Extended 2D myotube culture recapitulates postnatal fibre type plasticity

BMC Cell Biology

Kin-Chow Chang¹  Suresh V. Kuchipudi¹  Leah Goulding¹  Sujith Sebastian¹ 

[1] School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington LE12 5RD, UK

关键词: Oxidative;    Fast glycolytic;    Coordinated expression;    Linc-MYH;    lincRNA;    Six1;    Myotubes;    Porcine;    Fibre type switching;    Contraction;    Fusion;    Differentiation;    Myosin heavy chain;    Postnatal;   

Others  :  1230192
DOI  :  10.1186/s12860-015-0069-1

 received in 2015-07-28, accepted in 2015-09-08,  发布年份 2015

【 摘 要 】

Background

The traditional problems of performing skeletal muscle cell cultures derived from mammalian or avian species are limited myotube differentiation, and transient myotube persistence which greatly restricts the ability of myotubes to undergo phenotypic maturation. We report here on a major technical breakthrough in the establishment of a simple and effective method of extended porcine myotube cultures (beyond 50 days) in two-dimension (2D) that recapitulates key features of postnatal fibre types.

Results

Primary porcine muscle satellite cells (myoblasts) were isolated from the longissimus dorsi of 4 to 6 weeks old pigs for 2D cultures to optimise myotube formation, improve surface adherence and characterise myotube maturation. Over 95 % of isolated cells were myoblasts as evidenced by the expression of Pax3 and Pax7. Our relatively simple approach, based on modifications of existing surface coating reagents (Maxgel), and of proliferation and differentiation (Ultroser G) media, typically achieved by 5 days of differentiation fusion index of around 80 % manifested in an abundance of discrete myosin heavy chain (MyHC) slow and fast myotubes. There was little deterioration in myotube viability over 50 days, and the efficiency of myotube formation was maintained over seven myoblast passages. Regular spontaneous contractions of myotubes were frequently observed throughout culture. Myotubes in extended cultures were able to undergo phenotypic adaptation in response to different culture media, including the adoption of a dominant postnatal phenotype of fast-glycolytic MyHC 2x and 2b expression by about day 20 of differentiation. Furthermore, fast-glycolytic myotubes coincided with enhanced expression of the putative porcine long intergenic non-coding RNA (linc-MYH), which has recently been shown to be a key coordinator of MyHC 2b expression in vivo.

Conclusions

Our revised culture protocol allows the efficient differentiation and fusion of porcine myoblasts into myotubes and their prolonged adherence to the culture surface. Furthermore, we are able to recapitulate in 2D the maturation process of myotubes to resemble postnatal fibre types which represent a major technical advance in opening access to the in vitro study of coordinated postnatal muscle gene expression.

【 授权许可】

   
2015 Sebastian et al.

【 图 表 】

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【 参考文献 】

• [1]Goldring K, Partridge T, Watt D. Muscle stem cells. J Pathol. 2002; 197:457-467.
• [2]Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol Rev. 2011; 91:1447-1531.
• [3]Cornall L, Hryciw D, Mathai M, McAinch A. Generation and use of cultured human primary myotubes. Muscle biopsy. 1st ed. Sundaram C, editor. InTech, Croatia; 2012.
• [4]Owens J, Moreira K, Bain G. Characterization of primary human skeletal muscle cells from multiple commercial sources. In Vitro Cell Dev Biol. 2013; 49:695-705.
• [5]Partridge TA. Tissue culture of skeletal muscle. Basic cell culture protocols edition. In: Methods in molecular biology. Pollard JW, Walker JM, editors. Humana Press Inc, Totowa, NJ; 1997: p.131-144.
• [6]Kubis HP, Haller EA, Wetzel P, Gros G. Adult fast myosin pattern and Ca 2+ -induced slow myosin pattern in primary skeletal muscle culture. Proc Natl Acad Sci U S A. 1997; 94:4205-4210.
• [7]Abdul-Hussein S, van der Vwn PFM, Tajsharghi H. Expression profiles of muscle disease-asociated genes and their isoforms during differentiation of cultured human skeletal muscle cells. BMC Musculoskelet Disord. 2012; 13:262. BioMed Central Full Text
• [8]Guex AG, Kocher FM, Fortunato G, Körner E, Hegemann D, Carrel TP et al.. Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development. Acta Biomater. 2012; 8:1481-1489.
• [9]Sakar MS, Neal D, Boudou T, Borochin MA, Li Y, Weiss R et al.. Formation and optogenetic control of engineered 3D skeletal muscle bioactuators. Lab Chip. 2012; 12:4976-4985.
• [10]Das M, Wilson K, Molnar P, Hickman JJ. Differentiation of skeletal muscle and integration of myotubes with silicon microstrutures using serum-free medium and a synthetic silane substrate. Nat Protoc. 2007; 2:1795-1801.
• [11]Smith AST, Passey S, Greensmith L, Mudera V, Lewis MP. Characterization and optimization of a simple repeatable system for the long term in vitro culture of aligned myotubes in 3D. J Cell Biochem. 2012; 113:1044-1053.
• [12]Baqureo-Perez B, Kuchipudi SV, Nelli RK, Chang KC. A simplified but robust method for the isolation of avian and mammalian muscle satellite cells. BMC Cell Biol. 2012; 13:16. BioMed Central Full Text
• [13]da Costa N, Beuzen N, Johnston I, McGillivray C, Sun YM, Chang KC. The 5′-end of the porcine perinatal myosin heavy chain gene shows alternative splicing and is clustered with repeat elements. J Muscle Res Cell Motil. 2000; 21:183-188.
• [14]da Costa N, Blackley R, Alzuherri H, Chang KC. Quantifying the temporo-spatial expression of porcine postnatal skeletal myosin heavy chain genes. J Histochem Cytochem. 2002; 50:353-364.
• [15]Sun YM, da Costa N, Birrell R, Archibald AL, Alzuherri H, Chang KC. Molecular and quantitative characterisation of the porcine embryonic myosin heavy chain gene. J Muscle Res Cell Motil. 2001; 22:317-327.
• [16]von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA. Pax7 is critical for the normal function of satellite cells in adult skeletal muscle. Proc Natl Acad Sci U S A. 2013; 110:16474-16479.
• [17]Young AP, Wagers AJ. Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors. J Cell Sci. 2010; 123:2632-2639.
• [18]Doumit ME, Merkel RA. Conditions for isolation and culture of porcine myogenic satellite cells. Tissue Cell. 1992; 24:253-262.
• [19]Zhu H, Park S, Scheffler JM, Kuang S, Grant AL, Gerrard DE. Porcine satellite cells are restricted to a phenotype resembling their muscle of origin. J Anim Sci. 2013; 91:4684-4691.
• [20]Sakakibara I, Santolini M, Ferry A, Hakim V, Maire P. Six homeoproteins and a linc-RNA at the fast MYH locus lock fast myofiber terminal phenotype. PLoS Genet. 2014; 10:e1004386.
• [21]Grifone R, Laclef C, Spitz F, Lopez S, Demignon J, Guidotti JE et al.. Six1 and Eya1 expression can reprogram adult muscle from the slow-twitch phenotype into the fast-twitch phenotype. Mol Cell Biol. 2004; 24:6253-6267.
• [22]Hetzler KL, Collins BC, Shanely RA, Sue H, Kostek MC. The homeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle. Acta Physiol. 2014; 210:415-428.
• [23]Ulitsky I, Bartel DP. lincRNAs: genomics, evolution and mechanisms. Cell. 2013; 154:26-46.
• [24]Glass DJ. Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell Biol. 2005; 37:1974-1984.
• [25]Glass DJ. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol. 2003; 5:87-90.
• [26]Chang KC. Key signalling factors and pathways in the molecular determination of skeletal muscle phenotype. Animal. 2007; 1:681-698.
• [27]Chang KC, da Costa N, Blackley R, Southwood O, Evans G, Plastow G et al.. Relationships of myosin heavy chain fibre types to meat quality traits in traditional and modern pigs. Meat Sci. 2003; 64:93-103.
• [28]Zierath JR, Hawley JA. Skeletal muscle fiber type: influence on contractile and metabolic properties. PLoS Biol. 2004; 2:e348.
• [29]Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006; 444:840-846.
• [30]Ehlers ML, Celona B, Black BL. NFATc1 controls skeletal muscle fiber type and is a negative regulator of MyoD activity. Cell Rep. 2014; 8:1639-1648.
• [31]An D, Lessard SJ, Toyoda T, Lee MY, Koh HJ, Qi L et al.. Overexpression of TRB3 in muscle alters muscle fiber type and improves exercise capacity in mice. Am J Physiol Regul Integr Comp Physiol. 2014; 306:R925-R933.
• [32]Yeung F, Chung E, Guess MG, Bell ML, Leinwand LA. Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos. Nucleic Acids Res. 2012; 40:7303-7318.
• [33]Sharma M, Juvvuna PK, Kukreti H, McFarlene C. Mega roles of microRNAs in regulation of skeletal muscle health and disease. Front Physiol. 2014; 5:239.
• [34]Pandorf CE, Jiang W, Qin AX, Bodell PW, Baldwin KM, Haddad F. Regulation of an antisense RNA with transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2012; 302:R854-R867.
• [35]Wu W, Ren Z, Zhang L, Liu Y, Li H, Xiong Y. Overexpression of Six1 gene supresses proliferation and enhances expression of fast-type muscle genes in C2C12 myoblasts. Mol Cell Biochem. 2013; 380:23-32.
• [36]Russell SD, Cambon N, Nadal-Ginard B, Whalen RG. Thyroid hormone induces a nerve-independent precocious expression of fast myosin heavy chain mRNA in rat hindlimb skeletal muscle. J Biol Chem. 1988; 263:6370-6374.