Myotube Cultures Research Articles

Effects of Tart Cherry Extract and L-Arginine on Regeneration of Myotube Cultures after Mechanical Strain

Eccentrically-biased muscle loading produces temporary damage to muscles leading to repair and adaptation. The muscle adaptation process after exercise induced muscle damage results in temporary de-habilitation and compromised resiliency of the muscle. Both tart cherry extract (TCE) and L-arginine (L-Arg) have been shown to reduce strength loss due to muscle loading, but the mechanisms by which they operate are currently unknown. PURPOSE: To determine: a) the activation threshold of mechanical loading that induces muscle cell damage and regeneration, and b) to determine the influence of TCE and L-Arg on markers of damage, regeneration, and metabolism in cultured muscle cells. METHODS: A FlexCell™ FX5000 system was used to apply mechanical strain to C2C12 myotube cultures. The strain protocol consisted of a fixed magnitude of strain for 0, 2, 4, 6, 12, 18, or 24h. Cultures were then assayed at all time points for lactate dehydrogenase (LDH) release (damage), satellite cell proliferation (regeneration) using a Click-iT® EdU Imaging Kit and mitochondrial metabolism via XTT assay, in the presence or absence of TCE (5 ng/ml) or L-Arg (150 μm). RESULTS: Mechanical strain induced significant (p<0.05) LDH release from myotube cultures after 12h (64.4±31.5%), and 24h (60.5±20.3%) of strain, relative to controls. Satellite cell proliferation increased by (39.5±5.4%) at 6h, (87.5±6.1%) at 12, peaked at 18h (135 ±7.6%), and remained elevated at 24h (99.5 ±11%) post-strain, relative to controls (p<0.05). Mitochondrial metabolism was elevated in strained myotube cultures (vs. controls) at 18h (25.1 ±4.3%) and 24h (24 ±10.8%) post-injury (p<0.05). Changes in satellite cell activation (r=0.77, p=0.01) and metabolism (r=0.78, p=0.01) were highly correlated to LDH release. TCE (5 ng/ml) and L-Arg (150 μm); however, had no effect on damage, satellite cell activation, or metabolism, compared to untreated cultures. CONCLUSION: These findings demonstrate in vitro that the magnitude of the regenerative response to muscle damage is associated with the degree of the initial mechanical insult. Moreover, TCE and L-Arg do not lessen the initial mechanical insult or accelerate regeneration in this model of strain-induced muscle injury. Funding source: Combat Feeding Research & Engineering Program

Toxicity of Tart Cherry Extract of C2C12 and U937 Cell Lines

Oxidative stress and inflammation are implicated in secondary damage from eccentrically-biased muscle contractions. Research suggests that the antioxidant/anti-inflammatory compounds in tart cherry juice may accelerate strength recovery following damage; however, the underlying mechanisms remain unclear. PURPOSE: To investigate the effects of tart cherry extract (TCE) on skeletal muscle (C2C12) and immune cells (U937) and to determine maximum effective dose for subsequent muscle damage experiments.\ METHODS: TCE was purified using a water-based extraction method and evaluated for total phenolic content using the Folin-Ciocalteau’s assay. C2C12 myotube cultures were differentiated for 5d, and U937 monocytes were grown to 1x106 cells/mL and then preconditioned with TCE for 24-48h. Cultures were then incubated with 50% ethanol (EtOH) to induce cell death. Reactive oxygen species (ROS) scavenging activity of TCE was assessed using a DPPH assay. Toxicity of TCE and EtOH was assessed using an XTT assay. RESULTS: ROS inhibition was observed at all [TCE] (7-100μg/mL) in both C2C12 and U937 cell culture media (p<0.05), and ROS inhibition was highest in C2C12 media vs. U937 media (p<0.05). Treatment with EtOH decreased cell viability in both C2C12 (-81±5%) and U937 (-69±11%) cells, relative to untreated control cultures (p<0.05). Pretreatment of C2C12 cells with TCE (7-35μg/mL) for 48h attenuated EtOH-induced toxicity by 18±6%-26±5% (p<0.02), indicating a protective effect of TCE on myotube cultures. Higher [TCE] (50-100μg/mL) decreased viability by 14±3%-20±4%, compared with untreated controls (p<0.02), indicating a toxic threshold for TCE. Pretreatment with TCE for 24h (50-100μg/mL) and 48h (35-100μg/mL) rescued U937 cell viability after the EtOH challenge (p<0.02). Interestingly, U937 cell counts increased when preconditioned with TCE at [21.5-100μg/mL] for 24h vs. untreated controls (p<0.02), suggesting a stimulatory effect on cell proliferation. CONCLUSION: These data help establish an experimental range of TCE for future in vitro studies, and demonstrate that TCE has protective antioxidant effects on cultured skeletal muscle cells and monocytes. Funding Source: Combat Feeding Research & Engineering Program / Framingham State University Department of Chemistry and Food Science.

Cell Density and Joint microRNA-133a and microRNA-696 Inhibition Enhance Differentiation and Contractile Function of Engineered Human Skeletal Muscle Tissues

To utilize three-dimensional (3D) engineered human skeletal muscle tissue for translational studies and in vitro studies of drug toxicity, there is a need to promote differentiation and functional behavior. In this study, we identified conditions to promote contraction of engineered human skeletal muscle bundles and examined the effects of transient inhibition of microRNAs (miRs) on myogenic differentiation and function of two-dimensional (2D) and 3D cultures of human myotubes. In 2D cultures, simultaneously inhibiting both miR-133a, which promotes myoblast proliferation, and miR-696, which represses oxidative metabolism, resulted in an increase in sarcomeric α-actinin protein and the metabolic coactivator PGC-1α protein compared to transfection with a scrambled miR sequence (negative control). Although PGC-1α was elevated following joint inhibition of miRs 133a and 696, there was no difference in myosin heavy chain (MHC) protein isoforms. 3D engineered human skeletal muscle myobundles seeded with 5 × 10(6) human skeletal myoblasts (HSkM)/mL and cultured for 2 weeks after onset of differentiation consistently did not contract when stimulated electrically, whereas those seeded with myoblasts at 10 × 10(6) HSkM/mL or higher did contract. When HSkM were transfected with both anti-miRs and seeded into fibrin hydrogels and cultured for 2 weeks under static conditions, twitch and tetanic specific forces after electrical stimulation were greater than for myobundles prepared with HSkM transfected with scrambled sequences. Immunofluorescence and Western blots of 3D myobundles indicate that anti-miR-133a or anti-miR-696 treatment led to modest increases in slow MHC, but no consistent increase in fast MHC. Similar to results in 2D, only myobundles prepared with myoblasts treated with anti-miR-133a and anti-miR-696 produced an increase in PGC-1α mRNA. PGC-1α targets were differentially affected by the treatment. HIF-2α mRNA showed an expression pattern similar to that of PGC-1α mRNA, but COXII mRNA levels were not affected by the anti-miRs. Overall, joint inhibition of miR-133a and miR-696 accelerated differentiation, elevated the metabolic coactivator PGC-1α, and increased the contractile force in 3D engineered human skeletal muscle bundles.

Tumor necrosis factor-α impairs adiponectin signalling, mitochondrial biogenesis, and myogenesis in primary human myotubes cultures.

Skeletal muscle metabolic changes are common in patients with chronic heart failure (HF). Previously, we demonstrated a functional skeletal muscle adiponectin resistance in HF patients with reduced left ventricular ejection fraction (HFrEF). We aimed to examine the impact of adiponectin receptor 1 (AdipoR1) deficiency and TNF-α treatment on adiponectin signaling, proliferative capacity, myogenic differentiation, and mitochondrial biogenesis in primary human skeletal muscle cells. Primary cultures of myoblasts and myotubes were initiated from the musculus vastus lateralis of 10 HFrEF patients (left ventricular ejection fraction; 31.30 ± 2.89%) and 10 age- and gender-matched healthy controls. Healthy control cultures were transfected with siAdipoR1 and/or exposed to TNF-α (10 ng/ml; 72 h). Primary cultures from HFrEF patients preserved the features of adiponectin resistance in vivo. AdipoR1 mRNA was negatively correlated with time to reach maximal cell index (r = -0.7319, P = 0.003). SiRNA-mediated AdipoR1 silencing reduced pAMPK (P < 0.01), AMPK activation (P = 0.046), and myoblast proliferation rate (xCELLigence Real-Time Cellular Analysis; P < 0.0001). Moreover, TNF-α decreased the mRNA expression of genes involved in glucose (APPL1, P = 0.0002; AMPK, P = 0.021), lipid (PPARα, P = 0.025; ACADM, P = 0.003), and mitochondrial (FOXO3, P = 0.018) metabolism, impaired myogenesis (MyoD1, P = 0.053; myogenin, P = 0.048) and polarized cytokine secretion toward a growth-promoting phenotype (IL-10, IL-1β, IFN-γ, P < 0.05 for all; Meso Scale Discovery Technology). Major features of adiponectin resistance are retained in primary cultures from the skeletal muscle of HFrEF patients. In addition, our results suggest that an increased inflammatory constitution contributes to adiponectin resistance and confers alterations in skeletal muscle differentiation, growth, and function.

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

Calpain 3 deficiency affects SERCA expression and function in the skeletal muscle.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

Agents Which Inhibit NF-κB Signaling Block Spontaneous Contractile Activity and Negatively Influence Survival of Developing Myotubes.

Inhibiting the NF-κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF-κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF-κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF-α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF-κB signaling. The NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose- and time-dependent manner. Similarly, a vivo-morpholino designed to block translation of murine p65 (m-p65tb-vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m-p65tb-vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF-κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF-κB inhibitors in treating Duchenne and related muscular dystrophies. J. Cell. Physiol. 231: 788-797, 2016. © 2015 Wiley Periodicals, Inc.

Influence of statins on distinct circulating microRNAs during prolonged aerobic exercise.

Statins exacerbate exercise-induced skeletal muscle injury. Muscle-specific microRNAs (myomiRs) increase in plasma after prolonged exercise, but the patterns of myomiRs release after statin-associated muscle injury have not been examined. We examined the relationships between statin exposure, in vitro and in vivo muscle contraction, and expression of candidate circulating myomiRs. We measured plasma levels of myomiRs, circulating microRNA-1 (c-miR-1), c-miR-133a, c-miR-206, and c-miR-499-5p from 28 statin-using and 28 nonstatin-using runners before (PRE), immediately after (FINISH), and 24 h after they ran a 42-km footrace (the 2011 Boston marathon) (POST-24). To examine these cellular-regulation myomiRs, we used contracting mouse C2C12 myotubes in culture with and without statin exposure to compare intracellular and extracellular expression of these molecules. In marathoners, c-miR-1, c-miR-133a, and c-miR-206 increased at FINISH, returned to baseline at POST-24, and were unaffected by statin use. In contrast, c-miR-499-5p was unchanged at FINISH but increased at POST-24 among statin users compared with PRE and runners who did not take statins. In cultured C2C12 myotubes, extracellular c-miR-1, c-miR-133a, and c-miR-206 were significantly increased by muscle contraction regardless of statin use. In contrast, extracellular miR-499-5p was unaffected by either isolated statin exposure or isolated carbachol exposure but it was increased when muscle contraction was combined with statin exposure. In summary, we found that statin-potentiated muscle injury during exercise is accompanied by augmented extracellular release of miR-499-5p. Thus c-miR-499-5p may serve as a biomarker of statin-potentiated muscle damage.

Involvement of adiponectin in the pathogenesis of dystrophinopathy.

BackgroundThe hormone adiponectin (ApN) is decreased in the metabolic syndrome, where it plays a key pathogenic role. ApN also exerts some anti-inflammatory effects on skeletal muscles in mice exposed to acute or chronic inflammation. Here, we investigate whether ApN could be sufficiently potent to counteract a severe degenerative muscle disease, with an inflammatory component such as Duchenne muscular dystrophy (DMD).MethodsMdx mice (a DMD model caused by dystrophin mutation) were crossed with mice overexpressing ApN in order to generate mdx-ApN mice; only littermates were used. Different markers of inflammation/oxidative stress and components of signaling pathways were studied. Global force was assessed by in vivo functional tests, and muscle injury with Evans Blue Dye (EBD). Eventually, primary cultures of human myotubes were used.ResultsCirculating ApN was markedly diminished in mdx mice. Replenishment of ApN strikingly reduced muscle inflammation, oxidative stress, and enhanced the expression of myogenic differentiation markers along with that of utrophin A (a dystrophin analog) in mdx-ApN mice. Accordingly, mdx-ApN mice exhibited higher global force and endurance as well as decreased muscle damage as quantified by curtailed extravasation of EBD in myofibers. These beneficial effects of ApN were recapitulated in human myotubes. ApN mediates its protection via the adiponectin receptor 1 (AdipoR1, the main ApN receptor in muscle) and the AMPK-SIRT1-PGC-1α signaling pathway, leading to downregulation of the nuclear factor kappa B (NF-κB) and inflammatory genes, together with upregulation of utrophin.ConclusionsAdiponectin proves to be an extremely powerful hormone capable of protecting the skeletal muscle against inflammation and injury, thereby offering novel therapeutic perspectives for dystrophinopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0051-9) contains supplementary material, which is available to authorized users.

A differential response to newt regeneration extract by C2C12 and primary mammalian muscle cells.

BackgroundDedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate.MethodsWe isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation.ResultsWe have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.ConclusionsDedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0044-8) contains supplementary material, which is available to authorized users.

Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia.

Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass.

Symposia and Oral Abstracts

[i]In vitro[/i] myotubes derived from zebrafish myogenic precursor cells upregulate Pax−3 and −7 following starvation. The zebrafish[i] (Danio rerio)[/i] remains the teleost fish of choice for biological investigations due to the plethora of molecular tools available for use in this system. However, its somatic growth is not representative of other teleost fishes, most of which are able to grow throughout their lives (termed indeterminate growth) while the zebrafish possesses a rather limited growth pot protein turnover, with the balance between protein synthesis and degradation governing whether myofibers hypertrophy or atrophy. To better describe the potentially divergent mechanisms of skeletal muscle atrophy in this species, we developed a primary myotube culture system generated from isolated myogenic precursor cells (MPCs) from adult zebrafish. Using a media devoid of serum and amino acids, we induced the expression of many genes associated with autophagy. Interestingly, paired−box transcription factor (Pax3/7) expression increased concurrently with myocyte enhancing factor−2ca (Mef2c) upregulation when de novo myotubes were subjected to starvation. From these results, it suggests that zebrafish myotubes, cultured from primary myoblasts, can be induced to express early myogenic biomarkers using nutrient alterations.

Pregnancy-induced amelioration of muscular dystrophy phenotype in mdx mice via muscle membrane stabilization effect of glucocorticoid.

Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, mdx mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from mdx mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into mdx mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in mdx mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies.

Anti-Differentiation Effect of Oncogenic Met Receptor in Terminally-Differentiated Myotubes.

Activation of the hepatocyte growth factor/Met receptor is involved in muscle regeneration, through promotion of proliferation and inhibition of differentiation in myogenic stem cells (MSCs). We previously described that the specific expression of an oncogenic version of the Met receptor (Tpr–Met) in terminally-differentiated skeletal muscle causes muscle wasting in vivo. Here, we induced Tpr–Met in differentiated myotube cultures derived from the transgenic mouse. These cultures showed a reduced protein level of myosin heavy chain (MyHC), increased phosphorylation of Erk1,2 MAPK, the formation of giant sacs of myonuclei and the collapse of elongated myotubes. Treatment of the cultures with an inhibitor of the MAPK kinase pathway or with an inhibitor of the proteasome increased the expression levels of MyHC. In addition, the inhibition of the MAPK kinase pathway prevented the formation of myosacs and myotube collapse. Finally, we showed that induction of Tpr–Met in primary myotubes was unable to produce endoreplication in their nuclei. In conclusion, our data indicate that multinucleated, fused myotubes may be forced to disassemble their contractile apparatus by the Tpr–Met oncogenic factor, but they resist the stimulus toward the reactivation of the cell cycle.

Systemic inflammation in chronic obstructive pulmonary disease and lung cancer: common driver of pulmonary cachexia?

In this article, a putative role of systemic inflammation as a driver of pulmonary cachexia induced by either chronic obstructive pulmonary disease or nonsmall cell lung cancer is reviewed. Gaps in current translational research approaches are discussed and alternative strategies are proposed to provide new insights. Activation of the ubiquitin proteasome system has generally been considered a cause of pulmonary cachexia, but current animal models lack specificity and evidence is lacking in nonsmall cell lung cancer and conflicting in chronic obstructive pulmonary disease patients. Recent studies have shown activation of the autophagy-lysosome pathway in both nonsmall cell lung cancer and chronic obstructive pulmonary disease. Myonuclear loss, as a consequence of increased apoptotic events in myofibers, has been suggested in cancer-cachexia-associated muscle atrophy. Plasma transfer on myotube cultures can be used to detect early inflammatory signals in patients and presence of atrophy-inducing activity within the circulation. Comparative clinical research between nonsmall cell lung cancer and chronic obstructive pulmonary disease in different disease stages is useful to unravel disease-specific versus common denominators of pulmonary cachexia.

Redox proteins are constitutively secreted by skeletal muscle.

Myokines are skeletal muscle-derived hormones. In this study, using a C2C12 myotube contraction system, we sought to determine whether the skeletal muscle secreted thioredoxin (TRX) and related redox proteins. Redox proteins such as TRXs, peroxiredoxins, and glutaredoxins were detected in the C2C12 myotube culture medium in the absence of any stimulation. The amounts of TRXs, peroxiredoxins, and glutaredoxins secreted by the C2C12 myotubes were not affected by the contraction, unless the myotubes were injured. Because TRX-1 was known to be a secreted protein that lacks a signal peptide, we examined whether this protein was secreted via exosome vesicles. The results indicated that TRX-1 was not secreted via exosome vesicles. We concluded that TRX-1 and related redox proteins are myokines that are constitutively secreted by the skeletal muscle cells. Although the mechanism of TRX-1 secretion remains unclear, our findings suggest that the skeletal muscle is an endocrine organ and the redox proteins that are constitutively secreted from the skeletal muscle may exert antioxidant and systemic health-promoting effects.

SERONEGATIVE MYASTHENIA GRAVIS-CLINICAL/SEROLOGICAL ASPECTS

Acquired Myasthenia Gravis is an autoimmune channelopathy of the post-synaptic neuromuscular junction. The clinical diagnosis is supported by the detection of AChR, MuSK, or LRP4 antibodies. Despite clear autoimmune aetiology, a proportion of patients are seronegative (SNMG) and diagnostic confirmation can be challenging. We characterised clinical and serological features in 293 suspected SNMG patients. AChR, MuSK or LRP4 antibodies were detected using radioimmunoassays (RIA) and/or cell-based assays (CBA). SNMG sera were screened for binding to primary muscle cell lines (TE671 and CN21) and inhibition of agrin-induced AChR clustering in myotube cultures (C2C12). 212 patients were negative by RIA and CBA [M:F 1:1.4 median onset age 35.5 (1–63)]. Most patients had mild disease (MGFA ≤IIb) at onset (17/17) and follow-up (70% n=61/87). Neurophysiology was positive in 65% (n=28/43). Preliminary results suggested no functional effects on agrin-induced AChR clustering and some seronegative sera bound to primary muscle cell lines. Myasthenia gravis patients without AChR, MuSK or LPR4 antibodies have relatively mild disease. Neurophysiology was supportive in only 65% of patients. Our evidence suggests that some of these sera do bind to novel antigen(s) on muscle cells. The next stage will be to try to define the antigen(s) using immunoprecipitation and mass spectroscopy.

Stac3 inhibits myoblast differentiation into myotubes.

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.

Altered RIG‐I/DDX58‐mediated innate immunity in dermatomyositis

We investigated the molecular mechanisms involved in the pathogenesis of three inflammatory myopathies, dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM). We performed microarray experiments(†) using microdissected pathological muscle fibres from 15 patients with these disorders and five controls. Differentially expressed candidate genes were validated by immunohistochemistry on muscle biopsies, and the altered pathways were analysed in human myotube cultures. Up-regulation of genes involved in viral and nucleic acid recognition were found in the three myopathies but not in controls. In DM, retinoic acid-inducible gene 1 (RIG-I, DDX58) and the novel antiviral factor DDX60, which promotes RIG-I-mediated signalling, were significantly up-regulated, followed by IFIH1 (MDA5) and TLR3. Immunohistochemistry confirmed over-expression of RIG-I in pathological muscle fibres in 5/5 DM, 0/5 PM and 0/5 IBM patients, and in 0/5 controls. Stimulation of human myotubes with a ligand of RIG-I produced a significant secretion of interferon-β (IFNβ; p < 0.05) and up-regulation of class I MHC, RIG-I and TLR3 (p < 0.05) by IFNβ-dependent and TLR3-independent mechanisms. RIG-I-mediated innate immunity, triggered by a viral or damage signal, plays a significant role in the pathogenesis of DM, but not in that of PM or IBM.

Low doses of arginine butyrate derivatives improve dystrophic phenotype and restore membrane integrity in DMD models

A new approach to treating Duchenne muscular dystrophy was investigated by using the ester or amide covalent association of arginine [nitric oxide (NO) pathway] and butyrate [histone deacetylase (HDAC) inhibition] in mdx mice and patient myotubes. Two prodrugs were synthesized, and the beneficial effects on dystrophic phenotype were studied. Nerve excitability abnormalities detected in saline-treated mice were almost totally rescued in animals treated at low doses (50-100 mg/kg/d). Force and fatigue resistance were improved ≈60% and 3.5-fold, respectively, and the percentage of necrosis in heart sections was reduced ≈90% in the treated mice. A decrease of >50% in serum creatine kinase indicated an overall improvement in the muscles. Restoration of membrane integrity was studied directly by measuring the reduction (≈74%) of Evans blue incorporation in the limb muscles of the treated animals, the increase in utrophin level, and the normalization of lipid composition of the heart. In cultures of human myotubes (primary cells and cell line), both prodrugs and HDAC inhibitors increased by 2- to 4-fold the utrophin level, which was correctly localized at the membrane. β-Dystroglycan and embryonic myosin protein levels were also increased. Finally, a 50% reduction in the number of spontaneous Ca(2+) spikes was observed after treatment with NO synthase substrate and HDAC inhibitors. Overall, the beneficial effects were obtained with doses 10 (in vivo) and 5 (in vitro) times lower than those of the salt formulation. Altogether, these data constitute proof of principle of the beneficial effects of low doses of arginine butyrate derivatives on muscular dystrophy, enhancing the NO pathway and inhibiting HDAC.