Extended 2D myotube culture recapitulates postnatal fibre type plasticity
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.
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.
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.
KeywordsPostnatal Myosin heavy chain Differentiation Fusion Contraction Fibre type switching Porcine Myotubes Six1 lincRNA Linc-MYH Coordinated expression Fast glycolytic Oxidative
Myosin heavy chain
Long intergenic non-coding
Maxgel coating mixture
Green fluorescence protein
Skeletal muscle satellite cells as progenitor cells of multinucleated muscle fibres are one of the earliest recognised cells with stem cell-like properties . Isolated satellite cells in culture are activated to undergo rapid proliferation as myoblasts which then differentiate and fuse to form post-mitotic myotubes as early muscle fibres. Muscle fibres in vivo are highly adapted to undergo phenotypic changes in size (hypertrophy or atrophy) and metabolic capacity (ranging from highly oxidative to enhanced glycolytic fibre type) to meet changes in physiological demands or in response to disease . Newly formed myotubes undergo a maturation process of hypertrophy and metabolic remodelling during which the initial dominant embryonic and perinatal (neonatal) myosin heavy chain (MyHC) isoforms are replaced with the four main postnatal MyHC isoforms of slow, 2A, 2X and 2B, each sequentially corresponding to a fibre type of increasing glycolytic and decreasing oxidative capacity.
Although muscle satellite cells of different host species have been used in adherent two-dimension (2D) cultures for many years, to date such cultures can primarily only recapitulate the early stages of myogenesis and myotube formation [3, 4, 5]. Myoblasts often show limited efficiency of differentiation and fusion. Furthermore, as myotubes in cultures are prone to rapid loss, presumably through spontaneous contractions, their replacement by more newly-formed myotubes perpetuates an immature phenotype in culture . As a consequence to such technical limitations, muscle culture experiments are typically performed on immature myotubes over a narrow window of 3 to 7 days of differentiation [3, 7]. A variety of culture media and extracellular matrices, including the use of electrospun polycaprolactone polymer coating , have been reported to facilitate, with limited success, myoblast differentiation and fusion, and myotube attachment. Other attempts to extend the transient persistence of myotubes included the use of three-dimension (3D) cultures of murine C2C12 muscle cells on silicon wafers , rat myoblasts in cantilever arrays , and primary rabbit muscle cells on gelatin microbeads in suspension that allowed prolonged myotube adherence and fibre maturation for up to 5 weeks with the expression of adult fast MyHCs (2A, 2X and/or 2B isoform) . The use of 3D collagen mould in a chamber slide also improved primary rat myotube formation and reduced loss over a 3-week period . However, such culture methods and other similar approaches have limited practicalities requiring specialised culture platforms with reduced flexibility to conduct routine cellular manipulations.
We report on a major technical breakthrough in the long term culture of myotubes. We developed a simple and highly reproducible method for the extended 2D culture of myotubes based on the strategic use of primary porcine myoblasts; the pig is an excellent model species, owing to its physiological similarity to human and relative availability, and its own importance as target species. Our method, based on modified use of surface coating reagents (Maxgel), and of proliferation and differentiation (Ultroser G) media, allowed efficient differentiation and fusion of myoblasts into myotubes that remained adherent to the culture surface for over 7 weeks of differentiation. To our knowledge, we are able for the first time to recapitulate in vitro the maturation process of myotubes in 2D to resemble postnatal fibre types which is a major technical advance in the ability to study phenotype plasticity.
Culture of porcine myoblasts and myotubes
Porcine muscle satellite cells (myoblasts) were isolated from skeletal muscles (longissimus dorsi) of 4 to 6 weeks old commercial Large White-cross pigs as previously described . This work was approved by the School of Veterinary Medicine and Science ethical committee. Pigs were humanely euthanased according to Schedule 1 to the Animals (Scientific Procedures) Act 1986. All myoblasts and myotubes were grown on optimised coated surfaces. Into each well of a 12-well plate was applied 400 μl of 0.22 μm filter sterilised Maxgel coating mixture (MC+), comprising a 1:1 mix ratio of 1 % Maxgel ECM solution (Sigma-Aldrich, E0282-1ML; 1 in a 100 dilution with Dulbecco’s modified Eagle’s medium [DMEM]) and 2 % rat type I collagen solution (Sigma Aldrich, C3867; 1 ml rat collagen solution in 49 ml phosphate buffered saline [PBS]), which was left to fully dry overnight in a cell culture cabinet and rinsed with PBS before use. For other size plates or flasks, the volume of MC+ used was proportionally scaled.
Newly harvested satellite cells were grown in proliferation medium (PM), comprising SKGM-2 medium (Lonza, CC-3245) with added 10 % heat inactivated fetal calf serum (FCS) (Invitrogen, 10500–064), 2 % chick embryo extract (EGG Tech, 60650) and 1 % penicillin-streptomycin (P/S) (Invitrogen 15140-122), in a 37 °C incubator with gas mixture of 5 % CO2 and 5 % O2, with complete replacement of PM every 2 days. Myoblasts were passaged once at a ratio of 1:3 in PM before freezing in a mixture of 50 % FCS, 10 % dimethylsulphoxide and 40 % PM, and storing in liquid nitrogen. Depending on seeding density of thawed myoblasts, 0.5 million cells in a T75 flask should reach 60 to 70 % confluence by 3-4 days of culture.
At around 80 to 90 % confluence, the cells were rinsed and replaced with differentiation medium 1 (DM1) comprising DMEM high glucose (Invitrogen, 41965–039) with 0.4 % Ultroser G (Pall Corporation, 15950–017) and 1 % P/S. Ultroser G is a proprietary serum replacement containing a cocktail of undisclosed growth factors. Extensive myotubes should form by 3 to 4 days of differentiation. For long-term maintenance of the myotubes, from day 5–7 of differentiation, 25 % volume of the original DM1 was replaced every third day with fresh DM1, or differentiation medium 2 (DM2, DMEM high glucose with 2 % horse serum [Gibco, 26050-088] and 1 % P/S). In this way, good myotube integrity was readily maintained for several weeks.
Measurement of mitochondrial activity in myotubes, as an indication of cell viability, was performed in a 96-well format with a CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit (Promega) according to the manufacturer’s instructions. The MTS kit is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate). MTS is bioreduced by cells into a formazan product that is soluble in cell culture medium. The conversion of MTS (measured at 490 nm absorbance) into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product is directly proportional to the number or activity of living cells in culture.
Real time PCR
RNA extraction from myoblasts and myotubes was performed with an RNeasy fibrous tissue mini kit (Qiagen). TaqMan real-time PCR was used to quantify the expression of six porcine MyHC gene isoforms (MyHC embryonic, MyHC perinatal, MyHC slow/I, MyHC 2a, MyHC2x and MyHC 2b) using primers and TaqMan probes as previously described [13, 14, 15]. Forward and reverse primers for the SYBR Green detection of putative porcine long intergenic non-coding (linc)-MYH (exon 5) are 5′-GAGGCTCGGGAAGGAATCC-3′ and 5′-TGCCCTCTGGTGGTAAAAGC-3′. Forward and reverse primers for porcine Six1 and Eya1 detection are 5′-GTTCAAGAACCGAAGGCAAC-3′ and 5′-CCCCTTCCAGAGGAGAGAGT-3′, and 5′-CAGCTCTCCATATCCAGCACATT-3′ and 5′-TTTGTGGACGGCGTCGTA-3′ respectively. A relative standard curve was used to quantify the expression of each gene normalised to its corresponding 18S rRNA expression.
Culture of primary porcine muscle cells
Enhanced myotube differentiation and sustained viability
Extended myotube culture displayed phenotypic plasticity and postnatal phenotype
Rising putative linc-MYH expression coincided with accumulation of MyHC 2x and 2b in myotubes
The usefulness of myotubes in cultures to examine muscle gene regulation or response to external stimuli has hitherto been limited by the immature state of myotubes derived from a range of mammalian (human, pig, rabbit, cow, horse, mouse and rat) [3, 4, 6, 7, 12, 18, 19] and avian (chicken and duck) [5, 12] species. Most published experiments on myotubes are performed within about a week of differentiation, at a stage when adult MyHCs (namely MyHC 2X and 2B) are largely lacking. The principal reasons for this predicament are limited efficiency in myotube formation and continual loss of newly formed myotubes. The ability to culture myotubes long term in 2D to resemble the postnatal phenotype is of high biomedical value. The most important biomedical or veterinary muscle conditions (such as muscle hypertrophic growth , muscle wasting or atrophy , changes in fibre type composition affecting muscle performance  or even meat quality , and obesity-related insulin resistance [28, 29]) all involve skeletal muscle in the postnatal fibre state. To have a suitable in vitro platform to examine such basic underlying changes is clearly advantageous. Until now such a platform has been elusive in that myotube differentiation and maturation were major limiting factors. The present paper reports on such a technical breakthrough that promotes myotube differentiation and accommodates spontaneous myotube contractions without detachment which would facilitate the in vitro study of all aspects of postnatal muscle fibre biology.
In the first weeks of postnatal development in rats and mice, there is typically progressive loss of embryonic and perinatal MyHCs and accumulation of MyHC 2A, 2X and 2B isoforms in designated fast fibre populations . Our extended porcine myotube cultures in DM1 showed similar changes where reduction in embryonic and perinatal MyHC expression was accompanied by dominant expression of MyHC 2x and 2b (Fig. 3a). Furthermore, the pattern of relative MyHC expression at day 21 and 28 of differentiation in DM1 showed resemblance to the MyHC expression profile of a 22 week old pig longissimus dorsi muscle (Fig. 4b) . The divergence in MyHC profiles between DM1 and DM2 is biologically significant as it demonstrated the expression plasticity of postnatal MyHC genes through the use of different culture media. In vivo, early postnatal changes in MyHC gene expression that lead to the formation of adult fibre types are dependent on the establishment of corresponding fast and slow motor units, load bearing after birth, and thyroid hormone surge in the case of fast MyHC induction . The ability of our myotubes to adopt fast or slow MyHC profile in the absence of innervation indicates that the choice of particular culture conditions is also an important phenotype determinant. DM1 myotube culture over several weeks recapitulated a fast-like postnatal pattern of MyHC expression. We have therefore established a 2D culture platform that is conducive to the study of coordinated gene changes that govern fibre type and associated phenotypic alterations.
Acquiring a fundamental understanding of preferential up- or down-regulation of specific MyHC isoforms in vitro could facilitate our ability to manipulate phenotypic changes in vivo for beneficial biomedical and veterinary outcomes. The present culture platform opens up a convenient controlled environment to investigate a range of mechanisms and factors that are involved in the coordinated expression of muscle gene isoforms, such as the roles of transcription factors like NFATc1 , microRNAs [31, 32, 33], and anti-sense  and linc  RNAs in the differential regulation of MyHC and other fibre type-specific genes. We can systematically interrogate the role or effectiveness of individual genes or compounds on coordinated MyHC isoform switching or myotube development. As an exemplification, we found that the fast glycolytic myotube phenotype of elevated MyHC 2x and 2b expression (under DM1 culture condition) closely mirrored the rising profile of putative porcine linc-MYH RNA expression but not with that of Six1 and Eya1. In muscle, on the other hand, the fast phenotype has been shown to correlate with the up-regulation of linc-MYH  and the presence or over-expression of Six1 [21, 22, 35]. Our finding of an inverse relationship between linc-MYH RNA and Six1 expression (Fig. 5) suggests that factors other than Six1 could be responsible for the induction of linc-MYH RNA in growing myotubes.
Another research opportunity is to examine in vitro the role of thyroid hormone and other growth factors in the programming of fast phenotype to dissect the qualitative and quantitative changes of orchestrated gene expression during the transition [34, 36]. Access to largely pure cultures of myotubes of a particular phenotype would also make the isolation of myonuclei and subsequent study of chromatin modifications much simpler than the use of whole muscle tissues. Finally, extended myotube cultures would allow us to better scrutinise whether there are intrinsic differences in the conferment of myotube phenotype between satellite cells of fast and slow muscles from the same animal or between animals of different ages.
In conclusion, we have made a major technical breakthrough to be able to culture well differentiated porcine myotubes in 2D over an extended period of at least 50 days. Furthermore, we showed that cultured myotubes could be made to adopt a fast adult phenotype of dominant MyHC 2x and 2b expression. For the first time, to our knowledge, we are able to recapitulate the maturation process of myotubes in vitro, opening new opportunities to study coordinated postnatal muscle gene expression.
SS was supported by a PhD studentship jointly funded by University of Nottingham and Zoetis Inc. LG was supported by a Biotechnology and Biological Sciences Research Council-Zoetis CASE PhD studentship. We thank Zoe Redshaw and Paul Loughna for assistance in Pax immunocytochemistry and primary myoblasts isolation.
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