Perm1 regulates CaMKII activation and shapes skeletal muscle responses to endurance exercise training.
ABSTRACT: OBJECTIVE:Endurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise. METHODS:We performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function. RESULTS:We show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models. CONCLUSIONS:Our findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.
Project description:BACKGROUND: The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype. RESULTS: Fast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native ?/? CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. ?/? CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed ?/? CaMKII was functional as shown through increased threonine 287 phosphorylation of ?-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. ?/? CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres. CONCLUSION: Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.
Project description:The growth factor myostatin (Mstn) is a negative regulator of skeletal muscle mass. Mstn(-/-) muscles are hypertrophied, stronger, and more glycolytic than Mstn(+/+) muscles, suggesting that they might not perform endurance exercise as well as Mstn(+/+) mice. Indeed, it has previously been shown that treadmill exercise training reduces triceps weight in Mstn(-/-) mice. To analyze the response of Mstn(-/-) muscle to endurance exercise in detail, we carried out endurance training over 4 weeks to examine muscle mass, histology, and oxidative enzyme activity. We found that muscle mass was reduced with training in several muscles from both genotypes, with no evidence of muscle damage. Citrate synthase activity was increased with training in control and mutant mice. Non-trained Mstn(-/-) mice did, however, have lower maximal exercise capacity compared with Mstn(+/+) mice. These results show that Mstn(-/-) muscle retains the metabolic plasticity necessary to adapt normally to endurance training.
Project description:Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase II? (CaMKII?). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKII? signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKII? in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKII? signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKII? signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKII? activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKII? signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKII? signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKII? regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.
Project description:Diabetic myopathy refers to the manifestations in the skeletal muscle as a result of altered glucose homeostasis which reflects as fibrosis. Since physical exercise has been indicated a protective strategy for improving glucose metabolism in skeletal muscle, we tested a hypothesis under which the endurance exercise training could reverse the produced skeletal muscle fibrosis by diabetes. Eight-week-old male Wistar rats were randomly assigned into four groups including healthy control (HC), healthy trained (HT), diabetic control (DC), and diabetic trained (DT) groups. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 45?mg/kg). Rats in the HT and DT groups carried out an exercise program on a motorized treadmill for five days a week over six weeks. Skeletal muscle levels of NRG1and ErbB2 were measured by the Western blot method. Exercise training decreased blood glucose levels in the DT group. Induction of diabetes increased skeletal muscle fibrosis in both the fast extensor digitorum longus (EDL) and slow soleus muscles, while endurance training modified it in diabetic trained rats. Moreover, muscle NRG1and ErbB2 levels were increased in diabetic rats, while training modified muscle NRG1and ErbB2 levels in diabetic trained rats. Our study provides novel evidence that endurance training could modify skeletal muscle fibrosis through NRG1/ErbB2 modification in STZ-induced diabetic rats.
Project description:Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1?, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1? is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.
Project description:Mitochondrial oxidative metabolism and energy transduction pathways are critical for skeletal and cardiac muscle function. The expression of genes important for mitochondrial biogenesis and oxidative metabolism are under the control of members of the peroxisome proliferator-activated receptor ? coactivator 1 (PGC-1) family of transcriptional coactivators and the estrogen-related receptor (ERR) subfamily of nuclear receptors. Perturbations in PGC-1 and/or ERR activities have been associated with alterations in capacity for endurance exercise, rates of muscle atrophy, and cardiac function. The mechanism(s) by which PGC-1 and ERR proteins regulate muscle-specific transcriptional programs is not fully understood. We show here that PGC-1? and ERRs induce the expression of a so far uncharacterized muscle-specific protein, PGC-1- and ERR-induced regulator in muscle 1 (Perm1), which regulates the expression of selective PGC-1/ERR target genes. Perm1 is required for the basal as well as PGC-1?-enhanced expression of genes with roles in glucose and lipid metabolism, energy transfer, and contractile function. Silencing of Perm1 in cultured myotubes compromises respiratory capacity and diminishes PGC-1?-induced mitochondrial biogenesis. Our findings support a role for Perm1 acting downstream of PGC-1? and ERRs to regulate muscle-specific pathways important for energy metabolism and contractile function. Elucidating the function of Perm1 may enable novel approaches for the treatment of disorders with compromised skeletal muscle bioenergetics, such as mitochondrial myopathies and age-related/disease-associated muscle atrophies.
Project description:Diabetes and exercise training have been shown to involve interleukin 6 (IL-6) signaling in muscle. However, the relationship between the actions of these two stimuli on muscle IL-6 and their downstream components is still unknown. Thus, the effect of endurance training on the key components of muscle IL-6 signaling transduction was investigated in a rat model of type 2 diabetes.Diabetes was induced by streptozotocin (STZ) in male Wistar rats fed a high-fat diet, with normal rats acting as controls. The animals were left to conduct their normal activities or assigned to endurance training in a treadmill. At the end of 8 weeks, blood biochemical profiles, exercise performance, muscle oxidative capacity, glucose transporter 4 (GLUT4) protein distribution, and expressions of IL-6 and its downstream proteins were determined.Blood biochemical profiles of the diabetic rats were altered compared to normal rats, whereas endurance training improved blood chemistry and exercise performance. It also increased muscle oxidative capacity, and promoted GLUT4 subcellular localization to the membrane in muscles. Furthermore, protein expression of IL-6 receptor (IL-6Rα) was increased in both normal and diabetic rats after endurance training, but no significant changes in IL-6, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), or suppressor of cytokine signaling 3 (SOC3) were observed in muscles of normal and diabetic rats.IL-6 signaling pathway mediating muscle response to endurance training was conserved in type 2 diabetes. There was no link between training-induced IL-6 downstream targets in skeletal muscles and IL-6-induced type 2 diabetes.
Project description:The striated activator of rho-signaling (STARS) protein acts as a link between external stimuli and exercise adaptation such as muscle hypertrophy. However, the acute and long-term adaptational response of STARS is still unclear. This study aimed at investigating the acute and long-term endurance training response on the mRNA and protein expression of STARS and its related upstream and downstream factors in human skeletal muscle. mRNA and protein levels of STARS and related factors were assessed in skeletal muscle of healthy young men and women following an acute bout of endurance exercise (n = 15) or 12 weeks of one-legged training (n = 23). Muscle biopsies were obtained before (acute and long-term), at 30 min, 2, and 6 h following acute exercise, and at 24 h following both acute exercise and long-term training. Following acute exercise, STARS mRNA was significantly elevated 3.9-fold at 30 min returning back to baseline 24 h after exercise. STARS protein levels were numerically but nonsignificantly increased 7.2-fold at 24 h. No changes in STARS or ERR? mRNA or STARS protein expression were seen following long-term training. PGC-1? mRNA increased 1.7-fold following long-term training. MRTF-A mRNA was increased both following acute exercise and long-term training, in contrast to SRF mRNA and protein which did not change. STARS mRNA is acutely upregulated with exercise, but there is no cumulative effect to long-term training as seen in PGC-1? mRNA expression. Exercise intensity might play a role in manifestation of protein expression, suggesting a more complex regulation of STARS.
Project description:A single bout of exercise induces changes in gene expression in skeletal muscle. Regular exercise results in an adaptive response involving changes in muscle architecture and biochemistry, and is an effective way to manage and prevent common human diseases such as obesity, cardiovascular disorders and type II diabetes. However, the biomolecular mechanisms underlying such responses still need to be fully elucidated. Here we performed a transcriptome-wide analysis of skeletal muscle tissue in a large cohort of untrained Thoroughbred horses (n = 51) before and after a bout of high-intensity exercise and again after an extended period of training. We hypothesized that regular high-intensity exercise training primes the transcriptome for the demands of high-intensity exercise.An extensive set of genes was observed to be significantly differentially regulated in response to a single bout of high-intensity exercise in the untrained cohort (3241 genes) and following multiple bouts of high-intensity exercise training over a six-month period (3405 genes). Approximately one-third of these genes (1025) and several biological processes related to energy metabolism were common to both the exercise and training responses. We then developed a novel network-based computational analysis pipeline to test the hypothesis that these transcriptional changes also influence the contextual molecular interactome and its dynamics in response to exercise and training. The contextual network analysis identified several important hub genes, including the autophagosomal-related gene GABARAPL1, and dynamic functional modules, including those enriched for mitochondrial respiratory chain complexes I and V, that were differentially regulated and had their putative interactions 're-wired' in the exercise and/or training responses.Here we have generated for the first time, a comprehensive set of genes that are differentially expressed in Thoroughbred skeletal muscle in response to both exercise and training. These data indicate that consecutive bouts of high-intensity exercise result in a priming of the skeletal muscle transcriptome for the demands of the next exercise bout. Furthermore, this may also lead to an extensive 're-wiring' of the molecular interactome in both exercise and training and include key genes and functional modules related to autophagy and the mitochondrion.
Project description:The insertion/deletion polymorphism in the gene for the regulator of vascular tone, angiotensin-converting enzyme (ACE), is the prototype of a genetic influence on physical fitness and this involves an influence on capillary supply lines and dependent aerobic metabolism in skeletal muscle. The respective interaction of ACE-I/D genotype and training status on local metabolic and angiogenic reactions in exercised muscle is not known. Toward this end we characterized the metabolomic and angiogenic response in knee extensor muscle, m. vastus lateralis, in 18 untrained and 34 endurance-trained (physically active, [Formula: see text]O2max > 50 mL min-1 kg-1) white British men to an exhaustive bout of one-legged cycling exercise. We hypothesized that training status and ACE-I/D genotype affect supply-related muscle characteristics of exercise performance in correspondence to ACE expression and angiotensin 2 levels. ACE-I/D genotype and training status developed an interaction effect on the cross-sectional area (CSA) of m. vastus lateralis and mean CSA of slow type fibers, which correlated with peak power output (r ? 0.44). Genotype × training interactions in muscle also resolved for exercise-induced alterations of 22 metabolites, 8 lipids, glycogen concentration (p = 0.016), ACE transcript levels (p = 0.037), and by trend for the pro-angiogenic factor tenascin-C post exercise (p = 0.064). Capillary density (p = 0.001), capillary-to-fiber ratio (p = 0.010), systolic blood pressure (p = 0.014), and exercise-induced alterations in the pro-angiogenic protein VEGF (p = 0.043) depended on the ACE-I/D genotype alone. Our observations indicate that variability in aerobic performance in the studied subjects was in part reflected by an ACE-I/D-genotype-modulated metabolic phenotype of a major locomotor muscle. Repeated endurance exercise appeared to override this genetic influence in skeletal muscle by altering the ACE-related metabolic response and molecular aspects of the angiogenic response to endurance exercise.