Slow Myosin Heavy Chain Isoforms Research Articles

Effects of Short-Term Treatment of Rabbit Extraocular Muscle With Ciliary Neurotrophic Factor.

Little is known about the effect of ciliary neurotrophic factor (CNTF) on extraocular muscles, but microarray studies suggested CNTF might play a role in the development and/or maintenance of strabismus. The effect of short-term treatment of adult rabbit extraocular muscle with injected CNTF was examined for its ability to alter muscle characteristics. Eight adult New Zealand white rabbits received an injection into one superior rectus muscle of 2 µg/100 µL CNTF on 3 consecutive days. One week after the first injection, the rabbits were euthanized, and the treated and contralateral superior rectus muscles were assessed for force generation capacity and contraction characteristics using an in vitro stimulation protocol and compared to naïve control superior rectus muscles. All muscles were analyzed to determine mean cross-sectional areas and expression of slow twitch myosin heavy chain isoform. Short-term treatment of rabbit superior rectus muscles with CNTF resulted in a significant decrease in muscle force generation, but only at the higher stimulation frequencies. Significantly decreased myofiber cross-sectional areas of the treated muscles correlated with the decreased generated force. In addition, there were significant changes to contractile properties of the treated muscles, as well as a decrease in the number of myofibers expressing slow twitch myosin heavy chain. We show that short-term treatment of a single rabbit superior rectus muscle results in decreased myofiber size, decreased force, and altered contractile characteristics. Further studies are needed to determine if it can play a role in improving alignment in animal models of strabismus.

Apelin regulates skeletal muscle adaptation to exercise in a high-intensity interval training model.

Plasma apelin levels are reduced in aging and muscle wasting conditions. We aimed to investigate the significance of apelin signaling in cardiac and skeletal muscle responses to physiological stress. Apelin knockout (KO) and wild-type (WT) mice were subjected to high-intensity interval training (HIIT) by treadmill running. The effects of apelin on energy metabolism were studied in primary mouse skeletal muscle myotubes and cardiomyocytes. Apelin increased mitochondrial ATP production and mitochondrial coupling efficiency in myotubes and promoted the expression of mitochondrial genes both in primary myotubes and cardiomyocytes. HIIT induced mild concentric cardiac hypertrophy in WT mice, whereas eccentric growth was observed in the left ventricles of apelin KO mice. HIIT did not affect myofiber size in skeletal muscles of WT mice but decreased the myofiber size in apelin KO mice. The decrease in myofiber size resulted from a fiber type switch toward smaller slow-twitch type I fibers. The increased proportion of slow-twitch type I fibers in apelin KO mice was associated with upregulation of myosin heavy chain slow isoform expression, accompanied with upregulated expression of genes related to fatty acid transport and downregulated expression of genes related to glucose metabolism. Mechanistically, skeletal muscles of apelin KO mice showed defective induction of insulin-like growth factor-1 signaling in response to HIIT. In conclusion, apelin is required for proper skeletal and cardiac muscle adaptation to high-intensity exercise. Promoting apelinergic signaling may have benefits in aging- or disease-related muscle wasting conditions.NEW & NOTEWORTHY Apelin levels decline with age. This study demonstrates that in trained mice, apelin deficiency results in a switch from fast type II myofibers to slow oxidative type I myofibers. This is associated with a concomitant change in gene expression profile toward fatty acid utilization, indicating an aged-muscle phenotype in exercised apelin-deficient mice. These data are of importance in the design of exercise programs for aging individuals and could offer therapeutic target to maintain muscle mass.

Long non-coding RNA Tug1 modulates mitochondrial and myogenic responses to exercise in skeletal muscle

BackgroundMitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle.ResultsIn human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression (PPARGC1A). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca2+-mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca2+ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis.ConclusionsWe found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.

Bridging the muscle genome to phenome across multiple biological scales.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Myosin Heavy Chain Isoform Mrna Expression In Low- And High-capacity Running Rats

Differential expression of Myosin Heavy Chain (MHC) isoforms in skeletal muscle determines its metabolic and functional characteristics. Endurance exercise training is associated with fast-to-slow MHC isoform transition, and which is considered to mediate the beneficial effects of exercise on metabolic health. Rats genetically bred for low-capacity running (LCR) and high-capacity running (HCR) are characterized by the health benefits typically described after endurance exercise training. LCR and HCR rats differed in maximal running capacity 14 fold. PURPOSE: To quantify mRNA expression for slow (MHC-I) and fast (MCH-IIa, MCH-IIb) MHC isoforms in soleus, gastrocnemius and vastus skeletal muscle of LCR and HCR male adult rats, and determine if HCR rats have higher mRNA expression for slow, and lower mRNA expression for fast, MHC isoforms. METHODS: Muscle tissue from 5 HCR and 5 LCR rats was homogenized and mRNA was isolated. The mRNA content of MHC isoforms in muscle was quantified using reverse transcriptase polymerase chain reaction (RT-PCR), and standardized to the 18S mRNA content in muscle. Differences between groups were calculated using the comparative CT method (2-ΔΔCT). LCR were the control group. RESULTS: Expression of mRNA of the MHC isoforms differed between groups and the type of skeletal muscle analyzed. MHC-I mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference (2-ΔΔCT) was more pronounced for the gastrocnemius muscle [LCR: 1.0 ± 0.6 (mean±SE); HCR: 0.3 ± 0.2; P = 0.08]. Although MHC-IIa mRNA expression appeared lower in soleus and vastus in the HCR, it was higher in the HCR compared to LCR in the gastrocnemius muscle [LCR: 1.0 ± 1.0; HCR: 4.6 ± 3.6; P = 0.07]. MHC-IIb mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference was more pronounced for the soleus muscle [LCR: 1.0 ± 1.1; HCR: 0.2 ± 0.4; P = 0.08]. CONCLUSION: Genetic differences linked to the MHC transcriptome (i.e., decrease in MHC-I mRNA) in muscle do not explain the exercise performance observed in HCR rats. Changes in the muscle MHC transcriptome may not directly be responsible for the health benefits associated with exercise training. It remains to be determined on whether the differences we observed at the MHC mRNA level are translated to the MHC proteome level.

Evidence for Progressive Shifts Towards α‐Myosin Isoform with Paradoxical Increases in Cardiac Hemodynamic Loads in Post‐Natal Healthy Rats

IntroductionThe balance of fast (α) and slow (β) cardiac myosin heavy chain (MYHC) isoforms is known to change with both aging and disease. While pathological states with increased hemodynamic loads are often accompanied by shifts toward energy‐sparing β isoforms, the underlying mechanism(s) of these changes remain poorly understood. Neonatal rats are a promising model to study such cardiac MYHC remodeling, as they show a well‐documented transition (β to α) during post‐natal development. In these experiments, MYHC‐isoform switches and in vivo cardiac structure/hemodynamic changes were evaluated during post‐natal development.MethodsCardiac echocardiographic and systemic/left ventricular (LV) hemodynamics were evaluated in Sprague‐Dawley rats (n = 61) ranging from post‐natal day (PND) 3 to adulthood. Hemodynamics were obtained before/after challenges with sodium nitroprusside (SNP, 25 μg/kg IV) and/or phenylephrine (PE, 10 μg/kg IV) to study cardiovascular responsiveness. LV’s were also collected (n = 48) for myosin isoform analysis via SDS‐PAGE MYHC separating gels.ResultsAs expected, post‐natal rats showed a progressive decline in β‐MYHC expression from PND3 (1.29 ± 0.05 α:β) to adulthood when α‐MYHC was dominant (12.78 ± 2.51 α:β). Concomitantly, postnatal rats showed progressive increases (P<0.05) in systemic/LV pressures (MAP: 46 ± 1 at <PND14 to 103 ± 2 mmHg, P<0.05), LV mass (0.16 ± 0.02g at <PND14 to 0.84 ± 017g) and diastolic dimensions (79 ± 6 at <PND14 to 288 ± 14 μL), with vastly preserved heart rates. Pressor responses to PE increased during maturation, while all age groups were sensitive to SNP‐mediated vaso‐relaxation. Ejection fraction was preserved during postnatal maturation with young pups showing lower rates of LV pressure development (dPdtmax 4052 ± 125 at <PND14 vs. 9478 ± 231 mmHg/s, P<0.05) consistent with the early expression of β‐MYHC.ConclusionsTaken together, these data provide evidence of a maturing cardiovascular system in post‐natal rats showing progressive increases in systemic/ventricular pressures and cardiac dimensions, with paradoxical shifts towards α‐MYHC isoforms. In short, in rats, the cardiac β‐MYHC isoform consistently declines over maturation despite sustained increases in systemic load.

Ammonia Induces a Myostatin-Mediated Atrophy in Mammalian Myotubes, but Induces Hypertrophy in Avian Myotubes

Ammonia, a byproduct of protein catabolism that is generally regarded as toxic, is processed by the liver for excretion. In diseases resulting in hepatic insufficiency, circulating ammonia levels increase dramatically, ensuing secondary disorders. Sarcopenia, or loss of muscle mass, is commonly associated with hyperammonemia. In mammalian models of cirrhosis, increased myostatin is consistent, contributes to muscle autophagy, and reduces satellite cell activation and differentiation, whereas, avian species show a positive myogenic response to ammonia. The objective of the study was to elucidate the effect of ammonia in chicken, mouse, and rat derived myotubes. Primary myoblasts were isolated from the pectoralis major (breast) and biceps femoris (thigh) of embryonic day 17 chicken embryos, and from the hindlimbs of 3-day-old rat pups. C2C12 cells were used for mouse myoblasts. Myotubes were exposed to 10mM ammonium acetate (AA) or 10mM sodium acetate (SA) for 24 hours to determine myogenic response to ammonia. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly higher in mammalian myotubes compared to chicken myotubes (P < 0.001). Western blot analysis of myostatin protein confirmed a significant increase in ammonia treated rat myotubes, while chicken breast myotubes showed a significant decrease in myostatin (P < 0.05). Myotube diameter significantly increased in chicken breast and thigh cultures treated with ammonia, while diameter was significantly reduced in mouse and rat myotubes (P < 0.05). Intracellular glutamine is significantly higher in chicken thigh, but not breast, myotubes treated with AA compared to SA treated myotubes (P < 0.05). To investigate fiber type differences in ammonia metabolism, Western blot analysis of protein from AA and SA treated myotubes was examined for fast and slow myosin heavy chain isoforms. AA treatment resulted in a higher ratio of fast to slow isoforms of myosin heavy chain in both types of chicken myotubes, while fast isoforms were decreased in AA treated mouse and rat myotubes. These data demonstrate that chicken myotubes respond positively to ammonia while rodent myotubes respond negatively. Further, there is evidence that ammonia induces a fast fiber type shift in avian muscle, but a slow phenotype shift in mammalian muscle.

Plantar mechanical stimulation prevents calcineurin-NFATc1 inactivation and slow-to-fast fiber type shift in rat soleus muscle under hindlimb unloading.

The prevailing myosin isoform [myosin heavy chain (MyHC)] in a skeletal muscle determines contractile properties of the muscle. Under actual or simulated microgravity conditions such as human bed rest or rat hindlimb unloading, decrease in expression of MyHC of the slow type [MyHC I(β)] has been observed. It was demonstrated that increasing sensory input by performing plantar mechanical stimulation (PMS) on the soles of the feet results in an increase in neuromuscular activation of the lower limb muscles and may prevent slow-to-fast fiber type shift. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway is the main cascade regulating MyHC I(β) expression. The present study was aimed to analyze the states of the calcineurin-NFATc1 signaling cascade under conditions of PMS during rat hindlimb unloading. Male Wistar rats were randomly assigned to vivarium control groups and 1-day unloading (1HS), 3-day unloading (3HS), 1HS+PMS, and 3HS+PMS groups. We found that both 1 day and 3 days of unloading caused decrease in MyHC I(β) mRNA expression and decrease in glycogen synthase kinase-3β phosphorylation (Ser 9) that brought about the kinase activation, and these effects of unloading were prevented by PMS. Three days of unloading also caused increase in expression of calsarcin-2 (myozenin-I), which was found to be the endogenous calcineurin inhibitor. The level of calsarcin-2 expression in the 3HS+PMS group did not differ from the control group. Therefore, we conclude that PMS upregulates the calcineurin-NFATc1 signaling pathway and prevents unloading-induced MyHC I(β) decrease. NEW & NOTEWORTHY It is widely accepted that changes in the myosin phenotype during functional unloading (disuse) are determined by a decreased expression of the myosin heavy chain (MyHC) I(β) gene, and this decrease leads to changes of contractile and fatigue characteristics of soleus muscle. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) pathway is one of the most important signaling cascades regulating slow MyHC isoform expression. The present study is the first to show that plantar mechanical stimulation upregulates calcineurin-NFATc1 signaling in soleus muscles of hindlimb-unloaded rats.

Changing muscle function with sustained glial derived neurotrophic factor treatment of rabbit extraocular muscle

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2μg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

Mouse Hind Limb Skeletal Muscle Functional Adaptation in a Simulated Fine Branch Arboreal Habitat.

The musculoskeletal system is remarkably plastic during growth. The purpose of this study was to examine the muscular plasticity in functional and structural properties in a model known to result in significant developmental plasticity of the postcranial skeleton. Fifteen weanling C57BL/6 mice were raised to 16 weeks of age in one of two enclosures: a climbing enclosure that simulates a fine branch arboreal habitat and is traversed by steel wires crossing at 45° relative to horizontal at multiple intersections, and a control enclosure that resembles a parking deck with no wires but the same volume of habitable space. At killing, ex vivo contractility properties of the soleus (SOL) and extensor digitorum longus (EDL) muscles were examined. Our results demonstrate that EDL muscles of climbing mice contracted with higher specific forces and were comprised of muscle fibers with slower myosin heavy chain isoforms. EDL muscles also fatigued at a higher rate in climbing mice compared to controls. SOL muscle function is not affected by the climbing environment. Likewise, mass and architecture of both EDL and SOL muscles were not different between climbing and control mice. Our data demonstrate that functional adaptation does not require concomitant architectural adaptation in order to increase contractile force. Anat Rec, 301:434-440, 2018. © 2018 Wiley Periodicals, Inc.

Rapid decline in MyHC I(β) mRNA expression in rat soleus during hindlimb unloading is associated with AMPK dephosphorylation.

Inactivation of a skeletal muscle results in slow to fast myosin heavy chain (MyHC) shift. AMP-activated protein kinase (AMPK) can be implicated in the regulation of genes encoding the slow MyHC isoform. Here we report that AMPK dephosphorylation after 24h of mechanical unloading can contribute to histone deacetylase (HDAC) nuclear translocation; activation of AMPK prevents HDAC4 nuclear accumulation after 24h of unloading and AMPK dephosphorylation inhibits slow MyHC expression following 24h of unloading. Our data indicate that AMPK dephosphorylation during the first 24h of mechanical unloading has a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. One of the key events that occurs during skeletal muscle inactivation is a change in myosin phenotype, i.e. increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). It is known that calcineurin/nuclear factor of activated T-cells and AMP-activated protein kinase (AMPK) can regulate the expression of genes encoding MyHC slow isoform. Earlier, we found a significant decrease in phosphorylated AMPK in rat soleus after 24h of hindlimb unloading (HU). We hypothesized that a decrease in AMPK phosphorylation and subsequent histone deacetylase (HDAC) nuclear translocation can be one of the triggering events leading to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with AMPK activator (AICAR) for 6days before HU as well as during 24h of HU. We discovered that AICAR treatment prevented a decrease in pre-mRNA and mRNA expression of MyHC I as well as MyHC IIa mRNA expression. Twenty-four hours of hindlimb suspension resulted in HDAC4 accumulation in the nuclei of rat soleus but AICAR pretreatment prevented this accumulation. The results of the study indicate that AMPK dephosphorylation after 24h of HU had a significant impact on the MyHC I and MyHC IIa mRNA expression in rat soleus. AMPK dephosphorylation also contributed to HDAC4 translocation to the nuclei of soleus muscle fibres, suggesting an important role of HDAC4 as an epigenetic regulator in the process of myosin phenotype transformation.

Novel phenotypic variant in the MYH7 spectrum due to a stop-loss mutation in the C-terminal region: a case report

BackgroundDefects of the slow myosin heavy chain isoform coding MYH7 gene primarily cause skeletal myopathies including Laing Distal Myopathy, Myosin Storage Myopathy and are also responsible for cardiomyopathies. Scapuloperoneal and limb-girdle muscle weakness, congenital fiber type disproportion, multi-minicore disease were also reported in connection of MYH7. Pathogeneses of the defects in the head and proximal rod region of the protein are well described. However, the C-terminal mutations of the MYH7 gene are less known. Moreover, only two articles describe the phenotypic impact of the elongated mature protein product caused by termination signal loss.Case presentationHere we present a male patient with an unusual phenotypic variant of early-onset and predominant involvement of neck muscles with muscle biopsy indicating myopathy and sarcoplasmic storage material. Cardiomyopathic involvements could not be observed. Sequencing of MYH7 gene revealed a stop-loss mutation on the 3-prime end of the rod region, which causes the elongation of the mature protein.ConclusionsThe elongated protein likely disrupts the functions of the sarcomere by multiple functional abnormalities. This elongation could also affect the thick filament degradation leading to protein deposition and accumulation in the sarcomere, resulting in the severe myopathy of certain axial muscles. The phenotypic expression of the detected novel MYH7 genotype could strengthen and further expand our knowledge about mutations affecting the structure of MyHCI by termination signal loss in the MYH7 gene.

Mild heat stress enhances differentiation and proliferation of Japanese quail myoblasts and enhances slow muscle fiber characteristics

The objective of this study was to investigate the effect of mild heat stress on muscle fiber hyperplastic and hypertrophic growth in quail primary myogenic cells to better understand the mechanisms leading to increased skeletal muscle development in avian embryos incubated at a higher temperature. Compared to control cultures maintained at 37°C, incubation at 39°C enhanced myotube length (P < 0.01) and diameter (P < 0.001) at 3 days after differentiation (D3). This enlargement of the myotubes incubated at 39°C can be explained by differences in the fusion index (56.7 vs. 46.2%, P < 0.05) and nuclei number per myotube (18.1 vs. 10.8, P < 0.001) compared to the control cells at D3. Additionally, a higher density of myotubes at D3 in cultures exposed to a higher temperature were related to higher levels of Pax-7 (P < 0.05) compared to the control cells incubated continuously at 37°C. These results indicated a higher proliferative capacity in cells exposed to mild heat stress compared to the control cells. On the other hand, mild heat stress enhanced protein levels of slow myosin heavy chain isoform (P < 0.01) and cytochrome c oxidase subunit IV (P < 0.01) compared to the control cells at D3. These discrepancies in protein expression indicated maintenance of slow muscle fiber type characteristics in myotubes incubated at 39°C. Our results suggest that mild heat stress plays a significant role in myogenic mechanisms related to muscle mass and development.

Abnormally Small Neuromuscular Junctions in the Extraocular Muscles From Subjects With Idiopathic Nystagmus and Nystagmus Associated With Albinism.

PurposeInfantile nystagmus syndrome (INS) is often associated with abnormalities of axonal outgrowth and connectivity. To determine if this manifests in extraocular muscle innervation, specimens from children with idiopathic INS or INS and albinism were examined and compared to normal age-matched control extraocular muscles.MethodsExtraocular muscles removed during normal surgery on children with idiopathic INS or INS and albinism were immunostained for neuromuscular junctions, myofiber type, the immature form of the acetylcholine receptor, and brain-derived neurotrophic factor (BDNF) and compared to age-matched controls.ResultsMuscles from both the idiopathic INS and INS and albinism groups had neuromuscular junctions that were 35% to 71% smaller based on myofiber area and myofiber perimeter than found in age-matched controls, and this was seen on both fast and slow myosin heavy chain isoform–expressing myofibers (all P < 0.015). Muscles from subjects with INS and albinism showed a 7-fold increase in neuromuscular junction numbers on fast myofibers expressing the immature gamma subunit of the acetylcholine receptor. The extraocular muscles from both INS subgroups showed a significant increase in the number and size of slow myofibers compared to age-matched controls. Brain-derived neurotrophic factor was expressed in control muscle but was virtually absent in the INS muscles.ConclusionsThese studies suggest that, relative to the final common pathway, INS is not the same between different patient etiologies. It should be possible to modulate these final common pathway abnormalities, via exogenous application of appropriate drugs, with the hope that this type of treatment may reduce the involuntary oscillatory movements in these children.

MicroRNA-23a reduces slow myosin heavy chain isoforms composition through myocyte enhancer factor 2C (MEF2C) and potentially influences meat quality

MicroRNAs (miRNAs) are non-coding small RNAs that participate in the regulation of a variety of biological processes. Muscle fiber types were very important to meat quality traits, however, the molecular mechanism by which miRNAs regulate the muscle fiber type composition is not fully understood. The aim of this study was to investigate whether miRNA-23a can affect muscle fiber type composition. Luciferase reporter assays proved that miRNA-23a directly targets the 3′ untranslated region (UTRs) of MEF2c. Overexpression of miRNA-23a significantly suppressed the expression of MEF2c both in mRNA and protein levels, thus caused down-regulation of the expression of some key downstream genes of MEF2c (PGC1-α, NRF1 and mtTFA). More interestingly, overexpression of miRNA-23a significantly restrained the myogenic differentiation and decreased the ratio of slow myosin heavy chain in myoblasts (p<0.05). Our findings hinted a novel role of miRNA-23a in the epigenetic regulation of meat quality via decreasing the ratio of slow myosin heavy chain isoforms.

Size and Proportions of Slow-Twitch and Fast-Twitch Muscle Fibers in Human Costal Diaphragm.

Smaller diaphragmatic motor unit potentials (MUPs) compared to MUPs of limb muscles lead to the hypothesis that diaphragmatic muscle fibers, being the generators of MUPs, might be also smaller. We compared autopsy samples of costal diaphragm and vastus lateralis of healthy men with respect to fibers' size and expression of slow myosin heavy chain isoform (MyHC-1) and fast 2A isoform (MyHC-2A). Diaphragmatic fibers were smaller than fibers in vastus lateralis with regard to the mean minimal fiber diameter of slow-twitch (46.8 versus 72.2 μm, p < 0.001), fast-twitch (45.1 versus 62.4 μm, p < 0.001), and hybrid fibers (47.3 versus 65.0 μm, p < 0.01) as well as to the mean fiber cross-sectional areas of slow-twitch (2376.0 versus 5455.9 μm2, p < 0.001), fast-twitch (2258.7 versus 4189.7 μm2, p < 0.001), and hybrid fibers (2404.4 versus 4776.3 μm2, p < 0.01). The numerical proportion of slow-twitch fibers was higher (50.2 versus 36.3%, p < 0.01) in costal diaphragm and the numerical proportion of fast-twitch fibers (47.2 versus 58.7%, p < 0.01) was lower. The numerical proportion of hybrid fibers did not differ. Muscle fibers of costal diaphragm have specific characteristics which support increased resistance of diaphragm to fatigue.

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis

Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the preclinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6-wk-old animals with the natural phenol resveratrol (RSV; ~100 mg·kg(-1)·day(-1)) for 6 wk. Sirtuin 1 (SIRT1) activity and protein levels increased significantly, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ~500 mg·kg(-1)·day(-1) across a duration of 12 wk was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1α activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients.

Activation of alternative NF-κB signaling during recovery of disuse-induced loss of muscle oxidative phenotype

Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified, since the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-κB, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. Markers and regulators (including alternative NF-κB signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hindlimb suspension/reloading (HLS/RL) protocol. Expression levels of oxidative phosphorylation subunits and slow myosin heavy chain isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC) molecules PGC-1α and PGC-1β and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α increased upon RL. PPAR-δ, nuclear respiratory factor 1 (NRF-1), NRF-2α, and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased, and phosphorylation of P100 and subsequent processing to P52 were elevated, reflecting alternative NF-κB activation. We conclude that RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-κB signaling.

Fiber type composition of the architecturally distinct regions of human supraspinatus muscle: a cadaveric study.

The human supraspinatus muscle is clinically important as it is frequently injured in older adults and the elderly. We have previously shown that the supraspinatus has a complex architecture with two distinct regions each consisting of three parts. Further we have found dynamic changes in architectural parameters such as fiber bundle length markedly vary between these regions. Fiber types of the supraspinatus have not been thoroughly investigated throughout its volume and are of interest to clinicians treating supraspinatus pathologies. In this study we investigated the distribution of fiber types within the distinct regions and parts of supraspinatus. Samples of supraspinatus were excised from six distinct parts of each muscle from five formalin embalmed specimens (one male, four female; mean age 77±11.1 years) free of tendon pathology. Samples were frozen in liquid nitrogen and then cryosectioned. Serial sections were labeled using immunohistochemical techniques and antibodies against fast or slow myosin heavy chain isoforms. The mean percentage of Type I (slow) fibers ranged from 56.73% to 63.97%. Results demonstrated significant variations in fiber type distribution. The middle part of the anterior region has a significantly greater percentage of Type I fibers compared to that of the posterior. The superficial part of the anterior region has a greater percentage of Type II (fast) fibers compared to the middle and deep parts. Findings aid in highlighting the distinct functions of the anterior and posterior regions, and prompt the need to re-evaluate assessment and treatment techniques established on a limited understanding of the fiber type distribution.

Role of NO-synthase in regulation of protein metabolism of stretched rat m. soleus muscle during functional unloading

Gravitational unloading causes atrophy of muscle fibers and can lead to destruction of cytoskeletal and contractile proteins. Along with the atrophic changes, unloaded muscle frequently demonstrates significant shifts in the ratio of muscle fibers expressing fast and slow myosin heavy chain isoforms. Stretching of the m. soleus during hindlimb suspension prevents its atrophy. We supposed that neuronal NO-synthase (NOS) (which is attached to membrane dystrophin-sarcoglycan complex) can contribute to maintenance of protein metabolism in the muscle and prevent its atrophy when m. soleus is stretched. To test this hypothesis, we used Wistar rats (56 animals) in experiments with hindlimb suspension during 14 days. The group of hindlimb suspended rats with stretched m. soleus was injected with L-NAME to block NOS activity. We found that m. soleus mass and its protein content in hindlimb-suspended rats with stretched m. soleus were preserved due to prevention of protein degradation. NOS is involved in maintenance of expression of some muscle proteins. Proliferation of satellite cells in stretched m. soleus may be due to nNOS activity, but maintenance of muscle mass upon stretching is regulated not by NOS alone.