Abstract
Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2′, 7′- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.
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References
Amthor H, Nicholas G, McKinnell I, Kemp CF, Sharma M, Kambadur R, Patel K (2004) Follistatin complexes myostatin and antagonises myostatin-mediated inhibition of myogenesis. Dev Biol 270:19–30. doi:10.1016/j.ydbio.2004.01.046
Bonetto A, Penna F, Minero VG, Reffo P, Costamagna D, Bonelli G, Baccino FM, Costelli P (2011) Glutamine prevents myostatin hyperexpression and protein hypercatabolism induced in C2C12 myotubes by tumor necrosis factor-α. Amino Acids 40:585–594. doi:10.1007/s00726-010-0683-3
Campbell JW, Vorhaben JE (1976) Avian mitochondrial glutamine metabolism. J Biol Chem 251:781–786
Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibé B, Bouix J, Caiment F, Elsen J-M, Eychenne F, Larzul C, Laville E, Meish F, Milenkovic D, Tobin J, Charlier C, Georges M (2006) A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet 38:813–818. doi:10.1038/ng1810
Dasarathy S, Dodig M, Muc SM, Kalhan SC, McCullough AJ (2004) Skeletal muscle atrophy is associated with an increased expression of myostatin and impaired satellite cell function in the portacaval anastamosis rat. Am J Physiol Gastrointest Liver Physiol 287:G1124–G1130. doi:10.1152/ajpgi.00202.2004
Dasarathy S, Muc S, Runkana A, Mullen KD, Kaminsky-Russ K, McCullough AJ (2011) Alteration in body composition in the portacaval anastamosis rat is mediated by increased expression of myostatin. Am J Physiol Gastrointest Liver Physiol 301:G731–G738. doi:10.1152/ajpgi.00161.2011
Dejong CHC, Deutz NEP, Soeters PB (1994) Muscle ammonia and glutamine exchange during chronic liver insufficiency in the rat. J Hepatol 21:299–307. doi:10.1016/S0168-8278(05)80305-8
García PS, Cabbabe A, Kambadur R, Nicholas G, Csete M (2010) Elevated myostatin levels in patients with liver disease: a potential contributor to skeletal muscle wasting. Anesth Analg 111:707–709. doi:10.1213/ANE.0b013e3181eac1c9
Grobet L, Royo Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Ménissier F, Massabanda J, Fries R, Hanset R, Georges M (1997) A deletion in the bovine myostatin gene causes the double–muscled phenotype in cattle. Nat Genet 17:71–74. doi:10.1038/ng0997-71
Han HQ, Mitch WE (2011) Targeting the myostatin signaling pathway to treat muscle wasting diseases. Curr Opin Support Palliat Care 5:334–341. doi:10.1097/SPC.0b013e32834bddf9
Hartley RS, Bandman E, Yablonka-Reuveni Z (1992) Skeletal muscle satellite cells appear during late chicken embryogenesis. Dev Biol 153:206–216. doi:10.1016/0012-1606(92)90106-Q
He Y, Hakvoort TBM, Eleonore Köhler S, Vermeulen JLM, De Waart DR, De Theije C, Ten Have GAM, Van Eijk HMH, Kunne C, Labruyere WT, Houten SM, Sokolovic M, Ruijter JM, Deutz NEP, Lamers WH (2010) Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification. J Biol Chem 285:9516–9524. doi:10.1074/jbc.M109.092429
Ju C-R, Chen R-C (2012) Serum myostatin levels and skeletal muscle wasting in chronic obstructive pulmonary disease. Respir Med 106:102–108. doi:10.1016/j.rmed.2011.07.016
Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R (2002) Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 277:49831–49840. doi:10.1074/jbc.M204291200
Lee S (2004) Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol 20:61–86. doi:10.1146/cellbio.2004.20.issue-1
Lee SJ, McPherron AC (2001) Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A 98:9306–9311. doi:10.1073/pnas.151270098
Maier A (1993) Development of chicken intrafusal muscle fibers. Cell Tissue Res 274:383–391. doi:10.1007/BF00318757
McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83–90
McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci U S A 94:12457–12461. doi:10.1073/pnas.94.23.12457
Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, Parker HG, Ostrander EA (2007) A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genet 3:e79. doi:10.1371/journal.pgen.0030079
Murphy RA, Mourtzakis M, Chu QS, Reiman T, Mazurak VC (2010) Skeletal muscle depletion is associated with reduced plasma (n-3) fatty acids in non-small cell lung cancer patients. J Nutr 140:1602–1606. doi:10.3945/jn.110.123521.antineoplastic
Olde Damink SWM, Deutz NEP, Dejong CHC, Soeters PB, Jalan R (2002) Interorgan ammonia metabolism in liver failure. Neurochem Int 41:177–188. doi:10.1016/S0197-0186(02)00040-2
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. doi:10.1093/nar/29.9.e45
Qiu J, Thapaliya S, Runkana A, Yang Y, Tsien C, Mohan ML, Narayanan A, Eghtesad B, Mozdziak PE, McDonald C, Stark GR, Welle S, Naga Prasad SV, Dasarathy S (2013) Hyperammonemia in cirrhosis induces transcriptional regulation of myostatin by an NF-κB-mediated mechanism. Proc Natl Acad Sci U S A 110:18162–18167. doi:10.1073/pnas.1317049110
Qiu J, Tsien C, Thapalaya S, Narayanan A, Weihl CC, Ching JK, Eghtesad B, Singh K, Fu X, Dubyak G, McDonald C, Almasan A, Hazen SL, Naga Prasad SV, Dasarathy S (2012) Hyperammonemia-mediated autophagy in skeletal muscle contributes to sarcopenia of cirrhosis. Am J Physiol Endocrinol Metab 303:E983–E993. doi:10.1152/ajpendo.00183.2012
Salehian B, Mahabadi V, Bilas J, Taylor WE, Ma K (2006) The effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression. Metabolism 55:1239–1247. doi:10.1016/j.metabol.2006.05.009
Stern RA, Ashwell CM, Dasarathy S, Mozdziak PE (2015) The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos. Animal 9:992–999. doi:10.1017/S1751731115000117
Stockdale FE, Raman N, Baden H (1981) Myosin light chains and the developmental origin of fast muscle. Proc Natl Acad Sci U S A 78:931–935
Taylor WE, Bhasin S, Artaza J, Byhower F, Azam M, Willard DH, Kull FC, Gonzalez-Cadavid N (2001) Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am J Physiol - Endocrinol Metab 280:E221–E228. doi:10.1210/jc.82.6.1659
Thapaliya S, Runkana A, McMullen MR, Nagy LE, McDonald C, Naga Prasad SV, Dasarathy S (2014) Alcohol-induced autophagy contributes to loss in skeletal muscle mass. Autophagy 10:677–690. doi:10.4161/auto.27918
Zhou X, Wang JL, Lu J, Song Y, Kwak KS, Jiao Q, Rosenfeld R, Chen Q, Boone T, Simonet WS, Lacey DL, Goldberg AL, Han HQ (2010) Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 142:531–543. doi:10.1016/j.cell.2010.07.011
Acknowledgments
The authors would like to express their appreciation to Jonathan Duggins (North Carolina State University, Raleigh, NC) for assistance with statistical analysis.
Research funding and support was provided in part by NIH R01DK083414, R21AA022742, and P50AA02433-01-8236 (SD); and by NIH R01DK083414, USDA Regional Project 1184, and the North Carolina Agricultural Foundation (PEM).
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Stern, R.A., Dasarathy, S. & Mozdziak, P.E. Ammonia elicits a different myogenic response in avian and murine myotubes. In Vitro Cell.Dev.Biol.-Animal 53, 99–110 (2017). https://doi.org/10.1007/s11626-016-0088-z
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DOI: https://doi.org/10.1007/s11626-016-0088-z