 Neurohypophyseal hormones and skeletal muscle Eur J Transl Myol 30 (1): 53-57, 2020 - 53 - Neurohypophyseal hormones and skeletal muscle: a tale of two faces Alexandra Benoni, Alessandra Renzini, Giorgia Cavioli, Sergio Adamo Histology and Embryology Section, Dept. AHFOS, Sapienza University of Rome, Italy 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. Abstract The neurohypophyseal hormones vasopressin and oxytocin were invested, in recent years, with novel functions upon striated muscle, regulating its differentiation, trophism, and homeostasis. Recent studies highlight that these hormones not only target skeletal muscle but represent novel myokines. We discuss the possibility of exploiting the muscle hypertrophying activity of oxytocin to revert muscle atrophy, including cancer cachexia muscle wasting. Furthermore, the role of oxytocin in cardiac homeostasis and the possible role of cardiac atrophy as a concause of death in cachectic patients is discussed. Key Words: Vasopressin, Oxytocin, Muscle, Homeostasis. Eur J Transl Myol 30 (1): 53-57, 2020 Neurohypophyseal Hormones and Skeletal Muscle Homeostasis In the past few decades, the neurohypophyseal hormones (NH) vasopressin (AVP) and oxytocin (OT) emerged as novel myogenic regulators.1-5 In fact, besides their classical roles (uterus, mammary gland, vascular smooth muscle, kidney tubules, CNS),2 they were shown to target skeletal muscle. OT and AVP are mainly produced in the hypothalamus, transported to the neurohypophysis, and secreted into circulation. Seven- transmembrane-domain G-protein-coupled receptors (GPCR) and complex downstream signaling processes mediate their pleiotropic physiological functions.5 Functional receptors for NH mediate AVP and OT signals in myogenic cells (V1aR in the murine myogenic cell line L6, OTR in human satellite cells) and muscle fibers.3,6 Interestingly, despite possessing individual receptors, AVP and OT have been shown to cross- activate each other’s receptors.7 Skeletal muscle is a target of AVP and OT In vitro studies indicated that OT and AVP treatments dramatically increased the fusion of murine and avian myoblasts and human satellite cells.1,2,3 The addition of AVP to the culture medium of murine myoblasts induces the expression of Myf-5 and myogenin.1 Interaction of AVP and the V1a receptor rapidly increases intracellular [Ca2+] and stimulates the phospholipases C (PLC), D (PLD) and A2 (PLA2), producing intracellular messengers such as phosphatidic acid (PA), diacylglycerol (DAG) and InsP3 (Figure 1). As shown in Figure 1, PA (produced by the PLD-dependent hydrolysis of PtdCho) activates PDE4, which in turn promotes cAMP breakdown, thus inhibiting cAMP- dependent activation of PKA, a kinase known to negatively regulate myogenic differentiation.9 The increase in the intracellular level of Ca2+ interferes with the Calcium calmodulin Kinase (CaMK)/Calcineurin pathway inducing upregulation of skeletal muscle differentiation markers myogenin, MEF2 and GATA2 (zinc finger proteins) expression.10 The PI3K/Akt/mTOR pathway is well known for its role in cell growth, proliferation, differentiation, and muscle homeostasis. This pathway is recruited in response to NH in myoblasts, and its inhibition hampered the AVP-stimulated expression of both Akt and mTOR and increased the expression of the ubiquitin ligase atrogin-1 and FoxO (Figure 1).11 Satellite cells obtained from aged mice display lower OT receptor expression and myogenic potential when compared to satellite cells from young animals. By treating the “old” and “young” satellite cells with exogenous OT or with a selective oxytocin antagonist, respectively, OT was demonstrated to be required for effective muscle regeneration.12 In vivo, AVP signaling accelerates the regeneration of injured skeletal muscle and reverts TNF-induced muscle atrophy: overexpressing V1aR in the tibialis anterior decreased the p-NFkb-p65 protein expression, increased Pax7, myogenin and embryonic myosin heavy chain (eMHC) expressions after cardiotoxin injury, even in the presence of TNF.6 In summary, OT and AVP activate a complex array of signaling pathways to regulate skeletal muscle homeostasis (modulating different kinases, modifying the calcium level, through the CaMK/Calcineurin Neurohypophyseal hormones and skeletal muscle Eur J Transl Myol 30 (1): 53-57, 2020 - 54 - signaling to activate the myogenic regulatory factors expressions, and by stimulating the Akt/mTOR or the MAPK pathway to induce muscle cell proliferation, differentiation, contraction, and regeneration. Skeletal muscle is a source of AVP and OT Skeletal muscle is not only a target of neurohypophyseal hormones but also a source, acting in this way, as an autocrine/paracrine gland for its regulation. OT was demonstrated to be expressed by cultured human myoblasts.3 De Jager and collaborators revealed that cattle administrated continuously with anabolic steroids featured an augmented OT mRNA expression in skeletal muscle, followed by a ∼ 50-fold higher plasma level of OT.13 More recently, C2C12 myogenic cells were shown to express and secrete OT in the culture medium at a low level under basal conditions.8 These authors demonstrated that 17β-estradiol, which stimulates the differentiation of these myoblasts, transiently up- regulates OT gene expression and secretion rapidly after treatment. Increased expression of OT in livestock as a result of anabolizing treatments was reported by other groups.14 Interestingly, the estrogen control of OT expression appears to be mediated by ERR, an effect which can be further amplified by an OT/OT-R loop.14 Furthermore, physical activity was shown to physiologically stimulate AVP and OT secretion. Several groups demonstrated that exercise results in increased levels of AVP and OT in the hypothalamus as well as in plasma.14 Exercise plays beneficial effects on muscle physiology. The exercise-mediated stimulation of AVP and OT suggests that physical activity participates in their autocrine effect upon skeletal muscle triggering NH secretion by muscle and brain, thus regulating their receptors and contributing to muscle homeostasis. OT, a potential therapeutic agent against muscle atrophy Investigations have been conducted in aged mice to examine the potential of OT to reverse sarcopenia, which involves impaired skeletal muscle regeneration. A significant decrease of OT expression in aged mice in comparison to young mice has been reported.12 Indeed, a low level of OTR on satellite cells accompanies the decrease of OT circulating levels in the plasma. Moreover, exogenous OT administration in aged mice significantly increases muscle regeneration after cardiotoxin injury by increasing both the expansion and the differentiation of satellite cells through the MAPK/ERK pathway.12 Analysis of OT-knock out (KO) mice was conducted to better understand the OT role in regeneration and sarcopenia. Muscle regeneration was Fig 1. OT and AVP signaling in skeletal muscle (see acronyms list below). Neurohypophyseal hormones and skeletal muscle Eur J Transl Myol 30 (1): 53-57, 2020 - 55 - strongly impaired in OT-KO mice. 12-month-old KO mice, compared to wild type, displayed premature aging with muscle fibrosis and fat infiltration, a pattern characteristic of sarcopenia.12 As in sarcopenia, cancer-cachexia (CC) is characterized by muscle wasting, loss of fat and lean mass, and is associated with an adverse prognosis. Our group demonstrated that physical activity counteracts CC in vivo, increasing the survival of tumor-bearing mice and counteracting muscle atrophy.15 The effects of CC are dependent upon the presence of pro-inflammatory cytokines, among these TNF, which has been shown to hamper the differentiation potential of myogenic cells, ultimately leading to cachexia.16 Since NH have been demonstrated to stimulate myogenic differentiation, muscle trophism and regeneration, we believe that the use of OT may counteract CC-dependent skeletal muscle atrophy and thus, ameliorate the quality of life and response to treatment of cancer patients. Role of Oxytocin in Cardiovascular Regulation In the 90s, a group showed that OT is synthesized and released also by the heart. They found the concentration of OT higher in the atria and in the ventricles than in the uterus of rats.17 Furthermore, as in skeletal muscle, OT induces cardiomyocytes differentiation in vitro. Its addition to the culture medium of P19 cells (mouse embryonal carcinoma cells) increased the expression of MHC, and cardiac markers DHPR-α1 (dihydropyridine receptor α1) and ANP (atrial natriuretic peptide) (Figure 2). OT up-regulated OTR expression by 5–10%.18 Later,19 the establishment of the PI3K, Ca-CAMKK and AMPK pathways involvement in OT-dependent stimulation of glucose uptake was demonstrated (Figure 2). Therefore, OT contributes to cardiomyogenesis and CM differentiation. However, a recent study showed that overexpressing OTR specifically in the heart led to a decreased motor activity, irregular respiration and death because of severe heart failure.20 Indeed, chronic heart failure that affects more than 20 millions of people in the world, has been reported to induce cachexia. Conversely, it is possible to hypothesize that cardiac atrophy takes place in cachexia, and that heart failure may play a role in cachexia-associated mortality. This suggests a new therapeutic approach, using OT to ameliorate the cachectic patients’ quality of life and survival. In conclusion, OT could be a potential candidate to counteract muscle wasting implicated in cachexia. List of acronyms AKT – Serine-threonine Protein Kinase AMPK – AMP-activated Protein Kinase ANP - Atrial Natriuretic Peptide AVP – Vasopressin Fig 2. OT signaling in cardiac muscle (see acronyms list below). Neurohypophyseal hormones and skeletal muscle Eur J Transl Myol 30 (1): 53-57, 2020 - 56 - Ca-CAMKK – Ca2+/Calmodulin-dependent Protein Kinase CaMK – Calcium Calmodulin Kinase cAMP – Cyclic Adenosine Monophosphate CC – Cancer Cachexia CNS – Central Neural System DAG - Diacylglycerol DHPR-α1 – Dihydropyridine Receptor α1 eMHC - embryonic Myosin Heavy Chain ERK – Extracellular signal-regulated Kinase ERRα – Estrogen-related Receptor α FoxO – Forkhead box O G – G-protein GATA2 – GATA Binding Protein 2 GPCR - G-protein-coupled receptors InsP3 – Inositol Triphosphate MAPK – Mitogen-activated Protein Kinase MEF – Myocyte Enhancer Factor-2 MHC – Major Histocompatibility Complex mTOR – mammalian Target of Rapamycin Myf5 – Myogenic Factor 5 NH - Neurohypophyseal Hormones OT – Oxytocin OTR – Oxytocin Receptor PA – Phosphatidic Acid Pax7 – Paired box protein 7 PDE4 – Phosphodiesterase-4 PI3K – Phosphoinositide 3-kinase PKA – Protein Kinase A p-NFkb – phosphorylated Nuclear Factor-kappa B PLA2 – Phospholipases A2 PLC – Phospholipases C PLD – Phospholipases D PtdCho – Phosphatidylcholine TNF – Tumor Necrosis Factor V1aR - Vasopressin Receptor 1a Authors contributions AB and SA played a main role in the conception, study design, and data acquisition, while AR and GC participated in analyses of data and in drafting and finalizing the manuscript. Funding None Conflict of Interest The authors declare they have no financial, personal, or other conflicts of interest. Ethical Publication Statement We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Corresponding Author Sergio Adamo, Histology and Embryology Section, Dept. AHFOS, Sapienza University of Rome, Italy. ORCID iD: 0000-0002-1409-0452 Email: sergio.adamo@uniroma1.it E-mail and ORCID iD of co-authors Alexandra Benoni: alexandra.benoni@gmail.com ORCID iD: 0000-0003-3283-8660 Alessandra Renzini: alessandra.renzini@uniroma1.it ORCID iD: 0000-0002-4265-7188 Giorgia Cavioli: giocav96@gmail.com References 1. Nervi C, Benedetti L, Minasi A, et al. Arginine- Vasopressin Induces Differentiation of Skeletal Myogenic Cells and up-Regulation of Myogenin and Myf-5. Cell Growth Differ 1995;6:81–9. 2. Minotti S, Scicchitano BM, Nervi C, et al. Vasopressin and Insulin-like Growth Factors Synergistically Induce Myogenesis in Serum-Free Medium. Cell Growth Differ 1998;9:155–63. 3. Breton C, Haenggeli C, Barberis C, et al. Presence of Functional Oxytocin Receptors in Cultured Human Myoblasts. J Clin Endocrinol Metab 2002; 87:1415–18. doi: 10.1210/jcem.87.3.8537. 4. Costa A, Rossi E, Scicchitano BM, et. Neurohypophyseal Hormones: Novel Actors of Striated Muscle Development and Homeostasis. Eur J Transl Myol 2014;24(3):3790. doi: 10.4081/ejtm.2014.3790. 5. Barberis C, Mouillac B, Durroux T. Structural Bases of Vasopressin/Oxytocin Receptor Function. J Endocrinol 1998;156:223–9. doi: 10.1677/joe. 0.1560223. 6. Costa A, Toschi A, Murfuni I, Pelosi L, et al. M. Local Overexpression of V1a-Vasopressin Receptor Enhances Regeneration in Tumor Necrosis Factor-Induced Muscle Atrophy. Biomed Res Int 2014;2014:235426. doi: 10.1155/ 2014/235426. 7. Song Z, Albers H. E. Cross-Talk among Oxytocin and Arginine-Vasopressin Receptors: Relevance for Basic and Clinical Studies of the Brain and Periphery. Front Neuroendocrinol 201:51:14–24. doi:10.1016/j.yfrne.2017.10.004. 8. Berio E, Divari S, Cucuzza LS, et al. 17β-Estradiol Upregulates Oxytocin and the Oxytocin Receptor in C2C12 Myotubes. PeerJ 2017;2017:(3). doi: 10.7717/peerj.3124. 9. Naro F, De Arcangelis V, Sette C, et al. A Bimodal Modulation of the CAMP Pathway Is Involved in the Control of Myogenic Differentiation in L6 Cells. J Biol Chem 2003;278:49308–15. doi: 10.1074/jbc.M306941200. 10. Scicchitano B. M, Spath L, Musarò A, et al. Vasopressin-Dependent Myogenic Cell Differentiation Is Mediated by Both Ca2+/Calmodulin-Dependent Kinase and Calcineurin Pathways. Mol. Biol. Cell 2005;16:3632–41. doi: 10.1091/mbc.E05-01-0055. 11. Sorrentino S, Barbiera A, Proietti G, et al. Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-Dependent mailto:sergio.adamo@uniroma1.it mailto:%20giocav96@gmail.com Neurohypophyseal hormones and skeletal muscle Eur J Transl Myol 30 (1): 53-57, 2020 - 57 - Stimulation of Myogenic Differentiation. Int J Mol Sci 2019;20:(17). doi: 10.3390/ijms20174188. 12. Elabd C, Cousin W, Upadhyayula P, et al. Oxytocin Is an Age-Specific Circulating Hormone That Is Necessary for Muscle Maintenance and Regeneration. Nat Commun 2014;5:4082. doi: 10.1038/ncomms5082. 13. de Jager N, Hudson NJ, Reverter A, et al. Chronic Exposure to Anabolic Steroids Induces the Muscle Expression of Oxytocin and a More than Fifty Fold Increase in Circulating Oxytocin in Cattle. Physiol Genomics 2011;43: 467–78. doi: 10.1152/physiol genomics.00226.2010. 14. Adamo S, Pigna E, Lugarà Ret al. Skeletal Muscle: A Significant Novel Neurohypophyseal Hormone- Secreting Organ. Front Physiol 2019;9: doi: 10. 3389/fphys.2018.01885. 15. Pigna E, Berardi E, Aulino P, et al. Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer. Sci Rep 2016;6. doi: 10.1038/ srep26991. 16. Moresi V, Pristerà A, Scicchitano B. et al. Tumor Necrosis Factor-α Inhibition of Skeletal Muscle Regeneration Is Mediated by a Caspase-Dependent Stem Cell Response. Stem Cells 2008;26:997– 1008. doi: 10.1634/stemcells.2007-0493. 17. Jankowski M, Hajjar F, Al Kawas S, et al. Rat Heart: A Site of Oxytocin Production and Action. Proc Natl Acad Sci USA. 1998;95:14558–63. doi: 10.1073/pnas.95.24.14558. 18. Paquin J, Danalache BA, Jankowski M, et al. Oxytocin Induces Differentiation of P19 Embryonic Stem Cells to Cardiomyocytes. Proc Natl Acad Sci USA 2002;99: 9550–5. doi:10.1073/pnas.15230 2499. 19. Florian M, Jankowski M, Gutkowska J. Oxytocin Increases Glucose Uptake in Neonatal Rat Cardiomyocytes. Endocrinology 2010;151:482–91. doi: 10.1210/en.2009-0624. 20. Jung C, Wernly B, Bjursell M, et al. Cardiac- Specific Overexpression of Oxytocin Receptor Leads to Cardiomyopathy in Mice. J Card Fail 2018;24:470–8. doi: 10.1016/j.cardfail.2018.05. 004. Submitted: February 12, 2020 Revision received: February 18, 2020 Accepted for publication: February 18, 2020