Gene-panel and whole-exome analyses are now standard methodologies for mutation detection in Mendelian disease. However, the diagnostic yield achieved is at best 50%, leaving the genetic basis for ...disease unsolved in many individuals. New approaches are thus needed to narrow the diagnostic gap. Whole-genome sequencing is one potential strategy, but it currently has variant-interpretation challenges, particularly for non-coding changes. In this study we focus on transcriptome analysis, specifically total RNA sequencing (RNA-seq), by using monogenetic neuromuscular disorders as proof of principle. We examined a cohort of 25 exome and/or panel “negative” cases and provided genetic resolution in 36% (9/25). Causative mutations were identified in coding and non-coding exons, as well as in intronic regions, and the mutational pathomechanisms included transcriptional repression, exon skipping, and intron inclusion. We address a key barrier of transcriptome-based diagnostics: the need for source material with disease-representative expression patterns. We establish that blood-based RNA-seq is not adequate for neuromuscular diagnostics, whereas myotubes generated by transdifferentiation from an individual’s fibroblasts accurately reflect the muscle transcriptome and faithfully reveal disease-causing mutations. Our work confirms that RNA-seq can greatly improve diagnostic yield in genetically unresolved cases of Mendelian disease, defines strengths and challenges of the technology, and demonstrates the suitability of cell models for RNA-based diagnostics. Our data set the stage for development of RNA-seq as a powerful clinical diagnostic tool that can be applied to the large population of individuals with undiagnosed, rare diseases and provide a framework for establishing minimally invasive strategies for doing so.
Autophagy is a lysosomal degradation pathway with a role in the turnover of cell components via self-digestion. Over the past decade, it has been recognised as an essential process to maintain ...cellular and energy homeostasis. Nevertheless, little attention has been paid to this process in farm animals. In pigs, the role of autophagy in skeletal muscle homeostasis and more specifically on the formation of multinucleated muscle fibres needs to be determined. Primary culture of satellite cells, the resident muscle stem cells, is an appropriate model to investigate macroautophagy (hereafter autophagy), the main autophagy process. The objective of the current study was to evaluate tools to monitor autophagy in this cell model and to specify the role of autophagy on cell differentiation. Samples of longissimus muscle were collected from 3- to 4-day-old piglets. After isolation, satellite cells were plated in growth medium, allowed to proliferate up to 80% confluence and then placed in an appropriate culture medium to differentiate into myotubes. Cells were explored from day 0 to day 3 of differentiation. Autophagy-related proteins and Adenosine Mono Phosphate-activated protein kinase (AMPK), a major sensor for cell energy, were detected by Western blotting. Expression of genes related to autophagy were also quantified by qPCR. The Microtubule-associated protein 1 light-chain 3β forms ratio increased during cell differentiation whereas phosphatidylinositol 3-kinase and sequestosome 1 proteins decreased significantly. Mitochondrial protein expression also decreased significantly with satellite cell differentiation. Then, cell treatment with an inhibitor of autophagy flux, Bafilomycin A1, confirmed that autophagy was activated during the conversion of myoblasts into myotubes along with AMPK activation in our satellite cell culture model. In conclusion, we provided tools for porcine autophagy investigation in tissues or cells and demonstrated that basal autophagy and energy metabolism are concomitantly modulated during porcine myogenesis in vitro.
Exercise induces expression of the myokine irisin, which is known to promote browning of white adipose tissue and has been shown to mediate beneficial effects following exercise. Here we show that ...irisin induces expression of a number of pro-myogenic and exercise response genes in myotubes. Irisin increases myogenic differentiation and myoblast fusion via activation of IL6 signaling. Injection of irisin in mice induces significant hypertrophy and enhances grip strength of uninjured muscle. Following skeletal muscle injury, irisin injection improves regeneration and induces hypertrophy. The effects of irisin on hypertrophy are due to activation of satellite cells and enhanced protein synthesis. In addition, irisin injection rescues loss of skeletal muscle mass following denervation by enhancing satellite cell activation and reducing protein degradation. These data suggest that irisin functions as a pro-myogenic factor in mice.
Cancer‐associated cachexia is a wasting syndrome that results in dramatic loss of whole‐body weight, predominantly due to loss of skeletal muscle mass. It has been established that cachexia inducing ...cancer cells secrete proteins and extracellular vesicles (EVs) that can induce muscle atrophy. Though several studies examined these cancer‐cell derived factors, targeting some of these components have shown little or no clinical benefit. To develop new therapies, understanding of the dysregulated proteins and signaling pathways that regulate catabolic gene expression during muscle wasting is essential. Here, we sought to examine the effect of conditioned media (CM) that contain secreted factors and EVs from cachexia inducing C26 colon cancer cells on C2C12 myotubes using mass spectrometry‐based label‐free quantitative proteomics. We identified significant changes in the protein profile of C2C12 cells upon exposure to C26‐derived CM. Functional enrichment analysis revealed enrichment of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM treated myotubes. Furthermore, strong downregulation in muscle structural integrity and development and/or regenerative pathways were observed. Together, these enriched proteins in atrophied muscle could be utilized as potential muscle wasting markers and the dysregulated biological processes could be employed for therapeutic benefit in cancer‐induced muscle wasting.
As a posttranscriptional regulatory factor, microRNA (miRNA) plays an important role in the formation of myotubes. However, little is known about the mechanism of miRNA regulating myotube ...morphogenesis. Here, we aimed to characterize the function of miR‐455‐5p in myotube morphogenesis by inducing differentiation in C2C12 myoblasts containing murine Mylip fragments with the miR‐455‐5p target sequence. We found that miR‐455‐5p overexpression promoted the differentiation and hypertrophy of myotubes, while miR‐455‐5p inhibition led to the failure of myotube differentiation and formation of short myotubes. Furthermore, we demonstrated that miR‐455‐5p directly targeted the Mylip 3’‐untranslated region, which plays a key role in monitoring myotube morphogenesis. Interestingly, the expression and function of Mylip were opposite to those of miR‐455‐5p during myogenesis. Our data uncovered novel miR‐455‐5p targets and established a functional link between Mylip and myotube morphogenesis. Understanding the involvement of Mylip in myotube morphogenesis provides insight into the function of the gene regulatory network.
miR‐455‐5p overexpression promoted the differentiation and hypertrophy of myotubes, while miR‐455‐5p inhibition led to the failure of myotube differentiation and formation of short myotubes. miR‐455‐5p directly targeted the Mylip 3’‐untranslated region, which plays a key role in monitoring myotube morphogenesis.
Metallothionein (MT) 1 and 2 are ubiquitously expressed cysteine-rich, low molecular weight proteins. MT expression is upregulated in skeletal muscle during aging. MTs also play role in multiple ...types of skeletal muscle atrophy. Meanwhile, it has been reported that MT1 and MT2 gene deficiency increases myogenesis in MT knockout (MTKO) mice. However, little is known about the effect of MTs on muscle formation and atrophy. In this study, we investigated the effect of MT1 and MT2 gene knock-out using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system in an in vitro skeletal muscle differentiation model (C2C12 cell line). MT deficiency promoted myogenic differentiation and myotube formation in C2C12 cells. Muscle-specific transcription factors MyoD and myogenin were found to be upregulated at the late stage of myotube differentiation in MTKO cells. Furthermore, the fast-twitch myosin heavy chain (MyHC) protein expression was similar in MTKO and mock-transfected myotubes, but slow-MyHC expression was higher in MTKO cells than in mock cells. The MT gene deletion did not affect the number of fast MyHC-positive myotubes but increased the number of slow MyHC-positive myotubes. Treatment with the antioxidant N-acetylcysteine (NAC) inhibited the increase in the number of slow MyHC-positive myotubes as well as slow-MyHC expression in MTKO cells. In contrast, NAC treatment did not alter the number of fast MyHC-positive myotubes or the expression of fast-MyHC in MTKO cells. These results suggest that the antioxidant effects of MTs may be involved in slow-twitch myofiber formation in skeletal muscle.
Abstract We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a ...microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3–5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo . Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application.
This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice ...significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.
The aim of this study was to investigate the effect and mechanism of L-theanine (LT) on muscle fiber type transformation in C2C12 myotubes. Our data showed that LT exhibited significantly higher slow ...oxidative muscle fiber expression and lower glycolytic fibers expression. In addition, LT significantly increased the activities of malate dehydrogenase (MDH) and succinic dehydrogenase (SDH), and decreased lactate dehydrogenase (LDH) activity, the calcineurin (CaN) activity and the protein expressions of nuclear factor of activated T cell 1 (NFATc1), prospero-related homeobox1 (prox1), and calcineurin A (CnA) were significantly increased. However, inhibition of CaN activity by cyclosporine A (CsA) abolished LT-induced increase of slow oxidative muscle fiber expression and decrease of glycolytic fibers expression. Moreover, inhibition of prox1 expression by prox1-siRNA disrupted LT-induced activation of CaN signaling pathway and muscle fiber type transformation. Taken together, these results indicated that LT could promote skeletal muscle fiber type transformation from type II to type I via activation of prox1/CaN signaling pathway.
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