Age-related changes in human skeletal muscle transcriptome and proteome are more affected by chronic inflammation and physical inactivity than primary aging.

Evaluation of the influence of primary and secondary aging on the manifestation of molecular and cellular hallmarks of aging is a challenging and currently unresolved issue. Our study represents the first demonstration of the distinct role of primary aging and chronic inflammation/physical inactivity - the most important drivers of secondary aging, in the regulation of transcriptomic and proteomic profiles in human skeletal muscle. To achieve this purpose, young healthy people (n = 15), young (n = 8) and older (n = 37) patients with knee/hip osteoarthritis, a model to study the effect of long-term inactivity and chronic inflammation on the vastus lateralis muscle, were included in the study. It was revealed that widespread and substantial age-related changes in gene expression in older patients relative to young healthy people (~4000 genes regulating mitochondrial function, proteostasis, cell membrane, secretory and immune response) were related to the long-term physical inactivity and chronic inflammation rather than primary aging. Primary aging contributed mainly to the regulation of genes (~200) encoding nuclear proteins (regulators of DNA repair, RNA processing, and transcription), mitochondrial proteins (genes encoding respiratory enzymes, mitochondrial complex assembly factors, regulators of cristae formation and mitochondrial reactive oxygen species production), as well as regulators of proteostasis. It was found that proteins associated with aging were regulated mainly at the post-transcriptional level. The set of putative primary aging genes and their potential transcriptional regulators can be used as a resource for further targeted studies investigating the role of individual genes and related transcription factors in the emergence of a senescent cell phenotype.

Dysregulation of early gene response to a mixed meal in skeletal muscle in obesity and type 2 diabetes.

Obesity- and type 2 diabetes mellitus-induced changes in the expression of protein-coding genes in human skeletal muscle were extensively examined at baseline (after an overnight fast). We aimed to compare the early transcriptomic response to a typical single meal in skeletal muscle of metabolically healthy subjects and obese individuals without and with type 2 diabetes. Transcriptomic response (RNA-seq) to a mixed meal (nutritional drink, ∼25 kJ/kg of body mass) was examined in the vastus lateralis muscle (1 h after a meal) in 7 healthy subjects and 14 obese individuals without or with type 2 diabetes. In all obese individuals, the transcriptome response to a meal was dysregulated (suppressed and altered) and associated with different biological processes compared with healthy control. To search for potential transcription factors regulating transcriptomic response to a meal, the enrichment of transcription factor-binding sites in individual promoters of the human skeletal muscle was examined. In obese individuals, the transcriptomic response is associated with a different set of transcription factors than that in healthy subjects. In conclusion, metabolic disorders are associated with a defect in the regulation of mixed meal/insulin-mediated gene expression-insulin resistance in terms of gene expression. Importantly, this dysregulation occurs in obese individuals without type 2 diabetes, i.e., at the first stage of the development of metabolic disorders.NEW & NOTEWORTHY In skeletal muscle of metabolically healthy subjects, a typical single meal normalized to body mass induces activation of various transcription factors, expression of numerous receptor tyrosine kinases associated with the insulin signaling cascade, and transcription regulators. In skeletal muscle of obese individuals without and with type 2 diabetes, this signaling network is poorly regulated at the transcriptional level, indicating dysregulation of the early gene response to a mixed meal.

Rapid changes in transcriptomic profile and mitochondrial function in human soleus muscle after 3-day dry immersion.

We aimed to explore the effect of the 3-day dry immersion, a model of physical unloading, on mitochondrial function, transcriptomic and proteomic profiles in a slow-twitch soleus muscle of six healthy females. We registered that a marked reduction (25-34%) in the ADP-stimulated respiration in permeabilized muscle fibers was not accompanied by a decrease in the content of mitochondrial enzymes (mass spectrometry-based quantitative proteomics), hence, it is related to the disruption in regulation of respiration. We detected a widespread change in the transcriptomic profile (RNA-seq) upon dry immersion. Downregulated mRNAs were strongly associated with mitochondrial function, as well as with lipid metabolism, glycolysis, insulin signaling, and various transporters. Despite the substantial transcriptomic response, we found no effect on the content of highly abundant proteins (sarcomeric, mitochondrial, chaperon, and extracellular matrix-related, etc.) that may be explained by long half-life of these proteins. We suggest that during short-term disuse the content of some regulatory (and usually low abundant) proteins such as cytokines, receptors, transporters, and transcription regulators is largely determined by their mRNA concentration. These mRNAs revealed in our work may serve as putative targets for future studies aimed at developing approaches for the prevention of muscle deconditioning induced by disuse.NEW & NOTEWORTHY Three-day dry immersion (a model of physical unloading) substantially changes the transcriptomic profile in the human soleus muscle, a muscle with predominantly slow-twitch fibers and strong postural function; despite this, we found no effect on the muscle proteome (highly abundant proteins). Dry immersion markedly reduces ADP-stimulated respiration; this decline is not accompanied by a decrease in the content of mitochondrial proteins/respiratory enzymes, indicating the disruption in regulation of cellular respiration.

Signaling and Gene Expression in Skeletal Muscles in Type 2 Diabetes: Current Results and OMICS Perspectives.

Skeletal muscles mainly contribute to the emergence of insulin resistance, impaired glucose tolerance and the development of type 2 diabetes. Molecular mechanisms that regulate glucose uptake are diverse, including the insulin-dependent as most important, and others as also significant. They involve a wide range of proteins that control intracellular traffic and exposure of glucose transporters on the cell surface to create an extensive regulatory network. Here, we highlight advantages of the omics approaches to explore the insulin-regulated proteins and genes in human skeletal muscle with varying degrees of metabolic disorders. We discuss methodological aspects of the assessment of metabolic dysregulation and molecular responses of human skeletal muscle to insulin. The known molecular mechanisms of glucose uptake regulation and the first results of phosphoproteomic and transcriptomic studies are reviewed, which unveiled a large-scale array of insulin targets in muscle cells. They demonstrate that a clear depiction of changes that occur during metabolic dysfunction requires systemic and combined analysis at different levels of regulation, including signaling pathways, transcription factors, and gene expression. Such analysis seems promising to explore yet undescribed regulatory mechanisms of glucose uptake by skeletal muscle and identify the key regulators as potential therapeutic targets.

Alternative transcription start sites contribute to acute-stress-induced transcriptome response in human skeletal muscle.

BACKGROUND: More than half of human protein-coding genes have an alternative transcription start site (TSS). We aimed to investigate the contribution of alternative TSSs to the acute-stress-induced transcriptome response in human tissue (skeletal muscle) using the cap analysis of gene expression approach. TSSs were examined at baseline and during recovery after acute stress (a cycling exercise). RESULTS: We identified 44,680 CAGE TSS clusters (including 3764 first defined) belonging to 12,268 genes and annotated for the first time 290 TSSs belonging to 163 genes. The transcriptome dynamically changes during the first hours after acute stress; the change in the expression of 10% of genes was associated with the activation of alternative TSSs, indicating differential TSSs usage. The majority of the alternative TSSs do not increase proteome complexity suggesting that the function of thousands of alternative TSSs is associated with the fine regulation of mRNA isoform expression from a gene due to the transcription factor-specific activation of various alternative TSSs. We identified individual muscle promoter regions for each TSS using muscle open chromatin data (ATAC-seq and DNase-seq). Then, using the positional weight matrix approach we predicted time course activation of "classic" transcription factors involved in response of skeletal muscle to contractile activity, as well as diversity of less/un-investigated factors. CONCLUSIONS: Transcriptome response induced by acute stress related to activation of the alternative TSSs indicates that differential TSSs usage is an essential mechanism of fine regulation of gene response to stress stimulus. A comprehensive resource of accurate TSSs and individual promoter regions for each TSS in muscle was created. This resource together with the positional weight matrix approach can be used to accurate prediction of TFs in any gene(s) of interest involved in the response to various stimuli, interventions or pathological conditions in human skeletal muscle.

Transcriptomic Signatures and Upstream Regulation in Human Skeletal Muscle Adapted to Disuse and Aerobic Exercise.

Inactivity is associated with the development of numerous disorders. Regular aerobic exercise is broadly used as a key intervention to prevent and treat these pathological conditions. In our meta-analysis we aimed to identify and compare (i) the transcriptomic signatures related to disuse, regular and acute aerobic exercise in human skeletal muscle and (ii) the biological effects and transcription factors associated with these transcriptomic changes. A standardized workflow with robust cut-off criteria was used to analyze 27 transcriptomic datasets for the vastus lateralis muscle of healthy humans subjected to disuse, regular and acute aerobic exercise. We evaluated the role of transcriptional regulation in the phenotypic changes described in the literature. The responses to chronic interventions (disuse and regular training) partially correspond to the phenotypic effects. Acute exercise induces changes that are mainly related to the regulation of gene expression, including a strong enrichment of several transcription factors (most of which are related to the ATF/CREB/AP-1 superfamily) and a massive increase in the expression levels of genes encoding transcription factors and co-activators. Overall, the adaptation strategies of skeletal muscle to decreased and increased levels of physical activity differ in direction and demonstrate qualitative differences that are closely associated with the activation of different sets of transcription factors.

Regulation of Proteins in Human Skeletal Muscle: The Role of Transcription.

Regular low intensity aerobic exercise (aerobic training) provides effective protection against various metabolic disorders. Here, the roles played by transient transcriptome responses to acute exercise and by changes in baseline gene expression during up-regulation of protein content in human skeletal muscle were investigated after 2 months of aerobic training. Seven untrained males were involved in a 2 month aerobic cycling training program. Mass-spectrometry and RNA sequencing were used to evaluate proteome and transcriptome responses to training and acute exercise. We found that proteins with different functions are regulated differently at the transcriptional level; for example, a training-induced increase in the content of extracellular matrix-related proteins is regulated at the transcriptional level, while an increase in the content of mitochondrial proteins is not. An increase in the skeletal muscle content of several proteins (including mitochondrial proteins) was associated with increased protein stability, which is related to a chaperone-dependent mechanism and/or reduced regulation by proteolysis. These findings increase our understanding of the molecular mechanisms underlying regulation of protein expression in human skeletal muscle subjected to repeated stress (long term aerobic training) and may provide an opportunity to control the expression of specific proteins (e.g., extracellular matrix-related proteins, mitochondrial proteins) through physiological and/or pharmacological approaches.

Contractile activity-specific transcriptome response to acute endurance exercise and training in human skeletal muscle.

Reduction in daily activity leads to dramatic metabolic disorders, while regular aerobic exercise training is effective for preventing this problem. The purpose of this study was to identify genes that are directly related to contractile activity in human skeletal muscle, regardless of the level of fitness. Transcriptome changes after the one-legged knee extension exercise in exercised and contralateral nonexercised vastus lateralis muscle of seven men were evaluated by RNA-seq. Transcriptome change at baseline after 2 mo of aerobic training (5/wk, 1 h/day) was evaluated as well. Postexercise changes in the transcriptome of exercised muscle were associated with different factors, including circadian oscillations. To reveal transcriptome response specific for endurance-like contractile activity, differentially expressed genes between exercised and nonexercised muscle were evaluated at 1 and 4 h after the one-legged exercise. The contractile activity-specific transcriptome responses were associated only with an increase in gene expression and were regulated mainly by CREB/ATF/AP1-, MYC/MAX-, and E2F-related transcription factors. Endurance training-induced changes (an increase or decrease) in the transcriptome at baseline were more pronounced than transcriptome responses specific for acute contractile activity. Changes after training were associated with widely different biological processes than those after acute exercise and were regulated by different transcription factors (IRF- and STAT-related factors). In conclusion, adaptation to regular exercise is associated not only with a transient (over several hours) increase in expression of many contractile activity-specific genes, but also with a pronounced change (an increase or decrease) in expression of a large number of genes under baseline conditions.

Intensity-dependent gene expression after aerobic exercise in endurance-trained skeletal muscle.

We investigated acute exercise-induced gene expression in skeletal muscle adapted to aerobic training. Vastus lateralis muscle samples were taken in ten endurance-trained males prior to, and just after, 4 h, and 8 h after acute cycling sessions with different intensities, 70% and 50% V ˙ O 2 max . High-throughput RNA sequencing was applied in samples from two subjects to evaluate differentially expressed genes after intensive exercise (70% V ˙ O 2 max ), and then the changes in expression for selected genes were validated by quantitative PCR (qPCR). To define exercise-induced genes, we compared gene expression after acute exercise with different intensities, 70% and 50% V ˙ O 2 max , by qPCR. The transcriptome is dynamically changed during the first hours of recovery after intensive exercise (70% V ˙ O 2 max ). A computational approach revealed that the changes might be related to up- and down-regulation of the activity of transcription activators and repressors, respectively. The exercise increased expression of many genes encoding protein kinases, while genes encoding transcriptional regulators were both up- and down-regulated. Evaluation of the gene expression after exercise with different intensities revealed that some genes changed expression in an intensity-dependent manner, but others did not: the majority of genes encoding protein kinases, oxidative phosphorylation and activator protein (AP)-1-related genes significantly correlated with markers of exercise stress (power, blood lactate during exercise and post-exercise blood cortisol), while transcriptional repressors and circadian-related genes did not. Some of the changes in gene expression after exercise seemingly may be modulated by circadian rhythm.

Effect of aerobic training on baseline expression of signaling and respiratory proteins in human skeletal muscle.

Most studies examining the molecular mechanisms underlying adaptation of human skeletal muscles to aerobic exercise focused on the response to acute exercise. Here, we examined the effect of a 2-month aerobic training program on baseline parameters in human muscle. Ten untrained males performed a one-legged knee extension exercise for 1 h with the same relative intensity before and after a 2-month aerobic training program. Biopsy samples were taken from vastus lateralis muscle at rest before and after the 2 month training program (baseline samples). Additionally, biopsy samples were taken from the exercised leg 1 and 4 h after the one-legged continuous knee extension exercise. Aerobic training decreases baseline phosphorylation of FOXO1Ser256 , increases that of CaMKIIThr286 , CREB1Ser133 , increases baseline expression of mitochondrial proteins in respiratory complexes I-V, and some regulators of mitochondrial biogenesis (TFAM, NR4A3, and CRTC2). An increase in the baseline content of these proteins was not associated with a change in baseline expression of their genes. The increase in the baseline content of regulators of mitochondrial biogenesis (TFAM and NR4A3) was associated with a transient increase in transcription after acute exercise. Contrariwise, the increase in the baseline content of respiratory proteins does not seem to be regulated at the transcriptional level; rather, it is associated with other mechanisms. Adaptation of human skeletal muscle to regular aerobic exercise is associated not only with transient molecular responses to exercise, but also with changes in baseline phosphorylation and expression of regulatory proteins.

Regulation of PPARGC1A gene expression in trained and untrained human skeletal muscle.

Promoter-specific expression of the PPARGC1A gene in untrained and trained human skeletal muscle was investigated. Ten untrained males performed a one-legged knee extension exercise (for 60 min) with the same relative intensity both before and after 8 weeks of cycling training. Samples from the m. vastus lateralis of each leg were taken before and after exercise. Postexercise PPARGC1A gene expression via the canonical promoter increased by ~100% (P < 0.05) in exercised and nonexercised untrained muscles, but did not change in either leg after training program. In untrained and trained exercised muscle, PPARGC1A gene expression via the alternative promoter increased by two orders of magnitude (P < 0.01). We found increases in postexercise content of dephosphorylated (activated) CRTC2, a coactivator of CREB1, in untrained exercised muscle and in expression of CREB1-related genes in untrained and trained exercised muscle (P < 0.01-0.05); this may partially explain the increased expression of PPARGC1A via the alternative promoter. In addition, comparison of the regulatory regions of both promoters revealed unique conserved motifs in the alternative promoter that were associated with transcriptional repressors SNAI1 and HIC1. In conclusion, in untrained muscle, exercise-induced expression of the PPARGC1A gene via the canonical promoter may be regulated by systemic factors, while in trained muscle the canonical promoter shows constitutive expression at rest and after exercise. Exercise-induced expression of PPARGC1A via the alternative promoter relates to intramuscular factors and associates with activation of CRTC2-CREB1. Apparently, expression via the alternative promoter is regulated by other transcription factors, particularly repressors.