Knockout of Hsp70 genes significantly affects locomotion speed and gene expression in leg skeletal muscles of Drosophila melanogaster.

The functions of the Hsp70 genes were studied using a line of D. melanogaster with knockout of six these genes out of thirteen. Namely, effect of knockout of Hsp70 genes on negative geotaxis climbing (locomotor) speed and the ability to adapt to climbing training (0.5-1.5 h/day, 7 days/week, 19 days) were examined. Seven- and 23-day-old Hsp70- flies demonstrated a comparable reduction (2-fold) in locomotor speed and widespread changes in leg skeletal muscle transcriptome (RNA-seq), compared to w1118 flies. To identify the functions of genes related to decreased locomotor speed the overlapped differentially expressed genes at both time points were analyzed: the up-regulated genes encoded extracellular proteins, regulators of drug metabolism and antioxidant response, while down-regulated genes encoded regulators of carbohydrate metabolism and transmembrane proteins. Additionally, in Hsp70- flies, activation of transcription factors related to disruption of the fibril structure and heat shock response (Hsf) were predicted, using the position weight matrix approach. In the control flies, adaptation to chronic exercise training was associated mainly with gene response to a single exercise bout, while the predicted transcription factors were related to stress/immune (Hsf, NF-kB, etc.) and early gene response. In contrast, Hsp70- flies demonstrated no adaptation to training, as well as significantly impaired gene response to a single exercise bout. In conclusion, the knockout of Hsp70 genes not only reduced physical performance, but also disrupted adaptation to chronic physical training, which is associated with changes in leg skeletal muscle transcriptome and impaired gene response to a single exercise bout.

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.

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.

Promoter-specific regulation of PPARGC1A gene expression in human skeletal muscle.

The goal of this study was to identify unknown transcription start sites of the PPARGC1A (PGC-1α) gene in human skeletal muscle and investigate the promoter-specific regulation of PGC-1α gene expression in human skeletal muscle. Ten amateur endurance-trained athletes performed high- and low-intensity exercise sessions (70  min, 70% or 50% o2max). High-throughput RNA sequencing and exon-exon junction mapping were applied to analyse muscle samples obtained at rest and after exercise. PGC-1α promoter-specific expression and activation of regulators of PGC-1α gene expression (AMPK, p38 MAPK, CaMKII, PKA and CREB1) after exercise were evaluated using qPCR and western blot. Our study has demonstrated that during post-exercise recovery, human skeletal muscle expresses the PGC-1α gene via two promoters only. As previously described, the additional exon 7a that contains a stop codon was found in all samples. Importantly, only minor levels of other splice site variants were found (and not in all samples). Constitutive expression PGC-1α gene occurs via the canonical promoter, independent of exercise intensity and exercise-induced increase of AMPK(Thr172) phosphorylation level. Expression of PGC-1α gene via the alternative promoter is increased of two orders after exercise. This post-exercise expression is highly dependent on the intensity of exercise. There is an apparent association between expression via the alternative promoter and activation of CREB1.