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.

Physical inactivity by tail suspension alters markers of metabolism, structure, and autophagy of the mouse heart.

Sedentary behavior has become ingrained in our society and has been linked to cardiovascular diseases. Physical inactivity is the main characteristic of sedentary behavior. However, its impact on cardiovascular disease is not clear. Therefore, we investigated the effect of physical inactivity in an established mouse model on gene clusters associated with cardiac fibrosis, electrophysiology, cell regeneration, and tissue degradation/turnover. We investigated a sedentary group (CTR, n = 10) versus a tail suspension group (TS, n = 11) that caused hindlimb unloading and consequently physical inactivity. Through histological, protein content, and transcript analysis approaches, we found that cardiac fibrosis-related genes partly change, with significant TS-associated increases in Tgfb1, but without changes in Col1a1 and Fn1. These changes are not translated into fibrosis at tissue level. We further detected TS-mediated increases in protein degradation- (Trim63, p < 0.001; Fbxo32, p = 0.0947 as well as in biosynthesis-related [P70s6kb1, p < 0.01]). Corroborating these results, we found increased expression of autophagy markers such as Atg7 (p < 0.01) and ULK1 (p < 0.05). Two cardiomyocyte regeneration- and sarcomerogenesis-related genes, Yap (p = 0.0535) and Srf (p < 0.001), increased upon TS compared to CTR conditions. Finally, we found significant upregulation of Gja1 (p < 0.05) and a significant downregulation of Aqp1 (p < 0.05). Our data demonstrate that merely 2 weeks of reduced physical activity induce changes in genes associated with cardiac structure and electrophysiology. Hence, these data should find the basis for novel research directed to evaluate the interplay of cardiac functioning and physical inactivity.

Mechanosensors control skeletal muscle mass, molecular clocks, and metabolism.

BACKGROUND: Skeletal muscles (SkM) are mechanosensitive, with mechanical unloading resulting in muscle-devastating conditions and altered metabolic properties. However, it remains unexplored whether these atrophic conditions affect SkM mechanosensors and molecular clocks, both crucial for their homeostasis and consequent physiological metabolism. METHODS: We induced SkM atrophy through 14 days of hindlimb suspension (HS) in 10 male C57BL/6J mice and 10 controls (CTR). SkM histology, gene expressions and protein levels of mechanosensors, molecular clocks and metabolism-related players were examined in the m. Gastrocnemius and m. Soleus. Furthermore, we genetically reduced the expression of mechanosensors integrin-linked kinase (Ilk1) and kindlin-2 (Fermt2) in myogenic C2C12 cells and analyzed the gene expression of mechanosensors, clock components and metabolism-controlling genes. RESULTS: Upon hindlimb suspension, gene expression levels of both core molecular clocks and mechanosensors were moderately upregulated in m. Gastrocnemius but strongly downregulated in m. Soleus. Upon unloading, metabolism- and protein biosynthesis-related genes were moderately upregulated in m. Gastrocnemius but downregulated in m. Soleus. Furthermore, we identified very strong correlations between mechanosensors, metabolism- and circadian clock-regulating genes. Finally, genetically induced downregulations of mechanosensors Ilk1 and Fermt2 caused a downregulated mechanosensor, molecular clock and metabolism-related gene expression in the C2C12 model. CONCLUSIONS: Collectively, these data shed new lights on mechanisms that control muscle loss. Mechanosensors are identified to crucially control these processes, specifically through commanding molecular clock components and metabolism.

Omega-3 Supplementation Improves Isometric Strength But Not Muscle Anabolic and Catabolic Signaling in Response to Resistance Exercise in Healthy Older Adults.

Old skeletal muscle exhibits decreased anabolic sensitivity, eventually contributing to muscle wasting. Besides anabolism, also muscle inflammation and catabolism are critical players in regulating the old skeletal muscle's sensitivity. Omega-3 fatty acids (ω-3) are an interesting candidate to reverse anabolic insensitivity via anabolic actions. Yet, it remains unknown whether ω-3 also attenuates muscle inflammation and catabolism. The present study investigates the effect of ω-3 supplementation on muscle inflammation and metabolism (anabolism/catabolism) upon resistance exercise (RE). Twenty-three older adults (65-84 years; 8♀) were randomized to receive ω-3 (~3 g/d) or corn oil (placebo [PLAC]) and engaged in a 12-week RE program (3×/wk). Before and after intervention, muscle volume, strength, and systemic inflammation were assessed, and muscle biopsies were analyzed for markers of anabolism, catabolism, and inflammation. Isometric knee-extensor strength increased in ω-3 (+12.2%), but not in PLAC (-1.4%; pinteraction = .015), whereas leg press strength improved in both conditions (+27.1%; ptime < .001). RE, but not ω-3, decreased inflammatory (p65NF-κB) and catabolic (FOXO1, LC3b) markers, and improved muscle quality. Yet, muscle volume remained unaffected by RE and ω-3. Accordingly, muscle anabolism (mTORC1) and plasma C-reactive protein remained unchanged by RE and ω-3, whereas serum IL-6 tended to decrease in ω-3 (pinteraction = .07). These results show that, despite no changes in muscle volume, RE-induced gains in isometric strength can be further enhanced by ω-3. However, ω-3 did not improve RE-induced beneficial catabolic or inflammatory adaptations. Irrespective of muscle volume, gains in strength (primary criterion for sarcopenia) might be explained by changes in muscle quality due to muscle inflammatory or catabolic signaling.

Evidence for skeletal muscle fiber type-specific expressions of mechanosensors.

Mechanosensors govern muscle tissue integrity and constitute a subcellular structure known as costameres. Costameres physically link the muscle extracellular matrix to contractile and signaling 'hubs' inside muscle fibers mainly via integrins and are localized beneath sarcolemmas of muscle fibers. Costameres are the main mechanosensors converting mechanical cues into biological events. However, the fiber type-specific costamere architecture in muscles is unexplored. We hypothesized that fiber types differ in the expression of genes coding for costamere components. By coupling laser microdissection to a multiplex tandem qPCR approach, we demonstrate that type 1 and type 2 fibers indeed show substantial differences in their mechanosensor complexes. We confirmed these data by fiber type population-specific protein analysis and confocal microscopy-based localization studies. We further show that knockdown of the costamere gene integrin-linked kinase (Ilk) in muscle precursor cells results in significantly increased slow-myosin-coding Myh7 gene, while the fast-myosin-coding genes Myh1, Myh2, and Myh4 are downregulated. In parallel, protein synthesis-enhancing signaling molecules (p-mTORSer2448, p < 0.05; p-P70S6KThr389, tendency with p < 0.1) were reduced upon Ilk knockdown. However, overexpression of slow type-inducing NFATc1 in muscle precursor cells did not change Ilk or other costamere gene expressions. In addition, we demonstrate fiber type-specific costamere gene regulation upon mechanical loading and unloading conditions. Our data imply that costamere genes, such as Ilk, are involved in the control of muscle fiber characteristics. Further, they identify costameres as muscle fiber type-specific loading management 'hubs' and may explain adaptation differences of muscle fiber types to mechanical (un)loading.