Matrisome proteomics reveals novel mediators of muscle remodeling with aerobic exercise training.

Skeletal muscle has a unique ability to remodel in response to stimuli such as contraction and aerobic exercise training. Phenotypic changes in muscle that occur with training such as a switch to a more oxidative fiber type, and increased capillary density contribute to the well-known health benefits of aerobic exercise. The muscle matrisome likely plays an important role in muscle remodeling with exercise. However, due to technical limitations in studying muscle ECM proteins, which are highly insoluble, little is known about the muscle matrisome and how it contributes to muscle remodeling. Here, we utilized two-fraction methodology to extract muscle proteins, combined with multiplexed tandem mass tag proteomic technology to identify 161 unique ECM proteins in mouse skeletal muscle. In addition, we demonstrate that aerobic exercise training induces remodeling of a significant proportion of the muscle matrisome. We performed follow-up experiments to validate exercise-regulated ECM targets in a separate cohort of mice using Western blotting and immunofluorescence imaging. Our data demonstrate that changes in several key ECM targets are strongly associated with muscle remodeling processes such as increased capillary density in mice. We also identify LOXL1 as a novel muscle ECM target associated with aerobic capacity in humans. In addition, publically available data and databases were used for in silico modeling to determine the likely cellular sources of exercise-induced ECM remodeling targets and identify ECM interaction networks. This work greatly enhances our understanding of ECM content and function in skeletal muscle and demonstrates an important role for ECM remodeling in the adaptive response to exercise. The raw MS data have been deposited to the ProteomeXchange with identifier PXD053003.

Canagliflozin Prevents Hyperglycemia-Associated Muscle Extracellular Matrix Accumulation and Improves the Adaptive Response to Aerobic Exercise.

Chronic hyperglycemia is associated with low response to aerobic exercise training in rodent models and humans, including reduced aerobic exercise capacity and impaired oxidative remodeling in skeletal muscle. Here, we investigated whether glucose lowering with the sodium-glucose cotransporter 2 inhibitor (SGLT2i), canagliflozin (Cana; 30 mg/kg/day), could restore exercise training response in a model of hyperglycemia (low-dose streptozotocin [STZ]). Cana effectively prevented increased blood glucose in STZ-treated mice. After 6 weeks of voluntary wheel running, Cana-treated mice displayed improvements in aerobic exercise capacity, higher capillary density in striated muscle, and a more oxidative fiber-type in skeletal muscle. In contrast, these responses were blunted or absent in STZ-treated mice. Recent work implicates glucose-induced accumulation of skeletal muscle extracellular matrix (ECM) and hyperactivation of c-Jun N-terminal kinase (JNK)/SMAD2 mechanical signaling as potential mechanisms underlying poor exercise response. In line with this, muscle ECM accretion was prevented by Cana in STZ-treated mice. JNK/SMAD2 signaling with acute exercise was twofold higher in STZ compared with control but was normalized by Cana. In human participants, ECM accumulation was associated with increased JNK signaling, low VO2peak, and impaired metabolic health (oral glucose tolerance test-derived insulin sensitivity). These data demonstrate that hyperglycemia-associated impairments in exercise adaptation can be ameliorated by cotherapy with SGLT2i.

Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway.

KEY POINTS: Exercising at different times of day elicits different effects on exercise performance and metabolic health. However, the specific signals driving the observed time-of-day specific effects of exercise have not been fully identified. Exercise influences the skeletal muscle circadian clock, although the relative contribution of muscle contraction and extracellular signals is unknown. Here, we show that contraction acutely increases the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase-shifts Per2 rhythmicity in muscle cells. This contraction effect on core clock genes is mediated through a calcium-dependant mechanism; The results obtained in the present study suggest that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by muscle contraction. Contraction interventions may be used to mimic some time-of-day specific effects of exercise on metabolism and muscle performance. ABSTRACT: Exercise entrains the central and peripheral circadian clocks, although the mechanism by which exercise modulates expression of skeletal muscle clock genes is unclear. The present study aimed to determine whether skeletal muscle contraction alone could directly influence circadian rhythmicity and uncover the underlying mechanism by which contraction modulates clock gene expression. We investigated the expression of core clock genes in human skeletal muscle after acute exercise, as well as following in vitro contraction in mouse soleus muscle and cultured C2C12 skeletal muscle myotubes. Additionally, we interrogated the molecular pathways by which skeletal muscle contraction could influence clock gene expression. Contraction acutely increased the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase-shifted Per2 rhythmicity in C2C12 myotubes in vitro. Further investigation revealed that pharmacologically increasing cytosolic calcium concentrations by ionomycin treatment mimicked the effect of contraction on Per2 expression. Similarly, treatment with a calcium channel blocker, nifedipine, blocked the effect of electric pulse stimulation-induced contraction on Per2 expression. Increased calcium influx from contraction lead to binding of the phosphorylated form of cAMP response element-binding protein (CREB) to the Per2 promoter, suggesting a role of CREB in contraction-induced Per2 transcription. Thus, by dissociating the effect of muscle contraction alone from the whole effect of exercise, our investigations indicate that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by contraction.

Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

Increased aerobic exercise capacity, as a result of exercise training, has important health benefits. However, some individuals are resistant to improvements in exercise capacity, probably due to undetermined genetic and environmental factors. Here, we show that exercise-induced improvements in aerobic capacity are blunted and aerobic remodelling of skeletal muscle is impaired in several animal models associated with chronic hyperglycaemia. Our data point to chronic hyperglycaemia as a potential negative regulator of aerobic adaptation, in part, via glucose-mediated modifications of the extracellular matrix, impaired vascularization and aberrant mechanical signalling in muscle. We also observe low exercise capacity and enhanced c-Jun N-terminal kinase activation in response to exercise in humans with impaired glucose tolerance. Our work indicates that current shifts in dietary and metabolic health, associated with increasing incidence of hyperglycaemia, might impair muscular and organismal adaptations to exercise training, including aerobic capacity as one of its key health outcomes.

Endurance training remodels sperm-borne small RNA expression and methylation at neurological gene hotspots.

Remodeling of the sperm epigenome by lifestyle factors before conception could account for altered metabolism in the next generation offspring. Here, we hypothesized that endurance training changes the epigenome of human spermatozoa. Using small RNA (sRNA) sequencing and reduced representation bisulfite sequencing (RRBS), we, respectively, investigated sRNA expression and DNA methylation in pure fractions of motile spermatozoa collected from young healthy individuals before, after 6 weeks of endurance training and after 3 months without exercise. Expression of 8 PIWI interacting RNA were changed by exercise training. RRBS analysis revealed 330 differentially methylated regions (DMRs) after training and 303 DMRs after the detraining period, which were, in both conditions, enriched at close vicinity of transcription start sites. Ontology analysis of genes located at proximity of DMRs returned terms related to neurological function at the trained state and, to a much lesser extent, at the detrained state. Our study reveal that short-term endurance training induces marked remodeling of the sperm epigenome, and identify genes related to the development of the central nervous system as potential hot spots for epigenetic variation upon environmental stress.

Effect of Exercise Training on Signaling of Interleukin-6 in Skeletal Muscles of Type 2 Diabetic Rats.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Muscle Contraction Induces Acute Hydroxymethylation of the Exercise-Responsive Gene Nr4a3.

Exercise training triggers numerous positive adaptations through the regulation of genes controlling muscle structure and function. Epigenetic modifications, including DNA methylation, participate in transcriptional activation by allowing the recruitment of the transcription machinery to gene promoters. Exercise induces dynamic DNA demethylation at gene promoters; however, the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a muscle contraction model that allows for the collection of samples that are repeatedly stimulated over time is required to determine whether contraction-induced demethylation is preceded by changes in the hydroxymethylcytosine level. Here, we established an acute skeletal muscle contraction model to mimic the effects of acute exercise on gene expression. We used this model to investigate the effect of muscle contraction on DNA demethylation and hydroxymethylation. First, we performed an acute exercise study in healthy humans to identify an exercise-responsive gene that we could study in culture. We identified the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene with the highest fold-expression increase after acute exercise. We then refined an electrical pulse stimulation (EPS) protocol that could induce expression of the Nr4a3 gene in C2C12 myotubes. Using targeted bisulfite sequencing, we found that in response to EPS, a region of the Nr4a3 promoter is rapidly demethylated at 60 min and re-methylated at 120 min. Of interest, hydroxymethylation of the differentially methylated region of Nr4a3 promoter after EPS was elevated immediately after EPS, with lowest levels reached at 60 min after EPS. In conclusion, we have established a cell culture-based protocol to mimic the acute transcriptional responses to exercise. Furthermore, we provide insight into the mechanism by which the exercise-responsive gene Nr4a3 is demethylated after muscle contraction.