Skeletal Muscle Pathology Research Articles

Metabolism and Skeletal Muscle Homeostasis in Lung Disease.

There is increased awareness that patients with lung diseases develop muscle dysfunction. Muscle dysfunction is a major contributor to a decreased quality of life in patients with chronic pulmonary diseases. Furthermore, muscle dysfunction exacerbates lung disease outcome, as a decrease in muscle mass and function are associated with increased morbidity, often long after critical illness or lung disease has been resolved. As we are learning more about the role of metabolism in health and disease, we are appreciating more the direct role of metabolism in skeletal muscle homeostasis. Altered metabolism is associated with numerous skeletal muscle pathologies and, conversely, skeletal muscle diseases are associated with significant changes in metabolic pathways. In this review, we highlight the role of metabolism in the regulation of skeletal muscle homeostasis. Understanding the metabolic pathways that underlie skeletal muscle wasting is of significant clinical interest for critically ill patients as well as patients with chronic lung disease, in which proper skeletal muscle function is essential to disease outcome.

The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.

AimsCaveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability.Methods and resultsWe transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect.ConclusionThis study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.

Protective Efficacies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10 Infection.

Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, foot, and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, which can be complicated by severe neurological sequelae. The morbidity and mortality of CVA10-associated HFMD have been increasing in recent years, particularly in the pan-Pacific region. There are limited studies, however, on the pathogenesis and immunology of CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly 5-day-old ICR mice, which developed significant clinical signs, including reduced mobility, lower weight gain, and quadriplegia, with significant pathology in the brain, hind limb skeletal muscles, and lungs of infected mice in the moribund state. The severity of illness was associated with abnormally high expression of the proinflammatory cytokine interleukin 6 (IL-6). Antiviral assays demonstrated that ribavirin and gamma interferon administration could significantly inhibit CVA10 replication both in vitro and in vivo In addition, formaldehyde-inactivated CVA10 whole-virus vaccines induced immune responses in adult mice, and maternal neutralizing antibodies could be transmitted to neonatal mice, providing protection against CVA10 clinical strains. Furthermore, high-titer antisera were effective against CVA10 and could relieve early clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In summary, we present a novel murine model to study CVA10 pathology that will be extremely useful in developing effective antivirals and vaccines to diminish the burden of HFMD-associated disease.IMPORTANCE Hand, foot, and mouth disease cases in infancy, arising from coxsackievirus A10 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe disease and fatalities, however, can occur in some children, necessitating the development of vaccines and antiviral therapies. The present study has established a newborn-mouse model of CVA10 that, importantly, recapitulates many aspects of human disease with respect to the neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon could decrease viral titers in vitro and in vivo Whole-virus vaccines produced immune responses in adult mice, and immunized mothers conferred protection on neonates against challenge from CVA10 clinical strains. Passive immunization with high-titer antisera could also improve survival rates in newborn animals.

Early signs of architectural and biomechanical failure in isolated myofibers and immortalized myoblasts from desmin-mutant knock-in mice

In striated muscle, desmin intermediate filaments interlink the contractile myofibrillar apparatus with mitochondria, nuclei, and the sarcolemma. The desmin network’s pivotal role in myocytes is evident since mutations in the human desmin gene cause severe myopathies and cardiomyopathies. Here, we investigated skeletal muscle pathology in myofibers and myofibrils isolated from young hetero- and homozygous R349P desmin knock-in mice, which carry the orthologue of the most frequent human desmin missense mutation R350P. We demonstrate that mutant desmin alters myofibrillar cytoarchitecture, markedly disrupts the lateral sarcomere lattice and distorts myofibrillar angular axial orientation. Biomechanical assessment revealed a high predisposition to stretch-induced damage in fiber bundles of R349P mice. Notably, Ca2+-sensitivity and passive myofibrillar tension were decreased in heterozygous fiber bundles, but increased in homozygous fiber bundles compared to wildtype mice. In a parallel approach, we generated and subsequently subjected immortalized heterozygous R349P desmin knock-in myoblasts to magnetic tweezer experiments that revealed a significantly increased sarcolemmal lateral stiffness. Our data suggest that mutated desmin already markedly impedes myocyte structure and function at pre-symptomatic stages of myofibrillar myopathies.

BGP-15 Protects against Oxaliplatin-Induced Skeletal Myopathy and Mitochondrial Reactive Oxygen Species Production in Mice.

Chemotherapy is a leading intervention against cancer. Albeit highly effective, chemotherapy has a multitude of deleterious side-effects including skeletal muscle wasting and fatigue, which considerably reduces patient quality of life and survivability. As such, a defense against chemotherapy-induced skeletal muscle dysfunction is required. Here we investigate the effects of oxaliplatin (OXA) treatment in mice on the skeletal muscle and mitochondria, and the capacity for the Poly ADP-ribose polymerase (PARP) inhibitor, BGP-15, to ameliorate any pathological side-effects induced by OXA. To do so, we investigated the effects of 2 weeks of OXA (3 mg/kg) treatment with and without BGP-15 (15 mg/kg). OXA induced a 15% (p < 0.05) reduction in lean tissue mass without significant changes in food consumption or energy expenditure. OXA treatment also altered the muscle architecture, increasing collagen deposition, neutral lipid and Ca2+ accumulation; all of which were ameliorated with BGP-15 adjunct therapy. Here, we are the first to show that OXA penetrates the mitochondria, and, as a possible consequence of this, increases mtROS production. These data correspond with reduced diameter of isolated FDB fibers and shift in the fiber size distribution frequency of TA to the left. There was a tendency for reduction in intramuscular protein content, albeit apparently not via Murf1 (atrophy)- or p62 (autophagy)- dependent pathways. BGP-15 adjunct therapy protected against increased ROS production and improved mitochondrial viability 4-fold and preserved fiber diameter and number. Our study highlights BGP-15 as a potential adjunct therapy to address chemotherapy-induced skeletal muscle and mitochondrial pathology.

Pathologies related to depression in elderly patients

IntroductionThe occurrence of chronic diseases, motor limitation, cognitive impairment and social isolation could be related to late life depression.ObjectiveTo describe the pathologies related to elderly patients with depression. To study possible relationship between depression and others pathologies in elderly patients.MethodsThis retrospective study included 124 geriatric patients enrolled in a private long-term care institution with mean age of 86.2 ± 6.5 years old, mean weight of 60.28 ± 2.00 kg and mean period of hospitalisation of 4.4 ± 2.3 years. For data analysis, double-entry tables and tests of proportion Qui-square were used.ResultsCardiovascular, respiratory, endocrine metabolic, skeletal muscle, sense organs, hematological, digestive tract; neuropsychological and genitourinary, diseases were analyzed.We observed a significant correlation between depression and endocrine-metabolic (P value of 0.0003), sense organs (P value of 9.298 E-5) and skeletal muscle pathologies (P value of 6.843 E-6)ConclusionsWe observed in that population that depression could be prevalent in elderly patients with endocrine-metabolic, sense organs and skeletal muscle pathologies.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Short-term heat stress alters redox balance in porcine skeletal muscle.

Heat stress contributes to higher morbidity and mortality in humans and animals and is an agricultural economic challenge because it reduces livestock productivity. Redox balance and associated mitochondrial responses appear to play a central role in heat stress‐induced skeletal muscle pathology. We have previously reported increased oxidative stress and mitochondrial content in oxidative muscle following 12 h of heat stress. The purposes of this investigation were to characterize heat stress‐induced oxidative stress and changes in mitochondrial content and biogenic signaling in oxidative skeletal muscle. Crossbred gilts were randomly assigned to either thermal neutral (21°C; n = 8, control group) or heat stress (37°C) conditions for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). At the end, their respective environmental exposure, the red portion of the semitendinosus muscle (STR) was harvested. Heat stress increased concentration of malondialdehyde (MDA) following 2 and 4 h compared to thermal neutral and 6 h, which was similar to thermal neutral, and decreased linearly with time. Protein carbonyl content was not influenced by environment. Catalase activity was increased following 4 h of heat stress and superoxide dismutase activity was decreased following 6 h of heat stress compared to thermal neutral conditions. Heat stress‐mediated changes in antioxidant activity were independent of altered protein abundance or transcript expression. Mitochondrial content and mitochondrial biogenic signaling were similar between groups. These data demonstrate that heat stress caused a transient increase in oxidative stress that was countered by a compensatory change in catalase activity. These findings contribute to our growing understanding of the chronology of heat stress‐induced intracellular dysfunctions in skeletal muscle.

Cardiac transplantation in dermatomyositis: A case report and literature review

Background and objectivesCardiac involvement has been well recognized in patients with dermatomyositis (DM) and polymyositis (PM) with a variable frequency between 9 and 72%. However, clinically significant heart involvement in DM/PM is relatively infrequent and there have been rare reports of cardiac transplantation in DM. Our aims were to describe a case of severe cardiac involvement in DM requiring heart transplantation and review the literature of cardiac disease in DM and PM. MethodsA patient with dermatomyositis who was referred to our institution with severe heart failure is described. Pathology of the patient's skeletal and cardiac muscle is reviewed. A MEDLINE database search of reports of cardiac involvement in DM and PM was also conducted. ResultsA 36year-old man with DM presented with severe heart failure to our institution for evaluation of heart transplantation. After a three month hospitalization he underwent successful cardiac transplantation. Pathological examination of his explant heart revealed a pattern of inflammation and damage similar to DM in skeletal muscle. The patient is currently doing well, 20months post-transplant, and is maintained on tacrolimus, cellcept, rituximab, and low dose prednisone. To our knowledge, this is the first case report of heart transplantation in dermatomyositis in which the muscle pathology is similar in both heart and skeletal muscle. ConclusionsSevere cardiac involvement requiring transplantation is rare in dermatomyositis but does occur and appears to be related to a similar inflammatory process as noted in the skeletal muscle.

Airway smooth muscle dysfunction in Pompe (Gaa-/- ) mice.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.

Morpho-functional approaches to highlight skeletal muscle response to cell death inducers

Cell death has been long described, with continuously growing interest, in a variety of tissues and models. Its presence in muscle disorders stimulated us to study it in striated muscle tissue, in particular, in “ in vitro” differentiated myotubes. Apoptosis is a regulated mechanism of cell death which occurs in the absence of plasmalemma disruption, but with cell and organellar component swelling. It plays a crucial role in skeletal muscle pathology, in denervation and disuse [1]. Recently, “autophagy”, an intriguing phenomenon characterized by progressive deletion of cell components, could have a role in skeletal muscle death progression, also acting as a survival mechanism. Here, in vitro C2C12 skeletal muscle cells were exposed to etoposide, H2O2 or staurosporine and cell response has been investigated by means of morpho-functional approaches. Myotubes appeared more resistant than myoblasts to apoptotic induction. In particular, etoposide- or H2O2-treated myoblasts showed characteristic apoptotic features, visualized also in etoposide-treated myotubes characterized by a diffuse DNA cleavage presence. After H₂O₂ exposure, necrotic cells could be observed and myotubes exposed to staurosporine, evidenced late apoptotic features and secondary necrosis. The coexistence of normal and apoptotic nuclei within the same fiber has been demonstrated, in particular in the case of etoposide and staurosporine treatments. The deletion of a single nucleus can occur without the death of the entire myotube, evidencing that a multinucleated cell dies ‘more slowly’. After the majority of stimuli, autophagic vacuoles could be diffusely revealed in myotube cytoplasm. They could preserve muscle cell integrity counteracting chemical treatments, or could activate death pathways. It is the case of etoposide drug, which induced skeletal muscle apoptosis in the presence of an autophagic flux impairment. These findings reveal that apoptosis, necrosis and autophagy coexist in muscle biology and, ultrastructural analyses appear a useful approach for highlighting and describing these processes.

MicroRNA Dysregulation in Aging and Pathologies of the Skeletal Muscle.

Skeletal muscle is one of the biggest organs of the body with important mechanistic and metabolic functions. Muscle homeostasis is controlled by environmental, genetic, and epigenetic factors. Indeed, MiRNAs, small noncoding RNAs robust regulators of gene expression, have and have been shown to regulate muscle homeostasis on several levels: through controlling myogenesis, muscle growth (hypertrophy) and atrophy, as well as interactions of muscle with other tissues. Given the large number of MiRNA target genes and the important role of MiRNAs in most physiological processes and various diseases, MiRNAs may have an enormous potential as therapeutic targets against numerous disorders, including pathologies of muscle. The purpose of this review is to present the current knowledge of the role of MiRNAs in skeletal muscle homeostasis and pathologies and the potential of MiRNAs as therapeutics for skeletal muscle wasting, with particular focus on the age- and disease-related loss of muscle mass and function.

Outside the fiber: interstitial pathology of skeletal muscle in infantile Pompe disease

Outside the fiber: interstitial pathology of skeletal muscle in infantile Pompe disease

MAPK signaling pathways and HDAC3 activity are disrupted during emerin-null myogenic progenitor differentiation

ABSTRACTMutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo.

Effects of Benzothiazolamines on Voltage-Gated Sodium Channels.

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Nav) channels. Riluzole is clinically used as a neuroprotectant in amyotrophic lateral sclerosis. Inhibition of Nav channels by riluzole is voltage-dependent due to preferential binding to inactivated sodium channels. Yet the drug exerts little use-dependent block, probably because it lacks protonable amine. One important property is riluzole ability to inhibit persistent Na+ currents, which likely contributes to its neuroprotective activity. Lubeluzole showed promising neuroprotective effects in animal stroke models, but failed to show benefits in acute ischemic stroke in humans. One important concern is its propensity to prolong the cardiac QT interval, due to hERG K+ channel block. Lubeluzole very potently inhibits Nav channels in a voltage- and use-dependent manner, due to its great preferential affinity for inactivated channels and the presence of a protonable amine group. Patch-clamp experiments suggest that the binding sites of both drugs overlap the local anesthetic receptor within the ion-conducting pathway. Riluzole and lubeluzole displayed very potent antimyotonic activity in a rat model of myotonia, a pathological skeletal muscle condition characterized by high-frequency runs of action potentials. Such results well support the repurposing of riluzole as an antimyotonic drug, allowing the launch of a pilot study in myotonic patients. Riluzole, lubeluzole, and new Nav channel blockers built on the benzothiazolamine scaffold will certainly continue to be investigated for possible clinical applications.

Altered S-nitrosylation of p53 is responsible for impaired antioxidant response in skeletal muscle during aging.

p53 transcriptional activity has been proposed to regulate both homeostasis and sarcopenia of skeletal muscle during aging. However, the exact molecular function of p53 remains to be clearly defined. We demonstrated a requirement of nuclear p53 S-nitrosylation in inducing a nitric oxide/PGC-1α-mediated antioxidant pathway in skeletal muscle. Importantly, mutant form of p53-DNA binding domain (C124S) did not undergo nuclear S-nitrosylation and failed in inducing the expression of antioxidant genes (i.e. SOD2 and GCLC). Moreover, we found that during aging the nuclear S-nitrosylation of p53 significantly declines in gastrocnemius/soleus leading to an impairment of redox homeostasis of skeletal muscle. We suggested that decreased level of nuclear neuronal nitric oxide synthase (nNOS)/Syntrophin complex, which we observed during aging, could be responsible for impaired nuclear S-nitrosylation. Taken together, our data indicate that altered S-nitrosylation of p53 during aging could be a contributing factor of sarcopenia condition and of other skeletal muscle pathologies associated with oxidative/nitrosative stress.

Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model of cardiomyopathy caused by lamin A/C gene mutation

Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration. Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased cardiac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in humans.

From the Cover: Investigative Nonclinical Cardiovascular Safety and Toxicology Studies with BMS-986094, an NS5b RNA-Dependent RNA Polymerase Inhibitor.

BMS-986094, a 2'-C-methylguanosine prodrug that was in development for treatment of chronic hepatitis C infection was withdrawn from Phase 2 clinical trials because of unexpected cardiac and renal adverse events. Investigative nonclinical studies were conducted to extend the understanding of these findings using more comprehensive endpoints. BMS-986094 was given orally to female CD-1 mice (25 and 150 mg/kg/d) for 2 weeks (53/group) and to cynomolgus monkeys (15 and 30 mg/kg/d) for up to 6 weeks (2-3/sex/group for cardiovascular safety, and 5/sex/group for toxicology). Endpoints included toxicokinetics; echocardiography, telemetric hemodynamics and electrocardiography, and tissue injury biomarkers (monkey); and light and ultrastructural pathology of heart, kidney, and skeletal muscle (mouse/monkey). Dose-related and time-dependent findings included: severe toxicity in mice at 150 mg/kg/d and monkeys at 30 mg/kg/d; decreased left ventricular (LV) ejection fraction, fractional shortening, stroke volume, and dP/dt; LV dilatation, increased QTc interval, and T-wave flattening/inversion (monkeys at ≥ 15 mg/kg/d); cardiomyocyte degeneration (mice at 150 mg/kg/d and monkeys at ≥ 15 mg/kg/d) with myofilament lysis/myofbril disassembly; time-dependent proteinuria and increased urine β-2 microglobulin, calbindin, clusterin; kidney pallor macroscopically; and tubular dilatation (monkeys); tubular regeneration (mice 150 mg/kg/d); and acute proximal tubule degeneration ultrastructurally (mice/monkeys); and skeletal muscle degeneration with increased urine myoglobin and serum sTnI. These studies identified changes not described previously in studies of BMS-986094 including premonitory cardiovascular functional changes as well as additional biomarkers for muscle and renal toxicities. Although the mechanism of potential toxicities observed in BMS-986094 studies was not established, there was no evidence for direct mitochondrial toxicity.

α-Linolenic Acid Reduces TNF-Induced Apoptosis in C2C12 Myoblasts by Regulating Expression of Apoptotic Proteins.

Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions such as adjuvant strategy for preventing these conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested as having a positive impact on muscle diseases. We recently demonstrated that a diet enriched with plant derived n3-fatty acid, α-linolenic acid (ALA), exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we here investigate on the expression level of the anti- and pro-apoptotic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of tumor necrosis factor-α (TNF). The results demonstrated that ALA protective effect on C2C12 myoblasts was associated with a decrease in caspase-3 activity and an increase of the Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescuing Bcl-2 expression and to revert Bax translocation to mitochondria both affected in an opposite way by TNF, a major pro-inflammatory cytokine expressed in damaged skeletal muscle. Therefore, ALA counteracts inflammatory signals in the muscle microenvironment and may represent a valuable strategy for ameliorating skeletal muscle pathologies.

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington's Disease.

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

Analysis of Pathological Activities of CCN2/CTGF in Muscle Dystrophy.

CCN2 or connective tissue growth factor (CTGF) is a matricellular protein that regulates several cellular processes. In skeletal muscle, CTGF is a key modulator of fibrogenesis, is increased in pathological conditions such as muscular dystrophies, and plays a major role in the pathology outcome. Overexpression of CTGF in skeletal muscle of wild-type mice results in muscle damage, fibrosis, and reduction of strength. In contrast, a decrease in CTGF in dystrophic mice increases strength and reduces damage and fibrosis. Thus, CTGF is a relevant target to study in skeletal muscle pathology and its possible modulation by different treatments or potential new drugs to develop new strategies for the treatment of muscular dystrophies. We summarize the techniques used to detect CTGF in the skeletal muscle of dystrophic mdx mice.