Myotube Hypertrophy Research Articles

Testosterone Signals through mTOR and Androgen Receptor to Induce Muscle Hypertrophy

The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We addressed the question whether signal transduction pathways other than the androgen receptor (AR) are necessary to elicit hypertrophy in skeletal muscle myotubes. Cultured rat skeletal muscle myotubes were preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated by real time RT-PCR. Myotube size and sarcomerization were determined by confocal microscopy. Inhibition of AR was assessed by bicalutamide. Testosterone-induced myotube hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased α-actin mRNA and α-actin protein levels, with no changes in mRNA expression of atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min. This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition. Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as well as by AR inhibition. These results suggest a crosstalk between pathways involving fast intracellular signaling and the AR to explain testosterone-induced skeletal muscle hypertrophy.

O.15 Engineering a Wnt7a-based protein therapeutic for the treatment of muscular dystrophy

Satellite stem cells represent the endogenous source of regenerative potential in adult muscle after injury and in the context of chronic degenerative diseases. Therefore, enhancing the ability of satellite cells to regenerate muscle tissue represents a promising approach to therapeutic intervention following muscle injury, irrespective of etiology. Recent evidence from the laboratory of Fate Therapeutics’ scientific founder Michael Rudnicki has demonstrated that treating rodent models of muscular dystrophy with WNT7a confers significant functional improvement in muscle force through a combination of satellite stem cell expansion and direct myofiber hypertrophic activity. Wnt proteins are developmental morphogens that drive embryogenesis, tissue repair and regeneration. Despite their potential as regenerative molecules, Wnt proteins have not been previously developed as therapeutics; primarily due to challenges in scaled protein manufacture and formulation. Building on the academic observations of WNT7a activity, we have used rational protein design and structural engineering to produce and select WNT7a analogs with preferred pharmaceutical, manufacturing and formulation properties. These WNT7a analogs induced significant myotube hypertrophy in cultures from murine myoblasts or myoblasts from patients with muscular dystrophy. The direct administration of the pharmaceutical candidate WNT7a proteins to the tibialis anterior (TA) muscle of either wild-type or dystrophic (MDX) mice resulted in significant satellite stem cell expansion and fiber hypertrophy. In MDX mice this was associated with reduced inflammation and muscle fiber necrosis, and a significant increase in muscle specific force. Together, these results underscore the therapeutic potential for WNT7a, demonstrate the engineering of a viable drug candidate and provide evidence for its therapeutic potential in the treatment of muscular dystrophy and potentially other neuromuscular diseases.

Suppression of atrogin-1 and MuRF1 prevents dexamethasone-induced atrophy of cultured myotubes

ObjectiveThe mechanistic role of the ubiquitin ligases atrogin-1 and MuRF1 in glucocorticoid-induced muscle wasting is not fully understood. Here, we tested the hypothesis that glucocorticoid-induced muscle atrophy is at least in part linked to atrogin-1 and MuRF1 expression and that the ubiquitin ligases are regulated by compensatory mechanisms. MethodsThe expression of atrogin-1 and MuRF1 was suppressed individually or in combination in cultured L6 myotubes by using siRNA technique. Myotubes were treated with dexamethasone followed by determination of mRNA and protein levels for atrogin-1 and MuRF1, protein synthesis and degradation rates, and myotube morphology. ResultsSuppression of atrogin-1 resulted in increased expression of MuRF1 and vice versa, suggesting that the ubiquitin ligases are regulated by compensatory mechanisms. Simultaneous suppression of atrogin-1 and MuRF1 resulted in myotube hypertrophy, mainly reflecting stimulated protein synthesis, and prevented dexamethasone-induced myotube atrophy, mainly reflecting inhibited protein degradation. ConclusionsThe results provide evidence for a link between upregulated atrogin-1 and MuRF1 expression and glucocorticoid-induced muscle atrophy. The study also suggests that atrogin-1 and MuRF1 levels are regulated by compensatory mechanisms and that inhibition of both ubiquitin ligases may be needed to prevent glucocorticoid-induced muscle proteolysis and atrophy.

Impaired hypertrophy in myoblasts is improved with testosterone administration

We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have a reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7,26]). Cells were exposed to low serum conditions in the presence or absence of T (100nM)±PI3K inhibitor (LY294002) for 72h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (μm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32±2.56μm to 21.02±1.89μm and 14.58±2.66μm to 18.29±3.08μm (P≤0.05) respectively after 72h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P≤0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway.

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts.

The effects of endoplasmic reticulum stress response on duck decorin stimulate myotube hypertrophy in myoblasts

Decorin inhibits the transforming growth factor superfamily to stimulate skeletal muscle hypertrophy. The endoplasmic reticulum (ER) plays a crucial role in the synthesis and folding of proteins. The disruption of ER homeostasis affects protein folding and causes ER stress, which is associated with protein synthesis in muscle cells. The purpose of this study was to establish the mechanism by which decorin promotes myoblast proliferation and myotube hypertrophy as mediated by an ER stress-related pathway. Duck myoblasts were incubated for 24 h with tunicamycin. Our results show that tunicamycin triggered ER stress in myoblasts and led to an increase in protein synthesis via a decorin-dependent pathway. We then transfected the decorin gene into duck myoblasts in vitro, and the results demonstrated that overexpression of duck decorin increased myogenic determination factor and follistatin expression and decreased myogenin and myostatin expression. Moreover, the results demonstrated that overexpression of duck decorin led to higher expression of ER stress marker genes. This increased expression trend can be alleviated in vitro by 4-PBA, which is a chemical chaperone that inhibits palmitate-induced ER stress. Collectively, our data show that duck decorin promotes myoblast proliferation mediated by an ER stress-related pathway.

Regulation of IRS1/Akt insulin signaling by microRNA-128a during myogenesis

Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.

During post-natal human myogenesis, normal myotube size requires TRPC1 and TRPC4 mediated Ca2+ entry

Myogenesis involves expression of muscle-specific transcription factors such as myogenin and myocyte enhancer factor 2 (MEF2), and is essentially regulated by fluctuations of cytosolic Ca(2+) concentration. Recently we demonstrated that molecular players of store-operated Ca(2+) entry (SOCE), stromal interacting molecule (STIM) and Orai, were fundamental in the differentiation process of post-natal human myoblasts. Besides STIM and Orai proteins, the family of transient receptor potential canonical (TRPC) channels was shown to be part of SOCE in several cellular systems. In the present study, we investigated the role of TRPC channels in the human myogenesis process. We demonstrate, using an siRNA strategy or dominant negative TRPC overexpression, that TRPC1 and TRPC4 participate in SOCE, are necessary for MEF2 expression, and allow the fusion process to generate myotubes of normal size. Conversely, the overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes. Interestingly, in cells depleted of TRPC1 or TRPC4, the normalization of SOCE by increasing the extracellular calcium concentration or by overexpressing STIM1 or Orai1 was not sufficient to restore normal fusion process. A normal differentiation occurred only when TRPC channel was re-expressed. These findings indicate that Ca(2+) entry mediated specifically by TRPC1 and TRPC4 allow the formation of normal-sized myotubes.

Wnt7a treatment ameliorates muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder of childhood marked by progressive debilitating muscle weakness and wasting, and ultimately death in the second or third decade of life. Wnt7a signaling through its receptor Fzd7 accelerates and augments regeneration by stimulating satellite stem cell expansion through the planar cell polarity pathway, as well as myofiber hypertrophy through the AKT/mammalian target of rapamycin (mTOR) anabolic pathway. We investigated the therapeutic potential of the secreted factor Wnt7a for focal treatment of dystrophic DMD muscles using the mdx mouse model, and found that Wnt7a treatment efficiently induced satellite cell expansion and myofiber hypertrophy in treated mucles in mdx mice. Importantly, Wnt7a treatment resulted in a significant increase in muscle strength, as determined by generation of specific force. Furthermore, Wnt7a reduced the level of contractile damage, likely by inducing a shift in fiber type toward slow-twitch. Finally, we found that Wnt7a similarly induced myotube hypertrophy and a shift in fiber type toward slow-twitch in human primary myotubes. Taken together, our findings suggest that Wnt7a is a promising candidate for development as an ameliorative treatment for DMD.

Docosahexaenoic Acid Protects Muscle Cells from Palmitate-Induced Atrophy

Background. Accumulation of free fatty acids leads to lipid-toxicity-associated skeletal muscle atrophy. Palmitate treatment reduces myoblast and myotube growth and causes apoptosis in vitro. It is not known if omega-3 fatty acids will protect muscle cells against palmitate toxicity. Therefore, we examined the effects of docosahexaenoic acid (DHA) on skeletal muscle growth. Methods. Mouse myoblasts (C2C12) were differentiated to myotubes, and then treated with 0 or 0.5 mM palmitic acid or 0 or 0.1 mM DHA. Results. Intramyocellular lipid was increased in palmitate-treated cells but was prevented by DHA-palmitate cotreatment. Total AMPK increased in DHA+ palmitate-treated compared to palmitate only cells. RpS6 phosphorylation decreased after palmitate (−55%) and this was blunted by DHA+ palmitate (−35%) treatment. Palmitate treatment decreased PGC1α protein expression by 69%, but was increased 165% with DHA+ palmitate (P = 0.017) versus palmitate alone. While palmitate induced 25% and 90% atrophy in myotubes (after 48 hours and 96 hours, resp.), DHA+ palmitate treatment caused myotube hypertrophy of ~50% and 100% after 48 and 96 hours, respectively. Conclusion. These data show that DHA is protective against palmitate-induced atrophy. Although DHA did not activate the AMPK pathway, DHA treatment restored growth-signaling (i.e., rpS6) and rescued palmitate-induced muscle atrophy.

Progranulin compensates for blocked IGF-1 signaling to promote myotube hypertrophy in C2C12 myoblasts via the PI3K/Akt/mTOR pathway

It is well known that growth hormone (GH)-induced IGF-1 signaling plays a dominant role in postnatal muscle growth. Our previous studies have identified a growth factor, progranulin (PGRN), that is co-induced with IGF-1 upon GH administration. This result prompted us to explore the function of PGRN and its association with IGF-1. In the present study, we demonstrated that, similar to IGF-1, PGRN can promote C2C12 myotube hypertrophy via the PI(3)K/Akt/mTOR pathway. Moreover, PGRN can rescue the muscle atrophy phenotypes in C2C12 myotube when IGF-1 signaling is blocked. This result shows that PGRN can substitute for IGF-1 signaling in the regulation of muscle growth. Our findings provide new insights into IGF-1-modulated complicated networks that regulate muscle growth.

MmNEU3 sialidase over‐expression in C2C12 myoblasts delays differentiation and induces hypertrophic myotube formation

Several factors affect the skeletal muscle differentiation process, in particular modifications of cell-cell contact, cell adhesion, and plasma membrane characteristics. In order to support the role of the plasma membrane-associated sialidase NEU3 in skeletal muscle differentiation and to analyse which events of the process are mainly affected by this sialidase, we decided to stably over-express MmNEU3 in C2C12 cells by a lentiviral vector and to investigate cell behavior during the differentiation process. Vitally stained C2C12 and NEU3 over-expressing cells were counted to reveal modifications in differentiation induction. We found that NEU3 over-expressing cells remained proliferative longer than control cells and delayed the onset of differentiation. Expression of p21, myogenic transcription factors, and myosin heavy chain (MHC), assessed by real time PCR, confirmed this behavior. In particular, no MHC-positive myotubes were present in NEU3 over-expressing cells as compared to wild type C2C12 cells at day 3 of differentiation. Moreover, NEU3 over-expressing cells completed the differentiation process very quickly and formed hypertrophic myotubes. Analysis of MAPK/ERK pathway activation showed an increased ERK 1/2 phosphorylation in NEU3 over-expressing cells at the beginning of differentiation. We postulate that sialidase NEU3, decreasing plasma membrane ganglioside GM3 content, affects the EGF receptor and the downstream signaling pathways, promoting proliferation and delaying differentiation. Furthermore NEU3 improves myoblast fusion probably via neural-cell adhesion molecule (NCAM) desialylation. Therefore, this work further supports the central role of NEU3 as a key modulator in skeletal muscle differentiation, particularly in the myoblast fusion step.

Testosterone induces skeletal muscle hypertrophy via Akt/mTOR/S6K1 pathway and the androgen receptor

The anabolic hormone testosterone is associated with muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We studied whether signal transduction pathways and the androgen receptor are necessary to elicit hypertrophy in skeletal muscle myotubes. Testosterone induced an increase in myotube cross sectional area (control=134,5±10,2 μm2; testosterone=181,9±14,4 μm2) and in α‐actin protein levels at 12 hours. Morphological development of myotube sarcomerisation was more clearly established in testosterone stimulated myotubes at this time. Earlier times of testosterone stimulation showed increased transcription of α‐actin gene, concomitantly with no changes in atrogenes (MAFfbx and MuRF‐1) activity at 6 hours. Characteristic hypertrophy pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone at 5 and 15 minutes, respectively. S6K1 was phosphorilated at 60 minutes. This response was inhibited with LY294002, Akt‐inhibitor‐VIII and Rapamycin but not by PD98059. Similarly, the hypertrophic response at 12 hours was abolished with these inhibitors as well as partly by a siRNA against androgen receptor but not by PD98059. These results suggest interlink between pathways involving intracellular signal cascades and the classical genomic actions of androgens for testosterone‐induced skeletal muscle hypertrophy.FONDECYT 1090276, FONDAP 15010006

Hypertrophy in Skeletal Myotubes Induced by Junctophilin-2 Mutant, Y141H, Involves an Increase in Store-operated Ca2+ Entry via Orai1

Junctophilins (JPs) play an important role in the formation of junctional membrane complexes (JMC) in striated muscle by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. Researchers have found five JP2 mutants in humans with hypertrophic cardiomyopathy. Among these, Y141H and S165F are associated with severely altered Ca(2+) signaling in cardiomyocytes. We previously reported that S165F also induced both hypertrophy and altered intracellular Ca(2+) signaling in mouse skeletal myotubes. In the present study, we attempted to identify the dominant-negative role(s) of Y141H in primary mouse skeletal myotubes. Consistent with S165F, Y141H led to hypertrophy and altered Ca(2+) signaling (a decrease in the gain of excitation-contraction coupling and an increase in the resting level of myoplasmic Ca(2+)). However, unlike S165F, neither ryanodine receptor 1-mediated Ca(2+) release from the SR nor the phosphorylation of the mutated JP2 by protein kinase C was related to the altered Ca(2+) signaling by Y141H. Instead, abnormal JMC and increased SOCE via Orai1 were found, suggesting that the hypertrophy caused by Y141H progressed differently from S165F. Therefore JP2 can be linked to skeletal muscle hypertrophy via various Ca(2+) signaling pathways, and SOCE could be one of the causes of altered Ca(2+) signaling observed in muscle hypertrophy.

The SCF-Fbxo40 Complex Induces IRS1 Ubiquitination in Skeletal Muscle, Limiting IGF1 Signaling

Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle.

No muscle atrophy without GSK-3β. Focus on “Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy”

skeletal muscle atrophy is driven by an imbalance of protein synthesis and degradation, generally in favor of the latter. The ubiquitin-proteasome system (UPS) is commonly regarded as the major proteolytic system involved in breakdown of muscle protein under atrophy-inducing conditions ([6][1]), and

Electrical Impedance as a Novel Biomarker of Myotube Atrophy and Hypertrophy

Measuring myotube thickness is a physiological and unbiased approach for screening therapeutic compounds that prevent skeletal muscle atrophy or induce hypertrophy. However, an accurate cell thickness estimate is often quite challenging because of the extreme heterogeneity of the myotube cellular population and therefore the lack of a regular distribution of perturbed myotubes. Here the authors present a novel method to evaluate changes in myotube thickness via measuring cellular electrical impedance. They demonstrate that both qualitative and quantitative changes in electrical impedance as a function of cellular adhesion in real time correlate well with variation in myotube thickness caused by atrophy or hypertrophy agents. Conversely, pharmacologically blocking myotube hypertrophy prevents changes in electrical impedance. Thus, impedance can be used as a reliable and sensitive biomarker for myotube atrophy or hypertrophy. Application of this technique to drug screening might be beneficial in finding novel treatments preventing muscle atrophy and other diseases associated with any morphological change in cell shape.

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

Differently from S165F, Y141H Mutation of Junctophilin-2 affects Calcium Signaling in Skeletal Muscle

Junctophilins (JPs) play an important role in the formation of junctional membrane complexes in muscle cells by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. In humans with hypertrophic cardiomyopathy (HCM), mutations in JP2 are associated to altered Ca2+ signaling in cardiomyocytes. One of the HCM-related JP2 mutants, S165F also induces both hypertrophy and altered intracellular Ca2+ signaling in skeletal myotubes. Here, we tried to identify the dominant negative role of another HCM-related JP2 mutation, Y141H, in primary mouse skeletal myotubes in order to examine the effects of Y141H on skeletal muscle function. Consistent with S165F-expressing skeletal myotubes, over-expression of Y141H led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Immunoblot assays suggested that the increased resting cytosolic Ca2+ concentration is possibly due to the increased expression level of an extracellular Ca2+-entry channel, Orai1. Unlike S165F, single myotube Ca2+ imaging experiments with Y141H-expressing myotubes showed a reduction in the gain of excitation-contraction coupling without an alteration in both ryanodine receptor1-mediated Ca2+ release from the SR and the amount of SR Ca2+. Therefore, the hypertrophy in the Y141H-expressing skeletal myotubes would be differently progressed from that in the S165F-expressing skeletal myotubes.

Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.