Fiber-type Switching Research Articles

N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes

N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

Preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model

We previously demonstrated that transcutaneous CO2 application promotes muscle fiber-type switching, fracture healing, and osteogenesis by increasing blood flow and angiogenesis. Here, we aimed to investigate the preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model. Eleven-week-old male Sprague-Dawley rats were divided into hindlimb suspension (HS), HS with transcutaneous CO2 application (HSCO2), and control groups. HSCO2 rats were administered transcutaneous 100 % CO2 gas in their bilateral hindlimbs, five times a week for 20 min. After 3 weeks, we harvested the gastrocnemius, femur, and tibia for assessment. Histological analysis revealed a significant decrease in the gastrocnemius myofiber cross-sectional area in HS rats compared to the control rats, whereas HSCO2 rats exhibited a significant increase compared to HS rats. Micro-computed tomography showed significant bone atrophy in the trabecular and cortical bones of the femur in HS rats compared to those of the control rats, whereas significant improvement was noted in HSCO2 rats. Histological analysis of the proximal tibia revealed more marrow adipose tissue in the HS rats than in the control rats. However, in the HSCO2 rats, fewer marrow adipose tissue and osteoclasts were observed. Moreover, HSCO2 rats had more osteoblasts and higher expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) than the HS rats. The gastrocnemius and distal femur of HSCO2 rats also exhibited elevated PGC-1α and VEGF expression and upregulation of the myogenesis markers and osteogenesis markers compared to those of HS rats. This treatment effectively prevented disuse osteoporosis and muscle atrophy by promoting local angiogenesis and blood flow. PGC-1α is crucial for promoting this angiogenic pathway. Transcutaneous CO2 application may be a novel preventive procedure for disuse osteoporosis and muscle atrophy, complementing medication and rehabilitation.

Reduced exercise capacity occurs before intrinsic skeletal muscle dysfunction in experimental rat models of pulmonary hypertension.

Reduced exercise capacity in pulmonary hypertension (PH) significantly impacts quality of life. However, the cause of reduced exercise capacity in PH remains unclear. The objective of this study was to investigate whether intrinsic skeletal muscle changes are causative in reduced exercise capacity in PH using preclinical PH rat models with different PH severity. PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 0-4 weeks of normoxia exposure. Control s rats were injected with vehicle and housed in normoxia. Echocardiography was performed to assess cardiac function. Exercise capacity was assessed by VO2 max. Skeletal muscle structural changes (atrophy, fiber type switching, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7-weektimepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4-weektimepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Our data suggest that cardiopulmonary impairments in PH are the primary cause of reduced exercise capacity, which occurs before intrinsic skeletal muscle dysfunction.

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.

Lmod2 is necessary for effective skeletal muscle contraction.

Muscle contraction is a regulated process driven by the sliding of actin-thin filaments over myosin-thick filaments. Lmod2 is an actin filament length regulator and essential for life since human mutations and complete loss of Lmod2 in mice lead to dilated cardiomyopathy and death. To study the little-known role of Lmod2 in skeletal muscle, we created a mouse model with Lmod2 expressed exclusively in the heart but absent in skeletal muscle. Loss of Lmod2 in skeletal muscle results in decreased force production in fast- and slow-twitch muscles. Soleus muscle from rescued Lmod2 knockout mice have shorter thin filaments, increased Lmod3 levels, and present with a myosin fiber type switch from fast myosin heavy chain (MHC) IIA to the slower MHC I isoform. Since Lmod2 regulates thin-filament length in slow-twitch but not fast-twitch skeletal muscle and force deficits were observed in both muscle types, this work demonstrates that Lmod2 regulates skeletal muscle contraction, independent of its role in thin-filament length regulation.

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation.

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.

Investigation of genotype and allele distribution of ppargc1a-t19c locus in holstein cattle

This study aimed to investigate the genotype and allele distribution of the PPARGC1A-T19C locus in Holstein cattle. The PPARGC1A gene plays a crucial role in significant metabolic processes such as adaptive thermogenesis, gluconeogenesis, skeletal muscle fiber type switching, mitochondrial biogenesis, and adipogenesis. Additionally, it holds a key role in the activation of many hormone receptors and transcription factors, thereby emerging as a potential candidate gene for the milk traits of cattle. In this study, genotyping was performed using the PCR-RFLP method on 50 Holstein animal samples. The 205 bp PCR product was digested with the BsuRI restriction enzyme to reveal genotypic differentiation. Two different genotypes, TC and CC, were identified. The genotype frequencies were found to be 0.30 (TC) and 0.70 (CC). Allele frequencies were determined as 0.15 (T) and 0.85 (C). The Chi-square test showed that the PPARGC1A-T19C locus distribution was in Hardy-Weinberg equilibrium (p>0.05). In conclusion, these findings can be utilized to understand intra-breed genetic diversity and develop more effective selection strategies in the genetic improvement programs of the breed.

Loss of lysosomal acid lipase results in mitochondrial dysfunction and fiber switch in skeletal muscles of mice

ObjectiveLysosomal acid lipase (LAL) is the only enzyme known to hydrolyze cholesteryl esters (CE) and triacylglycerols in lysosomes at an acidic pH. Despite the importance of lysosomal hydrolysis in skeletal muscle (SM), research in this area is limited. We hypothesized that LAL may play an important role in SM development, function, and metabolism as a result of lipid and/or carbohydrate metabolism disruptions. ResultsMice with systemic LAL deficiency (Lal−/−) had markedly lower SM mass, cross-sectional area, and Feret diameter despite unchanged proteolysis or protein synthesis markers in all SM examined. In addition, Lal−/− SM showed increased total cholesterol and CE concentrations, especially during fasting and maturation. Regardless of increased glucose uptake, expression of the slow oxidative fiber marker MYH7 was markedly increased in Lal−/−SM, indicating a fiber switch from glycolytic, fast-twitch fibers to oxidative, slow-twitch fibers. Proteomic analysis of the oxidative and glycolytic parts of the SM confirmed the transition between fast- and slow-twitch fibers, consistent with the decreased Lal−/− muscle size due to the “fiber paradox”. Decreased oxidative capacity and ATP concentration were associated with reduced mitochondrial function of Lal−/− SM, particularly affecting oxidative phosphorylation, despite unchanged structure and number of mitochondria. Impairment in muscle function was reflected by increased exhaustion in the treadmill peak effort test in vivo. ConclusionWe conclude that whole-body loss of LAL is associated with a profound remodeling of the muscular phenotype, manifested by fiber type switch and a decline in muscle mass, most likely due to dysfunctional mitochondria and impaired energy metabolism, at least in mice.

Signaling pathways regulated by natural active ingredients in the fight against exercise fatigue-a review.

Exercise fatigue is a normal protective mechanism of the body. However, long-term fatigue hinders normal metabolism and exercise capacity. The generation and recovery from exercise fatigue involves alterations in multiple signaling pathways, mainly AMPK, PI3K/Akt, Nrf2/ARE, NF-κB, PINK1/Parkin, and BDNF/TrkB, as well as MAPK signaling pathways that mediate energy supply, reduction of metabolites, oxidative stress homeostasis, muscle fiber type switching, and central protective effects. In recent studies, a rich variety of natural active ingredients have been identified in traditional Chinese medicines and plant extracts with anti-fatigue effects, opening up the field of research in new anti-fatigue drugs. In this review we give an overview of the signaling pathways associated with the activity of natural food active ingredients against exercise fatigue. Such a comprehensive review is necessary to understand the potential of these materials as preventive measures and treatments of exercise fatigue. We expect the findings highlighted and discussed here will help guide the development of new health products and provide a theoretical and scientific basis for future research on exercise fatigue.

Effects of genetic selection for fast growth on the development of wooden breast myopathy in broilers

ABSTRACT 1. This study investigated the physiological and molecular mechanisms leading to wooden breast (WB) by comparing growth parameters, oxygen consumption rate, thyroid hormone and gene expression patterns in fast- versus slow-growing broiler lines (Cobb500 and L1986, respectively). 2. WB was observed in Cobb500 broilers only and was first diagnosed on d 21 post-hatch. Compared to the slow-growing L1986, Cobb500 showed a significantly higher growth rate, relative breast weight, breast thickness, meat pH and water-retention capacity (drip loss). Correspondingly, there was significantly lower relative heart weight, relative right ventricular weight, triiodothyronine and thyroxine concentrations and oxygen consumption rate. 3. Compared to No-WB Cobb500, the WB-affected samples exhibited higher relative breast weight, breast thickness and drip loss and lower plasma total thyroxine (T4) concentrations. 4. Selection for fast growth was associated with differential expression of genes involved in hypoxia (PLOD2), energy metabolism (FABP3, FABP4, CD36, and LPL), endoplasmic reticulum stress, muscle regeneration (CSRP3) and fibre-type switching (ANKRD1). WB-affected samples exhibited an upregulation of CSRP3, PLOD2 and ANKRD1, while CD36 was downregulated. Taken together, selection for fast growth and muscle gain is not matched by adequate cardiac and metabolic support systems.

Prolonged mechanical muscle loading increases mechanosensor gene and protein levels and causes a moderate fast-to-slow fiber type switch in mice.

Mechanosensing and subsequent mechanotransduction are indispensable for muscle plasticity. Nevertheless, a scarcity of literature exists regarding an all-encompassing understanding of the muscle mechanosensing machinery's response to prolonged loading, especially in conditions that resemble a natural physiological state of skeletal muscle. This study aimed to comprehensively explore the effects of prolonged mechanical loading on mechanosensitive components, skeletal muscle characteristics, and metabolism-related gene clusters. Twenty male C57BL/6J mice were randomly divided into two groups: control and prolonged mechanical loading. To induce prolonged mechanical loading on the triceps brachii (TRI) and biceps brachii (BIC) muscles, a 14-day period of tail suspension was implemented. In TRI only, prolonged mechanical loading caused a mild fast-to-slow fiber type shift together with increased mechanosensor gene and protein levels. It also increased transcription factors associated with slow muscle fibers while decreasing those related to fast-type muscle gene expression. Succinate dehydrogenase activity, a marker of muscle oxidative capacity, and genes involved in oxidative and mitochondrial turnover increased, whereas glycolytic-related genes decreased. Moreover, prolonged mechanical loading stimulated markers of muscle protein synthesis. Taken together, our data show a collective muscle-specific increase in mechanosensor gene and protein levels upon a period of prolonged mechanical loading in conditions that reflect a more natural physiological state of skeletal muscle in mice. We provide additional proof-of-concept that prolonged tail suspension-induced loading of the forelimbs triggers a muscle-specific fast-to-slow fiber type switch, and this coincides with increased protein synthesis-related signaling.NEW & NOTEWORTHY This study provides a comprehensive overview of the effects of prolonged loading on mechanosensitive components in conditions that better reflect the natural physiological state of skeletal muscle. Although the muscle mechanosensing machinery has been widely acknowledged for its responsiveness to altered loading, an inclusive understanding of its response to prolonged loading remains scarce. Our results show a fast-to-slow fiber type shift and an upregulation of mechanosensor gene and protein levels following prolonged loading.

Normobaric hypoxia shows enhanced FOXO1 signaling in obese mouse gastrocnemius muscle linked to metabolism and muscle structure and neuromuscular innervation.

Skeletal muscle relies on mitochondria for sustainable ATP production, which may be impacted by reduced oxygen availability (hypoxia). Compared with long-term hypoxia, the mechanistic in vivo response to acute hypoxia remains elusive. Therefore, we aimed to provide an integrated description of the Musculus gastrocnemius response to acute hypoxia. Fasted male C57BL/6JOlaHsd mice, fed a 40en% fat diet for six weeks, were exposed to 12% O2 normobaric hypoxia or normoxia (20.9% O2) for six hours (n = 12 per group). Whole-body energy metabolism and the transcriptome response of the M. gastrocnemius were analyzed and confirmed by acylcarnitine determination and Q-PCR. At the whole-body level, six hours of hypoxia reduced energy expenditure, increased blood glucose and tended to decreased the respiratory exchange ratio (RER). Whole-genome transcriptome analysis revealed upregulation of forkhead box-O (FOXO) signalling, including an increased expression of tribbles pseudokinase 3 (Trib3). Trib3 positively correlated with blood glucose levels. Upregulated carnitine palmitoyltransferase 1A negatively correlated with the RER, but the significantly increased in tissue C14-1, C16-0 and C18-1 acylcarnitines supported that β-oxidation was not regulated. The hypoxia-induced FOXO activation could also be connected to altered gene expression related to fiber-type switching, extracellular matrix remodeling, muscle differentiation and neuromuscular junction denervation. Our results suggest that a six-hour exposure of obese mice to 12% O2 normobaric hypoxia impacts M. gastrocnemius via FOXO1, initiating alterations that may contribute to muscle remodeling of which denervation is novel and warrants further investigation. The findings support an early role of hypoxia in tissue alterations in hypoxia-associated conditions such as aging and obesity.

Adipocyte Rnf20 ablation increases the fast-twitch fibers of skeletal muscle via lysophosphatidylcholine 16:0.

Both adipose tissue and skeletal muscle are highly dynamic tissues and interact at the metabolic and hormonal levels in response to internal and external stress, and they coordinate in maintaining whole-body metabolic homeostasis. In our previous study, we revealed that adipocyte-specific Rnf20 knockout mice (ASKO mice) exhibited lower fat mass but higher lean mass, providing a good model for investigating the adipose-muscle crosstalk and exploring the effect of the adipocyte Rnf20 gene on the physiology and metabolism of skeletal muscle. Here, we confirmed that ASKO mice exhibited the significantly increased body weight and gastrocnemius muscle weight. Fiber-type switching in the soleus muscle of ASKO mice was observed, as evidenced by the increased number of fast-twitch fibers and decreased number of slow-twitch fibers. Serum metabolites with significant alteration in abundance were identified by metabolomic analysis and the elevated lysophosphatidylcholine 16:0 [LysoPC (16:0)] was observed in ASKO mice. In addition, lipidome analysis of gonadal white adipose tissue revealed asignificant increase in LysoPCs and LysoPC (16:0) in ASKO mice. Furthermore, knockdown of Rnf20 gene in 3T3-L1 cells significantly increased the secretion of LysoPC, suggesting that LysoPC might be a critical metabolite in the adipose-muscle crosstalk of ASKO mice. Furthermore, in vitro study demonstrated that LysoPC (16:0) could induce the expression of fast-twitchmusclefibers related genes in differentiated C2C12 cells, indicating its potential role in adipose-muscle crosstalk. Taken together, these findings not only expand our understanding of the biological functions of Rnf20 gene in systemic lipid metabolism, but also provide insight into adipose tissue dysfunction-induced physiological alterations in skeletal muscle.

AAV1.NT-3 gene therapy in the SOD1KO mouse model of accelerated sarcopenia.

Sarcopenia, an age-related loss of muscle mass, is a critical factor that affects the health of the older adults. The SOD1KO mouse is deficient of Cu/Zn superoxide dismutase, used as an accelerated aging model. We previously showed that NT-3 improves muscle fibre size by activating the mTOR pathway, suggesting a potential for attenuating age-related muscle loss. This study assessed the therapeutic efficacy of AAV1.NT-3 in this accelerated aging model. Twelve 6months old SOD1KO mice were injected intramuscularly with a 1×1011 vg dose of AAV1.tMCK.NT-3, and 13 age-matched SOD1KO mice were used as controls. The treatment effect was evaluated using treadmill, rotarod and gait analyses as well as histological studies assessing changes in muscle fibre, and fibre type switch, in tibialis anterior, gastrocnemius, and triceps muscles, and myelin thickness by calculating G ratio in sciatic and tibial nerves. Molecular studies involved qPCR experiments to analyse the expression levels of mitochondrial and glycolysis markers and western blot experiments to assess the activity of mTORC1 pathway. Treatment resulted in a 36% (154.9 vs. 114.1; P<0.0001) and 76% increase (154.3 vs. 87.6; P<0.0001) in meters ran, with treadmill test at 3 and 6months post gene delivery. In addition, the treated cohort stayed on rotarod 30% (52.7s vs. 40.4s; P=0.0095) and 54% (50.4s vs. 32.7s; P=0.0007) longer, compared with untreated counterparts at 3 and 6months post injection. Gait analysis, performed at endpoint, showed that stride width was normalized to wild type levels (29.3mm) by an 11% decrease, compared with untreated cohort (28.6mm vs. 32.1mm; P=0.0014). Compared with wild-type, SOD1KO mice showed 9.4% and 11.4% fibre size decrease in tibialis anterior and gastrocnemius muscles, respectively, which were normalized to wild type levels with treatment. Fibre diameter increase was observed prominently in FTG fibre type. G ratio analysis revealed hypomyelination in the tibial (0.721) and sciatic (0.676) nerves of SOD1KO model, which was reversed in the NT-3 cohort (0.646 and 0.634, respectively). Fibre size increase correlated with the increase in the p-S6 and p-4E-BP1 levels, and in the glycolysis markers in tibialis anterior. Alterations observed in the mitochondrial markers were not rescued with treatment. Overall, response to NT-3 was subdued in gastrocnemius muscle. This study shows that AAV1.NT-3 gene therapy protected SOD1KO mouse from accelerated aging effects functionally and histologically. We further confirmed that NT-3 has potential to activate the mTOR and glycolytic pathways in muscle.

Abstract P3030: Reduced Exercise Capacity Occurs Before Intrinsic Changes In Skeletal Muscle In Experimental Rat Models Of Pulmonary Hypertension

Background: Pulmonary Hypertension (PH) is defined with mean pulmonary arterial pressure greater than 20 mmHg at rest. Reduced exercise capacity in PH significantly impacts quality of life. While the cause of reduced exercise capacity in PH remains unclear, it is suggested that regional skeletal muscle adaptions may play a role. The objective of this study was to investigate whether intrinsic changes in skeletal muscle determine exercise capacity in PH using preclinical PH rat models with different PH severity. Methods and Results: PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 1-4 weeks of normoxia exposure. Rats injected with vehicle and housed in normoxia were used as controls. Echocardiography and hemodynamics were performed to assess PH and cardiac function. Exercise capacity was assessed by VO 2 max. Skeletal muscle structural changes (atrophy, fiber type switch, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed in soleus muscles. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7 weeks timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4 weeks timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Conclusions: Our study demonstrated that reduced exercise capacity in PH occurs before intrinsic changes in skeletal muscle, suggesting that alterations in skeletal muscle are not causative to exercise intolerance in PH, while they may worsen exercise capacity collaboratively with primary factors in the late stage of PH. In addition, our study supports the notion that improving central cardiopulmonary impairments at the early stage of PH may be critical in preventing or delaying the occurrence of exercise limitation in settings of PH.

Thyroid Hormone Regulates the Lipid Content of Muscle Fibers, Thus Affecting Physical Exercise Performance.

Skeletal muscle (SkM) lipid composition plays an essential role in physiological muscle maintenance and exercise performance. Thyroid hormones (THs) regulate muscle formation and fuel energy utilization by modulating carbohydrates and lipid and protein metabolism. The best-known effects of THs in SkM include the promotion of mitochondrial biogenesis, the fiber-type switch from oxidative to glycolytic fibers, and enhanced angiogenesis. To assess the role of THs on the lipidic composition of SkM fibers, we performed lipidomic analyses of SkM cells and tissues, glucose tolerance experiments, and exercise performance tests. Our data demonstrated that TH treatment induces remodeling of the lipid profile and changes the proportion of fatty acids in SkM. In brief, THs significantly reduced the ratio of stearic/oleic acid in the muscle similar to what is induced by physical activity. The increased proportion of unsaturated fatty acids was linked to an improvement in insulin sensitivity and endurance exercise. These findings point to THs as critical endocrine factors affecting exercise performance and indicate that homeostatic maintenance of TH signals, by improving cell permeability and receptor stability at the cell membrane, is crucial for muscle physiology.

DNM2 levels normalization improves muscle phenotypes of a novel mouse model for moderate centronuclear myopathy

Dynamin 2 (DNM2) is a ubiquitously expressed GTPase regulating membrane trafficking and cytoskeleton dynamics. Heterozygous dominant mutations in DNM2 cause centronuclear myopathy (CNM), associated with muscle weakness and atrophy and histopathological hallmarks as fiber hypotrophy and organelles mis-position. Different severities range from the severe neonatal onset form to the moderate form with childhood onset and to the mild adult onset form. No therapy is approved for CNM. Here we aimed to validate and rescue a mouse model for the moderate form of DNM2-CNM harboring the common DNM2 R369W missense mutation. Dnm2R369W/+ mice presented with increased DNM2 protein level in muscle and moderate CNM-like phenotypes with force deficit, muscle and fiber hypotrophy, impaired mTOR signaling, and progressive mitochondria and nuclei mis-position with age. Molecular analyses revealed a fiber type switch toward oxidative metabolism correlating with decreased force and alteration of mitophagy markers paralleling mitochondria structural defects. Normalization of DNM2 levels through intramuscular injection of AAV-shDnm2 targeting Dnm2 mRNA significantly improved histopathology and muscle and myofiber hypotrophy. These results showed that the Dnm2R369W/+ mouse is a faithful model for the moderate form of DNM2-CNM and revealed that DNM2 normalization after a short 4-week treatment is sufficient to improve the CNM phenotypes.

Neuromuscular defects after infection with a beta coronavirus in mice

COVID-19 affects primarily the lung. However, several other systemic alterations, including muscle weakness, fatigue and myalgia have been reported and may contribute to the disease outcome. We hypothesize that changes in the neuromuscular system may contribute to the latter symptoms observed in COVID-19 patients. Here, we showed that C57BL/6J mice inoculated intranasally with the murine betacoronavirus hepatitis coronavirus 3 (MHV-3), a model for studying COVID-19 in BSL-2 conditions that emulates severe COVID-19, developed robust motor alterations in muscle strength and locomotor activity. The latter changes were accompanied by degeneration and loss of motoneurons that were associated with the presence of virus-like particles inside the motoneuron. At the neuromuscular junction level, there were signs of atrophy and fragmentation in synaptic elements of MHV-3-infected mice. Furthermore, there was muscle atrophy and fiber type switch with alteration in myokines levels in muscles of MHV-3-infected mice. Collectively, our results show that acute infection with a betacoronavirus leads to robust motor impairment accompanied by neuromuscular system alteration.

33-OR: Epigenetic Reader BRD4 Directs Fast-Twitch Myofiber Gene Programs Influencing Skeletal Muscle Endurance Capacity and Systemic Glucose Homeostasis

Skeletal myofibers exhibit remarkable diversity and plasticity in contractile and metabolic functions and play a key role in controlling systemic metabolism. Previous studies of muscle fiber-type switching have centered on the specification of slow-twitch myofibers. However, mechanisms that drive fast-twitch myofibers formation remain unclear. Here, we showed that BRD4, an epigenetic reader enriched in fast myofibers, plays a critical role in controlling fast-twitch myofiber identity as well as systemic glucose homeostasis. BRD4 levels are associated with fast-twitch myofiber gene expression in human muscle. Skeletal muscle-specific ablation of BRD4 in mice (Brd4f/fHSA-Cre, BRD4 mKO) resulted in downregulation of the fast-twitch myofiber gene program, increased number (~100%) of type I myofibers and mitochondrial oxidative capacity. This led to enhanced muscle endurance capacity. In adult mice, acute and inducible BRD4 deletion using a tamoxifen-inducible Cre elicited a similar fast-to-slow fiber type switch in muscle. Mechanically, genome-wide ChIP-seq and mRNA-seq analyses revealed that BRD4 directly binds to and activates transcriptional of fast-twitch myofiber genes through cooperating with MEF2. Furthermore, mice lacking muscle BRD4 were protected against high-fat-diet (HFD)-induced obesity, glucose intolerance and insulin resistance. HFD-fed BRD4 mKO mice had significantly less fat mass (∼50%) compared with HFD-fed WT controls. Histological and biochemical measurements confirmed a pronounced (~60%) decrease in fat deposition in BRD4 mKO livers. Thus, our results uncover a previously unrecognized mechanism underlying the determination of fast-twitch myofiber and provide new therapeutic opportunities for driving muscle fiber-type switch to combat metabolic diseases. Disclosure Z.Zhou: None. L.Liu: None. Z.Xu: None. T.Fu: None. L.Yang: None. M.Yan: None. Z.Gan: None.

Genetic deletion of Kvβ2 (AKR6) causes loss of muscle function and increased inflammation in mice.

The voltage-gated potassium channels (Kv) are complex ion channels with distinct roles in neurotransmission, electrical conductivity of the heart, and smooth and striated muscle functions. Previously, we demonstrated that deletion of Kvβ2 in mice results in decreased Pax7 protein levels, hindlimb muscles and body weights, and fiber type switching. In the present study, we tested the hypothesis that Kvβ2 regulates skeletal muscle function in mice. The young and old Kvβ2 knockout (KO) and wildtype (WT) mice were utilized to test the aging phenotype and skeletal muscle function. Consistent with our previous finding, we found a significant reduction in hindlimb skeletal muscles mass and body weight in young Kvβ2 KO mice, which was also significantly reduced in old Kvβ2 KO mice compared with age-matched WT mice. Forelimb grip strength, and the hindleg extensor digitorum longus (EDL) muscles force-frequency relations were significantly decreased in young and old Kvβ2 KO mice compared to age-matched WT mice. Analysis of transmission electron microscopy images of EDL muscles in young mice revealed a significant reduction in the sarcomere length for Kvβ2 KO vs. WT. Hematoxylin and eosin-stained tibialis anterior muscles cryosections displayed a significant decrease in the number of medium (2,000-4,000µm2) and largest (>4,000µm2) myofibers area in young Kvβ2 KO vs. WT mice. We also found a significant increase in fibrotic tissue area in young Kvβ2 KO mice compared with age-matched WT mice. Analysis of RNA Seq data of the gastrocnemius muscles (GAS) identified significant increase in genes involved in skeletal muscle development, proliferation and cell fate determination, atrophy, energy metabolism, muscle plasticity, inflammation, and a decrease in circadian core clock genes in young Kvβ2 KO vs. WT mice. Several genes were significantly upregulated (384 genes) and downregulated (40 genes) in young Kvβ2 KO mice compared to age-matched WT mice. Further, RT-qPCR analysis of the GAS muscles displayed a significant increase in pro-inflammatory marker Il6 expression in young Kvβ2 KO mice compared to age-matched WT mice. Overall, the present study shows that deletion of Kvβ2 leads to decreased muscles strength and increased inflammation.