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Research into the physiology of myosins - a personal odyssey 
Eur J Transl Myol 35 (2) 13688, 2025 doi: 10.4081/ejtm.2025.13688

My initial interest in neuroscience research 

I  have enjoyed doing research into muscle physiology 
throughout my academic life. My entry into muscle 

research was not a happy personal decision, but a 
traumatic one beyond my control. During my student days 
in the medical school at the University of Sydney, I was 
intrigued by the physiology of the nervous system. The 
highlight of 3rd year physiology course in 1959 was for 
me a guest lecture on synaptic transmission in 
motoneurons given by Professor Sir John Eccles, who was 
the Head of the Physiology Department at the John Curtin 
School of Medical Research at the Australian National 
University. I was fascinated by the lecture, and it strongly 

stimulated in me a desire to pursue a research career in 
neurophysiology after my medical graduation. There was 
an opportunity after the third year in the medical course 
to take a year off to do a BSc (Med) course. This involved 
a year of supervised research and the submission of a 
thesis on the research work. This course could give me a 
foretaste of research life and I decided to take it the 
following year.  
During the BSc (Med) year, I was very fortunate to be as-
signed to a neurophysiology laboratory and was supervised 
by Dr Bill Levick, working in the Brain Research Unit of 
the Head of Department, Prof. Peter Bishop, recording im-
pulses in single ciliary nerve fibres in response to light stim-
ulation. I enjoyed the taste of research life so much that I 

Abstract  
 
During my PhD, I worked on the neural regulation of mechanical properties fast and slow muscles. 
This led me to believe that myosins in fast and slow muscles are structurally distinct and that motor 
nerves regulate the expression of myosin genes. I devised a method for separating intact fast and 
slow myosins by gel electrophoresis and confirmed their neural regulation. The electrophoresis 
method was subsequently improved and used to analyse skeletal and cardiac myosin isoforms in 
various vertebrate species, including marsupials. This led to the discovery of neonatal myosin heavy 
chain (MyHC), α and β cardiac MyHCs and of the regulation of cardiac MyHCs by thyroid hormone. 
Antibodies were raised against 2A, 2X, 2B, masticatory and extraocular MyHCs and used to study 
the expression and regulation of MyHCs in jaw, laryngeal and Extraocular Muscle (EOM) fibres. 
Antibodies against masticatory myosin enabled the sequencing of masticatory MyHC and 
masticatory light chain 2 genes. Cross-bridge kinetics of fibres with different myosin isoforms were 
analysed. Different MyHC isoforms found in jaw-closing muscles across various species reflected 
evolutionary adaptations to diverse dietary intake, while MyHC expression changes in cardiac and 
laryngeal muscles with body mass reflected adaptations to changes in their specific metabolic rate. 
Transplantation experiments on masticatory and EOMs and cross-innervation experiments between 
laryngeal and somitic muscles revealed that their capacity to express masticatory or extraocular 
MyHC were myogenically determined but neural impulse patterns also influence MyHC expression. 
EOMs are the most complex, expressing 11 MyHC isoforms. Some EOM fibres express faster 
MyHCs in the endplate zone but slower MyHCs at the end segments, an arrangement helping to 
linearize the saccade. I suggested that during development, primary and secondary extraocular 
myotubes specify the synaptic inputs of the innervating neurons to generate impulse patterns which 
regulate the expression of their MyHCs.  
Key Words: myosin gene expression, muscle contraction, neural regulation, craniofacial muscles, 
cardiac contractility. 

Eur J Transl Myol 35 (2) 13688, 2025 doi: 10.4081/ejtm.2025.13688

Research into the physiology of myosins - a personal odyssey 
 
Joseph Foon Yoong Hoh 
 
Retired from the Discipline of Physiology, School of Medical Sciences, Faculty of Medicine and 
Health, The University of Sydney, NSW, Australia. 
 
This article is distributed under the terms of the Creative Commons Attribution Noncommercial 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. 

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firmly decided to pursue a research career after graduation 
instead of doing clinical medicine.  
In 1962, I visited Eccles’ laboratory at the John Curtin 
School of Medical Research and enquired about the pro-
spect of doing postgraduate studies under his supervision. 
He assured me that I could get an Australian National Uni-
versity scholarship to do a PhD in his Department if I ap-
plied after my medical graduation. In 1963 Eccles was 
awarded the Nobel Prize in Physiology or Medicine, and I 
was very happy at the prospect of having a Nobel Laureate 
as a mentor. After my graduation in1964, I did apply for the 
scholarship and was awarded it, to commence in 1965, but 
I decided to defer taking it up by a year to enable me to be 
home with my folks in Kuala Lumpur, Malaya, as I had 
then been away in Australia for 9 years. During that year I 
held a lectureship in the Department of Physiology at the 
new medical school of the University of Malaya.  
Opting to defer taking up the scholarship was a fateful deci-
sion! When I turned up in Canberra early in 1966, Eccles 
was 63 years old, and he had decided to avoid the man-
datory retirement at age 65 at the Australian National Uni-
versity by accepting an American offer to continue his 
research at the Institute of Biomedical Research in Chicago. 
He could not supervise my PhD as he would be leaving the 
Department within the next few months. Eccles appointed 
a muscle physiologist, Dr Russel Close, as my PhD super-
visor. My dream of doing brain research under his supervi-
sion was shattered. Furthermore, no one else in the 
Department could supervise me to do neurophysiological 
research. I was devastated.  
 
 
Diversion into muscle research 
I was assigned to work on a problem which Eccles and col-
leagues had pioneered a few years ago on the neural control 
of contractile properties of fast and slow skeletal muscles. 
They measured the isometric contraction times of muscles, 
the reciprocal of which is a crude indicator of muscle speed. 
They showed that all muscles of new-born kittens were uni-
formly slow, and that fast muscles acquired their speed pro-
gressively during the early postnatal period while the speed 
of developing slow muscle remained unchanged. However, 
after isolation of the lumbosacral spinal cord, the devel-
opment of fast muscle was unaffected but the developing 
slow muscle became nearly as fast as the fast muscle.1 Fur-
thermore, cross-innervation of fast and slow muscles in the 
adult resulted in reciprocal changes in their speeds, suggest-
ing that muscle speed was under neural control.2 Dr Close 
had been following up this work by measuring the force-ve-
locity properties of muscles, which best characterize muscle 
mechanics, during development and following cross-inner-
vation. He showed that the maximal velocities of shortening 
of fast and slow muscles the rat3 and mouse4 were virtually 
identical at birth. During the postnatal period, the speed of 
shortening of the developing fast muscle increased by 2.5-3 
times while that of slow muscle showed little change. Cross-
innervation of fast and slow muscles resulted in reciprocal 
changes in their speeds of shortening,5 indicating that the 
speeds of shortening of muscles were under neural control. 
My role was to extend this area of research and to explore 

whether other differences in properties between fast and 
slow muscles were also under neural regulation.  
Close and I first showed that force-velocity properties of 
developing fast muscles of new-born kittens had already di-
verged from that of slow muscle to some extent.6 We char-
acterized the different effects of temperature on the 
contractile properties of rat fast and slow muscles7 and the 
effects of tetanic contractions on the subsequent isometric 
twitches of fast and slow muscles. We showed that the iso-
metric twitch tension of fast muscle was enhanced about 
twofold by a prior tetanic stimulation, a phenomenon 
known as Post-Tetanic Potentiation (PTP).8 In contrast, 
slow muscle showed Post-Tetanic Depression (PTD), the 
isometric twitch was depressed by about 15% following a 
tetanus. We further showed that these post-tetanic effects 
were under neural control.9,10 Works contributing to the elu-
cidation of the molecular mechanisms of PTP and PTD will 
be referred to below. 
Cross-innervation studies on fast and slow avian mus-
cles11,12 surprisingly did not show any change in their me-
chanical properties. We showed that in another lower 
vertebrate, the toad, innervation also did not regulate me-
chanical properties of muscles. In the toad, fast-twitch and 
slow-tonic muscle fibres are innervated by nerve fibres with 
low and high thresholds for electrical stimulation, respec-
tively. Cross-innervation of fast-twitch and slow-tonic mus-
cle fibres with the alien nerve type did not change the 
mechanical properties of these muscle fibres.13 These ob-
servations show that neural regulation of muscle properties 
is an innovation that occurred during mammalian evolution, 
an advancement that allows adaptive changes in muscle 
properties in response to their pattern of use. 
I was initially bitterly disappointed at being deprived of the 
opportunity to pursue my passion for research in neuro-
physiology and for being diverted into muscle research. 
This turned out later to be a blessing in disguise. The role 
of myosin in muscle contraction was then fast emerging. 
Barany showed that the actin-activated ATPase activities of 
myosins from muscles of a wide range of speeds from di-
verse animal species were correlated with their maximal 
speeds of shortening.14 Close collaborated with Barany and 
showed that following cross-innervation of fast and slow 
muscles, the ATPase activities of their myosins were trans-
formed in proportion to their changes in speeds of short-
ening.15 There was thus the exciting prospect of tackling the 
neural regulation of muscle properties at the molecular 
level. At the end of my PhD in 1969, I became convinced 
that myosins in fast and slow muscles were structurally dis-
tinct, that myosin isoforms controlled the speeds of muscle 
contraction, and that motor nerves controlled the expression 
of myosin genes in the muscle fibres they innervate. These 
convictions set the direction of my future research. 
 
Development of methods for the electrophoretic 
analysis of intact myosin isoforms 
I accepted a post-doctoral research fellowship in the De-
partment of Pharmacology at the University of Illinois in 
Chicago in the laboratory of Dr Bernard Salafsky. I knew 
little about muscle biochemistry at that time, being a physi-
ologist learning to do biochemistry in a Pharmacology De-

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partment! During the two years of postdoctoral work, I was 
given a free hand in deciding the direction of the research. 
I initially showed that the intracellular K+ concentration was 
higher in rat fast muscle fibres compared with that of slow 
muscle fibres, and that this difference was under neural con-
trol.16 The main research objective was to develop specific 
antibodies against myosins extracted from fast and slow 
muscles to test the hypotheses regarding their structural dif-
ference and the neural regulation of myosin gene expres-
sion. I learned to prepare myosins from fast and slow 
muscles for use as antigens. To assess the purity of my myo-
sin preparations, I tried to use polyacrylamide gel electro-
phoresis, a technique routinely used by another staff 
member in the laboratory to analyse serum proteins. I was 
frustrated by the fact that myosin persistently precipitated 
at the top of the gel. Upon reading the literature on the prop-
erties of myosin, I realized that this was due to the low ionic 
strength of the tris/glycine electrophoresis buffer. A high 
ionic strength buffer would be needed to keep myosin in 
solution, but the high conductance of such a buffer would 
not be suitable for gel electrophoresis. I struggled with this 
dilemma and eventually came across the work of Brahms 
and Brezner, who showed that myosin could be dissolved 
in low ionic strength solutions of strongly negative polyva-
lent anions, such as ADP, ATP, and pyrophosphate.17 Simply 
adding 10 mmol ATP to the tris/glycine electrophoresis 
buffer solved the problem! It was a eureka moment!  
During my tenure of the postdoctoral fellowship, I managed 
to demonstrate that fast and slow muscle myosins could be 
resolved from each other using the ATP/tris/glycine elec-
trophoresis method, fast muscle myosin had a higher elec-
trophoretic mobility than slow muscle myosin. I devised a 
method for measuring the Ca2+-ATPase activity of the myo-
sins in the electrophoresis gel and showed that fast muscle 
myosin had a higher Ca2+-ATPase activity than slow muscle 
myosin. Cross-innervation of fast and slow muscles re-
versed these properties of their myosins.  
In 1971, I was appointed lecturer in the Department of 
Physiology at the University of Sydney. On my way to take 
up my appointment, I attended a conference in the UK 
where I presented the preliminary electrophoresis results. 
The successful electrophoresis of intact myosin stirred con-
siderable interest among the biochemists present. They 
quizzed me intensely regarding the electrophoretic method, 
but I was reluctant to divulge it for the following reasons. I 
would be going to an empty laboratory in a Physiology De-
partment which I knew would not have the infrastructure 
for biochemical research. I would therefore not have a level 
playing field in continuing the work in the competitive field 
of muscle biochemistry in which I was a novice. Further-
more, the work 18 was then still incomplete and had yet to 
be published. To this day I still feel uncomfortable about 
withholding scientific information from colleagues on that 
occasion.  
Research was difficult in the early years at the University 
of Sydney, as the Physiology Department lacked even very 
basic facilities for biochemical work. The spectrophoto-
meter, ultracentrifuge and other necessary expensive equip-
ment had to be acquired from research grants over several 
years. Fortunately, the electrophoresis method for intact 

myosin could cope with crude extracts of myosin. I was 
also fortunate to have a succession of talented BSc Honours 
and PhD students and research assistants to work in the la-
boratory. In the first few years, we improved on the myosin 
gel electrophoresis method simply by substituting 
ATP/tris/glycine buffer with 10 mmol pyrophosphate 
buffer. Initially this was a means to conserve research funds. 
Myosin isoform resolution was considerably improved with 
the pyrophosphate gel system. It opened the doorway for 
us to study myosin isoforms in various skeletal and cardiac 
muscles and their regulation. 
Myosin is a hexamer with a pair of Myosin Heavy Chains 
(MyHCs) and 2 pairs of Myosin Light Chains (MLCs). The 
two MyHCs interact to form two distinct domains: a head 
domain consisting of a pair of globular heads each bearing 
an essential MLC (MLC1) and a regulatory MLC (MLC2), 
and an a-helical coiled coil rod domain that is involved in 
the assembly of bipolar thick filaments. I often wondered 
how pyrophosphate anions can keep myosin in solution at 
low ionic strength. A conserved 29-residue region named 
assembly competent domain was identified near the C-ter-
minal of the MyHC as critical for filament assembly.19 Py-
rophosphate anions may interfere with the charge 
interactions between this region and a neighbouring myosin 
molecule involved in filament assembly. More recently, a 
highly conserved cluster of four positive amino acid resi-
dues near the assembly competent domain has also been 
shown to be critical for thick filament assembly.20 It seems 
more likely that the four negative charges of the pyrophos-
phate anion very effectively interact with this cluster of four 
positive residues at low ionic strength to prevent myosin 
polymerization.  
Pyrophosphate gel electrophoresis resolved avian fast myo-
sin into three components which differed in MLC compo-
sition. It resolved avian slow muscle into 2 components 
with identical MLC composition, suggesting that these 
components differed in MyHC composition.21,22 Fast mus-
cle myosins of rabbit,23 rat,24 human25 and mouse26 muscles 
also showed 3 components with different MLC composi-
tions. Myosin extracted from rabbit neonatal muscles 
showed 3 isoforms which migrated faster than those of limb 
fast myosin, with MLC composition similar to those of fast 
myosin components, suggesting that it was a new myosin 
isoform that differed in MyHC composition. We confirmed 
this by showing that the cyanogen bromide peptide map of 
the rabbit neonatal muscle MyHC was distinct from maps 
of MyHCs of fast and slow myosins, establishing it as a 
novel foetal or neonatal MyHC isoform.23 Whalen and col-
leagues subsequently discovered another novel MyHC iso-
form in developing muscle, the embryonic MyHC, which 
was expressed during muscle development prior to the ex-
pression of foetal/neonatal myosin.27 
 
Electrophoretic analyses of myosin isoforms  
in developing human skeletal muscles and  
in diseased muscles  
Using pyrophosphate gel electrophoresis, we studied the 
expression of embryonic and foetal myosins in human mus-
cles during development and in diseased human muscles. 
Human foetuses (16-20 weeks’ gestation) showed the pres-

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ence of embryonic and foetal myosin isoforms, all of which 
had higher electrophoretic mobilities than the three fast 
myosin isoforms. The embryonic and foetal isoforms were 
present in muscles from patients with Duchenne muscular 
dystrophy, consistent with the known presence of muscle 
regeneration in this disease. Foetal myosin isoforms were 
found in muscles from patients with spinal muscle atrophy 
in which there is a failure of muscle maturation.28 Analysis 
of muscles from the dystrophic mouse (129 REJ) revealed 
only a shift of myosins towards slower migrating isoforms, 
but embryonic and foetal isoforms were absent, consistent 
with the absence of muscle regeneration in these muscles.26 
 
 
Analysis of cardiac myosins and their regulation 
by thyroid hormone 
Analysis of cardiac myosins was most enjoyable and re-
warding. We showed by pyrophosphate gel electrophore-
sis that ventricular myosin in adult rats comprised 3 
electrophoretic components, V1, V2 and V3 in the order of 
decreasing electrophoretic mobility and Ca2+-activated AT-
Pase activity.29 These ventricular myosin isoforms had the 
same pair of ventricular MLCs. Analysis of the MyHCs 
of V1 and V3 showed that they have significant differences 
in amino acid composition and in their cyanogen bromide 
peptide maps. Since the peptide map of a mixture of V1 
and V3 MyHCs did not differ from that of MyHCs from 
myosin containing all three components, the results show 
that the MyHCs of V1 and V3 are structurally different, 
designated as α MyHC and β MyHC respectively, and that 
the MyHC compositions of V1, V2 and V3 are respectively 
αα, αβ and ββ.30   
We showed that cardiac myosin isoforms were regulated by 
thyroid hormone. Juvenile rats expressed purely V1, while 
V2 and V3 emerged as the animal matures. Thyroid hormone 
administration in the adult rat shifted isoform expression 
towards V1, while hypothyroidism induced by hypophysec-
tomy shifted ventricular myosin towards V3.

29 In vitro anal-
ysis of radioactive amino acid uptake into myosins by 
ventricular strips from hypophysectomized rats indicated 
that administration of thyroid hormone induced the synthe-
sis of α MyHC but suppressed β MyHC synthesis. This oc-
curred at the level of gene transcription since these actions 
were suppressed by actinomycin D.31 
In pursuing the functional significance of cardiac myosin 
isoforms, we showed that V1 and V3 isoforms differed ki-
netically by a factor of two, V1 having the higher actin-ac-
tivated ATPase activity.32 Cross-bridge cycling kinetics can 
be measured using dynamic stiffness analysis, which yields 
a stiffness minimum frequency, Fmin, which reflects cross-
bridge cycling kinetics. This parameter is sensitive to rate 
constants for the power stroke and the rate of cross-bridge 
detachment.33 In collaboration with Dr Gunther Rossmanith 
at Macquarie University, we showed that Fmin for V1 myo-
cardium was twice that of V3 myocardium.34 V1 myocar-
dium also had a higher maximal rate of tetanic stress 
development and heat production,35 and consumed twice as 
much ATP during isometric contractions as V3 
myocardium.36 Thus, V1 muscle is more powerful, while V3 
muscle is more economic. The functional significance of 

thyroid stimulation of V1 and suppression of V3 is clearly 
to enhance cardiac contractility and thus cardiac output to 
meet the metabolic demands due to the thyroid induced in-
crease in metabolic rate. This feature is a mammalian evo-
lutionary innovation since there is no evidence that thyroid 
hormone regulates avian cardiac myosin.37 
The expression of mammalian ventricular myosin isoforms 
is also influenced by hypertension and the associated car-
diac hypertrophy. Experimentally induced hypertension and 
cardiac hypertrophy increase the expression of V2 and V3, 
which regresses after normalization of blood pressure and 
ventricular mass.38 This ventricular myosin response during 
cardiac hypertrophy would serve to reduce the metabolic 
load on the hypertrophic heart.  
Myosin from the atrial myocardium in both eutherian 29 and 
marsupial 39 hearts migrate faster than V1 in pyrophosphate 
gel and has a higher Ca2+-ATPase activity than V1. Atrial 
myosin has the same αα MyHC composition as V1 but is 
associated with atrial-specific MLCs. The N-terminal of 
MLC1 interacts with actin during cross-bridge cycling. 
Atrial MLC1 (MLC1A) has a weaker interaction with actin 
compared with that of ventricular or slow MLC1 (MLC1S) 
of limb slow fibres.40 This difference in MLC1 isoform ex-
pression between atrial myocardium and V1 myocardium is 
associated with a 68% higher maximal velocity of short-
ening 41 and a Fmin 2.5-fold higher for atrial myocardium (L 
Turnbull, G Rossmanith and J Hoh, unpublished observa-
tions). The speed of atrial contraction will thus always be 
faster than ventricular contraction. The functional signifi-
cance of faster atrial contraction is that it leaves more time 
during the cardiac cycle for the energetically more demand-
ing ventricular contraction.  
 
 
Mechanisms of action of inotropic agents  
While thyroid hormone tonically controls cardiac contrac-
tility by regulating myosin gene expression, inotropic 
agents influence cardiac contractility phasically through 
cell signalling mechanisms. We showed that adrenaline 
enhances Fmin by about 50% in V1 myocardium and 25% 
in V3 myocardium, these effects were abolished by pro-
pranolol, a β-adrenergic blocker. This suggests that adren-
aline, which works by enhancing the intracellular level of 
cyclic AMP (cAMP), enhances the rate of cycling of 
cross-bridges in the ventricular myocardium.42 This en-
ables the ventricle to enhance contractility and cope with 
the reduced duration of the cardiac cycle due to the chro-
notropic effect of adrenaline. This effect on cross-bridge 
cycling has been confirmed by the work of others using 
other methods.43,44 We showed that dibutyryl cAMP, an 
agonist of cAMP, also enhanced Fmin. Likewise, agents 
that block cAMP phosphodiesterase, the enzyme that cat-
alyzes the hydrolysis of cAMP, namely, isobutylmethyl-
xanthine (IBMX) and caffeine, also increased Fmin. 
However, an inotropic agent that does not affect the intra-
cellular level of cAMP, namely, ouabain, had no effect on 
Fmin. These findings support the hypothesis that a β-adren-
ergic receptor-mediated increase in crossbridge cycling 
rate is due to an increase in intracellular cAMP level.45  
It is well established that elevation of intracellular cAMP 

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following β-adrenergic stimulation of the myocardium 
leads to the activation of protein kinase A, which phosphor-
ylates cardiac isoform of Myosin Binding Protein-C 
(MyBP-C), c-MyBP-C, and Cardiac Troponin I (c-TnI) but 
has no effect on ventricular Myosin Light Chain 2 (MLC2V) 
phosphorylation. Also, the chemical phosphatase 2,3-Bu-
tanedione Monoxime (BDM) is known to dephosphorylate 
the β-adrenergically mediated c-MyBP-C phosphorylation 
but leave c-TnI phosphorylation relatively intact.46 We 
showed that IBMX approximately doubled the Fmin value 
of rat papillary muscle, and this action was unaffected by 
the addition of BDM. In the presence of IBMX and BDM, 
the level of phosphorylation of c-MyBP-C was not en-
hanced, that of MyLC2v was reduced to 60% of control, but 
phosphorylation of c-TnI was markedly increased. This in-
crease in phosphorylation of c-TnI but not of c-MyBP-C 
associated with the increase in Fmin indicates that c-TnI 
phosphorylation is the molecular basis for the enhanced 
cross-bridge cycling seen during β-adrenergic stimulation 
of the heart.47 This agrees with the work of others using 
transgenic mice that express a non-phosphorylatable slow 
skeletal isoform of troponin I in the myocardium.48  
There is evidence that β-adrenergic stimulation also toni-
cally regulates cardiac contractility by modulating myosin 
isoform expression, since chronic administration of propra-
nolol, a β-adrenergic blocker, leads to enhanced expression 
of V2 and V3 myosin isoforms.49 
Endothelin is a powerful inotropic agent secreted by vas-
cular endothelial cells. It has been shown to increase the 
amplitude of cardiac twitch contraction without signifi-
cantly increasing the cytosolic calcium transient. We 
showed that endothelin had no effect on Fmin but caused an 
increase in the level of incorporation of 32Pi into ventricular 
MLC2V without a concurrent change in the level of incor-
poration of 32Pi into c-TnI seen following β-adrenergic 
stimulation. We concluded that endothelin exerts positive 
inotropic effects through an increase in divalent cation sen-
sitivity by increasing MLC2V phosphorylation.50 This action 
of endothelin is analogous to the mechanism of PTP of fast 
skeletal muscle in which tetanic stimulation induces the 
phosphorylation of fast MLC2 (MLC2F) which shifts OFF 
cross-bridges away from thick filaments to become ON 
cross-bridges (see later). 
 
 
Immunohistochemical analysis of limb muscle  
development  
We used immunohistochemical methods to study the de-
velopmental changes in myosin gene expression in fibres 
of fast and slow muscles in kittens.51 During limb muscle 
development, muscle fibres are formed in two rounds of 
myotube formation from myoblasts migrating from the so-
mites. Primary myotubes are first formed, increase in di-
ameter and get innervated before a second wave of 
myoblasts forming secondary myotubes around the primary 
myotubes. We found that primary and secondary myotubes 
were characterized by different patterns of myosin gene ex-
pression. Primary myotubes in future fast and slow muscles 
of new-born kittens stained strongly for β-slow myosin but 
weakly for foetal/embryonic myosins. These myotubes 

were termed slow primaries. During subsequent devel-
opment, foetal/embryonic myosins were withdrawn from 
slow primaries, and they continued to express β-slow myo-
sin in developing slow muscle under the influence of the 
slow motor nerve. Only a subpopulation of slow primaries 
in the developing fast muscle became slow fibres, the rest 
became fast fibres under the influence of the fast motor 
nerve. In the peripheral region of developing fast muscles, 
a subpopulation of primary myotubes, referred to as fast 
primary myotubes, expressed fast myosin in addition to 
β-slow and embryonic/foetal myosin. These fast primary 
myotubes end up expressing fast myosin at maturity under 
the influence of thyroid hormone.52  
The secondary myotubes stained strongly for foetal/em-
bryonic myosins but not β-slow myosin and could be di-
vided into two subpopulations: i) fast secondaries, in which 
foetal/embryonic myosins were replaced by fast myosin at 
maturity, and ii) slow secondaries, in which the foetal/em-
bryonic myosins were replaced by β-slow myosin. Fast sec-
ondaries were found principally in the developing fast 
muscle but formed a minor component of developing slow 
muscle. Slow secondaries were found principally in the de-
veloping slow muscle but formed only a minor component 
in the developing fast muscle.51 
It later emerged that the cellular basis of these differences 
in myosin gene expression in primary and secondary myo-
tubes lies in the fact that two distinct myoblast lineages with 
different patterns of gene expression during myogenesis 
emerge sequentially from the somites: embryonic myo-
blasts, which form primary myotubes, and foetal myoblasts, 
which form secondary myotubes. Embryonic myoblasts 
constitutively express genes characteristic of slow fibres 
during myogenesis in tissue culture, while foetal myoblasts 
express genes characteristic of fast fibres.53 The difference 
in the fate of slow primaries in the developing slow and fast 
muscles is now known to be due to the influences of the 
emerging different neural impulse patterns along the nerves 
to slow and fast muscles and the postnatal surge of thyroid 
hormone. Slow primaries in developing slow muscle re-
ceive Chronic Low Frequency Stimulation (CLFS) from 
the soleus nerve, which maintains β-slow myosin expres-
sion, while slow primaries in developing fast muscle re-
spond to the postnatal surge of thyroid hormone and express 
fast myosins in the absence of CLFS from the fast nerve.54  
Fibres of a given phenotype may respond differently to neu-
ral and thyroidal influences depending on their devel-
opmental origin. Surrounding the fast primaries in the 
periphery of fast muscles are fast secondaries which also 
express fast myosin in the adult, so that the peripheral fibres 
in a fast muscle are devoid of slow fibres. In response to 
hypothyroidism, only fast fibres of fast primary origin ex-
press β-slow myosin.52  
The above observations show that classification of fibres 
into phenotypes does not uniquely specify their response to 
modulating influences. Fibres should also be classified ac-
cording to their development origins, which has been re-
ferred to as their ontotype. The different properties of fibres 
of diverse ontotypes underlie the incomplete transformation 
of muscles following nerve cross-union and neural stimu-
lation, previously attributed to the vague notion of different 

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adaptive ranges of fast and slow muscles.55 The complex 
interplay of ontotype, neural and hormonal influences on 
myosin gene expression and their known molecular mech-
anisms during the development of limb muscle fibres have 
recently been reviewed.54  
 
 
Development of antibodies to specific myosin  
isoforms 
As masticatory and extraocular muscles express myosin 
isoforms not found in limb muscles, to study the expression 
of myosin isoforms in these muscles at the cellular level, 
we needed to employ immunohistochemical methods using 
antibodies specific to masticatory and EOM myosin iso-
forms. We first raised polyclonal antibodies against cat mas-
ticatory MLCs and used it to isolate a masticatory MLC2 
(MLC2M) clone from a cDNA library and to sequence the 
MLC2M gene.56 We also raised a polyclonal antibody 
against masticatory MyHC and used it to isolate a masti-
catory MyHC clone from the cDNA library in the course 
of sequencing the masticatory MyHC gene.57 This anti-mas-
ticatory MyHC antibody was also used to study the regen-
eration 58 and development 59,60 of cat jaw closer muscles.  
We next turned our attention to the production of Monoclo-
nal Antibodies (mabs). We initially produced mabs 4A6 and 
10A10 against Extraocular (EO) MyHC and used them to 
study EO MyHC expression in laryngeal muscles and 
EOMs.61,62 We also prepared mabs 3F10 and 1H2 to masti-
catory-specific isoforms of MyBP-C (m-MyBP-C) and tro-
pomyosin (m-Tm) respectively and used them to study the 
expression of these proteins in cell cultures63 and in trans-
planted masticatory muscles.64 
The pyrophosphate gel electrophoresis method can only dif-
ferentiate between native fast and slow myosin isoforms 
and their light chain-based isoforms but does not reveal the 
known MyHC heterogeneity of limb fast myosins. To study 
the expression of different isoforms of fast MyHCs, we 
raised mabs 2F7, 6H1 and 10F5 against MyHCs 2A, 2X 
and 2B respectively.65 The specificities of these mabs were 
verified in representatives of four orders of eutherians (ro-
dent, lagomorph, carnivore, and primate) as well as two or-
ders of marsupials (diprotodonts and dasyuromorphs). 
These mabs, together with mab 4A6 against EO MyHC, are 
powerful tools for studying mammalian muscle fibre types. 
We have deposited their clones at the Developmental 
Studies Hybridoma Bank at the University of Iowa to en-
able other investigators to access them. We used these mabs 
in our studies on MyHC gene expression in limb, laryngeal 
and EOMs in eutherian mammals and in limb muscles of 
marsupial mammals, as described below.  
 
 
Myosin gene expression in marsupial limb and 
cardiac muscles 
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electro-
phoresis (SDS-PAGE) of limb MyHCs from seven mar-
supial species, spanning two marsupial orders, revealed four 
MyHC components, each of which specifically cross-re-
acted in Western blots with a mab against a corresponding 

eutherian MyHC. For all seven marsupial species, the rel-
ative mobility of the band identified by each mab matched 
that in the rat, showing that the four MyHCs are homolo-
gous to eutherian β-slow, 2B, 2X and 2A MyHCs, respec-
tively, in the order of decreasing electrophoretic mobility.66 
Whereas small eutherian mammals like rats and mice ex-
press all 4 limb MyHCs, larger eutherians like the cat, dog 
and horse do not express 2B MyHC in their limb mus-
cles.54 Unlike eutherian mammals, large and small mar-
supial mammals express the full complement of limb 
MyHCs. 52,66,67 Remarkably, there is little or no 2B MyHC 
expression in the gastrocnemius and flexor digitorum pro-
fundus muscles in hopping marsupials, despite the pre-
dominance of fibres expressing 2B MyHC in most of their 
other hindlimb muscles studied.67 These two muscles are 
involved in elastic energy saving during hopping 68 and 
express high levels of 2X MyHC. In sharp contrast, non-
hopping marsupials strongly express 2B MyHC in their 
gastrocnemius muscles.67 We suggest that the suppression 
of 2B MyHC expression in these muscles of hopping mar-
supials and the general suppression of 2B MyHC in large 
eutherian mammals are due to the possibility that 2X fi-
bres are more efficient than 2B fibres in elastic energy sav-
ing. The general suppression of 2B MyHC in large 
eutherian mammals may be explained by the fact that in 
eutherians all four limbs and their muscles are involved 
in locomotion and elastic energy saving.  
We also analysed cardiac myosins and their subunit com-
positions in marsupial mammals.39 With pyrophosphate gel 
electrophoresis, ventricular myosin in macropodoids shows 
the same three isoforms, V1, V2 and V3, as in eutherian ven-
tricles.29,30 SDS-PAGE of marsupial ventricular myosins re-
veals two MyHCs, the faster isoform reacts with specific 
anti-α-MyHC antibody while the slower isoform reacts with 
anti-β-MyHC antibody. This shows that the MyHC com-
positions of marsupial V1, V2 and V3 to be αα, αβ and ββ, 
respectively, as for eutherian cardiac myosins.30 Interest-
ingly, in some members of the small Antechinus flavipes, 
we found 2 isoforms of α-MyHC with different Ca2+-AT-
Pase activities.69 Marsupial atrial myosin shows αα MyHC 
composition but diverged from V1 in having atrial-specific 
MLCs,39 as in eutherians.29 
We showed that marsupial ventricular myosins were also 
subject to thyroid regulation. Hypothyroidism in a small 
marsupial, which expressed virtually pure V1, led to a shift 
towards V3.

69 Analysis of ventricular myosin of the tammar 
wallaby during early pouch life revealed that it initially ex-
pressed pure V3 but started to shift towards V1 expression 
around the time of pouch exit,70 coincident with the devel-
opmental surge of thyroid hormone secretion. The hormone 
promotes skeletal muscle maturation and enhances general 
metabolism. The increase in V1 expression empowers the 
heart to meet the consequent enhanced cardiovascular de-
mand, enabling the joey to cope with the heightened activity 
of post pouch-exit life.  
 
 
Fibre types in jaw-closing muscles of carnivores 
Rowlerson and co-workers first reported the discovery of a 
new isoform of myosin found in jaw-closing muscles of the 

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cat.71 This myosin has a unique MyHC and a unique pair of 
specific MLCs, and was dubbed “superfast myosin” be-
cause cat jaw-closing muscle has a contraction time shorter 
than that of limb fast muscle and also had a very high AT-
Pase activity which normally connotes high contraction 
speed.14 Fibres expressing superfast myosin are associated 
with the development of very high stress and have a high 
tension cost.72 It is appropriately expressed predominantly 
in carnivores to enables them to deliver a very powerful bite 
during prey capture.73 However, we showed by dynamic 
stiffness analysis that the Fmin of cat masticatory muscle fi-
bres was not higher than that of limb fast fibres and thus 
were not “superfast”.74 Superfast myosin is thus currently 
referred to as masticatory myosin.  
A possible molecular basis for the high stress feature of 
masticatory fibres is its expression of m-MyBP-C.63,75 To 
appreciate this possibility and the molecular mechanisms 
for PTP and PTD later, a brief introduction to thick filament 
structure and the biology of MyBP-Cs is necessary. 
In the resting sarcomere, most myosin heads are bound to 
the core of the thick filament, forming an ordered helical 
array, the two heads of each myosin molecule are folded 
back against the thick filament, forming a stable interact-
ing head motif. In resting muscle fibres, these cross-
bridges are in a super-relaxed state with very low 
Mg-ATPase activity76 and are in the OFF state, being not 
competent to interact with activated thin filaments. Ho-
wever, a small proportion of myosin heads in resting mus-
cle fibres are shifted away from the thick filament in an 
ON state with higher Mg-ATPase activity and are compe-
tent to bind activated actin filaments.  
Recent research has shown that MyBP-C has a strong mod-
ulating influence on contractile function of skeletal and car-
diac muscles. MyBP-C is a component of the thick filament 
of striated muscles localized in the middle one-third of each 
half-thick filament. There are 4 fibre-type specific MyBP-
C isoforms: fast (f-MyBP-C), slow (s-MyBP-C), cardiac 
(c-MyBP-C) 77 and masticatory (m-MyBP-C).63,75 Slow 
muscle fibres express only s-MyBP-C, but fast fibres ex-
press both f-MyBP-C and s-MyBP-C, while m-MyBP-C is 
only expressed in masticatory fibres of carnivores. While 
the C-terminals of MyBP-Cs bind strongly to the thick fil-
ament, the N-terminals of MyBP-C can bind either thick or 
thin filament depending on their phosphorylation status.  
The role of c-MyBP-C in regulating muscle contractility 
has been more extensively investigated in cardiac muscle 
than in skeletal muscles. In cardiac muscle, unphosphory-
lated c-MyBP-C binds to both thick and thin filaments.78 
Unphosphorylated c-MyBP-C binding to the myosin cross-
bridge on the thick filament near the MLC2,79 stabilizing 
the OFF state of the cross-bridge. Upon β-adrenergic stim-
ulation of the myocardium, cAMP-activated protein kinase 
phosphorylates the N-terminal of c-MyBP-C (in addition 
to c-TnI referred to above), leading to the turning of OFF 
cross-bridges into the ON state, enhancing cardiac contrac-
tility.80 
In collaboration with Dr Maki Yamaguchi and colleagues 
at the Jikei University in Tokyo, we studied thick filament 
structure of masticatory fibres using X-ray diffraction 
analysis at the Spring8 Synchrotron Facility. We showed 

that in dog masticatory fibres, the proportion of cross-
bridges in the ON state was significantly higher than that 
in fast muscle fibres.81 This may be due to the presence of 
m-MyBP-C constitutively destabilizing the OFF state of 
masticatory myosin cross-bridges and turning them into 
the ON cross-bridges, thereby enhancing Ca2+ sensitivity 
and accelerating the cross-bridge attachment rate during 
activation which can contribute to the high stress feature 
of masticatory fibres.  
The high stress feature of masticatory fibres may also be 
due to a structural feature of MLC2M which may resemble 
phosphorylated MLC2F in destabilizing the OF cross-
bridges on the thick filament into the ON state,82 as de-
scribed below in connection with PTP. This could also 
lead to enhanced abundance of ON cross-bridges and en-
hanced Ca2+ sensitivity and rate of crossbridge attachment 
during contraction, leading to the high stress feature of 
masticatory fibres. 
Cat jaw-closing muscles also have some slow fibres which 
express a jaw-specific β-slow myosin which is immunohis-
tochemically distinct from limb slow myosin.83 The MyHC 
of this myosin is identical to that of limb β-slow myosin but 
its MLCs are identical to those of masticatory myosin.74 
This leads to jaw-slow fibres having very different kinetic 
properties compared with limb slow fibres. Masticatory 
MLC1 (MLC1M) has been shown to be identical to em-
bryonic MLC1E as well as atrial MLC1A.84 As referred to 
above in connection with atrial myosin, this MLC1A/E/M iso-
form has a weaker interaction with actin during the cross-
bridge cycle compared with the slow MLC1S of limb slow 
fibres.40 This increases the Fmin of jaw-slow fibres by ten-
fold compared with that of limb-slow fibres.74 The tempera-
ture sensitivity of Fmin of jaw-slow fibres is reduced relative 
to that of limb-slow fibres, indicating a reduced activation 
energy for the cross-bridge cycling, which reflects a re-
duced stability of the attached cross-bridge 74 resulting in 
an accelerated cross-bridge detachment rate, leading to a 
more rapid rate of relaxation. The main function of jaw-
slow fibres in jaw-closing muscles of carnivores is to pro-
duce a sustained force for holding the jaw against gravity. 
The rapid relaxation of jaw-slow fibres allows the carnivore 
to rapidly open the jaw wide prior to seizing a prey with a 
powerful bite.  
 
 
Development of masticatory muscles 
The finding of a jaw-specific isoform of myosin stimulated 
our interest in studying its expression during development, 
its regulation in adult life and the structure of its MyHC and 
MLC genes. Jaw-closers develop from cells in the dorsal 
portion of the first branchial arch which derive from the cra-
nial paraxial mesoderm. We analysed cat masseter muscle 
during perinatal development using immunohistochemical 
methods. This muscle, which has both masticatory and jaw-
slow fibres, has four developmentally distinct myotube 
types, each characterized by a specific sequence of myosin 
gene expression during perinatal development: i) slow pri-
maries, which initially express embryonic and β-slow 
MyHCs but not neonatal MyHC and end up expressing β-
slow MyHC at maturity, ii) masticatory primaries, which 

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initially express embryonic, neonatal and β-slow MyHCs 
and replace these with masticatory MyHC at maturity, iii) 
slow secondaries, which initially also express embryonic 
and neonatal MyHCs and replace these with β-slow MyHC 
with or without masticatory MyHC, and end up expressing 
only β-slow MyHC at maturity, and iv) masticatory second-
aries, which initially express embryonic and neonatal 
MyHCs which are progressively replaced by masticatory 
MyHC.60 These four myotubes types appear to be homolo-
gous to the four ontotypes of developing limb fast muscle. 
In the developing cat posterior temporalis, which is com-
posed homogeneously of masticatory fibres in the adult, 
we found only masticatory primary and masticatory sec-
ondary myotubes, both of which ended up expressing 
masticatory myosin at maturity.59 The posterior temporalis 
is homologous to the white region of limb fast muscle 
which is composed homogeneously of fast fibres which 
develop from fast primary and fast secondary myotubes.51 
I postulate that the postnatal expression of masticatory 
myosin is dependent on the postnatal surge of thyroid hor-
mone, just as the expression of fast myosin in developing 
limb fast muscle is thyroid hormone dependent.54 If so, 
masticatory fibres of masticatory primary myotube origin 
would be expected to revert to β-slow myosin expression 
under hypothyroidism, just as adult limb fast fibres of fast 
primary ontotype do.52 
 
 
Myogenic and neurogenic regulation of  
cat jaw-closing muscle  
We investigated whether the expression of masticatory 
myosin was neurogenically or myogenically regulated, 
using electrophoretic and immunohistochemical methods. 
Masticatory fibres of the cat temporalis muscle were trans-
planted into limb fast and slow muscle beds,58 and limb fast 
muscles were transplanted into the temporalis muscle bed.85 
The satellite cells in transplanted muscles underwent regen-
erative myogenesis and became reinnervated by the foreign 
nerves. Jaw muscle regenerates innervated by a fast limb 
muscle nerve expressed masticatory myosin but not limb 
fast muscle myosin, whereas the jaw muscle regenerates in-
nervated by a slow limb muscle nerve expressed not mas-
ticatory myosin, nor limb-slow myosin, but jaw-slow 
myosin. 58,64 Conversely, limb fast muscle regenerate inner-
vated by the jaw muscle nerve expressed limb fast myosin 
but not masticatory myosin.85 The transplantation experi-
ments also revealed that m-MyBP-C and m-Tm were co-
expressed with masticatory myosin in regenerated 
muscles.64 These results indicate that while the nerve may 
influence myosin gene expression, the phenotypic options 
for myosin gene expression during myogenesis is deter-
mined by the developmental origin of the myogenic cells. 
The term allotype has been introduced to classify muscles 
of diverse developmental origins with their unique pheno-
typic options for myofibrillar gene expression. This notion 
is supported by the observation that aneural regenerates of 
cat jaw muscle express masticatory myosin 86 and that myo-
tubes in tissue cultures of cat jaw satellite cells express mas-
ticatory myosin as well as m-MyBP-C and m-Tm.63  
The above experiments also reveal that motor nerves inner-

vating fast and slow muscles could influence myosin gene 
expression of fibres of masticatory muscle allotype. Since 
it is known that the conversion of a limb fast muscle into a 
slow one following cross-innervation by a slow nerve is due 
to the CLFS delivered by the slow nerve,87 it seems likely 
that the expression of jaw-slow myosin in masticatory mus-
cle transplants innervated by the nerve to limb slow muscle 
is also mediated by CLFS. This was confirmed by the ob-
servation that CLFS of masticatory muscle fibres trans-
formed them into jaw-slow fibres.88  
 
 
Sequencing of masticatory MyHC and  
masticatory MLC2 genes 
Using a polyclonal antibody raised against masticatory 
MLCs, we isolated a full-length cDNA clone encoding 
MLC2M gene (mylc2m) from an expression cDNA library 
prepared from cat masseter muscle, and fully sequenced 
it.56 The deduced amino acid sequence of MLC2M shares 
58% overall identity with limb mylc2f gene, indicating that 
superfast mylc2m gene has diverged considerably from 
limb fast mylc2f gene during evolution. However, it has a 
93% amino acid sequence identity with a known human 
MLC gene, MYL5,89 suggesting that MYL5 is a human 
homologue of mylc2m gene, consistent with the expression 
of masticatory myosin in primates.90 However, masti-
catory myosin is not expressed in human jaw-closers be-
cause of a frameshift mutation in the masticatory MyHC 
gene (MyHC-16).91 The possibility remains that human 
jaw muscle may express jaw-slow myosin, which would 
include MYL5. 
Screening the same cDNA library with an anti-masticatory 
myosin antibody combined with the use of RACE-PCR en-
abled us to obtain the full sequence of masticatory MyHC 
gene, MyHC-16.57 Sequence comparisons at the DNA and 
amino acid levels show that cat MyHC-16 gene has less 
than 70% homology to known mammalian striated muscle 
MyHC genes, compared with 87-96% between other mam-
malian fast isoforms themselves, indicating early evolution-
ary divergence from these MyHC genes.57 A phylogenetic 
tree of known mammalian MyHC gene sequences indicates 
that masticatory MyHC gene was the first mammalian 
MyHC gene to diverge from the invertebrate MyHC gene. 
 
 
Phylogenetic plasticity of mammalian jaw-closing 
muscles 
Masticatory myosin is expressed in the shark73 and croco-
dile,92 consistent with the ancient origin of the masticatory 
myosin heavy57 and light chain56 genes. Mammals evolved 
from carnivorous reptilian ancestors; the high-stress feature 
of masticatory fibres served these ancestors well in prey 
capture. We showed that the jaw-closers of some mammals 
continue to express masticatory myosin, e.g., dog, monkey, 
flying foxes and microbats, but the sheep expressed β-slow 
myosin.90 Masticatory myosin is expressed in the jaw-clos-
ing muscles of carnivorous eutherian and marsupial mam-
mals and some non-carnivorous mammals that consume 
nuts and hard vegetable matter.73  

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During mammalian evolution, new functional demands 
were made on jaw-closing muscles to cater for the wide 
range of diets and feeding habits. Mammals need to mas-
ticate food to facilitate rapid digestion and absorption of 
nutrients to support their high metabolic rate. This was 
achieved by replace masticatory myosin with cardiac and 
limb myosin isoforms.73 The range of myosins expressed 
in jaw-closers of eutherian mammals includes masticatory, 
limb fast, foetal, β-slow and α-cardiac myosins.73 The low 
tension-cost of β-slow myosin is highly appropriate for 
grazing animals like the sheep and cattle 90 that spend 
much time feeding and ruminating. Expression of all four 
limb fibre types in rodents 93 provides a wide range of ki-
netic properties to deal with their omnivorous diet.  
In jaw-closers of marsupials, the range of myosins ex-
pressed differs from that of eutherian mammals; they ex-
press only masticatory, β-slow and α-cardiac myosins. 
There are only 3 types of fibres in marsupial jaw-closers: 
masticatory fibres, α-cardiac fibres and α/β fibres which 
co-express α-cardiac and β-slow myosins.94 Masticatory 
fibres predominate in most marsupial species studied and 
are appropriate for predation or for chewing tough vege-
table matter. The dunnart and the antechinus are mar-
supial carnivores that feed primarily on large insects and 
other arthropods, and all their jaw-closer fibres are mas-
ticatory.94  
The jaw-closers of macropodids do not express masticatory 
myosin at all, but express α-cardiac myosin almost ho-
mogeneously.95 Since the bettong, which is closely related 
to kangaroos, expressed masticatory, β-slow and α-cardiac 
myosins, the ancestors of kangaroos presumably also ex-
pressed these myosins, but during evolution kangaroos have 
suppressed the expression of β-slow and masticatory myo-
sins, leaving pure α-cardiac myosin in their jaw-closers. 
Kangaroos are herbivorous foregut fermenters, in common 
with sheep and cattle, but they are not ruminants, and unlike 
sheep and cattle, they do not have a four-chambered stom-
ach to facilitate fermentation. While sheep and cattle ex-
press the economic β-slow myosin in their jaw-closers, 
kangaroos express the more powerful α-cardiac myosin, 
enabling them to finely masticate their herbaceous food for 
rapid digestion.95  
The jaw-closers of other marsupials studied express a mix-
ture of masticatory and α/β fibres, the relative abundance 
of α/β fibres decreased from 60% to zero in the order: ring-
tail possum > brushtail possum > bettong > bandicoot > 
dunnart/antechinus. These variations in jaw-closer fibre 
type are correlated with decreasing amounts of vegetable 
matter in the diets of these animals.94  
 
 
Expression and regulation of myosins in laryngeal 
muscles 
The intrinsic laryngeal muscles of mammals have evolved 
to perform a diversity of functions: airway protection, reg-
ulation of respiration and phonation. These functions re-
quire muscles with different kinetic properties. The speeds 
of contraction of two of these muscles, the Thyroarytenoid 
(TA) which closes the glottis, and the Cricothyroid (CT), 
which tightens the vocal fold during phonation, are differ-

ent, the TA has a shorter contraction time, similar in value 
to that of EOMs. In contrast, the CT has a longer contrac-
tion time, comparable to that of limb fast muscle.96 Sartore 
and colleagues reported the existence of a new Extraocular 
(EO) myosin isoform found in EOM which was immuno-
histochemically distinct from myosins found in somitic 
muscles.97 This EO myosin isoform may explain the high 
speed98 and the low maximal power output of EOMs.99 
These observations led us to suspect that apparent high 
speed of TA might be due to the express EO myosin. We 
used the mab 4A6 against EO MyHC and showed that in 
the rabbit, EO MyHC was expressed in the TA but not in 
the CT, which expressed limb myosin isoforms.61 As the 
TA is involved in closing the glottis, the functional signif-
icance of its expression of EO myosin is to enable the 
rapid closure of the glottis to enhance airway protection. 
We next explored whether EO MyHC expression in laryn-
geal muscles was neuronally or myogenically regulated. 
Rat TA muscle has a vocalis division (TA-V) subjacent to 
the vocal ligament, and a larger external division (TA-X) 
involved in closing the glottis. Immunohistochemical anal-
ysis revealed that all fibres in the rat TA-X co-expressed 2B 
and EO MyHCs, whereas TA-V had fewer of these fibres, 
most fibres expressing 2X MyHC. The recurrent laryngeal 
nerve, which supplies both TA divisions, was transacted and 
reunited to allow the random reinnervation of the TA mus-
cle fibres. Following this procedure, 16.5% of TA-X fibres 
expressed 2X MyHC de novo,100 presumably due to rein-
nervation of TA-X fibres by nerve fibres originally inner-
vating TA-V, suggesting that TA-X fibres were subject to 
neural regulation. To investigate the mechanisms regulating 
myosin gene expression in laryngeal muscles further, we 
cross-innervated the TA and the Sternohyoid (SH), a somitic 
muscle which expressed only limb myosin isoforms. The 
results showed that the SH nerve failed to suppress EO 
MyHC expression in 96% of TA fibres, while the recurrent 
laryngeal nerve failed to induce EO MyHC expression in 
the SH.101 The results show that the phenotypic options for 
MyHC expression of laryngeal and somitic muscles are 
myogenically determined, and that TA and SH differ in 
muscle allotype.  
 
 
Influence of body mass on myosin gene expression 
in cardiac and laryngeal muscles 
It is well established that in eutherians, ventricular myosin 
in small animals is purely V1, but in large animals it is 
purely V3, while animals of intermediate size express all 
three ventricular myosin isoforms. We showed that ven-
tricular myosins in ten species of marsupial mammals 
broadly reflected the body mass dependence of ventricular 
MyHC expression seen in eutherians.39 These differences 
in cardiac myosin expression with body mass reflect the 
need to adjust cardiac output to match changes in meta-
bolic rate per unit body mass, which is inversely related 
to body mass (see below). However, the kangaroos ex-
pressed considerably more α-cardiac MyHC for their body 
mass compared with eutherians of similar body mass. This 
is associated with the fact that kangaroos are highly ath-
letic animals. The high α-cardiac MyHC expression en-

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hances the contractility of their hearts to match their high 
metabolic scope and athletic lifestyle.   
Body mass of animals strongly influences their basal 
metabolic rates. The kinetic properties of the myocardium 
and of the skeletal muscles controlling the respiratory rate 
need to be adjusted to match changes in metabolic rate 
with body mass. McMahon, arguing theoretically from 
elastic similarity, concluded that metabolic rate should 
scale to (body mass)0.75 and physiological cycles should 
scale to (body mass)−0.25.102 Experimentally determined 
basal metabolic rates in eutherian103 and marsupial104 
mammals do scale approximately to (body mass)0.75, the 
specific metabolic rate per unit body mass would thus 
scales to (body mass)−0.25. Heart rate and respiratory 
frequency are similarly scaled to (body mass)−0.25,105 in ac-
cordance with McMahon’s theory, thus ensuring that the 
body gets adequately supplied with well oxygenated blood 
as animal body size changes. However, the kinetics of var-
ious myosin isoforms that constrain heart and respiratory 
rates scale to body mass with more modest negative ex-
ponents, being 0.175, 0.098, 0.048 and 0.041 for β-slow, 
2A, 2X and 2B myosins, respectively.106 This means that 
changes in kinetics of cardiac, fast and slow skeletal myo-
sins as body mass changes are not fast enough in small 
animals and too fast in large animals to match the changes 
in metabolic rate with body mass. In the ventricular myo-
cardium, this is compensated by the expression of the fast 
V1 myosin in small animals and a shift towards the slower 
V3 myosin as body mass increases.  
In laryngeal muscles, the disparity between respiratory 
rate and kinetics of myosins expressed in laryngeal mus-
cles as functions of body mass is compensated in small 
animals by recruiting faster myosins not expressed in their 
limb muscles: EO myosin in rats,100 2B myosin in cats,107 
and dogs.108 In larger animals like horses,109 baboons 107 
and humans,110 laryngeal muscle speed is reduced by ex-
pressing predominantly β-slow and 2A myosins. These 
variations in cardiac and skeletal myosin isoform expres-
sion across species illustrate the phylogenetic plasticity of 
both cardiac and skeletal muscles, i.e., their capacity to 
change the pattern of myosin gene expression in response 
to changing functional demands during phylogeny. 
 
 
Fibre types and myosin isoforms expressed  
in EOMs 
Muscle fibre types of EOMs and the MyHCs they express 
are the most complex among all skeletal muscles. EOMs 
are organized into two layers, a thin orbital layer facing 
the orbit and a more substantial global layer facing the 
globe. Both the orbital and global layers are made up of 
Singly-Innervated Fibres (SIFs) with large en plaque 
nerve endings, and Multiply-Innervated Fibres (MIFs) 
with small en grappe endings. The fibres in the orbital (o) 
and global (g) layers can be classified using ultrastructural 
and histochemical criteria into 6 fibre types: oSIF and 
oMIF in the orbital layer and gSIF red, gSIF intermediate, 
gSIF white and gMIF in the global layer. 
The EOMs express a complex mixture of 11 MyHC iso-
forms: the four isoforms found in adult limb muscles (2A, 

2X, 2B, β-slow MyHCs), foetal/neonatal-MyHC,111 em-
bryonic MyHC,112 α-cardiac MyHC113 and four EOM-spe-
cific isoforms: EO MyHC,114 slow-tonic MyHC,115 slow 
B MyHC116 and non-muscle myosin IIB MyHC.117  
The expression of such a wide range of MyHCs in EOMs 
is expected to generate fibres with a very wide range of 
mechanical properties. We compared the kinetic properties 
of single rabbit EOM fibres with those of limb fibres using 
dynamic stiffness analysis. Limb fast fibres have Fmin 
values ranging from 10 to 26 Hz while Fmin for limb slow 
fibres is 0.5 Hz. EOM fibres do have Fmin values in the 
range for limb fast fibres, but in addition have Fmin values 
below (4-9 Hz) and above (27-33 Hz) the range for limb 
fibres.118 The wider range of kinetic characteristics in 
EOM fibres compared with limb fibres can be attributed 
to their expression of embryonic and neonatal MyHCs 
(low Fmin) and EO MyHC (high Fmin) in addition to iso-
forms present in adult limb muscles. 
 
 
Distribution of MyHCs in EOM fibre types and 
functional significance 
We initially examined the distribution of MyHC isoforms 
in the levator palpebrae superioris and retractor bulbi 
which share their developmental origins with EOMs. 
These muscles in the rabbit express EO and limb fast 
MyHCs. The levator palpebrae superioris of monkey and 
cat also express EO MyHC but this isoform is absent in 
cat retractor bulbi and rat levator palpebrae superioris and 
retractor bulbi.114  
We next investigated the expression of MyHCs in EOM 
fibre types of the rat and rabbit. In the rat, gSIFs express 
2A, 2X or 2B MyHC and show no longitudinal variation 
in MyHC distribution. However, rat oSIF and oMIF both 
express faster MyHCs at the endplate zone and slower 
ones at the end-segments: oSIFs express EO MyHC at the 
end-plate zone and co-express β-slow and embryonic 
MyHCs at the end-segments while oMIFs express β-slow 
MyHC at the endplate zone and embryonic MyHC at the 
end-segments.119 The expression of EO MyHC at the cen-
tral segments of oSIFs in the rat emerge over the first few 
weeks postnatally.120 
Orbital fibres of the rabbit are also characterised by the 
expression of fast MyHCs in the endplate zone, flanked 
by segments with slower MyHCs. Rabbit oSIFs also ex-
press EO MyHC at the endplate zones, the flanking seg-
ments express embryonic and 2A MyHCs. The endplate 
zones of rabbit oMIFs express β-slow and embryonic 
MyHCs but express embryonic and slow-tonic MyHCs in 
the flanking end segments.62  
We propose that the functional significance of having ki-
netically fast central segments flanked by slower end seg-
ments in oMIFs and oSIFs is to linearize the rapid eye 
movement during a saccade. As the agonist starts to con-
tract at the beginning of the saccade, the rapid relaxation 
of the central segments of the orbital fibres of the antago-
nist reduces the load on the agonist to allow the initial ac-
celeration of the eyeball, while the kinetically slow end 
segments help to break the eyeball rotation towards the 
end of the saccade.62,121  

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Palisade endings of gMIFs and functional  
significance 
The gMIFs have structures called palisade ending at their 
myotendinous junctions. These structures resemble imma-
ture Golgi tendon organs and are innervated by the same 
motoneuron innervating the parent gMIF. Non-muscle 
myosin IIB is found at the centre of thick filaments of 
gMIFs. I suggest that the functional significance of the 
super-slow contracting non-muscle myosin IIB myosin is 
to permit the transmission of passive tension along gMIFs 
to palisade endings when the EOM is acting as an antago-
nist during a saccade. Activation of palisade endings elicits 
neural impulses that trigger axon reflexes on gMIFs, gen-
erating sustained tension that play important roles during 
fixation and vergence.121 Since gMIFs are polyneuronally 
innervated, the gMIF/palisade ending complex acts like a 
motoneuron with afferent synaptic inputs from several 
gMIF motoneurons, integrating not their endplate poten-
tials, but their tension contributions to stimulate palisade 
endings to generate neural impulses to gMIFs innervated 
by its motoneuron. Since each palisade ending can only in-
fluence tension output of the motor unit of which it is a part, 
and the control over this gMIF/palisade ending complex is 
vested in several gMIF motoneurons innervating the com-
plex, the system allows the gMIF motoneuron pool to ex-
ercise exquisitely fine control over EOM tension.   
Palisade endings are more abundant in the medial recti and 
in the inferior recti of front-eyed but not in lateral-eyed ani-
mals.122 These EOMs are involved in convergence move-
ments. The greater abundance of palisade endings would 
enable these EOMs to develop a strong axon reflex tension 
during convergence. This would allow the gMIF motoneu-
rons of these EOMs to exercise exquisitely fine control over 
ripple-free and highly fatigue-resistant tension of the gMIF 
of these EOMs, and thus the precision in controlling eye 
position during vergence.121 

 

 

Regulation of MyHC expression in EOMs 
To investigate mechanisms regulating the expression of 
MyHCs in EOMs, we i) transplanted EOMs into a limb fast 
muscle bed to allow it to be reinnervated by limb muscle 
nerve and ii) allowed EOMs to regenerate in situ to be re-
innervated by the oculomotor nerve. The results showed 
that the patterns of expression of MyHCs of the two types 
of regenerates were rather similar during the first few 
weeks, expressing the relatively slow embryonic, β-
slow/slow-tonic and 2A MyHCs, but differed substantially 
in the longer term. The in-situ regenerates at 10 weeks ad-
ditionally expressed the faster 2X, 2B and EO MyHCs. At 
22 weeks, fibres in in-situ regenerates expressing fast 2B 
and EOM MyHCs became progressively more dominant 
and clearly distinct from fibres expressing slow β-
slow/slow-tonic MyHCs. By 42 weeks, most of the fibres 
co-expressed EOM and 2B MyHCs, while expression of 
2X and 2A MyHCs weakened. In contrast, transplanted re-
generates innervated by limb fast muscle nerve only tran-
siently expressed 2X and 2B MyHCs after 20 weeks but 
never expressed EO MyHC even up to 42 weeks.121 

Contrary to earlier work,123 the above results showed that 
EOM satellite cells can express EO MyHC during myogen-
esis, thus supporting the notion that EOMs are a distinct al-
lotype. EOM regenerates differ from regenerating 
masticatory fibres which express masticatory MyHC even 
without innervation 86 and in tissue cultures of satellite cells 
of masticatory muscle.63 This is because fibres in jaw-
closers in carnivores have only 2 degrees of freedom for 
MyHC expression: masticatory or jaw-slow myosin, which 
can be adequately catered for by a default mechanism for 
masticatory myosin expression and a neural mechanism for 
controlling jaw-slow myosin expression by CLFS.88 EOMs 
require a much more sophisticated myogenic and neuro-
genic mechanisms for controlling the expression of 11 
MyHCs. The emergence of distinct fast and slow fibres in 
the in-situ regenerates suggested that there were distinct lin-
eages of satellite cells associate with MIFs and SIFs, which 
were derived from primary and secondary myotubes, re-
spectively. These results suggest that neural impulses from 
the limb muscle nerve can only induce limb muscle MyHC 
isoforms, the capacity of oculomotor nerve to induce EO 
MyHC can be linked to the fact that unlike limb fast nerve, 
it can deliver very high frequency impulses likely to be nec-
essary to induce EO MyHC expression.121 
Oculomotor neurons receive neurotrophins from muscle fi-
bres they innervate for their survival. These neurotrophins 
specify their synaptic input, and thus the impulse patterns 
they can generate.124 It is thus likely that synaptic inputs of 
slow-tonic and phasic motoneurons innervating MIFs and 
SIFs, respectively, are modulated during postnatal devel-
opment by different target-derived neurotrophins they re-
ceive from contact with primary and secondary myotubes 
respectively. In other words, primary and secondary myo-
tubes may specify the tonic and phasic impulse pattern ap-
propriate for their diverse functional roles in the mature 
EOM by influencing the synaptic inputs of their innervating 
ocular motoneurons. I propose that the very impulse pat-
terns driving the various ocular movements induce the ex-
pression of the appropriate MyHC compositions and thus 
the mechanical properties of different EOM motor units to 
accomplish their tasks.121  
 
 
Molecular mechanisms of post-tetanic potentiation 
and post-tetanic depression 
Since the documentation of the phenomena of PTP and 
PTD during my PhD, the molecular basis of PTP had been 
progressively unfolded.125 It is due to the phosphorylation 
of MLC2F by Ca2+-calmodulin-activated myosin light 
chain kinase activated by the elevated intracellular Ca2+ 
level during the tetanus. Phosphorylation of MLC2F 
causes cross-bridges that are bound to the thick filament 
in the OFF state to swing away from the thick filament. 
This transition has been shown by electron microscopy of 
isolated thick filaments with phosphorylated MLC2F.

126 In 
collaboration with Dr Maki Yamaguchi and colleagues, 
we show, using X-ray diffraction analysis of skinned rab-
bit fast fibres, that phosphorylation of MLC2F causes OFF 
cross-bridges to swing out towards the thin filament into 
the ON state.82 The enhanced number of ON cross-bridges 

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Eur J Transl Myol 35 (2) 13688, 2025 doi: 10.4081/ejtm.2025.13688

increases the Ca2+-sensitivity of the fibre and accelerates 
the rate of cross-bridge attachment at submaximal activat-
ing Ca2+ level,127 generating PTP.  
There has been no research on the molecular mechanism 
for PTD. A recent report showed that in slow muscle fi-
bres, phosphorylation of s-MyBP-C by cAMP-activated 
protein kinase enhanced the transient force overshoot at 
low levels of Ca2+ activation and increased the rate of 
force development at all Ca2+ activation levels during the 
release and re-stretch manoeuvres.128 It was proposed that 
unphosphorylated N-terminals of s-MyBP-C bind to thin 
filaments, thereby forming links between filaments which 
retard filament sliding, and that phosphorylation of N-ter-
minals of s-MyBP-C by cAMP-activated protein kinase 
detaches them from thin filaments to bind to thick fila-
ments, transforming OFF cross-bridges into the ON state. 
I proposed that this phosphorylation of s-MyBP-C is the 
molecular mechanism for the enhancement of slow mus-
cle force due to β-adrenergic stimulation,129 and that de-
phosphorylation of s-MyBP-C could provide a mechanism 
for PTD.130 The elevated intracellular Ca2+ following a tet-
anus in a slow muscle fibre is postulated to activate the 
Ca2+-calmodulin-activated phosphatase, calcineurin, to de-
phosphorylate s-MyBP-C. The N-terminal of dephospho-
rylated s-MyBP-C would then detach from thick filaments 
and attach to thin filaments, shifting ON cross-bridges into 
the OFF state and forming links between the filaments. 
This would reduce post-tetanic twitch force and the rate 
of force development, leading to PTD. 
 
 
Looking back 
In this sojourn I am thankful to Sir John Eccles for directing 
me into muscle research even though I initially deeply re-
gretted it. I was fortunate to have had the excellent guidance 
of Dr Russel Close, and to have a succession of talented 
Honours and PhD students and research assistants, in par-

ticular, Susan Hughes, Angela Hamilton, Robin Fitzsimons, 
Gary Yeoh, Han Qin, Christine Lucas, Lucia Kang and 
Wendy Zhong. I am also thankful to many national and in-
ternational collaborators for their valuable contributions 
over the years. I am extremely fortunate and grateful for the 
long-term and fruitful collaboration of Dr Gunther Ross-
manith, a mathematician and biophysicist, in analysing 
myosin cross-bridge kinetics of cardiac and skeletal muscle 
fibres. This enabled us to perform the important task of re-
lating the various myosin isoforms to their contractile 
speeds. I am also very grateful for the collaboration with 
Dr Maki Yamaguchi and colleagues at the Jikei University 
School of Medicine in Tokyo. This enabled us to use the 
synchrotron X-ray diffraction facilities at Spring8 to analyse 
cross-bridge orientation in masticatory muscle fibres and in 
fast skeletal fibres following the phosphorylation of MLC2F 
in connection with the mechanism for PTP. Finally, I am 
grateful to my wife for her love, patience and support over 
the years, which have been invaluable to my work.  
I have enjoyed attending many international conferences 
over the years. One of the most memorable was the 29th 
Congress of the International Union of Physiological 
Sciences (IUPS) held in Sydney in 1983. On that occasion 
I invited a few muscle scientists, including Sir Andrew 
Huxley and Dr John Solaro, to dinner at home, and had an 
enjoyable evening in their company. In 2001, the 34th Con-
gress of the IUPS was held in New Zealand and I took the 
opportunity to contribute to the muscle research community 
by organizing a satellite meeting in Sydney on “Muscle 
fibre types: development, function and regulation”. It was 
attended by 30 delegates from Europe, US, Japan, China 
and Australia (Figure 1). An unforgettable fact was that the 
meeting took place a few days before the devastation of 
New York on September 11. Another memorable meeting 
was in Umea organized by Prof. Lars-Eric Thornell in the 
summer of 2002 (Figure 2). Being in Umea was the closest 
to the artic circle I have ever been, where one could read a 

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Figure 1. Group photograph of attendees at the IUPS Satellite Meeting 2001 in Sydney.



Research into the physiology of myosins - a personal odyssey 
Eur J Transl Myol 35 (2) 13688, 2025 doi: 10.4081/ejtm.2025.13688

book at midnight without artificial lighting! In 2003, I was 
honoured to have been invited by muscle scientists at the 
University of Padua to deliver some lectures on my work. 

I was accompanied by my wife, and we thoroughly enjoyed 
the hospitality of our hosts in Padua (Figure 3), most mem-
orable was a horse-meat dinner in the home of Prof. Fran-
cesco Mascarello! The trip gave us a wonderful opportunity 
to tour Venice, Florence, Assisi and Rome, which we thor-
oughly enjoyed. I retired in 2004 and spent much time writ-
ing up unpublished works and reviews but regret not being 
able to continue to probe issue raised by the work done and 
test hypotheses proposed. 
 
 
List of abbreviations 
BDM, 2,3-butanedione monoxime 
CLFS, chronic low frequency stimulation 
CT, cricothyroid 
EOM, extraocular muscle 
Fmin, stiffness minimum frequency 
IBMX, Isobutylmethylxanthine 
Mab, monoclonal antibody 
MIFs, multiply-innervated fibres 
MLC, myosin light chain 
m-Tm, masticatory tropomyosin 

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Figure 2. Group photograph of attendees at the Muscle Meeting 2002 in Umea.

Figure 3. With hosts at the University of Padua.



Research into the physiology of myosins - a personal odyssey 
Eur J Transl Myol 35 (2) 13688, 2025 doi: 10.4081/ejtm.2025.13688

m-TnI, masticatory troponin I 
MyHC, myosin heavy chain 
MyBP-C, myosin binding protein-C 
PTD, post-tetanic depression 
PTP, post-tetanic potentiation 
SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel 

electrophoresis  
SIFs, singly-innervated fibres 
TA, thyroarytenoid 
 
 
Acknowledgements 
The research described in this memoir had been supported 
by grants from the National Health and Medical Research 
Council of Australia, the Australian Research Council, the 
National Heart Foundation of Australia and the Muscular 
Dystrophy Association of America. 

 
 
Conflict of interest 
There is no conflict of interest to declare. 
 
 
Corresponding author 
Joseph F Y Hoh, retired from the Discipline of Physiology, 
School of Medical Sciences, Faculty of Medicine and 
Health, The University of Sydney, NSW, Australia. 
ORCID ID: 0000-0002-5085-3349 
E-mail: joeh@iinet.net.au 
 
 
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