Generation and Use of Cultured Human Primary Myotubes

Cornall, Lauren M; Hryciw, Deanne H.; Mathai, Michael L.; McAinch, Andrew J.

doi:10.5772/33534

Cited by 10 publications

(7 citation statements)

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“…We next tested the hypothesis that, as in C2C12 cells, miR-711 may be a mediator of the anti-inflammatory action of ApN in C and DMD myotubes. To mimic the inflammatory microenvironment which prevails in DMD, we challenged the myotubes by an inflammatory stimulus (TNFα/interferon gamma (IFNγ)) [ 18 ], as already described [ 19 ] (Fig. 10b, c ).…”

Section: Resultsmentioning

confidence: 99%

Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

et al. 2018

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BackgroundThe hormone adiponectin (ApN) exerts powerful anti-inflammatory effects on skeletal muscle and can reverse devastating myopathies, like Duchenne muscular dystrophy (DMD), where inflammation exacerbates disease progression. The NLRP3 inflammasome plays a key role in the inflammation process, and its aberrant activation leads to several inflammatory or immune diseases. Here we investigated the expression of the NLRP inflammasome in skeletal muscle and its contribution to DMD.ResultsWe find that NLRP3 is expressed in skeletal muscle and show that ApN downregulates NLRP3 via its anti-inflammatory mediator, miR-711. This repression occurs both in vitro in C2C12 myotubes and in vivo after either local (via muscle electrotransfer) or systemic (by using transgenic mice) ApN supplementation. To explore the role of the NLRP3 inflammasome in a murine model of DMD, we crossed mdx mice with Nlrp3-knockout mice. In mdx mice, all components of the inflammasome were upregulated in muscle, and the complex was overactivated. By contrast, in mdx mice lacking Nlrp3, there was a reduction in caspase-1 activation, inflammation and oxidative stress in dystrophic muscle, and these mice showed higher global muscle force/endurance than regular mdx mice as well as decreased muscle damage. To investigate the relevance of NLPR3 regulation in a human disease context, we characterized NLRP3 expression in primary cultures of myotubes from DMD subjects and found a threefold increase compared to control subjects. This overexpression was attenuated by ApN or miR-711 mimic treatments.ConclusionsThe NLRP3 inflammasome plays a key pathogenic role in DMD and muscle inflammation, thereby opening new therapeutic perspectives for these and other related disorders.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0501-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

et al. 2018

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“…Rectus abdominis muscle samples, collected during gastric band insertion, were used to establish myotube cultures (obese or obese and type 2 diabetic (obese diabetic); n = 8/group) (Cornall et al, 2012;McAinch et al, 2007). Biochemical analysis of fasting blood samples obtained within 3 months of surgery were performed by Melbourne Pathology (Abbotsford, Australia) ( Table 2).…”

Section: Human Primary Skeletal Muscle Myotube Donorsmentioning

confidence: 99%

GPR119 regulates genetic markers of fatty acid oxidation in cultured skeletal muscle myotubes

Cornall

Mathai

Hryciw

et al. 2013

Molecular and Cellular Endocrinology

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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 – 5 ]. Myoblasts often show limited efficiency of differentiation and fusion.…”

Section: Introductionmentioning

confidence: 99%

“…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 [ 6 ]. 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 [ 8 ], have been reported to facilitate, with limited success, myoblast differentiation and fusion, and myotube attachment.…”

Section: Introductionmentioning

confidence: 99%

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Extended 2D myotube culture recapitulates postnatal fibre type plasticity

et al. 2015

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BackgroundThe 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.ResultsPrimary 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0069-1) contains supplementary material, which is available to authorized users.

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Generation and Use of Cultured Human Primary Myotubes

Cited by 10 publications

References 97 publications

Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

GPR119 regulates genetic markers of fatty acid oxidation in cultured skeletal muscle myotubes

Extended 2D myotube culture recapitulates postnatal fibre type plasticity

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