Muscle Myotubes Research Articles

Expressed Sequence Tags for Bovine Muscle Satellite Cells, Myotube Formed-Cells and Adipocyte-Like Cells

BackgroundMuscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.ResultsA total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.ConclusionIn this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

BMS-986001, an HIV Nucleoside Reverse Transcriptase Inhibitor, Does Not Degrade Mitochondrial DNA in Long-Term Primary Cultures of Cells Isolated from Human Kidney, Muscle, and Adipose Tissue

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) remain the cornerstone of HIV treatment; however, they are associated with toxicities attributed in part to inhibition of mitochondrial DNA (mtDNA) polymerase γ. In this study, we compared the in vitro toxicity profiles of structurally similar NRTIs (BMS-986001 to stavudine and tenofovir to adefovir) that differ by the presence of an acetylene or methyl group, respectively. Primary cultures of human renal proximal tubule epithelium, skeletal muscle myotubes, and differentiated adipocytes were exposed to the NRTIs at the maximum concentration (Cmax) reported for the clinically approved dose (investigational dose for BMS-986001, 600 mg) and a high equimolar concentration (200 μM) for 19 days. After 19 days, BMS-986001 did not significantly decrease mtDNA or cell protein at either concentration in any cell line. In contrast, stavudine significantly decreased mtDNA in all cultures (1.5- to 2.5-fold) (except at Cmax in renal cells) and cell protein in renal cells (1.4- to 2.4-fold). By day 19, at 200 μM, tenofovir significantly reduced mtDNA in adipocytes (1.9-fold) and adefovir significantly decreased mtDNA in all cultures (3.7- to 10.2-fold); however, no significant reduction in mtDNA was observed at Cmax in any cell line. Adefovir also significantly reduced cell protein at both concentrations in renal cells (2.2- to 2.8-fold) and at 200 μM in muscle cells (2.0-fold). In conclusion, BMS-986001 and tenofovir were considerably less cytotoxic than their respective structural analogs, demonstrating that small structural differences can contribute to significant differences in toxicity.

Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.

The metabolic modulator trimetazidine triggers autophagy and counteracts stress‐induced atrophy in skeletal muscle myotubes

It has recently been demonstrated that trimetazidine (TMZ), an anti-ischemic antianginal agent, is also able to improve exercise performance in patients with peripheral arterial disease. TMZ is a metabolic modulator, and the mechanisms underlying its cytoprotective anti-ischemic activity could be ascribed, at least in cardiomyocytes, to optimization of metabolism. However, regarding the cytoprotection exerted by TMZ on skeletal muscle and allowing the improvement of exercise performance, no information is yet available. In the present study, we investigated in detail the protective effects of this drug on in vitro skeletal muscle models of atrophy. Experiments carried out with murine C2C12 myotubes treated with TMZ revealed that this drug could efficiently counteract the cytopathic effects induced by the proinflammatory cytokine tumor necrosis factor-α and by the withdrawal of growth factors. Indeed, TMZ significantly counteracted the reduction in myotube size induced by these treatments. TMZ also increased myosin heavy chain expression and induced hypertrophy in C2C12 myotubes, both effects strongly suggesting a role of TMZ in counteracting atrophy in vitro. In particular, we found that TMZ was able to activate the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin 2 pathway and to reduce the stress-induced transcriptional upregulation of atrogin-1, muscle ring finger protein 1, and myostatin, all of which are key molecules involved in muscle wasting. Moreover, this is the first demonstration that TMZ induces autophagy, a key mechanism involved in muscle mass regulation. On the basis of these results, it can be hypothesized that the improvement in exercise performance previously observed in patients could be ascribed to a cytoprotective mechanism exerted by TMZ on skeletal muscle integrity.

Lipid in skeletal muscle myotubes is associated to the donors' insulin sensitivity and physical activity phenotypes

ObjectiveThis study investigated the relationship between in-vitro lipid content in myotubes and in-vivo whole body phenotypes of the donors such as insulin sensitivity, intramyocellular lipids (IMCL), physical activity and oxidative capacity.Design and MethodsSix physically active donors were compared to 6 sedentary lean and 6 T2DM. Lipid content was measured in tissues and myotubes by immunohistochemistry. Ceramides, triacylglycerols (TAGs) and diacylglycerols (DAGs) were measured by LC-MS-MS and GC-FID. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (80mU/min/m2), maximal mitochondrial capacity (ATPmax) by 31P-MRS, physical fitness by VO2max and physical activity level (PAL) by accelerometers.ResultsMyotubes cultured from physically active donors had higher lipid content (0.047±0.003 vs. 0.032±0.001 and 0.033±0.001AU; p<0.001) than myotubes from lean and T2DM donors. Lipid content in myotubes was not associated with IMCL in muscle tissue but importantly, correlated with in-vivo measures of ATPmax (r=0.74; p<0.001), insulin sensitivity (r=0.54; p<0.05), type-I fibers (r=0.50; p<0.05) and PAL (r=0.92; p<0.0001). DAGs and ceramides in myotubes were inversely associated with insulin sensitivity (r=−0.55, r=−0.73; p<0.05) and ATPmax (r=−0.74, r=−0.85; p<0.01).ConclusionsThese results indicate that cultured human myotubes can be used in mechanistic studies to study the in vitro impact of interventions on phenotypes such as mitochondrial capacity, insulin sensitivity, and physical activity.

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.

P.3.6 Antioxidant capacity is impaired in hyposialylated myotubes of GNE myopathy

GNE myopathy (also known as distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy) is a rare autosomal recessive myopathy characterized by skeletal muscle atrophy and weakness that preferentially involve the distal muscles. The causative <i>GNE</i> gene encodes an essential enzyme in the biosynthesis of sialic acid and hyposialylation is thought to be a key factor in the underlying pathomechanism. We previously found that muscle atrophy appears in the young age of GNE myopathy model mice and is preventable by antioxidant administration, suggesting that oxidative stress would be associated with this primary myopathic phenotype. To link hyposialylation with the pathologic features, we investigated cellular response to oxidative stress in GNE myopathy myotubes. Skeletal muscle myotubes were prepared from Gne^−/−hGNED176VTg mice and littermate controls. Intracellular reactive oxygen species (ROS) and cell viability were evaluated. First, we found that intracellular ROS generation was more profound in GNE myopathy myotubes compared to littermates. Then the susceptibility of GNE myopathy myotubes to increasing concentrations of menadione, a generator of ROS, with manipulating cellular sialylation was analyzed. We found that hyposialylated myotubes cultured in serum-free media are more vulnerable to menadione-induced oxidative stress. Dose-dependent increase in intracellular oxidants and cell mortality were weakened with the addition of NeuAc to culture media, which implies that normalization of sialylation levels improved intracellular antioxidant capacities. Our results provide a clue how hyposialylation makes the pathognomonic change in GNE myopathy and suggest that oxidative stress can be a therapeutic target of this progressive disease.

Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device

Rigorous analysis of muscle function in in vitro systems is needed for both acute and chronic biomedical applications. Forces generated by skeletal myotubes on bio-microelectromechanical cantilevers were calculated using a modified version of Stoney's thin-film equation and finite element analysis (FEA), then analyzed for regression to physical parameters. The Stoney's equation results closely matched the more intensive FEA and the force correlated to cross-sectional area (CSA). Normalizing force to measured CSA significantly improved the statistical sensitivity and now allows for close comparison of in vitro data to in vivo measurements for applications in exercise physiology, robotics, and modeling neuromuscular diseases.

Astragalus polysaccharide improves muscle atrophy from dexamethasone- and peroxide-induced injury in vitro

Astragalus polysaccharide (APS) is an important bioactive component of Astragalus membranaceus Bunge (Leguminosae) that has been used in traditional Chinese medicine for treating muscle wasting, a serious complication with complex mechanism manifested as myofibers atrophy and satellite cells apoptosis. In this study, the anti-atrophy and anti-apoptotic activity of Astragalus polysaccharide (APS) was characterized in C2C12 skeletal muscle myotubes and myoblasts. APS inhibited dexamethasone-induced atrophy by restoring phosphorylation of Akt, m-TOR, P70s6k, rpS6 and FoxO3A/FoxO1. The targets that protected C2C12 myoblasts from damage by H2O2 were promoting cells proliferation and inhibiting cells apoptosis. The protective mechanisms involved mitochondrial pathway and death receptor pathway. Moreover, Antioxidant effect of APS was also detected in this work. Our findings suggested that APS could be explored as a protective and perhaps as a therapeutic agent in the management of muscle wasting.

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

Vitamin D attenuates nucleoside reverse transcriptase inhibitor induced human skeletal muscle mitochondria DNA depletion

To evaluate the impact of the active metabolite of vitamin D, 1α,25-dihydroxycholecalciferol (1,25D3), on nucleoside reverse transcriptase inhibitor (NRTI) induced mitochondrial DNA (mtDNA) depletion in human skeletal muscle myoblasts and myotubes. mtDNA was quantified in human skeletal muscle myoblasts and myotubes following 1,25D3 and NRTI treatment using real-time PCR. Human skeletal muscle myoblasts and myotubes were treated with didanosine (ddI), stavudine (d4T), zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) alone or in combination either in the presence or absence of 1,25D3 for 5 days. Cells were harvested, DNA extracted and mtDNA quantified. ddI and ddI-d4T significantly decreased both myoblast and myotube mtDNA in the absence of 1,25D3 compared with untreated controls (P≤0.029). In addition, the ZDV-3TC combination resulted in a 47% decrease in myotube mtDNA (P=0.005). 1,25D3 increased myotube mtDNA levels in ddI, ZDV, 3TC, ABC, ddI-d4T, d4T-3TC, ZDV-3TC, ZDV-ABC and ZDV-3TC-ABC-containing regimens and myoblast mtDNA levels in ddI, d4T, ZDV, 3TC, ddI-d4T, ZDV-3TC and ZDV-ABC-containing regimens. Of note, 1,25D3 protected against myotube mtDNA depletion following ZDV-3TC treatment, rendering them similar to 1,25D3 untreated controls (P=0.62), and increased both myotube and myoblast mtDNA two to three-fold in ddI-containing regimens (P<0.05). 1,25D3 confers a protective effect against NRTI-induced mitochondrial toxicity in skeletal muscle myoblasts and myotubes. These findings support a protective role for vitamin D in preventing mitochondrial toxicity and suggest that supplemental vitamin D may protect against NRTI-associated mitochondrial toxicity.

Heat shock factor‐1 regulates IL‐6 promoter activity in C2C12 muscle myotubes

Hyperthermia stimulates interleukin‐6 (IL‐6) mRNA and protein production and also potentiates the IL‐6 response to epinephrine (EPI) and lipopolysaccharide (LPS), but the signaling pathways are not known. In this study we specifically tested the role of heat shock factor‐1 (HSF‐1) in these responses using a mouse IL‐6 promoter reporter construct (mIL‐6.Luc) in C2C12 myotubes. IL‐6 promoter activity was inhibited by co‐transfection of dominant negative (d.n.) HSF‐1 or stimulated using a constitutively active (c.a.) HSF‐1 construct. The d.n. HSF‐1 significantly diminished IL‐6 promoter activity at both 37°C and 42°C (P &lt; 0.05). Furthermore, the d.n. HSF‐1 significantly reduced the IL‐6 transcriptional responses to EPI (100 ng/ml) and LPS (1 ug/ml) at both 37°C and 42°C (P &lt; 0.05). In contrast, co‐transfection with c.a. HSF‐1 significantly elevated IL‐6. Luc reporter activity at 37°C and amplified the response to LPS (P&lt; 0.05); however, it had no influence on the response to EPI. Results demonstrate that hyperthermia is an important functional stimulus for IL‐6 promoter activity, which employs HSF‐1 activation. In addition, the interactions of overexpressed HSF‐1 with stress signals (from EPI or LPS) and the IL‐6 promoter have different regulatory outcomes. The overexpression of HSF‐1 does not amplify the IL‐6 promoter's response to EPI. The results confirm the important role of HSF‐1 in IL‐6 regulation and suggest that hyperthermia may also recruit other parallel signaling pathways besides HSF‐1 that influence IL‐6 regulation. Supported by: AHA #11GRNT7990119

Muscle mTORC1 suppression by IL-6 during cancer cachexia: a role for AMPK

Although catabolic signaling has a well-established role in muscle wasting during cancer cachexia, the suppression of anabolic signaling also warrants further investigation. In cachectic tumor-bearing mice, circulating IL-6 levels are associated with suppressed muscle protein synthesis and mTORC1 signaling. We have found AMPK and IGF-I/insulin signaling, two well-known regulators of the mammalian target of rapamycin (mTOR), are altered with the progression of cachexia. How IL-6 can induce suppression of mTORC1 signaling remains to be established. The purpose of this study was to examine mTOR complex 1 (mTORC1) activation and regulation by IL-6 during cancer cachexia. IL-6 effects on mTOR activation were examined in Apc(Min/+) mouse skeletal muscle and C2C12 myotubes. Systemic IL-6 overexpression in Apc(Min/+) mice produced a dose-dependent suppression of mTOR signaling that corresponded to induction of STAT3 and AMPK phosphorylation. This result was also evident in IL-6-treated myotubes. Basal mTOR activation and mTOR responsiveness to glucose administration were suppressed in cachectic skeletal muscle. However, insulin induction of mTOR activity was maintained in IL-6-treated myotubes. Whereas IL-6 suppression of myotube mTOR activity was rescued by AMPK inhibition, inhibition of STAT3 signaling was not sufficient to rescue IL-6 suppression of mTOR activity. Last, treadmill exercise training was able to prevent IL-6-induced inhibition of mTOR signaling in Apc(Min/+) mice independently of activated STAT. In conclusion, we report dose-dependent suppression of mTOR activity by IL-6 and suppressed mTOR responsiveness to glucose administration in Apc(Min/+) mice. IL-6 suppression of mTOR activity was dependent on AMPK activation and independent of STAT signaling in myotubes.

Sirtuin 1-mediated Effects of Exercise and Resveratrol on Mitochondrial Biogenesis

The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.

The peculiar apoptotic behavior of skeletal muscle cells.

Apoptosis plays an active role in maintaining skeletal muscle homeostasis. Its deregulation is involved in several skeletal muscle disorders such as dystrophies, myopathies, disuse and sarcopenia. The aim of this work was to study in vitro the apoptotic behavior induced by etoposide, staurosporine and hydrogen peroxide in the C2C12 skeletal muscle cell line, comparing myoblast vs myotube sensitivity, investigated by means of morphological and cytofluorimetric analyses. Myotubes appeared more resistant than myoblasts to apoptotic induction. In myoblasts treated with etoposide, nuclei with chromatin condensation were observed, in the presence of a diffuse DNA fragmentation, as shown by confocal microscopy. The latter also appeared in myotubes, where apoptotic and normal nuclei coexisted inside the same syncytium. After staurosporine treatment, myobalsts evidenced late apoptotic features and a high number of TUNEL-positive nuclei. Secondary necrosis appeared in myotubes, where myonuclei with cleaved DNA again coexisted with normal myonuclei. After H₂O₂ exposure, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, autophagic granules appeared abundantly in myotubes after each treatment. In myotubes, mitochondria were better preserved than in myoblasts since those which were damaged were probably degraded through autophagic processes. These findings demonstrate a scarce sensitivity of myotubes to apoptotic stimuli due to acquisition of an apoptosis-resistant phenotype during differentiation. The presence of nuclear-dependent "territorial" death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to apoptosis induction.

Evaluating Cellular Repair Potential - Lessons from Skeletal Muscle

The ability to repair disruptions in the plasma membrane is critical for maintaining cellular integrity and health; this is especially true for cells that routinely experience membrane damage as a result of activity such as skeletal muscle. Dysferlinopathies are a class of late onset muscle wasting diseases associated with the absence or truncation of the protein dysferlin. Dysferlin is postulated to have a role in membrane repair and the absence of dysferlin in both humans and animal models is associated with muscle damage. We are developing techniques to evaluate the repair potential of dysferlin null skeletal muscle fibers and myotubes using fluorescence assays of dye-leakage and calcium activity. In isolated muscle fibers or myotubes containing fluorescent dye and subjected to IR laser wounding, both the rate and duration of dye-leakage are measured by confocal microscopy. Upon wounding repair-competent fibers a “puff” of dye is released at the wound site and dye leakage ceases within 60 seconds. Under conditions where resealing is inhibited (i.e. low extracellular [Ca2+]) a large amount of dye is lost; leakage continues for >10 minutes. Changes in intracellular [Ca2+]free are measured using a combination of the calcium indicator dyes fluo-4 and Fura Red. Upon wounding, a rise in [Ca2+]free is observed at the wound site and either returns to the pre-wound level following successful resealing, or in the absence of resealing remains high and spreads through the entire fiber or mytube. Dysferlin null muscle, is repair competent but may differ in the developed calcium load, contractile response, and wounding threshold compared to dysferlin positive muscle. Since our overall goal is to develop interventions that boost cellular repair potential, quantifiable metrics of this complicated cellular process are required when testing the efficacy of repair-targeted treatments.

Mitsugumin 53 Attenuates the Activity of Sarcoplasmic Reticulum Ca2+-ATPase 1a (SERCA1a) in Skeletal Muscle

Mitsugumin 53 (MG53) is a member of membrane repair system in skeletal muscle. However, role(s) of MG53 in unique functions of skeletal muscle has not been addressed although MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were searched among proteins mediating skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knock-down in mouse primary skeletal myotubes increased Ca2+-uptake through SERCA1a (more than 35%) at micromolar Ca2+ but not at nanomolar Ca2+, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. In-silico studies suggest that the binding of MG53 to SERCA1a is mediated by unique ways compared with bindings by other proteins containing TRIM or PRY domains.

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca2+-uptake through SERCA1a (more than 35%) at micromolar Ca2+ but not at nanomolar Ca2+, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.

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

Gene knockout and agonist studies indicate that activation of the G protein-coupled receptor, GPR119, protects against diet-induced obesity and insulin resistance. It is not known if GPR119 activation in skeletal muscle mediates these effects. To address this uncertainty, we measured GPR119 expression in skeletal muscle and determined the effects of PSN632408, a GPR119 agonist, on the expression of genes and proteins required for fatty acid and glucose oxidation in cultured myotubes. GPR119 expression was readily detected in rat skeletal muscle and mRNAs were induced by 12weeks of high-fat feeding. Treatment of cultured mouse C2C12 myotubes with 5μM PSN632408 or 0.5mM palmitate reduced expression of mRNAs encoding fatty acid oxidation genes to similar extents. More so, treatment with PSN632408 decreased AMPKα (Thr172 phosphorylation) activity in the absence of palmitate and ACC (Ser79 phosphorylation) activity in the presence of palmitate. In human primary myotubes PSN632408 decreased expression of PDK4 and AMPKα2 mRNA in myotubes derived from obese donors. These data suggest GPR119 activation in skeletal muscle may impair fatty acid and glucose oxidation.

CB1 antagonism mediates adiponectin signalling in obese and obese diabetic derived human skeletal muscle myotubes

CB1 antagonism mediates adiponectin signalling in obese and obese diabetic derived human skeletal muscle myotubes