Myostatin功能缺失性突变可导致梅山猪的II型肌纤维增多

doi:10.1016/S2095-3119(21)63669-9

摘要/Abstract

摘要：

Myostatin (MSTN)是骨骼肌生长发育的负调控因子。MSTN^-/-小鼠的骨骼肌明显肥大，同时II型肌纤维显著增多而I型肌纤维显著减少。然而，MSTN^-/-猪的肌纤维类型的变化情况，以及MSTN是调控肌纤维类型的机制目前尚不清楚，特别是在猪这样的大型动物中。本研究中，我们对实验室前期获得的MSTN^-/-猪的多个发育阶段的骨骼肌的肌纤维类型的变化情况进行了综合分析，结果发现与野生型猪相比，MSTN^-/-猪的骨骼肌总量和IIb型肌纤维的数量均显著上调(P<0.01)，而I型和Ⅱa型肌纤维的数量则显著下调(P<0.01)。此外，为了进一步探究MSTN在胚胎期对肌纤维类型的影响及调控机制，我们从前期获得的转录组差异表达基因数据中选取了部分调控肌纤维类型相关的基因（包括Myf5, Mef2d, MyoD 和 Six1），并通过荧光定量PCR方法检测其表达情况。我们发现肌纤维亚型的标志基因：Myh7、 Myh2、 Myh4 和Myh1 （分别对应I、IIa、IIb、IIx型肌纤维）在胚胎期65天的骨骼肌中已有表达，而且与野生型个体相比，MSTN^-/-个体的Myh7 表达量显著下调 (P<0.01)，Myh4 (P<0.001) 和Myh1 (P<0.05) 的表达量则显著上调；同时，MSTN^-/-个体骨骼肌中Myf5 (P<0.05), Mef2d (P<0.01) 和 Six1 (P<0.05)的表达量显著上调预示MSTN在胚胎发育早期即参与肌纤维类型定向发育的调控。由此可见，MSTN^-/-猪骨骼肌中的II型肌纤维增多，而且这一增多开始于胚胎期。该研究结果不仅能够为猪肉品质的改善提供有价值的参考依据，而且可为人类骨骼肌的发育和疾病治疗提供理论基础。

Abstract: Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. The skeletal muscle in MSTN–/– mice is significantly hypertrophied, with muscle fiber type II increasing significantly while muscle fiber type I decreasing. However, it is still not clear how the skeletal muscle types change in MSTN^–/– pigs, and how the mechanism for MSTN regulates fiber types, especially in large animals like pigs. This study conducted a comprehensive analysis of the composition of skeletal muscle fibers in MSTN^–/– pigs produced in our laboratory. It was observed that, compared with wild-type (WT) pigs, both the total mass of skeletal muscle and type IIb muscle fibers increased significantly (P<0.01), while the type I and type IIa muscle fibers decreased significantly (P<0.01), in MSTN^–/– Meishan pigs. In addition, to explore the influence of MSTN on muscle fiber type and its regulation mechanism in the embryonic stage, this study selected a few genes (Myf5, Mef2d, MyoD and Six1) associated with muscle fiber type and validated their expression by quantitative RT-PCR. Herein, it was found that Myh7, Myh2, Myh4 and Myh1 can be detected in the skeletal muscle of pigs at 65 days of gestation (dg). Compared with WT pigs, in MSTN^–/– Meishan pigs, Myh7 decreased significantly (P<0.01), while Myh4 (P<0.001) and Myh1 (P<0.05) increased significantly. Meanwhile, the increased expression of Myf5 (P<0.05), Mef2d (P<0.01) and Six1 (P<0.05) in MSTN^–/– Meishan pigs suggested that MSTN should regulate the directional development of muscle fiber types in the early stage of embryonic development. Thus, at the embryonic stage, the type II muscle fibers began to increase in MSTN^–/– pigs. These results can provide valuable information not only for pig meat quality improvement, but also for the study of human skeletal muscle development and disease treatment.

Key words: MSTN , Meishan pigs , muscle fiber type , muscle fiber development

. Myostatin功能缺失性突变可导致梅山猪的II型肌纤维增多 [J]. Journal of Integrative Agriculture, 2022, 21(1): 188-198.

QIAN Li-li, XIE Jing-yi, GAO Ting, CAI Chun-bo, JIANG Sheng-wang, BI Han-fang, XIE Shan-shan, CUI Wen-tao. Targeted myostatin loss-of-function mutation increases type II muscle fibers in Meishan pigs[J]. Journal of Integrative Agriculture, 2022, 21(1): 188-198.

参考文献

Bataille S, Chauveau P, Fouque D, Aparicio M, Koppe L. 2020. Myostatin and muscle atrophy during chronic kidney disease. Nephrol Dial Transplant. [2020-9-24]. https://doi.org/10.1093/ndt/gfaa129
Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J, Meilhac S, Montarras D, Rocancourt D, Relaix F. 2003. The formation of skeletal muscle: From somite to limb. Journal of Anatomy, 202, 59–68.
Buckingham M, Rigby P W J. 2014. Gene regulatory networks and transcriptional mechanisms that control myogenesis. Developmental Cell, 28, 225–238.
Cai C B, Qian L L, Jiang S W, Sun Y D, Wang Q Q, Ma D Z, Xiao G J, Li B, Xie S S, Gao T, Chen Y X, Liu J, An X R, Cui W T, Li K. 2017. Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs. Oncotarget, 8, 34911–34922.
Chikuni K, Tanabe R, Muroya S, Nakajima I. 2001. Differences in molecular structure among the porcine myosin heavy chain-2a, -2x, and -2b isoforms. Meat Science, 57, 311–317.
Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibe 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. Nature Genetics, 38, 813–818.
Davies A S. 1972. Postnatal changes in the histochemical fibre types of porcine skeletal muscle. Journal of Anatomy, 113, 213–240.
Elashry M I, Otto A, Matsakas A, El-Morsy S E, Patel K. 2009. Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice. Rejuvenation Research, 12, 269–281.
Girgenrath S, Song K, Whittemore L A. 2005. Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow- and fast-type skeletal muscle. Muscle & Nerve, 31, 34–40.
Groenen M A M, Archibald A L, Uenishi H, Tuggle C K, Takeuchi Y, Rothschild M F, Rogel-Gaillard C, Park C, Milan D, Megens H J, Li S T, Larkin D M, Kim H, Frantz L A F, Caccamo M, Ahn H, Aken B L, Anselmo A, Anthon C, Auvil L, et al. 2012. Analyses of pig genomes provide insight into porcine demography and evolution. Nature, 491, 393–398.
Hennebry A, Berry C, Siriett V, O’Callaghan P, Chau L, Watson T, Sharma M, Kambadur R. 2009. Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression. American Journal of Physiology (Cell Physiology), 296, C525–C534.
Hindi S M, Tajrishi M M, Kumar A. 2013. Signaling mechanisms in mammalian myoblast fusion. Science Signaling, 6, re2.
Horak V. 1995. Fibre type differentiation during postnatal development of miniature pig skeletal muscles. Reproduction Nutrition Development, 35, 725–736.
Kambadur R, Sharma M, Smith T P, Bass J J. 1997. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Research, 7, 910–916.
Karlsson A H, Klont R E, Fernandez X. 1999. Skeletal muscle fibres as factors for pork quality. Livestock Production Science, 60, 255–269.
Kim J H, Kim J H, Sutikno L A, Lee S B, Jin D H, Hong Y K, Kim Y S, Jin H J. 2019. Identification of the minimum region of flatfish myostatin propeptide (Pep45–65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals. PLoS ONE, 14, e0215298.
Krivickas L S, Walsh R, Amato A A. 2009. Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029. Muscle Nerve, 39, 3–9.
Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. 2002. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. Journal of Biological Chemistry, 277, 49831–49840.
Lee S J, McPherron A C. 2001. Regulation of myostatin activity and muscle growth. Proceedings of the National Academy of Sciences of the United States of America, 98, 9306–9311.
Lee Y S, Lee S J. 2013. Regulation of GDF-11 and myostatin activity by GASP-1 and GASP-2. Proceedings of the National Academy of Sciences of the United States of America, 110, E3713–E3722.
Li B, Xie S S, Cai C B, Qian L L, Jiang S W, Ma D Z, Xiao G J, Gao T, Yang J Z, Cui W T. 2017. MicroRNA-95 promotes myogenic differentiation by downregulation of aminoacyl-tRNA synthase complex-interacting multifunctional protein 2. Oncotarget, 8, 111356–111368.
Liu D, Qiao X R, Ge Z J, Shang Y, Li Y, Wang W D, Chen M H, Si S Y, Chen S Z. 2019. IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway. Skelet Muscle, 9, 8.
McPherron A C, Huynh T V, Lee S J. 2009. Redundancy of myostatin and growth/differentiation factor 11 function. BMC Developmental Biology, 9, 24.
McPherron A C, Lawler A M, Lee S J. 1997. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 387, 83–90.
McPherron A C, Lee S J. 1997. Double muscling in cattle due to mutations in the myostatin gene. Proceedings of the National Academy of Sciences of the United States of America, 94, 12457–12461.
McPherron A C, Lee S J. 2002. Suppression of body fat accumulation in myostatin-deficient mice. Journal of Clinical Investigation, 109, 595–601.
Molkentin J D, Black B L, Martin J F, Olson E N. 1995. Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell, 83, 1125–1136.
Mosher D S, Quignon P, Bustamante C D, Sutter N B, Mellersh C S, Parker H G, Ostrander E A. 2007. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genetics, 3, e79.
Niro C, Demignon J, Vincent S, Liu Y B, Giordani J, Sgarioto N, Favier M, Guillet-Deniau I, Blais A, Maire P. 2010. Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome. Developmental Biology, 338, 168–182.
NRC (National Research Council). 2012. Nutrient Requirements of Swine. 11th ed. National Academies Press, Washington, D.C.
Patruno M, Caliaro F, Maccatrozzo L, Sacchetto R, Martinello T, Toniolo L, Reggiani C, Mascarello F. 2008. Myostatin shows a specific expression pattern in pig skeletal and extraocular muscles during pre- and post-natal growth. Differentiation, 76, 168–181.
Prather R S, Hawley R J, Carter D B, Lai L, Greenstein J L. 2003. Transgenic swine for biomedicine and agriculture. Theriogenology, 59, 115–123.
Qian L L, Tang M X, Yang J Z, Wang Q Q, Cai C B, Jiang S W, Li H G, Jiang K, Gao P F, Ma D Z, Chen Y X, An X R, Li K, Cui W T. 2015. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Scientific Reports, 5, 14435.
Relaix F, Rocancourt D, Mansouri A, Buckingham M. 2005. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature, 435, 948–953.
Richard A F, Demignon J, Sakakibara I, Pujol J, Favier M, Strochlic L, Grand F L, Sgarioto N, Guernec A, Schmitt A, Cagnard N, Huang R, Legay C, Guillet-Deniau I, Maire P. 2011. Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression. Developmental Biology, 359, 303–320.
Saez L, Leinwand L A. 1986. Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle. Nucleic Acids Research, 14, 2951–2969.
Sakakibara I, Wurmser M, Dos Santos M, Santolini M, Ducommun S, Davaze R, Guernec A, Sakamoto K, Maire P. 2016. Six1 homeoprotein drives myofiber type IIA specialization in soleus muscle. Skeletal Muscle, 6, 30.
Schiaffino S, Reggiani C. 1996. Molecular diversity of myofibrillar proteins: Gene regulation and functional significance. Physiological Reviews, 76, 371–423.
Schiaffino S, Reggiani C. 2011. Fiber types in mammalian skeletal muscles. Physiological Reviews, 91, 1447–1531.
Schuelke M, Wagner K R, Stolz L E, Hubner C, Riebel T, Komen W, Braun T, Tobin J F, Lee S E. 2004. Myostatin mutation associated with gross muscle hypertrophy in a child. The New England Journal of Medicine, 350, 2682–2688.
Stavaux D, Art T, McEntee K, Reznick M, Lekeux P. 1994. Muscle fibre type and size, and muscle capillary density in young double-muscled blue Belgian cattle. Zentralbl Veterinarmed (A), 41, 229–236.
Swatland H J. 1973. Muscle growth in the fetal and neonatal pig. Journal of Animal Science, 37, 536–545.
Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. 2000. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. The Journal of Biological Chemistry, 275, 40235–40243.
Wagner K R, Fleckenstein J L, Amato A A, Barohn R J, Bushby K, Escolar D M, Flanigan K M, Pestronk A, Tawil R, Wolfe G I, Eagle M, Florence J M, King W M, Pandya S, Straub V, Juneau P, Meyers K, Csimma C, Araujo T, Allen R, et al. 2008. A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy. Annals Neurology, 63, 561–571.
Watts R, McAinch A J, Dixon J B, O’Brien P E, Cameron-Smith D. 2013. Increased smad signaling and reduced mrf expression in skeletal muscle from obese subjects. Obesity, 21, 525–528.
Wegner J, Albrecht E, Fiedler I, Teuscher F, Papstein H J, Ender K. 2000. Growth- and breed-related changes of muscle fiber characteristics in cattle. Journal of Animal Science, 78, 1485–1496.
Weiss A, Leinwand L A. 1996. The mammalian myosin heavy chain gene family. Annual Review of Cell and Developmental Biology, 12, 417–439.
Whyte J J, Prather R S. 2011. Genetic modifications of pigs for medicine and agriculture. Molecular Reproduction and Development, 78, 879–891.
Wigmore P M, Stickland N C. 1983. Muscle development in large and small pig fetuses. Journal of Anatomy, 137, 235–245.
Wydro R M, Nguyen H T, Gubits R M, Nadal-Ginard B. 1983. Characterization of sarcomeric myosin heavy chain genes. Journal of Biological Chemistry, 258, 670–678.
Xie S S, Li X, Qian L L, Cai C B, Xiao G J, Jiang S W, Li B, Gao T, Cui W T. 2019. An integrated analysis of mRNA and miRNA in skeletal muscle from myostatin-edited Meishan pigs. Genome, 62, 305–315.
Zhang G X, Zhang T, Wei Y, Ding F X, Zhang L, Wang J Y. 2015. Functional identification of an exon 1 substitution in the myostatin gene and its expression in breast and leg muscle of the Bian chicken. British Poultry Science, 56, 639–644.