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Mucolipidosis type IV is a rare autosomal recessive lysosomal storage disorder caused by bi-allelic pathogenic variants in the gene MCOLN1. This encodes for mucolipin-1 (ML1), an endo-lysosomal transmembrane Ca++ channel involved in vesicular trafficking. Although experimental models suggest that defects in mucolipin-1 can cause muscular dystrophy, putatively due to defective lysosomal-mediated sarcolemma repair, the role of mucolipin-1 in human muscle is still poorly deciphered. Elevation of creatine kinase (CK) had been reported in a few cases in the past but comprehensive descriptions of muscle pathology are lacking. Here we report a 7-year-old boy who underwent muscle biopsy due to persistently elevated CK levels (780-15,000 UI/L). Muscle pathology revealed features of a lysosomal storage myopathy with mild regenerative changes. Next generation sequencing confirmed homozygous nonsense variants in MCOLN1. This is a comprehensive pathological description of ML1-related myopathy, supporting the role of mucolipin-1 in muscle homoeostasis.

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm(2) in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF‐β in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

Raw meat is characterized by various natural cellular processes that lead to its chemical content alteration. The purpose of the present study is to evaluate autolysis induced histologic alterations in skeletal muscle tissue of cow elk. In this study the authors used skeletal muscle tissue of 2-year-old cow elk. The tissue was sampled from m. quadriceps femoris. The study results showed that in 48 hours post mortem linear striation in muscle tissues was expressed. But 72 hours post mortem the integrity of muscle fibers degraded and they became inconsistently stained. The obtained data showed that post mortem alterations were characterized by significant degradation and separation of muscle fibers and nuclei and nuclear structures lysis.

Membrane theory of muscle weakness in hereditary muscular dystrophy was described with accent of predominated Ca 2+ ions in skeletal muscle due to destruction of the sarcolemma and other membranes of the different organelles of the skeletal muscle. Showed the role of CT, MRI and phosphorus NMR-spectroscopy as well as the noninvasive the analysis cell-free fetus DNA in maternal plasma for prenatal diagnostic of muscular dystrophies. The different a trial attempts of treatment of muscular dystrophy was described.

The glucose transporter GLUT4 is critical for skeletal muscle glucose uptake in response to insulin and muscle contraction/exercise. Exercise increases GLUT4 translocation to the sarcolemma and t-tubule and, over the longer term, total GLUT4 protein content. Here, we review key aspects of GLUT4 biology in relation to exercise, with a focus on exercise-induced GLUT4 translocation, postexercise metabolism and muscle insulin sensitivity, and exercise effects on GLUT4 expression.

PURPOSE The beneficial effects of exercise training on the cardiovascular system are well known. Because our knowledge of exercise-induced vascular function is still limited, we aimed to uncover the molecular mechanisms conditioning the improved vascular relaxation in muscular arteries. METHODS Male Wistar-Kyoto rats with the same ability to run on a treadmill after maximal exercise tests were allocated to the following two groups: trained (Tr) (treadmill, 50%-60% of maximal capacity, 5 d·wk) and untrained (UnTr). After 13 wk, the femoral arteries were harvested and used for functional, structural, and molecular analyses. RESULTS Acetylcholine (ACh)-induced relaxation and nitric oxide (NO) production were enhanced in arteries from Tr rats compared with UnTr rats. Tr arteries exhibited reduced microRNA (miRNA)-124a expression (whose target is caveolin-1), increased the density of caveolae aligned along the sarcolemma and reduced ACh-induced relaxation in the presence of methyl-β-cyclodextrin, which disrupts caveolae. Higher endothelial NO synthase (eNOS) expression with lower miRNA-155 expression and the posttranslational modification of eNOS (phosphorylation of stimulatory Ser1177 and dephosphorylation of inhibitory Thr495) by the PI3-kinase/Akt1/2/3 pathway also contributed to the higher NO production induced by exercise training. Furthermore, increased Cu/Zn- and extracellular-superoxide dismutase expression and enhanced effects of their pharmacological scavenger activity on the ACh-induced response were observed in Tr arteries. CONCLUSIONS The results of the present study provide a molecular basis for exercise-induced NO bioavailability in healthy femoral arteries. Increased caveolae domain and eNOS expression/activity in Tr arteries are associated with downregulation of miRNA-124a and -155, as well as are involved with higher antioxidant defense, subsequently inducing a favorable endothelium-dependent milieu in Tr arteries.

Objective Hypokalaemic periodic paralysis (HypoPP) is caused by mutations of Cav1.1, and Nav1.4 which result in an aberrant gating pore current. Hyperkalaemic periodic paralysis (HyperPP) is due to a gain-of-function mutation of the main alpha pore of Nav1.4. This study used muscle velocity recovery cycles (MVRCs) to investigate changes in interictal muscle membrane properties in vivo. Methods MVRCs and responses to trains of stimuli were recorded in tibialis anterior and compared in patients with HyperPP(n = 7), HypoPP (n = 10), and normal controls (n = 26). Results Muscle relative refractory period was increased, and early supernormality reduced in HypoPP, consistent with depolarisation of the interictal resting membrane potential. In HyperPP the mean supernormality and residual supernormality to multiple conditioning stimuli were increased, consistent with increased inward sodium current and delayed repolarisation, predisposing to spontaneous myotonic discharges. Conclusions The in vivo findings suggest the interictal resting membrane potential is depolarized in HypoPP, and mostly normal in HyperPP. The MVRC findings in HyperPP are consistent with presence of a window current, previously proposed on the basis of in vitro expression studies. Although clinically similar, HyperPP was electrophysiologically distinct from paramyotonia congenita. Significance MVRCs provide important in vivo data that complements expression studies of ion channel mutations.

Aims Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit β5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases. Methods We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry. Results All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies. Conclusions These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism.

Key points Fetuses with intrauterine growth restriction (IUGR) have reduced muscle mass that persists postnatally, which may contribute to their increased risk for adult onset metabolic diseases, such as diabetes and obesity. Amino acid transporter-mediated histidine uptake and system L amino acid transporter activity were similar in sarcolemmal membranes isolated from control and IUGR hindlimb skeletal muscle. Activity of Na+ K+ -ATPase, which is responsible for establishing the sodium gradient necessary for system A and N amino acid transporter function, was significantly reduced in IUGR skeletal muscle sarcolemma compared to control. ATP content was lower in IUGR skeletal muscle. Expression and phosphorylation of proteins in the mechanistic target of rapamycin pathway were similar in control and IUGR skeletal muscle homogenate. Our data suggest that lower Na+ K+ -ATPase activity, which reduces the driving force for active amino acid transport, and lower ATP availability contribute to reduced amino acid uptake and protein synthesis in IUGR fetal skeletal muscle. Abstract Fetuses with intrauterine growth restriction (IUGR) have lower muscle mass that persists postnatally. Using a sheep model of placental insufficiency and IUGR, we have previously demonstrated lower net total uptake of amino acids by the fetal hindlimb and lower skeletal muscle protein synthesis rates. To investigate the mechanisms underlying these changes, we tested the hypothesis that ex vivo amino acid transporter and Na+ K+ -ATPase activity is reduced, and ex vivo ATP levels are lower in hindlimb skeletal muscle of the IUGR fetus. We developed a novel protocol to measure transporter-mediated histidine uptake, system L amino acid transporter activity and Na+ K+ -ATPase activity using sarcolemmal membranes isolated from hindlimb muscle of control (CON, n = 11-12) and IUGR (n = 12) late gestation fetal sheep. We also determined ATP content and the activity of insulin and mechanistic target of rapamycin (mTOR) signalling, which are involved in regulating cellular amino acid uptake and protein synthesis, by measuring the expression and phosphorylation of AKT, 4E-BP1, eIF2α, AMPKα, p70 S6 kinase and rpS6 in muscle homogenates. Transporter-mediated histidine uptake and system L activity were similar in control and IUGR sarcolemma, although ex vivo Na+ K+ -ATPase activity was lower by 64% (P = 0.019) in IUGR sarcolemma. ATP content was lower by 25% (P = 0.007) in IUGR muscle. Insulin, AMPK, and mTOR signalling activity was similar in control and IUGR muscle. We speculate that reduced muscle sarcolemmal Na+ K+ -ATPase activity and lower ATP content diminishes the sodium gradient in vivo, resulting in a reduced driving force for sodium-dependent transporters and subsequently lower muscle amino acid uptake.

To study patients with sporadic inclusion body myositis (sIBM) with muscle velocity recovery cycles (MVRC) to assess muscle membrane excitability, pathophysiological mechanisms and potential biomarkers of this disorder.MVRC were recorded from 20 individuals with sIBM from tibialis anterior (TA) and rectus femoris (RF) muscles. Excitability parameters were compared with MVRC data obtained from 22 normal controls50 years.Muscle relative refractory period was prolonged in both TA (6.4 ms vs 4.4 ms, P 0.001) and RF (7.1 ms vs 3.9 ms, P 0.001) of sIBM affected muscle when compared to controls. Early supernormality was reduced in both TA (3.6% vs 8.8% P = 0.001) and in RF (mean 5.4% vs 13% P 0.001). Late supernormality was only decreased significantly in sIBM affected TA (1.8% vs 3.6% P = 0.001) but not in RF. No consistent correlations between MVRC parameters and clinical markers of sIBM disease severity were found.The resting sarcolemmal muscle membrane potential of sIBM muscle is depolarized relative to that of normal controls, which may be related to intramuscular amyloid deposition in sIBM.Sarcolemmal depolarization may play a role in muscle dysfunction and weakness observed in sIBM patients.

(31)Phosphorus magnetic resonance spectroscopy ((31)P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid–base status, and free intracellular magnesium ([Mg(2+)]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine (31)P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on (31)P-MRS indices in dystrophic muscle. In vivo (31)P-MRS spectra were acquired from the posterior hindlimb muscles in young (4–10 weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by (31)P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg(2+)] were calculated. (1)H(2)O-T(2) was measured using single-voxel (1)H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated (1)H(2)O-T(2) (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg(2+)] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg(2+)] and a lower (p = 0.048) intracellular [H(+)] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that (31)P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.

Desminopathy the most common intermediate filament disease in humans. Desmin is an essential part of the filamentous network that aligns myofibrils, anchors nuclei and mitochondria, and connects the z-discs and the sarcolemma. We created a rat model with a mutation in R349P DES, analog to the most frequent R350P DES missense mutation in humans. To examine the effects of a chronic, physiological exercise stimulus on desminopathic muscle, we subjected R349P DES rats and their wildtype (WT) and heterozygous littermates to a treadmill running regime. We saw significantly lower running capacity in DES rats that worsened over the course of the study. We found indicators of increased autophagic and proteasome activity with running in DES compared to WT. Stable isotope labeling and LC-MS analysis displayed distinct adaptations of the proteomes of WT and DES animals at baseline as well as with exercise: While key proteins of glycolysis, mitochondria and thick filaments increased their synthetic activity with running in WT, these proteins were higher at baseline in DES and did not change with running. The results suggest an impairment in adaption to chronic exercise in DES muscle and a subsequent exacerbation in the functional and histopathological phenotype.

Background: The potassium channel encoded by the ether-a-gogo-related gene 1A (erg1a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis, however, to our knowledge, ERG1A has not been reported in human skeletal muscle. Methods and Results: Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%, p &lt, 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% (p &lt, 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle. Conclusions: The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic neuromuscular disorder. The variability in neurologic deficits in DMD patients may be explained by the fact that (1) dystrophin containing complexes in the brain are more stable than dystrophin containing complexes in the muscle (2) neurons are not affected by the same stresses as muscle and (3) neurons have a greater capacity to buffer increases in intracellular calcium levels. In the muscle, the loss of dystrophin and subsequent loss of dystrophin-associated proteins (DAPs) affects the stability of the dystrophin-glycoprotein complex and calcium ion channels. It causes the sarcolemma of the muscle to tear and calcium ion leak. The subsequent calcium influx leads to calcium dependant proteolysis. In the brain, the structure of the dystrophin-containing complexes is completely different from the muscle. There are several dystrophin isoforms that combine with a completely different set of proteins compared to the muscle to form several different dystrophin-containing complexes. In addition, the loss of dystrophin does not affect the expression of DAPs. The heterogeneity of dystrophin-containing complexes and the continued expression of DAPs will result in more stable dystrophin-containing complexes in the DMD brain. Muscles are under more stress than neurons as they undergo contractions. This combined with txhe fact that the neurons have a better ability to buffer increases in calcium would suggest that neurons are less likely to be damaged despite the loss of dystrophin.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild-type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P

The highly organised transverse tubule (t-tubule) network consisting of invaginations of the cell sarcolemma facilitates synchronous cardiac myocyte contraction. This study aimed to investigate post-myocardial infarction (MI) t-tubule remodelling in infarct border and remote regions in a translationally relevant ischaemia reperfusion injury MI model. Six adult sheep were used in this study (n=3 MI, n=3 control). Eight weeks after MI, left ventricular tissue was collected from the remote and border MI regions and from control sheep, processed and imaged using 3D scanning electron microscopy. The t-tubule network was manually segmented using 3dmod. One-way ANOVA with Tukey’s post-hoc correction, unpaired t-tests or Mann-Whitney U test were used where appropriate. Marked disorganisation of the t-tubule network was observed in the border region following MI. Quantitative analysis revealed that in comparison to the control sheep myocardium, the MI border zone had a decreased t-tubule count (0.07 ± 0.007 tubules per μm3 in control vs 0.05 ± 0.004 tubules per μm3 in border; p = 0.02) and showed t-tubule dilation (405 ± 22 nm in control vs 533 ± 30 nm in border; p = 0.02). Whilst there was minimal disorganisation and loss of t-tubules in the MI remote region, we observed increased t-tubule length as a fraction of the cell diameter (0.41 ± 0.04 in control vs 0.56 ± 0.04 in remote; p = 0.045). In addition to gross t-tubule remodelling, we also noted post-MI fragmentation of t-tubules, particularly in the border region. In comparison to control, the number of t-tubule fragments per μm3 was increased in the post MI heart (control, 0.17 ± 0.1 fragments per μm3; border, 2.21 ± 0.7 fragments per μm3; remote, 1.20 ± 0.4 fragments per μm3; p = 0.04 border vs control; p = 0.02 remote vs control). The volume occupied by fragments as a percentage of the cell volume was also higher following MI (control, 0.003 ± 0.002 %; border, 0.071 ± 0.023 %; remote 0.014 ± 0.005 %; p = 0.003 border vs control; p = 0.013 border vs remote). Whilst there was no difference in fragments density between the remote and border regions, there was an increase in the volume of cell occupied by fragments in the MI border region compared to remote. This is explained by a larger average fragment volume in the border region (0.04 ± 0.006 μm3 in border vs 0.01 ± 0.002 μm3 in remote; p Our research shows remodelling of the t-tubule network in the post-MI sheep myocardium. We noted reduced t-tubule count, t-tubule fragmentation, and dilation of remaining t-tubules. Importantly our work shows that these changes occur in a regional manner, being most pronounced in the border region. These changes may reflect regional wall stresses post-MI, and we speculate that our observations may result in region-specific changes to systolic calcium and contractility post-MI. Conflict of Interest Authors declare that there is no conflict of interest.

Strabismus is an ocular disorder characterized by partial or complete inability to keep eye alignment. It represents a very common ocular problem at ophthalmology clinics worldwide. The current study aimed to show the most encountered ultrastructural changes in extraocular muscles (EOMs) collected from patients with different forms of strabismus. Nine specimens of EOMs were collected from five patients during strabismus correction surgery and processed for light and electron microscopy examinations. Histologically, skeletal muscle fibers in normal EOMs appeared tight and normally arranged with clear striations. In strabismic muscles, the fibers appeared disarranged, and atrophied, swollen and disintegrated in some situations. By transmission electron microscopy, normal EOMs were formed of skeletal muscle fibers with intact basal membrane and sarcolemma, tightly aligned myofibrils with well-arranged sarcomeres, Z line and H zone, and normally distributed mitochondria. On the other hand, strabismic EOMs revealed vacuolation and degeneration of myofibrils, accumulation of lipid droplets, subsarcolemmal inclusions and clustering of mitochondria. EOMs obtained from a Down syndrome patient with V-pattern infantile esotropia showed extensive vacuolation and disintegration of myofibrils, and extra- and intracellular deposition of collagen fibers. Interestingly, some skeletal muscle cells exhibited features of autophagic cell death with a trial of engulfing process by neighboring cells. In conclusion, our study traces some characteristic ultrastructural changes in strabismic EOMs, most notably, extensive vacuolation, clustering of mitochondria, degeneration of myofibrils and autophagic changes. These changes might be emphasized as possibly secondary to strabismus.

The purpose of the study was the histological changes analysis in the gastrocnemius muscle under the conditions of experimental ischemia of different duration complicated by chronic alcoholization. The experiment was conducted on inbred Wistar line rats with 140 – 160 g body weight. The animals were divided into four groups: control; animals with ischemia (1, 2 and 3 hours ischemia duration); alcoholized animals and alcoholized animals with experimentally induced 1, 2, 3 hours ischemia. Chronic alcohol intoxication of the animals was achieved by intragastric delivery of 40 % ethyl alcohol for 30 days. Unilateral vascular ischemia was induced by tourniquet ligating of the main arteries. Histologic processing of the tissue was carried out according to standard histological methods, stained with Van Gison method. Morphometric indices (thickness of muscle fibers and the size of interfibrillary space) were measured using VideoTest Morphology 5,0 software. It was found that the level of expression of morphological changes directly depends on the duration of ischemia. With its increase, destructive changes of the gastrocnemius muscle become more evident: invaginations on the sarcolmes of fibers are more clearly visible, myofibrils are disorganized, the amount of collagen increases and the scar formation starts. After three-hour ishemia the cytological signs of necrosis are observed, but in many areas the typical cross-striped and the normal structure of muscle fibers was preserved. Under the conditions of chronic alcoholization and ischemia of different duration, the structure and course of partially atrophied after alcoholic myopathy fibers are broken, degenerative changes in the integrity of sarcolemes are observed, some fibers decrease the volume of myofibrils, there endomysium swells and its necrosis starts, myofibrils dissociate. The presence of neutrophils between myofibrils and diffuse hemorrhage has been observed, nevertheless, in some sites a typical cross-striped structure was preserved. The obtained results show the negative synergy of myotoxic effects of ethyl alcohol and the destructive effects of vascular ischemia on fibers of rat m. gastrocnemius.

INTRODUCTION The sarcolemmal resting membrane potential (RMP) affects muscle excitability, contractility, and force generation. However, there are limited In vivo data on the normal RMP of the human sarcolemma between muscles. We hypothesize that the in vivo RMP may differ between human muscles with different physiological roles. METHODS Muscle velocity recovery cycles were recorded from a proximal antigravity muscle, the rectus femoris, and compared with paired recordings from a distal non-antigravity muscle, the tibialis anterior, in 34 normal individuals. RESULTS Significant differences in muscle relative refractory period (3.55 millseconds vs 3.87 milliseconds, P = .002), early supernormality (14.22% vs 10.50%, P

Background:Some studies have linked the use of paracetamol (PAR) with adverse effects like wheezing, exacerbation of asthma symptoms and other respiratory problems. Other studies are inconclusive or deny this correlation. This makes the association between PAR and airway hypersensitivity very controversial and still under debate. Objective: This work investigated if chronic treatment with PAR in rats could directly affect the contraction and relaxation for different stimulus in isolated airways. Methods:Rats were treated for 2 weeks with PAR (400 mg/Kg, v.o.). The blood was collected for biochemical analysis (alanine aminotransferase (ALT), aspartate aminotransferase (AST), TBARs reaction and glutathione) and isolated tracheal rings were prepared in organ bath to measure isometric tone after contractile and relaxant stimulus. Results: Hepatic enzymes (ALT, AST) and lipid peroxidation were increased after PAR-treatment, while glutathione was decreased. Rats do not present any alteration in airway myocytes responsiveness, either to contractile or relaxant stimulus (i.e. cholinergic agonist, membrane depolarization, Ca2+ influx across sarcolemma, internal Ca2+ release from sarcoplasmic reticulum, Ca2+ channel blocking, β-agonist and NOmediating relaxation). Conclusion:Despite increased oxidative stress and reduced antioxidant defense, chronic treatment with PAR does not induce airway hypersensitivity or risk of asthma in rats.

Highlights • In myocardial ischemia, the integrity of the cardiac sarcolemma is severely stressed in the critical earliest moments upon reperfusion. Bolstering sarcolemma integrity improves myocyte survival. • This review focuses on cardiac sarcolemma stability and its role as a therapeutic target in ischemia-reperfusion injury. • Synthetic block copolymers have been shown to interface with the muscle membrane to confer membrane stabilization during stress. • Integrated multidisciplinary research teams, spanning cardiology, physiology, chemistry, and chemical engineering are essential to guide future mechanistic and translational studies of novel chemical-based membrane stabilizers for preserving viable heart muscle during ischemia-reperfusion injury in human patients., Summary The phospholipid bilayer membrane that surrounds each cell in the body represents the first and last line of defense for preserving overall cell viability. In several forms of cardiac and skeletal muscle disease, deficits in the integrity of the muscle membrane play a central role in disease pathogenesis. In Duchenne muscular dystrophy, an inherited and uniformly fatal disease of progressive muscle deterioration, muscle membrane instability is the primary cause of disease, including significant heart disease, for which there is no cure or highly effective treatment. Further, in multiple clinical forms of myocardial ischemia-reperfusion injury, the cardiac sarcolemma is damaged and this plays a key role in disease etiology. In this review, cardiac muscle membrane stability is addressed, with a focus on synthetic block copolymers as a unique chemical-based approach to stabilize damaged muscle membranes. Recent advances using clinically relevant small and large animal models of heart disease are discussed. In addition, mechanistic insights into the copolymer-muscle membrane interface, featuring atomistic, molecular, and physiological structure-function approaches are highlighted. Collectively, muscle membrane instability contributes significantly to morbidity and mortality in prominent acquired and inherited heart diseases. In this context, chemical-based muscle membrane stabilizers provide a novel therapeutic approach for a myriad of heart diseases wherein the integrity of the cardiac muscle membrane is at risk., Central Illustration

The loss of dystrophin produces a mechanically fragile sarcolemma, causing muscle membrane disruption and muscle loss. The degree to which exercise alters muscular dystrophy has been evaluated in humans with Duchenne Muscular Dystrophy (DMD) and in mouse models including the mdx mouse but with inconsistent findings. We now examined two different levels of exercise, moderate and low intensity, in the mdx mouse model in the DBA2J background. mdx mice at 4–5 months of age were subjected to two different doses of exercise. We found a dose-dependent benefit for low and moderate exercise, defined as 4 m/min or 8 m/min, for 30 minutes three times a week. After six months, exercised mdx mice showed improved tetanic and specific force compared to the sedentary group. We also observed increased respiratory capacity manifesting as greater minute volume, as well as enhanced cardiac function mitigating the decline of fractional shortening that is normally seen. Exercised mdx mice also showed a dose-dependent increase in serum adiponectin with a concomitant reduced adipocyte cross sectional area. These findings identify moderate intensity exercise as a means to improve muscle performance in the mdx DBA2J mice and suggest serum adiponectin as a biomarker for beneficial exercise effect in DMD.

Duchenne muscle dystrophy (DMD), characterized by progressive loss of muscle architecture and function, is caused by lack of dystrophin expression in the sarcolemma of myofibers. Recurrent muscle damages in DMD patients and DMD mouse model, mdx, lead to chronic inflammation, which further exacerbate the muscle histopathology. It is critical to find a successful therapy that will improve the histopathology of muscles of DMD patients and restore skeletal muscle function. TIPE2 (tumor necrosis factor α-induced-protein 8-like 2), identified as a negative regulator of immune response, has been found to be expressed in various types of immune cells including macrophages. However, whether and how TIPE2 plays a role in the DMD-related inflammation remains unknown. In this study, we found the basal expression levels of TIPE2 in skeletal muscle from mdx mice are significantly lower than wild-type (WT) mice. To investigate the potential beneficial effect of TIPE2 in muscular dystrophy, we performed intramuscular injection of adeno-associated virus 9 carrying the TIPE2 gene in mdx mice. Our results indicate that the restoration of TIPE2 ameliorates muscular dystrophy phenotype through a reduction in inflammation and fibrosis. In addition, TIPE2 overexpression dramatically decreased the proliferation and migration rate of macrophages, as well as repressed the secretion of pro-inflammatory factors induced by tumor necrotic factor alpha. Taken together, our results indicate that a reduction of TIPE2 expression is observed in dystrophic skeletal muscle, when compared to WT and more importantly that TIPE2 gene delivery may provide as a novel anti-inflammatory therapy to alleviate the muscle weakness in DMD patients.

Background Niemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo. Methods Utilizing the NPDA/B mouse model ASM−/− and wild type (WT) littermates, we performed excitation-contraction coupling/Ca2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury. Results ASM−/− flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca2+]i in response to electrical stimulation and early failure in sustaining [Ca2+]i during repeated tetanic contractions. When injured mechanically by needle passage, ASM−/− flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM−/− mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury. Conclusions Skeletal muscle fibers from ASM−/− mice have an impairment in intracellular Ca2+ handling that results in reduced Ca2+ mobilization and a more rapid decline in peak Ca2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction-induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease. Electronic supplementary material The online version of this article (10.1186/s13395-018-0187-5) contains supplementary material, which is available to authorized users.

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca2+ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca2+ handling, including increased resting Ca2+ and slow Ca2+ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca2+ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca2+ cycling. We tested the hypothesis that enhanced cardiac Ca2+ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca2+ transient peak height and faster Ca2+ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca2+ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca2+ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca2+ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca2+ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca2+ handling in dystrophic cardiomyopathy. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/unregulated-ca2-cycling-exacerbates-dmd-cardiomyopathy/ .

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in dystrophin gene, affecting 1/3000 males. Gene therapy has been proven to ameliorate dystrophic pathology. To investigate therapeutic benefits from long-term effect of human mini-dystrophin and functional outcomes, transgenic mdx mice (Tg-mdx) containing a single copy of human mini-dystrophin (∆hDys3849) gene, five rods (Rods1-2, Rods22-24), and two hinges (H1 and H4) driven by a truncated creatine-kinase promoter (dMCK) in a recombinant adeno-associated viral vector (rAAV) backbone, were generated and used to determine gene expression and improvement of muscle function. Human mini-dystrophin gene expression was found in a majority of the skeletal muscles, but no expression in cardiac muscle. Dystrophin-associated glycoproteins (DAGs) such as sarcoglycans and nNOS were restored at the sarcolemma and coincided with human mini-dystrophin gene expression at the ages of 6, 10, and 20 months; Morphology of dystrophic muscle expressing the human mini-dystrophin gene was improved and central nuclei were reduced. Myofiber membrane integrity was improved by Evans blue dye test. Improvement in treadmill running and grip force was observed in transgenic mice at 6 months. Tetanic force and specific force of tibialis anterior (TA) muscle were significantly increased at the ages of 6, 10, and 20 months. Pseudohypertrophy was not found in TA muscle at 10 and 20 months when compared with wild-type C57 (WT) group. This study demonstrated that the long-term effects of human mini-dystrophin effectively ameliorated pathology and improved the functions of the dystrophic muscles in the transgenic DMD mouse model.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7 days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg−1 day−1) (CONL), 7 days of tail‐traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading‐induced loss of muscle fiber cross‐sectional area (CSA) and fiber‐type shift. Losartan mitigated unloading‐induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a., Angiotensin receptor blockade (ARB) reduced unloading‐induced atrophy of skeletal muscle. Losartan mitigated assembly of the Nox2 complex, and thus oxidative stress ARB attenuated loss of sarcolemmal nNOSµ and nuclear levels of FoxO3a.

Made available in DSpace on 2022-04-29T08:35:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 This study investigated the dependence of contraction from extracellular Ca2+, the presence of a functional sarcoplasmic reticulum (SR), and the effects of β-adrenergic stimulation using isometric cardiac muscle preparations. Moreover, the expression of Ca2+-handling proteins such as SR-Ca2+-ATPase (SERCA), phospholamban (PLN), and Na+/Ca2+ exchanger (NCX) were also evaluated in the ventricular tissue of adult African sharptooth catfish, Clarias gariepinus, a facultative air-breathing fish. In summary, we observed that (1) contractility was strongly regulated by extracellular Ca2+; (2) inhibition of SR Ca2+-release by application of ryanodine reduced steady-state force production; (3) ventricular myocardium exhibited clear post-rest decay, even in the presence of ryanodine, indicating a decrease in SR Ca2+ content and NCX as the main pathway for Ca2+ extrusion; (4) a positive force-frequency relationship was observed above 60 bpm (1.0 Hz); (5) ventricular tissue was responsive to β-adrenergic stimulation, which caused significant increases in twitch force, kept a linear force-frequency relationship from 12 to 96 bpm (0.2 to Hz), and improved the cardiac pumping capacity (CPC); and (6) African catfish myocardium exhibited similar expression patterns of NCX, SERCA, and PLN, corroborating our findings that both mechanisms for Ca2+ transport across the SR and sarcolemma contribute to Ca2+ activator. In conclusion, this fish species displays great physiological plasticity of E-C coupling, able to improve the ability to maintain cardiac performance under physiological conditions to ecological and/or adverse environmental conditions, such as hypoxic air-breathing activity. Department of Physiological Sciences Federal University of São Carlos (UFSCar), Via Washington Luís km 235, 13565-905 São Carlos Joint Graduate Program in Physiological Sciences Federal University of São Carlos – UFSCar/São Paulo State University, UNESP Campus Araraquara Joint Graduate Program in Physiological Sciences Federal University of São Carlos – UFSCar/São Paulo State University, UNESP Campus Araraquara

Mitochondrial dysfunction is considered to be a major cause of sarcopenia, defined as age-related muscle fiber atrophy and muscle weakness, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in aging muscles. However, the role of mitochondrial dysfunction in sarcopenia is not fully elucidated. Although previous studies have suggested that aging has a fiber type-specific effect on mitochondrial function, little is known about mitochondrial changes in individual fiber types. Here, we used C57BL/6NCr female mice to identify fiber type-specific pathological changes, examine the significance of pathological changes in sarcopenia, and identify possible mechanisms behind mitochondrial changes in slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL). We observed reduced type I fiber-specific mitochondrial respiratory enzyme activity, impaired respiration, and subsarcolemmal mitochondrial accumulation in aged SOL, which was different from RRFs. These pathological alterations were not directly associated with fiber atrophy. Additionally, we found increased oxidative stress markers in aged SOL, suggesting that oxidative stress is involved in the pathological and functional changes in mitochondria. Meanwhile, obvious mitochondrial changes were not seen in aged EDL. Thus, age-related mitochondrial dysfunction is specific to the fiber type and may correlate with the muscle quality rather than the muscle mass.

Background: Immobilization stress occurs in various clinical situations as chronic inflammatory and neuromuscular diseases leading to disuse muscle atrophy. Recovery of skeletal muscle after immobilization is slow and incomplete. Platelet-rich plasma (PRP) was previously used in improving traumatic muscle injuries, yet, its role in accelerating muscle recovery upon immobilization stress has not been studied. Aim of the work: to study the possible adjuvant effect of PRP on skeletal muscle atrophy during recovery after immobilization stress in rat using different histological and immunohistochemical teschniques.Material and Methods: Thirty-six adult male rats were equally divided into four groups; control, immobilization stress (restrained in reduced sized cages for 4 weeks), recovery alone (4 weeks), and recovery & PRP groups. Skeletal muscle specimens were processed for histological and immunohistochemical studies.Results: Immobilized group showed splitting of the muscle fibers, internalization of the nuclei, loss of striation, undulated disrupted sarcolemma with signs of inflammation. Ultrastructural examination revealed myocytes with indented nuclei, mitochondria of abnormal shape and size, atrophied myofibrils with loss of myofilaments and splitting of myofibrils. A significant reduction of both mean muscle fiber cross sectional area and area percentage was reported together with a significant downregulation in desmin immunohistochemical expression. Recovery group revealed persistence of histological and immunohistochemical alterations. Recovery & PRP group showed a near normal morphology together with a non-significant difference in desmin immunohistochemical expression compared to control.Conclusion: PRP offered a promising improvement of skeletal muscle atrophy after immobilization stress. It is recommended to apply adjuvant PRP therapy with recovery rather than recovery alone.

Duchenne muscular dystrophy (DMD) is a life-threatening neuromuscular disease caused by the lack of dystrophin, resulting in progressive muscle wasting and locomotor dysfunctions. By adulthood, almost all patients also develop cardiomyopathy, which is the primary cause of death in DMD. Although there has been extensive effort in creating animal models to study treatment strategies for DMD, most fail to recapitulate the complete skeletal and cardiac disease manifestations that are presented in affected patients. Here, we generated a mouse model mirroring a patient deletion mutation of exons 52-54 (Dmd Δ52-54). The Dmd Δ52-54 mutation led to the absence of dystrophin, resulting in progressive muscle deterioration with weakened muscle strength. Moreover, Dmd Δ52-54 mice present with early-onset hypertrophic cardiomyopathy, which is absent in current pre-clinical dystrophin-deficient mouse models. Therefore, Dmd Δ52-54 presents itself as an excellent pre-clinical model to evaluate the impact on skeletal and cardiac muscles for both mutation-dependent and -independent approaches., Summary: The Dmd Δ52-54 mouse model, which carries a deletion of Dmd exons 52-54, emulates Duchenne muscular dystrophy disease progression in skeletal muscle and has early onset of cardiac functional abnormalities.

Dystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy., Editor's choice: The newly established rat model carrying in-frame mutations in the Dmd gene exhibits the dystrophic phenotype and abnormal dystrophin expression profile, similar to patients with Becker muscular dystrophy.

Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 μg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

Abundant evidence reveals that activation of the renin-angiotensin system promotes skeletal muscle atrophy in several conditions including congestive heart failure, chronic kidney disease, and prolonged mechanical ventilation. However, controversy exists about whether circulating angiotensin II (AngII) promotes skeletal muscle atrophy by direct or indirect effects, the centerpiece of this debate is the issue of whether skeletal muscle fibers express AngII type 1 receptors (AT1Rs). While some investigators assert that skeletal muscle expresses AT1Rs, others argue that skeletal muscle fibers do not contain AT1Rs. These discordant findings in the literature are likely the result of study design flaws and additional research using a rigorous experimental approach is required to resolve this issue. We tested the hypothesis that AT1Rs are expressed in both human and rat skeletal muscle fibers. Our premise was tested using a rigorous, multi-technique experimental design. First, we established both the location and abundance of AT1Rs on human and rat skeletal muscle fibers by means of an AngII ligand-binding assay. Second, using a new and highly selective AT1R antibody, we carried out Western blotting and determined the abundance of AT1R protein within isolated single muscle fibers from humans and rats. Finally, we confirmed the presence of AT1R mRNA in isolated single muscle fibers from rats. Our results support the hypothesis that AT1Rs are present in both human and rat skeletal muscle fibers. Moreover, our experiments provide the first evidence that AT1Rs are more abundant in fast, type II muscle fibers as compared with slow, type I fibers. Together, these discoveries provide the foundation for an improved understanding of the mechanism(s) responsible for AngII-induced skeletal muscle atrophy.

When muscle biopsies first began to be used routinely in research on exercise physiology five decades ago, it soon become clear that the muscle content of glycogen is an important determinant of exercise performance. Glycogen particles are stored in distinct pools within the muscles, but the role of each pool during exercise and how this is affected by diet is unknown. Here, the effects of diet and exercise on these pools, as well as their relationship to endurance during prolonged cycling were examined. We demonstrate here that an improved endurance capacity with high carbohydrate loading is associated with a temporal shift in the utilization of the distinct stores of glycogen pools and is closely linked to the content of the glycogen pool closest to actin and myosin (intramyofibrillar glycogen). These findings highlight the functional importance of distinguishing between different subcellular microcompartments of glycogen in individual muscle fibres ABSTRACT: In muscle cells, glycogen is stored in three distinct subcellular pools: between or within myofibrils (inter- and intramyofibrillar glycogen, respectively) or beneath the sarcolemma (subsarcolemmal glycogen) and these pools may well have different functions. Here, we investigated the effect of diet and exercise on the content of these distinct pools and their relation to endurance capacity in type 1 and 2 muscle fibres. Following consumption of three different diets (MIX, high in carbohydrate = HIGH, or low in carbohydrate = LOW) for 72 h, 11 men cycled at 75% of VCO2 max until exhaustion. The volumetric content of the glycogen pools in muscle biopsies obtained before, during, and after exercise were quantified by transmission electron micrograph. Mean (SD) time to exhaustion was 150 (30), 112 (22), and 69 (18) minutes in the HIGH, MIX, and LOW trials, respectively (P < 0.001). As shown by multiple regression analyses, the intramyofibrillar glycogen content in type 1 fibres, particularly after 60 min of exercise, correlated the strongest with time to exhaustion. In the HIGH trial, intramyofibrillar glycogen was spared during the initial 60 min of exercise, which was associated with above levels and utilisation of subsarcolemmal glycogen above normal. In all trials, utilisation of subsarcolemmal and intramyofibrillar glycogen was more pronounced than that of intermyofibrillar glycogen in relative terms. In conclusion, the muscle pool of intramyofibrillar glycogen appears to be the most important for endurance capacity in humans. In addition, a local abundance of subsarcolemmal glycogen reduces the utilization of intramyofibrillar glycogen during exercise. This article is protected by copyright. All rights reserved.

Background Bisphosphonates (BPs) are widely used as anti-bone-resorptive agents. Despite the great benefits of BPs, they may cause local and systemic adverse side effects. Objectives The aim of this study was to evaluate the histopathological effect zoledronic acid (ZA), which belongs to BPs, has on the intrinsic tongue muscles in a rat model. Material and methods A total of 30 adult male albino rats were divided into 3 groups (10 rats each): group I served as a control; group II was given an intraperitoneal (i.p.) injection of 0.2 mg/kg of ZA once per week for 3 weeks; and group III received the same dosage of ZA, but for 8 weeks. After the animals were euthanized, the tongue tissue was dissected and examined histologically, histochemically and immunohistochemically. Results Histologically, a normal architecture of the muscle fascicles was observed in the control group. Group II showed degenerated muscle fibers with an indistinct sarcolemma. In group III, the muscle fibers were degenerated with severe sarcoplasmic dissolution. The histochemical examination using Masson's trichrome (MT) demonstrated a significant increase in collagen fibers in groups II and III as compared to the control group. The immunohistochemical results revealed a statistically significantly higher expression of nuclear factor kappa B (NF‑κB) in the ZA-treated groups (II and III) as compared to the control group, with the highest mean value recorded in group III. Conclusions Zoledronic acid induced histopathological changes to the intrinsic tongue muscles, and this effect was exaggerated with a longer duration of administration.

We herein report a 56-year-old Japanese woman who had been diagnosed with hereditary angioedema. She experienced progressing muscle weakness and pain in the upper and lower extremities. Blood tests revealed a marked increase in creatine kinase levels; however, myositis-specific autoantibodies were not detected. Serum C1-inhibitor activity and C4 levels were low. A muscle biopsy showed mild muscle fiber necrosis and C5b-9 deposition in the endomysial capillary vessel walls and sarcolemma, mimicking necrotizing myopathy. These results suggest that C1-inhibitor deficiency induces myositis-like symptoms through the activation of the complement pathway and deposition of the membrane attack complex in the muscles.

AIMS/HYPOTHESIS: Subcellular localisation is an important factor in the known impact of bioactive lipids, such as diacylglycerol and sphingolipids, on insulin sensitivity in skeletal muscle; yet, the role of localised intramuscular triacylglycerol (IMTG) is yet to be described. Excess accumulation of IMTG in skeletal muscle is associated with insulin resistance, and we hypothesised that differences in subcellular localisation and composition of IMTG would relate to metabolic health status in humans. METHODS: We evaluated subcellular localisation of IMTG in lean participants, endurance-trained athletes, individuals with obesity and individuals with type 2 diabetes using LC-MS/MS of fractionated muscle biopsies and insulin clamps. RESULTS: Insulin sensitivity was significantly different between each group (athletes>lean>obese>type 2 diabetes; p

Objective Exercise is a cornerstone in the management of skeletal muscle insulin-resistance. A well-established benefit of a single bout of exercise is increased insulin sensitivity for hours post-exercise in the previously exercised musculature. Although rodent studies suggest that the insulin-sensitization phenomenon involves enhanced insulin-stimulated GLUT4 cell surface translocation and might involve intramuscular redistribution of GLUT4, the conservation to humans is unknown. Methods Healthy young males underwent an insulin-sensitizing one-legged kicking exercise bout for 1 h followed by fatigue bouts to exhaustion. Muscle biopsies were obtained 4 h post-exercise before and after a 2-hour hyperinsulinemic-euglycemic clamp. Results A detailed microscopy-based analysis of GLUT4 distribution within seven different myocellular compartments revealed that prior exercise increased GLUT4 localization in insulin-responsive storage vesicles and T-tubuli. Furthermore, insulin-stimulated GLUT4 localization was augmented at the sarcolemma and in the endosomal compartments. Conclusions An intracellular redistribution of GLUT4 post-exercise is proposed as a molecular mechanism contributing to the insulin-sensitizing effect of prior exercise in human skeletal muscle., Graphical abstract Image 1, Highlights • Intramyocellular GLUT4 is redistributed 4 h after exercise in humans. • GLUT4 content is increased in GLUT4 storage vesicles and T-tubuli post-exercise. • Prior exercise + insulin increases sarcolemmal and endosomal GLUT4. • GLUT4 redistribution may thus contribute to post-exercise muscle insulin-sensitization.

Although the presence of cardiac dysfunction and cardiomyopathy in chronic diabetes has been recognized, the pathophysiology of diabetes-induced metabolic and subcellular changes as well as the therapeutic approaches for the prevention of diabetic cardiomyopathy are not fully understood. Cardiac dysfunction in chronic diabetes has been shown to be associated with Ca2+-handling abnormalities, increase in the availability of intracellular free Ca2+ and impaired sensitivity of myofibrils to Ca2+. Metabolic derangements, including depressed high-energy phosphate stores due to insulin deficiency or insulin resistance, as well as hormone imbalance and ultrastructural alterations, are also known to occur in the diabetic heart. It is pointed out that the activation of the sympathetic nervous system and renin–angiotensin system generates oxidative stress, which produces defects in subcellular organelles including sarcolemma, sarcoplasmic reticulum and myofibrils. Such subcellular remodeling plays a critical role in the pathogenesis of diabetic cardiomyopathy. In fact, blockade of the effects of neurohormonal systems has been observed to attenuate oxidative stress and occurrence of subcellular remodeling as well as metabolic abnormalities in the diabetic heart. This review is intended to describe some of the subcellular and metabolic changes that result in cardiac dysfunction in chronic diabetes. In addition, the therapeutic values of some pharmacological, metabolic and antioxidant interventions will be discussed. It is proposed that a combination therapy employing some metabolic agents or antioxidants with insulin may constitute an efficacious approach for the prevention of diabetic cardiomyopathy.

The highly organized transverse T-tubule membrane system represents the ultrastructural substrate for excitation–contraction coupling in ventricular myocytes. While the architecture and function of T-tubules have been well described in animal models, there is limited morpho-functional data on T-tubules in human myocardium. Hypertrophic cardiomyopathy (HCM) is a primary disease of the heart muscle, characterized by different clinical presentations at the various stages of its progression. Most HCM patients, indeed, show a compensated hypertrophic disease (“non-failing hypertrophic phase”), with preserved left ventricular function, and only a small subset of individuals evolves into heart failure (“end stage HCM”). In terms of T-tubule remodeling, the “end-stage” disease does not differ from other forms of heart failure. In this review we aim to recapitulate the main structural features of T-tubules during the “non-failing hypertrophic stage” of human HCM by revisiting data obtained from human myectomy samples. Moreover, by comparing pathological changes observed in myectomy samples with those introduced by acute (experimentally induced) detubulation, we discuss the role of T-tubular disruption as a part of the complex excitation–contraction coupling remodeling process that occurs during disease progression. Lastly, we highlight how T-tubule morpho-functional changes may be related to patient genotype and we discuss the possibility of a primitive remodeling of the T-tubule system in rare HCM forms associated with genes coding for proteins implicated in T-tubule structural integrity, formation and maintenance.

Neuronal nitric oxide synthase (nNOS) is involved in nitric oxide (NO) production and suggested to play a crucial role in blood flow regulation of skeletal muscle. During activation of the muscle, NO helps attenuate the sympathetic vasoconstriction to accommodate increased metabolic demands, a phenomenon known as functional sympatholysis. In inherited myopathies such as the dystrophinopathies Duchenne and Becker muscle dystrophies (DMD and BMD), nNOS is lost from the sarcolemma. The loss of nNOS may cause functional ischemia contributing to skeletal and cardiac muscle cell injury. Effects of NO is augmented by inhibiting degradation of the second messenger cyclic guanosine monophosphate (cGMP) using sildenafil and tadalafil, both of which inhibit the enzyme phosphodiesterase 5 (PDE5). In animal models of DMD, PDE5-inhibitors prevent functional ischemia, reduce post-exercise skeletal muscle pathology and fatigue, show amelioration of cardiac muscle cell damage and increase cardiac performance. However, effect on clinical outcomes in DMD and BMD patients have been disappointing with minor effects on upper limb performance and none on ambulation. This review aims to summarize the current knowledge of nNOS function related to functional sympatholysis in skeletal muscle and studies on PDE5-inhibitor treatment in nNOS-deficient animal models and patients.

Following heterotopic transplantation, the rat heart undergoes atrophy and exhibits delayed cardiac relaxation without any changes in contraction and systolic Ca (2+) transients. Furthermore, the sarcoplasmic reticular Ca (2+) uptake and release activities were reduced and Ca (2+) influx through L-type Ca (2+) channels was increased in the atrophied heart. Since Ca (2+) movements at sarcolemma are intimately involved in the regulation of intracellular Ca (2+) concentration, the present study was undertaken to test if sarcolemma plays any role to maintain cardiac function in the atrophied heart.The characteristics of sarcolemmal Ca (2+) pump and Na (+) –Ca (2+) exchange activities were examined in 8 weeks heterotopically isotransplanted rat hearts which did not support hemodynamic load and underwent atrophy. Sarcolemmal ATP (adenosine triphosphate)-dependent Ca (2+) uptake and Ca (2+) -stimulated ATPase (adenosine triphosphatase) activities were increased without any changes in Na (+) –K (+) ATPase activities in the transplanted hearts. Although no alterations in the Na (+) -dependent Ca (2+) uptake were evident, Na (+) -induced Ca (2+) release was increased in the transplanted heart sarcolemmal vesicles. The increase in Na (+) -induced Ca (2+) release was observed at different times of incubation as well as at 5, 20, and 40 mM Na (+) . The sarcolemma from transplanted hearts also showed higher contents of phosphatidic acid, sphingomyelin, and cholesterol.These results indicate that increases in the sarcolemmal, Ca (2+) transport activities in unloaded heart may provide an insight into adaptive mechanism to maintain normal contractile behavior of the atrophic heart.

BackgroundThe activating signal cointegrator 1 (ASC-1) complex acts as a transcriptional coactivator for a variety of transcription factors and consists of four subunits: ASCC1, ASCC2, ASCC3 and TRIP4. A single homozygous mutation in ASCC1 has recently been reported in two families with a severe muscle and bone disorder.ObjectiveWe aim to contribute to a better understanding of the ASCC1-related disorder.MethodsHere, we provide a clinical, histological and genetic description of three additional ASCC1 families.ResultsAll patients presented with severe prenatal-onset muscle weakness, neonatal hypotonia and arthrogryposis, and congenital bone fractures. The muscle biopsies from the affected infants revealed intense oxidative rims beneath the sarcolemma and scattered remnants of sarcomeres with enlarged Z-bands, potentially representing a histopathological hallmark of the disorder. Sequencing identified recessive nonsense or frameshift mutations in ASCC1, including two novel mutations.ConclusionOverall, this work expands the ASCC1 mutation spectrum, sheds light on the muscle histology of the disorder and emphasises the physiological importance of the ASC-1 complex in fetal muscle and bone development.

Key points We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS-derived NO-dependent and NO-independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO-independent sympatholysis was not affected in the canine DMD model. Abstract The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane-associated nNOS to blood flow regulation in dystrophin-deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO-independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD.

BACKGROUND This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

The arrhythmogenic potential of &beta, 1-adrenoceptor autoantibodies (&beta, 1-AA), as well as antiarrhythmic properties of omega-3 in heart diseases, have been reported while underlying mechanisms are poorly understood. We aimed to test our hypothesis that omega-3 (eicosapentaenoic acid-EPA, docosahexaenoic acid-DHA) may inhibit matrix metalloproteinase (MMP-2) activity to prevent cleavage of &beta, 1-AR and formation of &beta, 1-AA resulting in attenuation of pro-arrhythmic connexin-43 (Cx43) and protein kinase C (PKC) signaling in the diseased heart. We have demonstrated that the appearance and increase of &beta, 1-AA in blood serum of male and female 12-month-old spontaneously hypertensive rats (SHR) was associated with an increase of inducible ventricular fibrillation (VF) comparing to normotensive controls. In contrast, supplementation of hypertensive rats with omega-3 for two months suppressed &beta, 1-AA levels and reduced incidence of VF. Suppression of &beta, 1-AA was accompanied by a decrease of elevated myocardial MMP-2 activity, preservation of cardiac cell membrane integrity and Cx43 topology. Moreover, omega-3 abrogated decline in expression of total Cx43 as well as its phosphorylated forms at serine 368 along with PKC-&epsilon, while decreased pro-fibrotic PKC-&delta, levels in hypertensive rat heart regardless the sex. The implication of MMP-2 in the action of omega-3 was also demonstrated in cultured cardiomyocytes in which desensitization of &beta, 1-AR due to permanent activation of &beta, 1-AR with isoproterenol was prevented by MMP-2 inhibitor or EPA. Collectively, these data support the notion that omega-3 via suppression of &beta, 1-AA mechanistically controlled by MMP-2 may attenuate abnormal of Cx43 and PKC-&epsilon, signaling, thus, abolish arrhythmia substrate and protect rats with an advanced stage of hypertension from malignant arrhythmias.