  Gerstenberger  (1),  Jonathan  C.  Jarvis  (3),  Gerhard  Friedrich  (1)   (1)  ENT  University  Hospital  Graz,  Department  of  Phoniatrics,  Medical   University   of   Graz,   Austria;   (2).   The   Royal   Veterinary   College,   Department  of  Clinical  Sciences  and  Services,  London,  UK,  3.  Faculty   of  Science,  John  Moores  University,  Liverpool,  UK   E-­‐mail:  Markus.Gugatschka@klinikum-­‐graz.at   Muscle  atrophy  as  part  of  the  ageing  process  also  affects  the  larynx,   where   it   constitutes   the   major   cause   of   presbyphonia.   Current   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   56 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   treatment  options  are  mainly  conservative  or  phonosurgically  based   and   are   far   from  being   satisfactory.   Electrical   stimulation  of  motor   neurons   constitutes   a   promising   strategy.  Materials   and  Methods:   Using  aged  sheep  as  an  animal  model  electrical  chronic  stimulation   of  laryngeal  muscles  was  achieved  via  a  mini-­‐electrode  that  targeted   the   right   recurrent   laryngeal   nerve   (RLN;   unilateral   stimulation).   Functional  electrical  stimulation  (FES)  implants  were  programmed  to   deliver   a   pattern   able   to   evoke   supramaximal   muscle   stimulation   over   a   period   of   29   days.   At   the   end   of   the   study,   vocalis   and   posterior   crico-­‐arytenoid   muscles   were   excised   and   analyzed   molecularly   and   histologically.   To   quantify   the   expression   levels   of   genes   related   to   distinct   muscle   fiber   types,   a   real-­‐time   PCR   (RT-­‐ qPCR)  analysis  pipeline  was  newly  established.  Results:  First   results   showed   a   shift   towards   larger   muscle   fiber   diameters   of   the   stimulated   side,   compared   to   the  unstimulated   control   side.  Based   on   this,   chronic   electrical   stimulation   of   the   RLN   can   induce   hypertrophy   of   the   vocalis   muscle   even   after   a   relatively   short   stimulation  period  of  29  days.  Upcoming   trials  will   focus  on   longer   stimulation  periods  as  well  as  more  intense  stimulation  algorithms.   1.   Kletzien   H,   Russell   JA,   Connor   NP.   The   effects   of   treadmill   running   on   aging   laryngeal   muscle   structure.   Laryngoscope.   2015  Aug  8.  doi:  10.1002/lary.25520.   2.   Suzuki   T,   Connor   NP,   Lee   K,   Bless   DM,   Ford   CN,   Inagi   K.   Age-­‐ related  alterations  in  myosin  heavy  chain  isoforms  in  rat  intrinsic   laryngeal  muscles.  Ann  Otol  Rhinol  Laryngol.  2002;111:962-­‐7.   3.   Martins  RH,  Benito  Pessin  AB,  Nassib  DJ,  Branco  A,  Rodrigues  SA,   Matheus   SM.   Aging   voice   and   the   laryngeal   muscle   atrophy.   Laryngoscope.  2015;125:2518-­‐21.11.   *****    Workshop:  ES  in  Neuromuscular  Disorders,   Jonathan  C.  Jarvis,  Feliciano  Protasi,  Chairs     Method  for  selective  surface  stimulation  of  denervated   muscles  in  the  larynx   Berit  Schneider-­‐Stickler  (1),  Matthias  Leonhard  (2),  Lukas  Kneisz   (2),  Winfried  Mayr  (3)   (1)   Medical   University   of   Vienna,   Austria;   (2),   Med-­‐El,   Innsbruck,   Austria;   (3)   Center   of  Medical   Physics   and   Biomedical   Engineering,   Medical  University  of  Vienna,  Austria.   E-­‐mail:   berit.schneider@meduniwien.ac.at     Vocal  fold  paralysis  is  a  pathological  motion  impairment  of  one  vocal   fold,   mostly   caused   by   laryngeal   nerve   damage.   If   the   vocal   fold   does  not  reinnervate  a  flaccid  paralysis  occurs  due  to  denervation  of   the   vocalis   muscle   and   its   atrophy.1   Patients   with   unilateral   vocal   cord   paralysis   suffer   from   hoarse   and   weak   voice   since   there   is   always   a   remaining   glottic   gap  during   phonation.   Today’s   standard   treatment   of   unilateral   paralysis   includes   surgical   medialization   through  either   injection  augmentation  or   laryngoplastic   framework   surgery.2  We  want  to  investigate  whether  it  is  possible  to  selectively   stimulate   the   denervated   muscle   fibers   of   the   vocalis   without   causing   pain   or   excitation   of   sensory   nerve   fibers   or   activation   of   innervated  muscles  in  the  neck  region.  The  goal  is  1.  a  verivication  of   functionality   for   screening   and   2.   a   strengthening   and   increase   in   total  volume  of  the  target  muscle  on  order  to  improve  voice  quality   in   patients   with   unilateral   paralysis.   In   combination   with   voice   therapy  also  electrical   stimulation  of   laryngeal  muscles  has  alraedy   been  used   in  order   to  achieve  hypertrophy.3  Furthermore   research   with   functional   electrical   stimulation   of   patients   with   long   time   denervated   limb   muscles   showed   very   promising   results.   4   The   selective  stimulation  of  denervated  muscles  has  been  investigated  in   rabbits   with   unilateral   paresis   of   the   recurrent   laryngeal   nerve.   It   could  be  shown  that  with  triangular  ramping  and  very  long  pulses  (>   200ms)   the   afferent   nerve   fibers   where   not   stimulated   but   only   denervated  muscle,  with  change  in  muscle  fibers  confirmed  through   histology.5,6  It  is  to  be  investigated  if  these  findings  can  be  repeated   with   surface   electrodes  positioned   in   the  neck   area   and   successful   stimulation   of   the   denervated   vocalis   muscle   can   be   performed   without  causing  pain  and  excessive  contraction  of  neighboring  neck   muscles   rendering   treatment   impossible.   The   optimal   stimulation   parameters   for   this   application   and   ideal   position   of   the   surface   electrodes  have  yet  to  be  investigated.   1.   Hirano   M,   Kirchner   J,   Bless   D,   Neurolaryngology,   recent   advances",  SPG  1986   2.   Sulica  L,  Blitzer  A.  Vocal  Fold  Paralysis,  Springer  2006   3.     Kruse  E.  Die  Reizstrombeh.andlung  als  integraler  Bestandteil  der   logopadischen  Stimmtherapie.   Sprache  Stimme  Gehör  1989;13:   64-­‐70.   4.   Mayr   W,   Hofer   C,   Bijak   M,   Rafolt   D,   Unger   E,   Reichel   M,   Sauermann   S,   Lanmueller   H,   Kern   H.   Functional   Electrical   Stimulation   (   FES   )   of   Denervated   Muscles :   Existing   and   Prospective   Technological   Solutions   Eur   J   Transl   Myol   2002;12:1–4.   5.   Martin   F,   Witt   TN.   “Elektrodiagnostische   und   histometrische   Untersuchungen   über   den   Einfluß   von   Reizstrom   auf   die   Atrophie   der   denervierten   Kehlkopfmuskulatur   im   Tierexperiment.,”  Laryngol.  Rhinol.  Otol.  (Stuttg)  1983;62.   6.   Schleier   E,   Streubel   H-­‐G.   Beziehungen   zwischen   diagnostischer   Aussage   und   therapeutischem   Ergebnis   bei   Rekurrenslähmung,   Folia  phoniat  1980;32:323-­‐33   *****   Determining  optimal  settings  for  selective  surface   stimulation  in  order  to  recruit  paralyzed  facial  muscles   under  non  painful  conditions   Gerd  Fabian  Volk(1),  Orlando  Guntinas-­‐Lichius(1),  Tobias  Schmid   (1),  Lukas  Kneisz  (2),  Matthias  Ladurner  (2),  Winfried  Mayr  (3)   (1)   ENT   Jena,  Germany   (2)  MEDEL   Innsbruck,  Austria;   (3)  Center  of   Medical   Physics   and   Biomedical   Engineering,  Medical   University   of   Vienna,  Austria.   E-­‐mail:  fabian.volk@med.uni-­‐jena.de     Facial   nerve   paralysis   as   a   peripheral   nerve   injury   results   in   neuromuscular  atrophy.  The  symptoms  include  significant  aesthetic,   functional  and  often   life-­‐altering  consequences.  Several  procedures   such  as  Nerve  Grafting,  Facial  Reanimation  and  Rehabilitation  have   been   developed   to   treat   functional   and   cosmetic   aspects   of   this   disease.[1]  Nerve  grafting  is  a  sophisticated  surgery,  which  requires   experience   but   offers   promising   results.   Although   cable   grafting   is   state  of  the  art,  the  method  suffers  the  disadvantage  of   long  nerve   regrowth  time.  [2]Facial  Pacing  systems  too  show  promising  results   to  treat  facial  paralysis.  [3]  [4]  Former  research  showed  good  results   stimulating   denervated   extremity   muscles   using   FES.[5]   Nevertheless  this  field  of  research  is  still  lacking  optimal  stimulation   settings   to   selectively   recruit   denervated   atrophic   or   simply   age-­‐ related   atrophic   facial   muscles   under   non   painful   conditions.   Methods:   Several   Devices   are   considered   to   investigate   optimal   stimulation   settings.   To   encourage   noninvasive   screening  methods   for  facial  pacing,  surface  electrodes  are  used  to  estimate  the  optimal   settings   for   stimulations.   The   use   of   surface   electrodes   causes   the   need  for  optimized  electrode  positioning,  which  is  also  investigated.   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   57 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Results:   Martin   et   al.   [6]   showed   that   recruitment   of   denervated   muscles   requires   exponentially   shaped   pulses   with   long   phase   durations(>200ms).     The   outcome   of   our   investigation   confirms   these   findings   as   well,   showing   best   performance   when   recruiting   paralyzed   facial   human   muscles   with   biphasic   long-­‐duration   impulses.   It   is   crucial   to   position   the   surface   electrodes   appropriately   in   order   to   avoid   stimulation   of   innervated  muscles,   for   instance   the  masseter  muscle.   Conclusions:   Surface   electrodes,   combined   with   the   optimal   stimulation   settings,   offer   a   screening   possibility   for   facial   pacing.   Since   muscles   affected   by   age-­‐related   atrophy   could   be   recruited   too,   further   research   is   necessary   to   show   effectiveness   of   training   using   the   determined   exponential   patterns.   1   Divi   V,   Deschler   DG.   Re-­‐animation   and   rehabilitation   of   the   paralyzed   face   in   head   and   neck   cancer   patients.   Clin   Anat   2012;25:99–107.   2   Guntinas-­‐Lichius   O,   Streppel   M,   Stennert   E.   Postoperative   functional   evaluation   of   different   reanimation   techniques   for   facial  nerve  repair.  Am  J  Surg  2006;191:61–7.   3   Frigerio   A,   Hadlock   TA,   Murray   EH,   Heaton   JT.   Infrared-­‐Based   Blink-­‐Detecting  Glasses  for  Facial  Pacing.  JAMA  Facial  Plast  Surg   2014;16:211.   4   Gittins   J,   Martin   K,   Sheldrick   J,   Reddy   A,   Thean   L.   Electrical   stimulation  as  a  therapeutic  option  to  improve  eyelid  function  in   chronic   facial   nerve   disorders,”   Investig   Ophthalmol   Vis   Sci   1999;40;547–54.   5.   Mayr   W,   Hofer   Ch,   Bijak   M,   Rafolt   D,   Unger   E,   Reichel   M,   Sauermann   S   Lanmueller   H,   Kern   H.   Functional   Electrical   Stimulation   (FES)   of   Denervated   Muscles :   Existing   and   Prospective   Technological   Solutions.   Eur   J   Transl   Myol   2002;12:1–4,.   6.   Martin   F,   Witt   TN.   Elektrodiagnostische   und   histometrische   Untersuchungen   über   den   Einfluß   von   Reizstrom   auf   die   Atrophie   der   denervierten   Kehlkopfmuskulatur   im   Tierexperiment.  Laryngol.  Rhinol  Otol  (Stuttg)  1983;62,.     *****   Vagus  stimulation  and  other  ES  managements   Jonathan  C.  Jarvis,  Francesco  Piccione,  Chairs   Percutaneous  Auricular  Vagus  Nerve  Stimulation   Eugenijus  Kaniusas  (1),  Stefan  Kampusch  (1),  Jozsef  Constantin   Széles  (2)   (1)  Institute  of  Electrodynamics,  Microwave  and  Circuit  Engineering,   TU   Wien,   Austria;   (2)   University   Clinic   for   Surgery,   Medical   University  Vienna,  Austria.   E-­‐mail:  kaniusas@tuwien.ac.at     Artificial   stimulation   of   the   vagus   nerve,   the   main   nerve   of   the   parasympathetic   nervous   system,   gained   importance   in   the   last   years.   Due   to   the   modulatory   interaction   with   the   autonomous   nervous   system,   the   stimulation   re-­‐establishes   the   sympathovagal   balance,   counteracts   over-­‐inflammation   responses   or   improves   peripheral   perfusion.1-­‐3   Thus,   the   clinical   applications   have   a   wide   range   from   depression   to   acute/chronic   pain   or   cardiovascular   dysfunction   up   to   various   neurological   disorders.3-­‐7   Neuromodulation   is   mediated   via   either   implanted   cervical,   transcutaneous   cervical/auricular   or   percutaneous   auricular   stimulation   devices.   While   implanted   stimulation   devices   are   interrelated   with   high   risks/costs   and   transcutaneous   devices   lack   precision   and   require   strong   stimuli   during   their   operation,   percutaneous   devices   seem   to   avoid   these   drawbacks.8   Current   percutaneous   stimulators   use   needle   electrodes   in   the   auricle   to   stimulate  afferent  nerve  fibers  by  the  use  of  simple  monophasic  or   biphasic   stimulation   patterns   with   the   need   for   an   additional   reference   electrode.5   No   adaptation   of   the   stimuli   is   possible   to   account   for   the   specific   pathology   to   be   treated   as   well   as   the   current  physiological  state  of  the  patient.8  Our  group  has  developed   a   multi-­‐punctual   percutaneous   stimulator   which   operates   three   independent  stimulation  channels  without  any  additional  reference   electrode.8  Stimulation  patterns,  with  specific  triphasic  pulses,  seem   to  reduce  adaptation  processes  and  to  establish  a  pathology  specific   efficient   stimulation.   The   pattern   can   be   advantageously   adapted   throughout   the   stimulation   duration   to   account   for   the   current   treatment   state   and   physiological   state   of   the   patient   [9].   Specific   and  precise  positioning  of  needles   -­‐  based  on  electrical  and  optical   approaches10   -­‐   close   to   auricular   nerves   is   performed,   which   is   of   high   importance   for  efficient  nerve  stimulation.  Preliminary  studies   of  our  group  show  positive  effects  of  this  percutaneous  stimulation   on  heart   rate   variability,   cerebral/peripheral   blood  perfusion,  pain,   sleep,  diabetic  food  syndrome  and  cervical  dystonia.1,6,8,9,11,12   1.   Kampusch  S,  Thürk  F,  Kaniusas  E,  Széles  JC.  Autonomous  nervous   system   modulation   by   percutaneous   auricular   vagus   nerve   stimulation.   2015   IEEE   Sensors   Applications   Symposium   Proc:   2015;79-­‐84.   2.     Borovikova   LV,   Ivanova   S,   Zhang   M,   et   al.   Vagus   nerve   stimulation   attenuates   the   systemic   inflammatory   response   to   endotoxin.  Nature  2000;405(6785):458-­‐62.   3     Payrits   T,   Ernst   A,   Ladits   E,   et   al.   Vagal   stimulation   -­‐   a   new   possibility   for   conservative   treatment   of   peripheral   arterial   occlusion  disease.  Zentralbl  Chir  2011;136:431-­‐5.   4     Groves   DA,   Brown   VJ.   Vagal   nerve   stimulation:   a   review   of   its   applications   and  potential  mechanisms   that  mediate   its   clinical   effects.  Neurosci  Biobehav  Rev  2005;29:493-­‐500.   5     Sator-­‐Katzenschlager  SM,  Scharbert  G,  Kozek-­‐Langenecker  SA,  et   al.   The   short   and   long   term   benefit   in   chronic   low   back   pain   through   adjuvant   electrical   versus   manual   auricular   acupuncture.  Anesth  Analg  2004;98:1359-­‐64.   6.   Kampusch   S,   Kaniusas   E,   Széles   JC.  Modulation   of  muscle   tone   and   sympathovagal   balance   in   cervical   dystonia   using   percutaneous   stimulation   of   the   auricular   vagus   nerve.   Artif   Organs  2015;39:E202-­‐12,.   7.   Ellrich   J.   Transcutaneous   vagus   nerve   stimulation.   Eur   Neurol   Rev  2011;6:  254-­‐6.   8.   Kampusch   S,   Kaniusas   E,   Széles   JC.   New   approaches   in   multi-­‐ punctual  percutaneous  stimulation  of  the  auricular  vagus  nerve.   Proc   of   the   6th   Intern   IEEE   EMBS   Conf   on   Neural   Engineering   2013:263-­‐6.   9.   Kaniusas   E,   Széles   JC,   Materna   T,   Varoneckas   G.   Adaptive   auricular   electrical   stimulation   controlled   by   vital   biosignals.   BIODEVICES  Proc  2009:304-­‐9.   10.  Kaniusas  E,  Varoneckas  G,  Mahr  B,  Széles  JC.  Optic  visualization   of   auricular   nerves   and   blood   vessels:   optimisation   and   validation.  IEEE  Trans  Instrum  Meas  2011;60:3253-­‐8.   11.  Széles   JC,   Varoneckas   G,   Kaniusas   E.   Auricular   electrical   stimulation   (P-­‐STIM)   for   insomnia   treatment   using   remote   control.  Med-­‐e-­‐Tel  Proc  2010:747-­‐51.   12.  Széles   JC,   Kampusch   S,   Kaniusas   E.   Peripheral   blood   perfusion   controlled  by  auricular  vagus  nerve  stimulation.  Proc  of  the  17th   Intern  Conf  on  Biomedical  Engineering  2013:  73-­‐7.   *****   “Noisy”  electrical  stimulation  patterns  facilitate  the  activity   of  cultured  muscle  cells   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   58 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Marina  Sciancalepore  (1),  Giuliano  Taccola  (2),  Gaia  Ziraldo  (1),   Tamara  Coslovich  (2),  Paola  Lorenzon  (1)   (1)  Dept.   Life   Sciences   and  BRAIN  Center,  University   of   Trieste;   (2)   Dept.  Neuroscience,  SISSA,  Trieste,  Italy   E-­‐mail:  msciancalepore@units.it   Use   of   electrical   stimulation   (ES)   of   skeletal   muscle   as   a   tool   to   restore   normal   control   of  movement   and   ability   to   perform  motor   tasks  has   lately   received   increasing  attention.   Its   capability   to  elicit   muscle  tissue  contractions  through  the  delivery  of  current  impulses   is   commonly   exploited   in   clinical   settings,1   when   damage   to   the   nervous   system,   either   central   or   peripheral,   produces   rapid   denervation   of  muscle,   resulting   in  weakness   or   paralysis.   Possible   mechanisms   of   muscle   fibre   recruitment   have   previously   been   studied   using   stereotyped   electrical   pulses   delivered   at   variable   pulse   frequency,   width   and   current   amplitude.2   However,     these   protocols  often  exhibit  several  significant  limitations,  resulting  in  an   overall  decreased  efficiency  of  contraction,  ultimately  leading  to  the   development   of   muscular   fatigue,   as   well   as   the   elicitation   of   unpleasant   symptoms.   In   the   present   study,   the   influence   that   different  parameters  of  ES  protocols  exert  on  the  efficacy  of  skeletal   muscle   cell   contractions   was   investigated   in   skeletal   myotubes   in   culture.  The  efficiency  of  a  “noisy”  stimulus  waveform,  derived  from   human   muscle   electromyogram   (EMG)   recordings,   used   as   templates   for   the   delivery   of   ES,  was   compared  with   conventional   stereotyped   1   Hz   and   40   Hz   electrical   stimulation   delivery.3   EMG   traces  obtained  by  recording  human  gastrocnemius  medialis  muscle   activity  during  sessions  of  real  overground  locomotion,  were  used  to   design   the   “noisy”   stimulation   pattern   (EMGstim).   ES   protocol   efficiency  in  inducing  contractile  activity  of  cultured  skeletal  muscle   cells   was   compared   by   measuring   intracellular   Ca2+   dynamics   and   patch-­‐clamp   electrophysiological   recordings.   Collected   data   demonstrated   that   EMGstim   was   more   efficient   in   inducing   myotube   cell   action   potential   firing,   [Ca2+]i   changes   and   contractions,   when   compared   with   more   conventional   electrical   stimulation   using   stereotyped   rectangular   pulses.   Furthermore,   it   was   demonstrated   that   EMGstim   strength   was   also   considerably   lower   than   the   minimum   current   amplitude   required   to   induce   contractions   via   canonical   stimulation   protocols.   These   results   demonstrate  the  peculiar  properties  of  the  “noisy”  EMGstim  pattern   to   enhance   the   efficiency   of   muscle   cell   recruitment,   minimizing   charge   transfer   and   therefore   preventing   possible   tissue   damage.   We  suggest  this  could  represent  a  promising  new  approach  for  the   optimization   of   ES   protocols   and   for   future   design   of   electrical   devices   to   stimulate   the   rehabilitation/recovery   of   weakened   or   injured  muscles  in  human  patients.   1.   Huang   H,   Sun   T,   Chen   L,   et   al.   Consensus   of   clinical   neurorestorative   progress   in   patients   with   complete   chronic   spinal  cord  injury.  Cell  Transplant  2014;23  Suppl  1:S5-­‐17.   2.   Crago  PE,  Peckham  PH,  Thrope  GB.  Modulation  of  muscle  force   by   recruitment   during   intramuscular   stimulation.   IEEE   Trans   Biomed  Eng  1980;27:679–84.   3.   Sciancalepore  M,  Coslovich  T,  Lorenzon  P,  Ziraldo  G,  Taccola  G.   Extracellular  stimulation  with  human  ‘‘noisy’’  electromyographic   patterns   facilitates   myotube   activity.   J   Muscle   Res   Cell   Motil   2015;36:349–57.   *****   Evaluation  of  tSCS  treatment  for  the  alleviation  of  lower   limb  spasticity   Halla  Kristin  Gudfinnsdottir  (1),  Jose  Luis  Vargas  Luna  (1,3),  Vilborg   Gudmundsdottir  (2),  Gigja  Magnusdottir  (2),  Gudbjorg  Kristin   Ludvigsdottir  (2),  Thordur  Helgason  (1,2)   (1)  Reykjavik  University,  Iceland;  (2)  Landspitali  –  University  Hospital,   Reykjavik,   Iceland;   (3)   Tecnológico   de   Monterrey,   Monterrey,   Mexico   E-­‐mail:  thordur@landspitali.is     Spinal   cord   injury   is   a   traumatic   injury   of   descending   spinal   cord   tracts  that  alters  the  spinal  neural  circuitry.1-­‐3  Spasticity  is  a  common   result  of  spinal  cord  injury  (SCI)  and  can  restrict  daily  living  activities,   cause  pain  and  fatigue  and,  therefore,  decrease  the  quality  of  life  for   SCI   individuals.4-­‐5  The  aim  of  this  study   is  to  evaluate  the  effects  of   transcutaneous   spinal   cord   stimulation   (tSCS)   on   individuals   with   post-­‐traumatic   SCI   for   the   alleviation   of   lower   limb   spasticity.   Methods:  In  total,  8  subjects,  5  males  and  3  females,  aged  between   31  –  63  years  old  (M  =  49,9;  SD  =  11,5)  were  studied,  with  complete-­‐   and  incomplete  SCI.  The  evaluation  of  the  effects  of  tSCS  was  done   by   means   of   electrophysiological   evaluation   and   evaluation   of   residual   motor   control   functions.   The   protocol   consisted   of   four   stages:  first  assessment/evaluation  (control  data),  application  of  30-­‐ min  tSCS,  a  second  assessment  immediately  after  the  treatment  and   a   third   assessment   two   hours   after   stimulation.     The   assessments   consist   of   the   evaluation   of   the   spasticity   level   through   the   Ashworth   scale,   clonus   beet   quantification,   10-­‐m   walking   test   (if   possible),   electrophysiological   evaluation   (Brain   Motor   Control   Assessment,  BMCA  [1])  and  the  Wartenberg  pendulum  test  (WPT).6   Results:   The   index   of   spasticity   R2n,   derived   from   the  WPT   is   the   primary  variable  and  the  results  of  the  WPT  show  increase  in  muscle   tone   in   four   subjects   while   the   others   presented   average   index   values   ≥   1,   indicating   non-­‐spastic   conditions.   During   the   BMCA,   there  was  a  significant  difference  of  the  normalized  EMG  activity  of   all  muscles  before  the  stimulation  and  immediately  after  stimulation   for   all   participants,  which   indicates   amelioration   of   intrinsic   phasic   and   extrinsic   spasticity.   Enhancement   of   motor   control   was   also   observed.  Conclusion:  The  similarity  of  the  effects  of  tSCS  with  those   induced  by  epidural  SCS,  strongly  suggests  that  both  techniques  are   able   to   activate   similar   neural   structures.   From  our   results  we   can   see  that  the  application  of  low-­‐intensity  tSCS  for  30  minutes  leads  to   the   alleviation   of   lower   limb   spasticity   regardless   of   the   clinical   profile  of  the  subjects  and  enhancement  of  voluntary  motor  control   in  the  motor  incomplete  SCI  subjects.   1   Hofstoetter   US,   McKay   WB,   Tansey   KE,   Mayr   W,   Kern   H,   Minassian  K.  Modification  of  spasticity  by  transcutaneous  spinal   cord   stimulation   in   individuals   with   incomplete   spinal   cord   injury.  J  Spinal  Cord  Med  2014;37:202–11.   2   Sheean   G.   The   pathophysiology   of   spasticity.   Eur   J   Neurol   2002;9:3–9.   3   Biering-­‐Sørensen  F,Nielsen  JB,  Klinge  K.  Spasticity-­‐assessment:  a   review.Spinal  Cord  2006;44:708–22.   4    Adams  MM,  Hicks  AL.  Spasticity  after   spinal   cord   injury.   Spinal   Cord  2005;43:577–86.   5   Sköld   C,   Levi   R,   Seiger   Å.   Spasticity   after   traumatic   spinal   cord   injury:   Nature,   severity,   and   location.   Arch   Phys   Med   Rehabil   1999;80:1548  –  57.   6   Bajd  T,  Vodovnik  L.  Pendulum  testing  of  spasticity.  J  Biomed  Eng   1984;6:  9–16.   *****   Quantifying  muscle  degeneration  from  nonlinear  trimodal   regression  analysis  of  radiodensitometric  CT  distributions   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   59 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   Kyle  J.  Edmunds  (1),  Iris  Árnadóttir  (1),  Magnus  K.  Gíslason  (1),   Ugo  Carraro  (2),  Paolo  Gargiulo  (1,3)   (1)  Institute  for  Biomedical  and  Neural  Engineering,  Reykjavík   University,  Reykjavík,  Iceland;  (2)  IRRCS  Fondazione  Ospedale  San   Camillo,  Venezia,  Italy;  (3)  Department  of  Rehabilitation,  Landspítali,   Reykjavík,  Iceland   E-­‐mail:  Kyle  Edmunds  kylejedmunds@gmail.com     Whether   via   sarcopenia,   cachexia,   or   sequela   of   trauma,   the   degeneration   of   muscle   has   been   consistently   identified   as   an   independent   risk   factor   for   mortality.1   Many   recent   investigations   have   realized   the   quantitative   potential   of   CT   image   analysis   to   describe   skeletal   muscle   volume   and   quality.2-­‐4   However,   the   optimum   metric   for   assessing   these   data   remains   debated.   Identifying   a   novel   quantitative   method   for   muscle   assessment   in   this   regard   would   allow   for   the   generalizability   of   such   studies   to   clinical  practice  and  therefore  aid  in  the  indication  of  compensatory   targets   for   clinical   intervention.   While   there   is   much   extant   literature   reporting   the   use   of   average   HU   values   to   investigate   muscle   quality   and   its   utility   as   a   comorbidity   index,   standardized   methods   for   this   analysis   have   yet   to   be   defined,   and   no   existing   studies   have   explored   the   utility   of   an   entire   radiodensitometric   distribution.   Herein,   we   hypothesize   that   rigorously   quantifying   entire  HU  distributions  can  elicit  much  more   information  regarding   muscle   quality   than   extant   methods   that,   to   date,   only   utilize   average  HU  attenuation  values.  This  study  reports  the  development   and  use  of  this  method,  wherein  we  assess  upper  leg  muscle  quality   utilizing   nonlinear   trimodal   regression   analysis   with   radiodensitometric   distributions   from   computed   tomography   (CT)   scans   of   a   healthy   young   adult,   a   healthy   elderly   subject,   and   a   spinal   cord   injury   patient   exhibiting   complete   lower  motor   neuron   denervation.   Results   from   this   assessment   highlight   the   utility   of   entire   HU   attenuation   value   distributions   and   identify   novel   parameters   from   these   analyses   that   could   provide   further   insight   into  how  muscle  degeneration  can  be  optimally  quantified.   1.   Metter,  E.  J.,  Talbot,  L.  a,  Schrager,  M,  Conwit  R.  Skeletal  muscle   strength   as   a   predictor   of   all-­‐cause  mortality   in   healthy  men.   J   Gerontol  A  Biol  Sci  Med  Sci  57:B359–65.   2.    Hicks   GE,   Simonsick,   EM,   Harris   TB,   Newman   AB,   Weiner   DK,   Nevitt  M,  Tylavsky  F.  Cross-­‐sectional  associations  between  trunk   muscle   composition,   back   pain,   and   physical   function   in   the   health,   aging   and   body   composition   study.   The   Journals   of   Gerontology.  Series  A,  Biological  Sciences  and  Medical  Sciences,   2005;60:882–7.   3.   Sur  MD,  Namm  JP,  Hemmerich  JA,  Buschmann  MM,  Roggin  KK,   Dale  W.   Radiographic   Sarcopenia   and   Self-­‐reported   Exhaustion   Independently   Predict   NSQIP   Serious   Complications   After   Pancreaticoduodenectomy   in   Older   Adults.   Ann   Surg   Oncol   2015;22:3897-­‐904.  doi:  10.1245/s10434-­‐015-­‐4763-­‐1.  Epub  2015   Aug  5.   4.   Lang   T,   Cauley   J,   Tylavsky   F,   Bauer   D,   Cummings   S,     Harris   TB.   Computed   tomographic   measurements   of   thigh   muscle   cross-­‐ sectional   area   and   attenuation   coefficient   predict   hip   fracture:   the  health,  aging,  and  body  composition  study.  J  Bone  Miner  Res   2010;25:513-­‐9.  doi:  10.1359/jbmr.090807.   5.   Rantanen  T.  Muscle  strength  and  body  mass  index  as  long-­‐term   predictors   of   mortality   in   initialle   healthy   men.   J   Gerontol   2000;55A:168–73.   6.   Newman  AB,   Kupelian  V,  Visser  M,   Simonsick,   EM,  Goodpaster   BH,  Kritchevsky  SB,  Harris  TB.  Strength,  but  not  muscle  mass,   is   associated   with   mortality   in   the   health,   aging   and   body   composition   study   cohort.   J   Gerontol   A   Biol   Sci   Med   Sci   2006;61:72–7.   *****   Biomarkers  in  aging  and  neuromuscular  disorders   S  Masiero,  U  Carraro,  Chairs   Ca2+-­‐handling  biomarkers  of  skeletal  muscle  plasticity  for   neurorehabilitation   Simone  Mosole  (1,2,3,4),  Sandra  Zampieri  (1,4),  Angie  Caon  (1,2),   Sandra  Furlan  (5),  Hanna  Fruhmann  (4),  Helmut  Kern  (4),  Pompeo   Volpe  (1,2),  Ugo  Carraro  (6),  Alessandra  Nori  (1,2,4)   (1)   Laboratory   of   Translational   Myology   of   the   Interdepartmental   Research   Center   of   Myology,   Department   of   Biomedical   Science,   University  of  Padova;  (2)  Pathophysiology  of  Striated  Muscles,  Dept.   Biomedical   Sciences,  University  of  Padua;   (3)  Plastic  Surgery  Clinics   of   the  Department  of  Neurosciences,  University  of  Padua,   Italy;   (4)   Ludwig   Boltzmann   Institute   of   Electrical   Stimulation   and   Physical   Rehabilitation,  Department  of  Physical  Medicine  and  Rehabilitation,   Wilhelminenspital   Wien,   Austria;   (5)   Neuroscience   Institute   of   the   Italian   C.N.R.,   c/o  Department   of   Biomedical   Science,  University   of   Padova,   Italy;   (6)   IRCCS   Fondazione   Ospedale   San   Camillo,   Venice,   Italy   E-­‐mail:  alessandra.nori@unipd.it   Physical   activity   plays   an   important   role   in   preventing   muscle   atrophy   and   chronic   diseases   in   adults   and   the   elderly.   Voluntary   physical   exercise   is   not   always   feasible   and  other   therapies   should   be   applied   such   as   electrical   stimulation   (ES).1   The   process   of   calcium  storage,  uptake  and  release  (EC-­‐coupling)  and,  in  a  broader   framework,   Ca2+   cycling   is   essential   in   activity-­‐induced   muscle   adaptation.2   De-­‐codification   of   Ca   signals   is   accomplished   by   an   heterogeneous   class   of   decoders   such   as   transcription   factors   (i.   e   NFATc1,  PGC1α)  kinases  (CaMks)  and  phosphatases  (Calcineurin).3,4   We   investigated   the   effects   of   either   passive   ES   (acute   or   long-­‐ lasting)  or  voluntary  physical  exercise   (leg  press,  LP),  on  expression   of  Ca2+  handling  proteins  of   the  sarcoplasmic   reticulum  and  on   the   activation  of  key  Ca2+  signal  decoders  in  human  vastus  lateralis  (VL)   of   elderly   sedentary   persons.   Muscle   sections   and   total   homogenates   were   obtained   from   biopsies   performed   before   and   seven  days  after  nine  weeks  of  ES,  before  and  30  minutes  after  one   session  of  ES  and  before  and  seven  days  after  LP  volitional  exercise   on  a  group  of  volunteers.1,5  Expression  of  Sarcalumenin,  SERCA  and   p-­‐CaMKII   were   evaluated   by   western   blot   while   NFATc1/PGC1α   nuclear   translocation   and  muscle   remodeling   were   determined   by   immunofluoresence.  Evidence  of  kinase  and  phosphatase  activation   after  both  ES  and  LP  were  obtained.  NFATc1  translocation  to  nuclei   30  minutes  after  one  ES  session  training  was  obtained,  after  9  weeks   of  ES  NFATc1  translocation   lasted  at   least  7  days.  Moreover,  mixed   SERCA  2/MHCII   fibers  and  Ca2+  handling  proteins  Sarcalumenin  and   SERCA  2  increased  after  ES.  Conclusions.  These  results  show  that  ES   influences  expression  of  muscle  components  deputed  to  Ca2+  cycling   and   promotes   fiber   remodeling   essential   to   improve   muscle   performance   in   old   sedentary   people.   This  work   identifies   a   set   of   molecules  which  are  modifiable  by  ES,  easy  to  measure  and  gender   and   age   independent   suitable   as   biomarkers   for   skeletal   muscle   response  to  neurorehabilitation.   1.   Kern   H,   Barberi   L,   Löfler   S,   et   al.   Electrical   Stimulation   Counteracts   Muscle   Decline   in   Seniors.   Front   Aging   Neurosci   2014;  6:189.   European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   60 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy   2-­‐   Gundersen   K.   Excitation-­‐transcription   coupling   in   skeletal   muscle:   the   molecular   pathways   of   exercise.   Biol   Rev   Camb   Philos  Soc  2011;  86:  564–600.   3-­‐   Dyar   KA,   Ciciliot   S,   Tagliazucchi   GM,   et   al.   The   calcineurin-­‐NFAT   pathway   controls   activity-­‐dependent   circadian   gene   expression   in   slow   skeletal   muscle.   Mol   Metab   2015;201:823-­‐33.   eCollection.   4-­‐   Silvennoinen  M,   Ahtiainen   J   P,   Hulmi   J   J   et   al.   PGC-­‐1   isoforms   and   their   target   genes   are   expressed   differently   in   human   skeletal   muscle   following   resistance   and   endurance   exercise   Physiol  Rep  2015;3:e12563   5.   Zampieri   S,  Mosole  S,   Löfler  S,  et  al.  Physical  exercise   in  Aging:   Nine  weeks   of   leg   press   or   electrical   stimulation   training   in   70   years  old  sedentary  elderly  people  Eur  J  Transl  Myol  -­‐  Basic  Appl   Myol  2015;25:237-­‐42.   *****   Biomarkers  of  skeletal  muscle  and  bone  regeneration-­‐ adaptation  to  neurorehabilitation  training  strategies   Barbara  Zavan  (1),  Chiara  Gardin  (1),  Letizia  Ferroni  (1),  Simone   Mosole  (1),  Ugo  Carraro  (2)   (1)   Department   of   Biomedical   Sciences,   University   of   Padova,   Padova;  (2)  IRCCS  Fondazione  Ospedale  San  Camillo,  Venice,  Italy   E-­‐mail:  barbara.zavan@unipd.it   Most   organisms   experience   changes   in   regenerative   abilities   through   their   lifespan.   The   principles   that   underlie   the   decline   in   regenerative  abilities  through  lifespan  are  currently  being  unraveled.   However,   it   is   already  clear   that  both  cell-­‐intrinsic   (such  as   cellular   senescence)   as   well   as   cell-­‐extrinsic   (such   as   alterations   in   the   regenerative   environment)   factors   play   significant   roles.   During   aging,  numerous  tissues  exhibit  a  progressive  decline  in  homeostasis   and   regeneration   that   results   in   tissue  malfunction,   pathology   and   degeneration.  With   age,  both   stem  and  progenitor   cells   undergo  a   series  of  alterations  including  loss  of  self-­‐renewal  capacities,  altered   proliferative   activity,   declines   in   functionality   and   potency.   These   changes   have   been   shown   to   contribute   to   the   dysfunction   and   degeneration   of   a   number   of   tissues   and   systems   including   most   epithelia  and  endothelia,  blood,   skeletal  and  cardiac  muscle,  bone,   cartilage,   the   peripheral   and   central   nervous   system   (CNS),   and   organs   such   as   the   pancreas,   liver,   kidney   and   lungs.   The   regenerative   capacity   in   the   skeletal   muscle   system   experiences   a   marked   decline   with   age   in   many   organisms,   as   reflected   by   a   decrease   in   the   generation   of   muscle   fibres   and   an   increase   in   fibrotic   tissue  upon  muscle   injury.   In  humans,   this   is   an  underlying   cause  of  sarcopenia,  the  loss  of  muscle  mass  that  accompanies  late   aging.   The   decline   in   muscle   regenerative   potential   is   largely   attributed  to  changes  in  satellite  cells,  the  muscle  stem  cells,  which   undergo   age-­‐related   declines   in   proliferative   and   myogenic   capacities.   Indeed,   satellite   cell   numbers   decline   gradually   in   mammalian  muscles  with  advancing  age.  Age-­‐specific  changes  have   also  been  reported  for  mesenchymal  stem  cells  (MSCs),  stromal  cells   that   can   differentiate   into   multiple   cell   types   such   as   osteoblasts,   chondrocytes,   and   adipocytes.   Alterations   include   a   loss   in   chondrogenic  potential   leading  to  impaired  chondrocyte  formation,   which   results   in   decreased   cartilage   repair   in   aged   mammals.   Furthermore,   studies   in   human-­‐derived   bone   marrow   MSCs   revealed  age-­‐dependent  decreases   in  their  capacity  to  differentiate   to  osteoblasts,  which   are   related   to   increases   in   the   level   of  MSCs   senescence   and   apoptosis   upon   aging.   Together,   these   alterations   contribute   to   conditions   such   as   osteoporosis   and   reduced   bone   repair  capacity  that  are  characteristic  of  human  aging.  A  number  of   cellular   and  molecular  mechanisms   have   been   associated  with   the   decline   in   regenerative   abilities   observed   during   aging   in   humans.   These  include  intrinsic  factors  such  as  genomic  instability  (including   telomere   attrition),  mitochondrial   dysfunction,   epigenetic   changes,   loss   of   proteostasis   and   metabolic   alterations,   as   well   as   cell-­‐ extrinsic   factors   such   as   disruption   of   the   regeneration   niche   and   alterations   in   systemic   signals.   Though   most   of   these   factors   can   contribute   to   age-­‐related   impairment   in   regenerative   capacity,   a   consensus   on   their   relative   importance   in   this   process   is   currently   lacking.  Furthermore,  emerging  evidence  suggests  a  high  degree  of   interconnectivity   between   them,   stressing   the   importance   of   identifying   the   common   denominators.   The   advances   in   our   understanding   of   the   factors   that   modulate   the   decline   in   regenerative   abilities   have   pinpointed   areas   of   potential   clinical   relevance.   In   this   view  examining   the   influence  of   systemic   factors   on   aged   progenitor   cells   from   tissues   activated   during   neurorehabilitation  training  may  prove  to  be  clinically  relevant.  Our   activity  will  be  focused  on  the  study  of  3  different  markers  present   in   blood   and   tissue   biopsies,   such   as   long   and   small   noncoding   RNAs,1-­‐3  growth  factors,4-­‐6  and  transcription  factors,  such  as  NFAT.7,8   1.   Bates  DJ1,  Liang  R,  Li  N,  Wang  E.  The  impact  of  noncoding  RNA   on  the  biochemical  and  molecular  mechanisms  of  aging.  Biochim   Biophys   Acta   2009;1790:970-­‐9.   doi:   10.1016/j.bbagen.2009.03.028.  Epub  2009  Apr  2.   2   Ciesla   M,   Skrzypek   K,   Kozakowska   M,   Loboda   A,   Jozkowicz   A,   Dulak  J.  MicroRNAs  as  biomarkers  of  disease  onset.  Anal  Bioanal   Chem   2011;401:2051-­‐61.   doi:   10.1007/s00216-­‐011-­‐5001-­‐8.   Epub  2011  May  6.   3.   Kim   J,   Kim   KM,  Noh   JH,   Yoon   JH,   Abdelmohsen   K,   Gorospe  M.   Long  noncoding  RNAs  in  diseases  of  aging.  Biochim  Biophys  Acta   2016;1859:209-­‐21.   doi:   10.1016/j.bbagrm.2015.06.013.   Epub   2015  Jul  2.   4.   Loffredo   FS,   Steinhauser   ML,   Jay   SM,   et   al..   Growth   differentiation  factor  11  is  a  circulating  factor  that  reverses  age-­‐ related   cardiac   hypertrophy.   Cell.   2013;153(4):828-­‐39.   doi:   10.1016/j.cell.2013.04.015.   5.   Sinha  M1,   Jang  YC,  Oh  J,  et  al.  Restoring  systemic  GDF11   levels   reverses   age-­‐related   dysfunction   in   mouse   skeletal   muscle.   Science.  2014;344(6184):649-­‐52.  doi:  10.1126/science.1251152.   Epub  2014  May  5.   6.   Katsimpardi   L,   Litterman   NK,   Schein   PA,   et   al.   Vascular   and   neurogenic   rejuvenation   of   the   aging   mouse   brain   by   young   systemic   factors.   Science   2014;344(6184):630-­‐4.   doi:   10.1126/science.1251141.  Epub  2014  May  5.   7.   Serrano-­‐Pérez   MC1,   Fernández   M,   Neria   F,   et   al.   NFAT   transcription   factors   regulate   survival,   proliferation,   migration,   and   differentiation   of   neural   precursor   cells.   Glia.   2015   Jun;63(6):987-­‐1004.  doi:  10.1002/glia.22797.  Epub  2015  Mar  2.   8.   Qin  W,  Pan   J,  Wu  Y,  Bauman  WA,  Cardozo  C.  Anabolic   steroids   activate   calcineurin-­‐NFAT   signaling   and   thereby   increase   myotube   size   and   reduce   denervation   atrophy.   Mol   Cell   Endocrinol.   2015;399:336-­‐45.   doi:   10.1016/j.mce.2014.09.025.   Epub  2014  Oct  29.   *****         European  Journal  of  Translational  Myology  -­‐  eISSN  2037-­‐7460  –    BAM  On-­‐Line   2016Spring  PaduaMuscleDays  April  13  to  16  -­‐  ABSTRACTS  -­‐  Eur  J  Transl  Myol  -­‐  Basic  Appl  Myol  2016;  26  (1):37-­‐60     This  article  is  distributed  under  the  terms  of  the  Creative  Commons  AttributionNoncommercial  License  (CC  BY-­‐NC  4.0)  which  permits  any  noncommercial  use,distribution,  and  reproduction  in  any  medium,  provided  the  original  author(s)  and  source  are  credited..       EJTM  WebSite  -­‐  http://www.ejtm.eu     eISSN  2037-­‐7460  -­‐  ISSN  2037–7452   BAM  On-­‐Line    -­‐  http://www.bio.unipd.it/bam/bam.html     Organizing  Secretariat:  Ugo  Carraro,  IRCCS  Fondazione  Ospedale  San  Camillo,  Via  Alberoni  70,  I-­‐30126  Venezia-­‐Lido,  Italy    Phone:  +39  338  15  75745;  E-­‐mail:  ugo.carraro@ospedalesancamillo.net   61 Interdepartmental  Research  Center  of  Myology  (CIR-­‐Myo)   Department  of  Biomedical  Sciences,   University  of  Padua,  Italy