Fast-twitch Extensor Digitorum Longus Research Articles

Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability.

Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle "increasing" allele C (n = 15) or "decreasing" allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/- and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the "increasing" compared with the "decreasing" CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/- mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

Tirasemtiv enhances submaximal muscle tension in an Acta1:p.Asp286Gly mouse model of nemaline myopathy.

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant.

Effects of sclerostin injection on soleus and extensor digitorum longus muscle tissue in male mice.

Sclerostin, a potent inhibitor of the Wnt signaling pathway, plays a critical role in bone homeostasis. Evidence suggests that sclerostin may also be involved in crosstalk between other tissues, including muscle. This pilot study attempted to examine the effects of sclerostin on soleus and extensor digitorum longus (EDL) muscle tissue from male mice that were given continuous recombinant sclerostin injections for 4 weeks. A total of 48 10-week-old male C57BL/6J mice were assigned to be sedentary or perform 1 h treadmill running per day for 4 weeks and administered subcutaneous injections of either saline or recombinant sclerostin 5 days/week. Sclerostin injection led to a reduction in the soleus myosin heavy chain (MHC) I, MHC I/IIA, MHC IIA/X, and MHC IIB cross-sectional area (p< 0.05) with no exercise effects on these reductions. In contrast, there were no effects of sclerostin injections or exercise on the fast-twitch EDL muscle in terms of size, MHC protein, or markers of Wnt signaling. These findings provide preliminary evidence of sclerostin's endocrine role in muscle via decreases in myofiber cross-sectional area, which seems to be independent of fiber type but muscle type-specific. More studies, however, are needed to confirm these preliminary results.

Weakened Contractile Performance and Mitochondrial Respiratory Complex Activity in Skeletal Muscle Improve during Interbout Arousal in Hibernating Daurian Ground Squirrel, Spermophilus dauricus

Mammalian hibernation is composed of multiple episodes of torpor bout, separated by phases of interbout arousal. During torpor, the skeletal muscles of mammals are undoubtedly inactive, but it has been proven to mitigate disuse atrophy. While interbout arousal has been implicated in the prevention of muscle atrophy, the underlying mechanisms sustaining muscle contraction remain to be explored. In the present study, Daurian ground squirrels (Spermophilus dauricus) were divided into four groups: pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation (POST). The contractile performance of slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL) was detected both in situ and in vitro. Concurrently, mitochondrial respiratory chain complex activity in these muscles was quantified. Our findings revealed that in situ contractile properties of both muscles, including force, power output, time duration, and force development/relaxation rates of twitch contraction, and force and power output of tetanic contraction declined in the TOR group compared to the PRE group, but improved in the IBA and POST groups. Fatigue resistance of muscles, determined by the power output of repetitive tetanic contractions in situ, decreased in the TOR group but recovered in the IBA and POST groups. In vitro studies demonstrated that tetanic contraction power output in isolated muscles increased with muscle temperature in both TOR and IBA groups. However, at the same temperature, power output was consistently lower in the TOR group compared to the IBA group. Moreover, the activity of the mitochondrial respiratory chain complex, especially Complexes I and II, decreased in the TOR group but showed recovery in the IBA and POST groups. These findings suggest that both the contractile performance and fatigue resistance of mammalian skeletal muscle are compromised during torpor but can be improved during interbout arousal and post-hibernation. The rebound in body temperature and rise in mitochondrial respiratory chain complex activity in skeletal muscle are involved in enhancing contractile performance and fatigue resistance. This study suggests that interbout arousal functions as a vital temporal interval during which skeletal muscles can transition from the inactivity induced by torpor to a state of restored contractile functionality. Thus, interbout arousal serves as a behavioral safeguard against disuse-induced damage to skeletal muscles during hibernation.

Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+ -handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild-type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200μM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200μM) on maximal force in slow-twitch soleus were lower than found in the fast-twitch EDL muscle. In single interosseous myofibres, HOCl (10μM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl-induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD. KEY POINTS: Duchenne muscular dystrophy is a fatal genetic disease with pathological mechanisms which involve the complex interaction of chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations. Hypochlorous acid can be endogenously produced by neutrophils via the enzyme myeloperoxidase. Both neutrophil and myeloperoxidase activity are increased in dystrophic mice. This study found that hypochlorous acid decreased muscle force production and increased cytosolic Ca2+ concentrations in isolated muscles from wild-type and dystrophic mice at relatively low concentrations of hypochlorous acid. These results indicate that hypochlorous acid may be key in the Duchenne muscular dystrophy disease pathology and may provide a unifying link between the chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations observed in Duchenne muscular dystrophy. Hypochlorous acid production may be a potential target for therapeutic treatments of Duchenne muscular dystrophy.

Exercise training induces depot-specific remodeling of protein secretion in skeletal muscle and adipose tissue of obese male mice.

Acute exercise induces changes in circulating proteins, which are known to alter metabolism and systemic energy balance. Skeletal muscle is a primary contributor to changes in the plasma proteome with acute exercise. An important consideration when assessing the endocrine function of muscle is the presence of different fiber types, which show distinct functional and metabolic properties and likely secrete different proteins. Similarly, adipokines are important regulators of systemic metabolism and have been shown to differ between depots. Given the health-promoting effects of exercise, we proposed that understanding depot-specific remodeling of protein secretion in muscle and adipose tissue would provide new insights into intertissue communication and uncover novel regulators of energy homeostasis. Here, we examined the effect of endurance exercise training on protein secretion from fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscle and visceral and subcutaneous adipose tissue. High-fat diet-fed mice were exercise trained for 6 wk, whereas a Control group remained sedentary. Secreted proteins from excised EDL and soleus muscle, inguinal, and epididymal adipose tissues were detected using mass spectrometry. We detected 575 and 784 secreted proteins from EDL and soleus muscle and 738 and 920 proteins from inguinal and epididymal adipose tissue, respectively. Of these, 331 proteins were secreted from all tissues, whereas secretion of many other proteins was tissue and depot specific. Exercise training led to substantial remodeling of protein secretion from EDL, whereas soleus showed only minor changes. Myokines released exclusively from EDL or soleus were associated with glycogen metabolism and cellular stress response, respectively. Adipokine secretion was completely refractory to exercise regulation in both adipose depots. This study provides an in-depth resource of protein secretion from muscle and adipose tissue, and its regulation following exercise training, and identifies distinct depot-specific secretion patterns that are related to the metabolic properties of the tissue of origin.NEW & NOTEWORTHY The present study examines the effects of exercise training on protein secretion from fast-twitch and slow-twitch muscle as well as visceral and subcutaneous adipose tissue of obese mice. Although exercise training leads to substantial remodeling of protein secretion from fast-twitch muscle, adipose tissue is completely refractory to exercise regulation.

Slow or fast: Implications of myofibre type and associated differences for manifestation of neuromuscular disorders.

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow- to fast-twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow-twitch soleus and fast-twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre-type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders.

Soleus muscle contains a higher concentration of lipid metabolites than extensor digitorum longus in rats with lipopolysaccharide-induced acute muscle atrophy

Muscle atrophy is one of the most important and frequent problems for critically ill patients. The purpose of this study was to evaluate the effect of lipid mediators on acute muscle atrophy. Skeletal muscle fiber-specific analysis of lipid mediators in endotoxemic rats was therefore performed. Male Wistar rats were intraperitoneally injected with lipopolysaccharide (LPS). Slow-twitch soleus muscle and fast-twitch extensor digitorum longus (EDL) muscle were harvested 0, 6, and 24h after LPS injection. Lipid mediators were profiled using liquid chromatography-tandem mass spectrometry, and free fatty acid (FFA) concentrations were measured using gas chromatography-mass spectrometry. Muscles were weighed and their cross-sectional areas were evaluated. Expression levels of mRNAs encoding inflammatory cytokines, autophagy-related transcription factors, and members of the ubiquitin-proteasome system were measured using real-time PCR. Before LPS injection, the concentrations of all FFAs, including arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, and all measured lipid mediators were higher in soleus muscle than in EDL muscle, especially those of pro-inflammatory prostaglandin E2 (PGE2) and leukotriene B4. LPS injection, increased PGE2 and D2 and decreased FFAs in soleus muscle but did not change in EDL muscle. The concentrations of specialized pro-resolving mediators E-series hydroxy-eicosapentaenoic acid and D-series hydroxy-docosahexaenoic acid were higher in soleus muscle. Muscle cross-sectional area decreased and the expression level of atrogin-1 was upregulated in EDL muscle, but both were unchanged in soleus muscle. After LPS injection, a discrepancy involving an increased PGE2 concentration and decreased muscle atrophy was identified in this acute muscle atrophy model of critical illness. Concentrations of FFAs and lipid mediators were higher in soleus muscle than in EDL muscle, and LPS injection rapidly increased concentrations of pro-inflammatory lipid mediators. However, muscle atrophy with upregulation of autophagy-related transcription factors was observed in EDL muscle but not in soleus muscle.

A chronic high-fat diet does not exacerbate muscle atrophy in fast-twitch skeletal muscle of aged mice.

What is the central question of this study? Ageing leads to a loss of mass in skeletal muscle, but the effect of obesity on ageing-related muscle wasting is unclear. In this study, we aimed to demonstrate the specific effect of obesity on fast-twitch skeletal muscle in ageing. What is the main finding and its importance? Our findings show that the obesity induced by long-term ingestion of a high-fat diet does not aggravate muscle wasting in fast-twitch skeletal muscle of aged mice, indicating that the present study provides morphological characteristics for skeletal muscle of sarcopenic obesity. Obesity and ageing reduce muscle mass and lead to deficits in muscle maintenance, but it is not known whether obesity accelerates muscle wasting additively in the setting of ageing. We investigated morphological characteristics in fast-twitch extensor digitorum longus (EDL) muscle of mice fed a low-fat diet (LFD) or a high-fat diet (HFD) for 4 or 20 months. The fast-twitch EDL muscle was harvested, and the muscle fibre-type composition, individual muscle cross-sectional area and myotube diameter were measured. We found an increase in the percentage of type IIa and IIx myosin heavy chain fibres in the whole EDL muscle, but a decrease in type IIB myosin heavy chain in both HFD protocols. The cross-sectional area and myofibre diameter were lower in both groups of aged mice (after 20 months of LFD or HFD) compared with young mice (after 4 months of the diets), but there were no differences between mice fed LFD or HFD for 20 months. These data suggest that long-term feeding of HFD does not aggravate muscle wasting in fast-twitch EDL muscle of male mice.

Redox status biomarkers in the fast-twitch extensor digitorum longus resulting from the hypoxic exercise.

ABSTRACTThe fast-twitch muscle may be affected from over-produced reactive oxygen species (ROS) during hypoxia/hypoxic exercise. The study aims to investigate redox status biomarkers in the fast-twitch extensor digitorum longus (EDL) muscle after hypoxic exercise. Male Sprague Dawley rats (eight-week-old) were randomly divided into six groups of the experimental “live high train high (LHTH), live high train low (LHTL) and live low train low (LLTL)” and their respective controls. Before the EDLs’ extraction, the animals exercised for a 4-week familiarization period, then they exercised for four-weeks at different altitudes. The LHTH group had higher ratios of lipid hydroperoxides (LHPs) than the experimental groups of LHTL (p=0.004) and LLTL (p=0.002), while having no difference than its control ‘LH’. Similarly, a higher percentage of advanced oxidation protein products (AOPP) was determined in the LHTH than the LHTL (p=0.041) and LLTL (p=0.048). Furthermore, oxidation of thiol fractions was the lowest in the LHTH and LH. However, redox biomarkers and thiol fractions illustrated no significant change in the LHTL and LLTL that might ensure redox homeostasis due to higher oxygen consumption. The study shows that not hypoxic exercise/exercise, but hypoxia might itself lead to a redox imbalance in the fast-twitch EDL muscle.

Skeletal muscle sympathetic denervation disrupts the neuromuscular junction postterminal organization: A single-cell quantitative approach

The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.

Endurance exercise attenuates juvenile irradiation-induced skeletal muscle functional decline and mitochondrial stress

BackgroundRadiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function.MethodsWe utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca2+ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR).ResultsIrradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca2+ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca2+ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca2+ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis.ConclusionsJuvenile irradiation stunted muscle development, disrupted proper Ca2+ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca2+ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation.

The peak force-resting membrane potential relationships of mouse fast- and slow-twitch muscle.

We endeavored to understand the factors determining the peak force-resting membrane potential (EM) relationships of isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles from mice (25°C), especially in relation to fatigue. Interrelationships between intracellular K+ activity ([Formula: see text]), extracellular K+ concentration ([K+]o), resting EM, action potentials, and force were studied. The large resting EM variation was mainly due to the variability of [Formula: see text]. Action potential overshoot-resting EM relationships determined at 4 and 8-10 mM [K+]o after short (<5 min) and prolonged (>50 min) depolarization periods revealed a constant overshoot from -90 to -70 mV providing a safety margin. Overshoot decline with depolarization beyond -70 mV was less after short than prolonged depolarization. Inexcitable fibers occurred only with prolonged depolarization. The overshoot decline during action potential trains (2 s) exceeded that during short depolarizations. Concomitant lower extracellular [Na+] and raised [K+]o depressed the overshoot in an additive manner and peak force in a synergistic manner. Raised [K+]o-induced force loss was exacerbated with transverse wire versus parallel plate stimulation in soleus, implicating action potential propagation failure in the surface membrane. Increasing stimulus pulse parameters restored tetanic force at 9-10 mM [K+]o in soleus but not EDL, indicative of action potential failure within trains. The peak tetanic force-resting EM relationships (determined with resting EM from deeper rather than surface fibers) were dynamic and showed pronounced force depression over -69 to -60 mV in both muscle types, implicating that such depolarization contributes to fatigue. The K+-Na+ interaction shifted this relationship toward less depolarized potentials, suggesting that the combined ionic effect is physiologically important during fatigue.

Carotenoid transporter CD36 expression depends on hypoxia-inducible factor-1α in mouse soleus muscles.

Dietary β-carotene induces muscle hypertrophy and prevents muscle atrophy in red slow-twitch soleus muscles, but not in white fast-twitch extensor digitorum longus (EDL) muscles and gastrocnemius muscles. However, it remains unclear why these beneficial effects of β-carotene are elicited in soleus muscles. To address this issue, we focused on carotenoid transporters in skeletal muscles. In mice, Cd36 mRNA levels were higher in red muscle than in white muscle. The siRNA-mediated knockdown of CD36 decreased β-carotene uptake in C2C12 myotubes. In soleus muscles, CD36 knockdown inhibited β-carotene-induced increase in muscle mass. Intravenous injection of the hypoxia marker pimonidazole produced more pimonidazole-bound proteins in soleus muscles than in EDL muscles, and the hypoxia-inducible factor-1 (HIF-1) α protein level was higher in soleus muscles than in EDL muscles. In C2C12 myotubes, hypoxia increased the expression of CD36 and HIF-1α at the protein and mRNA levels, and HIF-1α knockdown reduced hypoxia-induced increase in Cd36 mRNA level. In soleus muscles, HIF-1α knockdown reduced Cd36 mRNA level. These results indicate that CD36 is predominantly involved in β-carotene-induced increase in soleus muscle mass of mice. Furthermore, we demonstrate that CD36 expression depends on HIF-1α in the soleus muscles of mice, even under normal physiological conditions.

Lifespan Analysis of Dystrophic mdx Fast-Twitch Muscle Morphology and Its Impact on Contractile Function.

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model, we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast-twitch Extensor Digitorum Longus muscles from animals 4–22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a “tipping point” is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

Modified UCN2 peptide treatment improves skeletal muscle mass and function in mouse models of obesity‐induced insulin resistance

BackgroundType 2 diabetes and obesity are often seen concurrently with skeletal muscle wasting, leading to further derangements in function and metabolism. Muscle wasting remains an unmet need for metabolic disease, and new approaches are warranted. The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin releasing factor receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance, glucose metabolism, and muscle mass. The aim of this study was to investigate the effects of modified UCN2 peptides as a pharmaceutical therapy to counteract the loss of skeletal muscle mass associated with obesity and casting immobilization.MethodsHigh‐fat‐fed mice (C57Bl/6J; 26 weeks old) and ob/ob mice (11 weeks old) were injected daily with a PEGylated (Compound A) and non‐PEGylated (Compound B) modified human UCN2 at 0.3 mg/kg subcutaneously for 14 days. A separate group of chow‐fed C57Bl/6J mice (12 weeks old) was subjected to hindlimb cast immobilization and, after 1 week, received daily injections with Compound A. In vivo functional tests were performed to measure protein synthesis rates and skeletal muscle function. Ex vivo functional and molecular tests were performed to measure contractile force and signal transduction of catabolic and anabolic pathways in skeletal muscle.ResultsSkeletal muscles (extensor digitorum longus, soleus, and tibialis anterior) from high‐fat‐fed mice treated with Compound A were ~14% heavier than muscles from vehicle‐treated mice. Chronic treatment with modified UCN2 peptides altered the expression of structural genes and transcription factors in skeletal muscle in high‐fat diet‐induced obesity including down‐regulation of Trim63 and up‐regulation of Nr4a2 and Igf1 (P < 0.05 vs. vehicle). Signal transduction via both catabolic and anabolic pathways was increased in tibialis anterior muscle, with increased phosphorylation of ribosomal protein S6 at Ser235/236, FOXO1 at Ser256, and ULK1 at Ser317, suggesting that UCN2 treatment modulates protein synthesis and degradation pathways (P < 0.05 vs. vehicle). Acutely, a single injection of Compound A in drug‐naïve mice had no effect on the rate of protein synthesis in skeletal muscle, as measured via the surface sensing of translation method, while the expression of Nr4a3 and Ppargc1a4 was increased (P < 0.05 vs. vehicle). Compound A treatment prevented the loss of force production from disuse due to casting. Compound B treatment increased time to fatigue during ex vivo contractions of fast‐twitch extensor digitorum longus muscle. Compound A and B treatment increased lean mass and rates of skeletal muscle protein synthesis in ob/ob mice.ConclusionsModified human UCN2 is a pharmacological candidate for the prevention of the loss of skeletal muscle mass associated with obesity and immobilization.

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle.

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk-old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 min of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 h after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 h after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation-initiation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.NEW & NOTEWORTHY The application of cyclic ex vivo dynamic muscle contractions delivered in an intermittent pattern may be suitable for the exploration of skeletal muscle regulatory responses to endurance-type contractile activity. In the present study, it is demonstrated that the response to such stimulus of some nodal myocellular signaling proteins related to either metabolic or anabolic signaling events may be influenced by muscle phenotype, whereas the response of others appears to be independent of phenotype.

Attractin deficiency causes metabolic and morphological abnormalities in slow-twitch muscle

Skeletal muscle fibers are classified as slow-twitch and fast-twitch fibers, which have different reactive oxygen species (ROS) metabolism and mitochondrial biogenesis. Recently, Attractin (Atrn), which encodes secreted (sAtrn) and transmembrane (mAtrn)-type proteins, has been shown to be involved in free radical scavenging. Although Atrn has been found in skeletal muscle, little is known about the expression levels and function of Atrn in each muscle fiber type. Therefore, we investigatesAtrn and mAtrn expression levels in the slow-twitch soleus (sol) and fast-twitch extensor digitorum longus (EDL) muscles as well as the morphology and expression levels of antioxidant enzymes and functional mitochondrial markers using Atrn-deficient muscles. Both types of Atrn were expressed in the sol and EDL. mAtrn was mainly expressed in the adult sol, whereas sAtrn expression levels did not differ between muscle types. Moreover, mAtrn in the sol was abundantly localized in the subsarcolemmal area, especially in the myoplasm near mitochondria. Atrn-deficient Zitter rats showed muscle fiber atrophy, myofibril misalignment, mitochondrial swelling and vacuolation in the sol but not EDL. Furthermore, the Atrn-deficient sol exhibited a marked reduction in antioxidant enzyme SOD1, GPx1, catalaseand Prx6and mitochondrial functional protein, UCP2, expression. Even Atrn-deficient EDL showed a significant reduction in Prx3, Prx6, UCP2and UCP3 expression. These data indicate that Atrn-deficiency disturbs ROS metabolism in skeletal muscles. In particular, mAtrn is involved in metabolism in the slow-twitch sol muscle and mAtrn-deficiency may cause ROS imbalance, resulting in morphological abnormalities in the muscle.

Lifelong Ulk1-Mediated Autophagy Deficiency in Muscle Induces Mitochondrial Dysfunction and Contractile Weakness

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1′s dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice

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.