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Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise., (© 2024. The Author(s).)

Previous research has shown that leucine (Leu) can stimulate and enhance the proliferation of equine skeletal muscle satellite cells (SCs). The gene expression profile associated with Leu-induced proliferation of equine SCs has also been documented. However, the specific role of Leu in regulating the expression of slow-twitch muscle fibers (slow-MyHC) and mitochondrial function in equine SCs, as well as the underlying mechanism, remains unclear. During this investigation, equine SCs underwent culturing in differentiation medium and were subjected to varying concentrations of Leu (0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM) over a span of 3 days. AMP-activated protein kinase (AMPK) inhibitor Compound C and mammalian target of rapamycin complex (mTOR) inhibitor Rapamycin were utilized to explore its underlying mechanism. Here we showed that the expression of slow-MyHC at 2 mM Leu level was significantly higher than the concentration levels of 0 mM,0.5 mM and 10 mM (P <0.01), and there was no significant difference compared to other groups (P > 0.05); the basal respiration, maximum respiration, standby respiration and the expression of slow-MyHC, PGC-1α, Cytc, ND1, TFAM, and COX1 were significantly increased with Leu supplementation (P < 0.01). We also found that Leu up-regulated the expression of key proteins on AMPK and mTOR signaling pathways, including LKB1, p-LKB1, AMPK, p-AMPK, S6, p-S6, 4EBP1, p-4EBP1, mTOR and p-mTOR (P < 0.05 or P < 0.01). Notably, when we treated the equine SCs with the AMPK inhibitor Compound C and the mTOR inhibitor Rapamycin, we observed a reduction in the beneficial effects of Leu on the expression of genes related to slow-MyHC and signaling pathway-related gene expressions. This study provides novel evidence that Leu promotes slow-MyHC expression and enhances mitochondrial function in equine SCs through the AMPK/mTOR signaling pathways, shedding light on the underlying mechanisms involved in these processes for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Skeletal muscle comprises slow and fast myofibers, with slow myofibers excelling in aerobic metabolism and endurance. Quercetin, a polyphenol, is reported to induce slow myofibers in rodent skeletal muscle both in vitro and in vivo. However, its effect on human myofiber types remains unexplored. In this study, we evaluated quercetin's impact on slow myofiber induction using human skeletal muscle satellite cells. In a two-dimensional culture, quercetin enhanced gene expression, contributing to muscle differentiation, and significantly expanded the area of slow-type myosin heavy chain positive cells. It also elevated the gene expression of Pgc1α, an inducer of slow myofibers. Conversely, quercetin did not affect mitochondrial abundance, fission, or fusion, but it did increase the gene expression of Cox7A2L, which aids in promoting mitochondrial supercomplexity and endurance, and Mb, which contributes to oxidative phosphorylation. In a three-dimensional culture, quercetin significantly extended the time to peak tension and half relaxation time of the engineered human skeletal muscle tissues constructed on microdevices. Moreover, quercetin enhanced the muscle endurance of the tissues and curbed the rise in lactate secretion from the exercised tissues. These findings suggest that quercetin may induce slow myofibers in human skeletal muscle., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

This study aimed to evaluate the influence of combined intermittent fasting (IF) and high-intensity interval training (HIIT) on morphology, caspase-independent apoptosis signaling pathway, and myostatin expression in soleus and gastrocnemius (white portion) muscles from healthy rats. Sixty-day-old male Wistar rats (n = 60) were divided into four groups: control (C), IF, high-intensity-interval training (T), and high-intensity-interval training and intermittent fasting (T-IF). The C and T groups received ad libitum chow daily; IF and T-IF received the same standard chow every other day. Animals from T and T-IF underwent a HIIT protocol five times a week for 12 weeks. IF reduced gastrocnemius mass and increased pro-apoptotic proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in soleus and cleaved-to-non-cleaved PARP-1 ratio and myostatin expression in gastrocnemius white portion. HIIT increased AIF and apoptosis repressor with caspase recruitment domain expression in soleus and cleaved-to-total PARP-1 ratio in gastrocnemius muscle white portion. The combination of IF and HIIT reduced fiber cross-sectional area in both muscles, increased EndoG and AIF expression, and decreased cleaved-to-non-cleaved PARP-1 ratio in gastrocnemius muscle white portion. Muscle responses to IF and HIIT are directly impacted by the muscle fiber type composition and are modulated, at least in part, by myostatin and caspase-independent apoptosis signaling., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Insulin resistance (IR) is a risk factor for the development of several major metabolic diseases. Muscle fiber composition is established early in life and is associated with insulin sensitivity. Hence, muscle fiber composition was used to identify early defects in the development of IR in healthy young individuals in the absence of clinical manifestations. Biopsies were obtained from the thigh muscle, followed by an intravenous glucose tolerance test. Indices of insulin action were calculated and cardiovascular measurements, analyses of blood and muscle were performed. Whole body insulin sensitivity (SI galvin ) was positively related to expression of type I muscle fibers ( r = 0.49; P < 0.001) and negatively related to resting heart rate (HR, r = -0.39; P < 0.001), which was also negatively related to expression of type I muscle fibers ( r = -0.41; P < 0.001). Muscle protein expression of endothelial nitric oxide synthase (eNOS), whose activation results in vasodilation, was measured in two subsets of subjects expressing a high percentage of type I fibers (59 ± 6%; HR = 57 ± 9 beats/min; SI galvin = 1.8 ± 0.7 units) or low percentage of type I fibers (30 ± 6%; HR = 71 ± 11; SI galvin = 0.8 ± 0.3 units; P < 0.001 for all variables vs. first group). eNOS expression was 1 ) higher in subjects with high type I expression; 2 ) almost twofold higher in pools of type I versus II fibers; 3 ) only detected in capillaries surrounding muscle fibers; and 4 ) linearly associated with SI galvin . These data demonstrate that an altered function of the autonomic nervous system and a compromised capacity for vasodilation in the microvasculature occur early in the development of IR. NEW & NOTEWORTHY Insulin resistance (IR) is a risk factor for the development of several metabolic diseases. In healthy young individuals, an elevated heart rate (HR) correlates with low insulin sensitivity and high expression of type II skeletal muscle fibers, which express low levels of endothelial nitric oxide synthase (eNOS) and, hence, a limited capacity to induce vasodilation in response to insulin. Early targeting of the autonomic nervous system and microvasculature may attenuate development of diseases stemming from insulin resistance.

The skeletal muscle is the main organ responsible for insulin action, and glucose disposal and metabolism. Endurance and/or resistance training raises the number of mitochondria in diabetic muscles. The details of these adaptations, including mitochondrial adaptations of the slow and fast muscles in diabetes, are unclear. This study aimed to determine whether exercise training in streptozotocin (STZ)-induced mice leads to differential adaptations in the slow and fast muscles, and improving glucose clearance. Eight-week-old mice were randomly distributed into normal control (CON), diabetes (DM), and diabetes and exercise (DM+Ex) groups. In the DM and DM+Ex groups, mice received a freshly prepared STZ (100 mg/kg) intraperitoneal injection on two consecutive days. Two weeks after the injection, the mice in the groups ran on a treadmill for 60 min at 20 m/min for a week and subsequently at 25 m/min for 5 weeks (5 days/week). The analyses indicated that running training at low speed (25 m/min) enhanced mitochondrial enzyme activity and expression of lactate and glucose transporters in the plantaris (low-oxidative) muscle that improved whole-body glucose metabolism in STZ-induced diabetic mice. There were no differences in glucose transporter expression levels in the soleus (high-oxidative) muscle. The endurance running exercise at 20-25 m/min was sufficient to induce mitochondrial adaptation in the low-oxidative muscles, but not in the high-oxidative muscles, of diabetic mice. In conclusion, the present study indicated that running training at 25 m/min improved glucose metabolism by increasing the mitochondrial enzyme activity and glucose transporter 4 and monocarboxylate transporter 4 protein contents in the low-oxidative muscles in STZ-induced diabetic mice.

This study conducted a thorough analysis of the myofiber type composition in the extensor digitorum longus muscle (EDL) and soleus muscle (SOL) of Kazakh horses, across different genders (male and female). The results showed significant differences in myofiber type composition between EDL and SOL, with a higher proportion of Type I fibers in SOL muscles and a greater prevalence of Type II fibers in EDL muscles. Additionally, the myofiber diameter in Kazakh horses was relatively small, potentially related to the tenderness and edible quality of their muscles. Using high-throughput sequencing technology, we constructed 32 cDNA sequencing libraries and obtained high-quality read data. Gene expression analysis revealed 278 and 372 differentially expressed genes (DEGs) in EDL and SOL muscles, respectively, including genes related to muscle contraction, metabolism, and development. Intersection analysis of DEGs between genders showed that 60 DEGs were significantly different in both male and female horses. GO annotation and KEGG analysis further elucidated the roles of these DEGs in muscle structure, function, and cellular signaling. Protein-protein interaction (PPI) network analysis and identification of hub genes provided new insights into the molecular mechanisms underlying muscle growth and development. Finally, the reliability of the DEGs data was validated through quantitative real-time PCR (qRT-PCR). This study not only enhances our understanding of the biological characteristics of horse muscles but also provides potential molecular targets for improving horse muscle performance and health., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Aging and obesity pose significant threats to public health and are major contributors to muscle atrophy. The trends in muscle fiber types under these conditions and the transcriptional differences between different muscle fiber types remain unclear. Here, we demonstrate distinct responses of fast/glycolytic fibers and slow/oxidative fibers to aging and obesity. We found that in muscles dominated by oxidative fibers, the proportion of oxidative fibers remains unchanged during aging and obesity. However, in muscles dominated by glycolytic fibers, despite the low content of oxidative fibers, a significant decrease in proportion of oxidative fibers was observed. Consistently, our study uncovered that during aging and obesity, fast/glycolytic fibers specifically increased the expression of genes associated with muscle atrophy and inflammation, including Dkk3, Ccl8, Cxcl10, Cxcl13, Fbxo32, Depp1, and Chac1, while slow/oxidative fibers exhibit elevated expression of antioxidant protein Nqo-1 and downregulation of Tfrc. Additionally, we noted substantial differences in the expression of calcium-related signaling pathways between fast/glycolytic fibers and slow/oxidative fibers in response to aging and obesity. Treatment with a calcium channel inhibitor thapsigargin significantly increased the abundance of oxidative fibers. Our study provides additional evidence to support the transcriptomic differences in muscle fiber types under pathophysiological conditions, thereby establishing a theoretical basis for modulating muscle fiber types in disease treatment., (© 2024. The Author(s).)

The composition of skeletal muscle fiber types affects the quality of livestock meat and human athletic performance and health. L-arginine (Arg), a semi-essential amino acid, has been observed to promote the formation of slow-twitch muscle fibers in animal models. However, the precise molecular mechanisms are still unclear. This study investigates the role of Arg in skeletal muscle fiber composition and mitochondrial function through the mTOR signaling pathway. In vivo, 4-week C56BL/6J male mice were divided into three treatment groups and fed a basal diet supplemented with different concentrations of Arg in their drinking water. The trial lasted 7 weeks. The results show that Arg supplementation significantly improved endurance exercise performance, along with increased SDH enzyme activity and upregulated expression of the MyHC I, MyHC IIA, PGC-1α, and NRF1 genes in the gastrocnemius (GAS) and quadriceps (QUA) muscles compared to the control group. In addition, Arg activated the mTOR signaling pathway in the skeletal muscle of mice. In vitro experiments using cultured C2C12 myotubes demonstrated that Arg elevated the expression of slow-fiber genes (MyHC I and Tnnt1) as well as mitochondrial genes (PGC-1α, TFAM, MEF2C, and NRF1), whereas the effects of Arg were inhibited by the mTOR inhibitor rapamycin. In conclusion, these findings suggest that Arg modulates skeletal muscle fiber type towards slow-twitch fibers and enhances mitochondrial functions by upregulating gene expression through the mTOR signaling pathway.

The conversion of fast-twitch fibers into slow-twitch fibers within skeletal muscle plays a crucial role in improving physical stamina and safeguarding against metabolic disorders in individuals. Grape seed proanthocyanidin extract (GSPE) possesses numerous pharmacological and health advantages, effectively inhibiting the onset of chronic illnesses. However, there is a lack of research on the specific mechanisms by which GSPE influences muscle physiology and gut microbiota. This study aims to investigate the role of gut microbiota and their metabolites in GSPE regulation of skeletal muscle fiber type conversion. In this experiment, 54 male BALB/c mice were randomly divided into three groups: basal diet, basal diet supplemented with GSPE, and basal diet supplemented with GSPE and antibiotics. During the feeding period, glucose tolerance and forced swimming tests were performed. After euthanasia, samples of muscle and feces were collected for analysis. The results showed that GSPE increased the muscle mass and anti-fatigue capacity of the mice, as well as the expression of slow-twitch fibers. However, the beneficial effects of GSPE on skeletal muscle fibers disappeared after adding antibiotics to eliminate intestinal microorganisms, suggesting that GSPE may play a role by regulating intestinal microbial structure. In addition, GSPE increased the relative abundance of Blautia, Muribaculaceae, and Enterorhabdus, as well as butyrate production. Importantly, these gut microbes exhibited a significant positive correlation with the expression of slow-twitch muscle fibers. In conclusion, supplementation with GSPE can increase the levels of slow-twitch fibers by modulating the gut microbiota, consequently prolonging the duration of exercise before exhaustion. PRACTICAL APPLICATION: This research suggests that grape seed proanthocyanidin extract (GSPE) has potential applications in improving physical stamina and preventing metabolic disorders. By influencing the gut microbiota and increasing butyric acid production, GSPE contributes to the conversion of fast-twitch muscle fibers into slow-twitch fibers, thereby enhancing anti-fatigue capacity and exercise endurance. While further studies are needed, incorporating GSPE into dietary supplements or functional foods could support individuals seeking to optimize their exercise performance and overall metabolic health., (© 2024 Institute of Food Technologists.)

There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

Homeostasis is essential for muscle repair and regeneration after skeletal muscle exercise. This study investigated the role of methyltransferase-like 21C (METTL21C) in skeletal muscle of mice after exercise and the potential mechanism. First, muscle samples were collected at 2, 4 and 6 weeks after exercise, and liver glycogen, muscle glycogen, blood lactic acid and triglyceride were assessed. Moreover, the expression levels of autophagy markers and METTL21C in skeletal muscle were analyzed. The results showed that the expression levels of METTL21C and MYH7 in the gastrocnemius muscle of mice in the exercise group were significantly higher after exercise than those in the control group, which suggested that long-term exercise promoted the formation of slow-twitch muscle fibers in mouse skeletal muscle. Likewise, the autophagy capacity was enhanced with the prolongation of exercise in muscles. The findings were confirmed in mouse C2C12 cells. We discovered that knockdown of Mettl21c reduced the expression of MYH7 and the autophagy level in mouse myoblasts. These findings indicate that METTL21C promotes skeletal muscle homeostasis after exercise by enhancing autophagy, and also contributes to myogenic differentiation and the formation of slow muscle fibers.

Sarcopenia is a musculoskeletal disease that reduces muscle mass and strength in older individuals. The study investigates the effects of azilsartan (AZL) on skeletal muscle loss in natural sarcopenic rats. Male Sprague-Dawley rats aged 4-6 months and 18-21 months were selected as young-matched control and natural-aged (sarcopenic) rats, respectively. Rats were allocated into young and old control (YC and OC) and young and old AZL treatment (YT and OT) groups, which received vehicles and AZL (8 mg/kg, orally) for 6 weeks. Rats were then sacrificed after muscle function analysis. Serum and gastrocnemius (GN) muscles were isolated for further endpoints. AZL significantly improved muscle grip strength and antioxidant levels in sarcopenic rats. AZL also restored the levels of insulin, testosterone, and muscle biomarkers such as myostatin and creatinine kinase in sarcopenic rats. Furthermore, AZL treatment improved the cellular and ultrastructure of GN muscle and prevented the shift of type II (glycolytic) myofibers to type I (oxidative) myofibers. The results showed that AZL intervention restored protein synthesis in natural sarcopenic rats by increasing p-Akt-1 and decreasing muscle RING-finger protein-1 and tumor necrosis factor alpha immunoexpressions. In conclusion, the present findings showed that AZL could be an effective intervention in treating age-related muscle impairments., Competing Interests: There is no competing interest, as stated by the authors.

Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality., (© 2024 Wiley Periodicals LLC.)

Plasma apelin levels are reduced in aging and muscle wasting conditions. We aimed to investigate the significance of apelin signaling in cardiac and skeletal muscle responses to physiological stress. Apelin knockout (KO) and wild-type (WT) mice were subjected to high-intensity interval training (HIIT) by treadmill running. The effects of apelin on energy metabolism were studied in primary mouse skeletal muscle myotubes and cardiomyocytes. Apelin increased mitochondrial ATP production and mitochondrial coupling efficiency in myotubes and promoted the expression of mitochondrial genes both in primary myotubes and cardiomyocytes. HIIT induced mild concentric cardiac hypertrophy in WT mice, whereas eccentric growth was observed in the left ventricles of apelin KO mice. HIIT did not affect myofiber size in skeletal muscles of WT mice but decreased the myofiber size in apelin KO mice. The decrease in myofiber size resulted from a fiber type switch toward smaller slow-twitch type I fibers. The increased proportion of slow-twitch type I fibers in apelin KO mice was associated with upregulation of myosin heavy chain slow isoform expression, accompanied with upregulated expression of genes related to fatty acid transport and downregulated expression of genes related to glucose metabolism. Mechanistically, skeletal muscles of apelin KO mice showed defective induction of insulin-like growth factor-1 signaling in response to HIIT. In conclusion, apelin is required for proper skeletal and cardiac muscle adaptation to high-intensity exercise. Promoting apelinergic signaling may have benefits in aging- or disease-related muscle wasting conditions. NEW & NOTEWORTHY Apelin levels decline with age. This study demonstrates that in trained mice, apelin deficiency results in a switch from fast type II myofibers to slow oxidative type I myofibers. This is associated with a concomitant change in gene expression profile toward fatty acid utilization, indicating an aged-muscle phenotype in exercised apelin-deficient mice. These data are of importance in the design of exercise programs for aging individuals and could offer therapeutic target to maintain muscle mass.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25 . Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

The sleep-breathing condition obstructive sleep apnea (OSA) is characterized by repetitive upper airway collapse, which can exacerbate oxidative stress and free radical generation, thereby detrimentally impacting both motor and sensory nerve function and inducing muscular damage. OSA development is promoted by increasing proportions of fast-twitch muscle fibers in the genioglossus. Orientin, a water-soluble dietary C-glycosyl flavonoid with antioxidant properties, increased the expression of slow myosin heavy chain (MyHC) and signaling factors associated with AMP-activated protein kinase (AMPK) activation both in vivo and in vitro . Inhibiting AMPK signaling diminished the effects of orientin on slow MyHC, fast MyHC, and Sirt1 expression. Overall, orientin enhanced type I muscle fibers in the genioglossus, enhanced antioxidant capacity, increased mitochondrial biogenesis through AMPK signaling, and ultimately improved fatigue resistance in C2C12 myotubes and mouse genioglossus. These findings suggest that orientin may contribute to upper airway stability in patients with OSA, potentially preventing airway collapse.

Objective: Human skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic., Methods: Here, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity., Results: Maximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid-or complex I respiration., Conclusion: Mitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sebastian Edman reports financial support was provided by Elisabeth och Gunnar Liljedahls foundation. William Apro reports financial support was provided by Centrum för idrottsforskning. Filip Larsen reports financial support was provided by Centrum för idrottsforskning., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The proportion of the different types of fibers in a given skeletal muscle contributes to its overall metabolic and functional characteristics. Greater proportion of type I muscle fibers is associated with favorable oxidative metabolism and function of the muscle. Humans with obesity have a lower proportion of type I muscle fibers. We discuss how lower proportion of type I fibers in skeletal muscle of humans with obesity may explain metabolic and functional abnormalities reported in these individuals. These include lower muscle glucose disposal rate, mitochondrial content, protein synthesis, and quality/contractile function, as well as increased risk for heart disease, lower levels of physical activity, and propensity for weight gain/resistance to weight loss. We delineate future research directions and the need to examine hybrid muscle fiber populations, which are indicative of a transitory state of fiber phenotype within skeletal muscle. We also describe methodologies for precisely characterizing muscle fibers and gene expression at the single muscle fiber level to enhance our understanding of the regulation of muscle fiber phenotype in obesity. By contextualizing research in the field of muscle fiber type in obesity, we lay a foundation for future advancements and pave the way for translation of this knowledge to address impaired metabolism and function in obesity.

Skeletal muscle is the tissue directly involved in insulin-stimulated glucose uptake. Glucose is the primary energy substrate for contracting muscles, and proper metabolism of glucose is essential for health. Contractile activity and the associated Ca 2+ signaling regulate functional capacity and muscle mass. A high concentration of Ca 2+ and the presence of calmodulin (CaM) leads to the activation of calcineurin (CaN), a protein with serine-threonine phosphatase activity. The signaling pathway linked with CaN and transcription factors like the nuclear factor of activated T cells (NFAT) is essential for skeletal muscle development and reprogramming of fast-twitch to slow-twitch fibers. CaN activation may promote metabolic adaptations in muscle cells, resulting in better insulin-stimulated glucose transport. The molecular mechanisms underlying the altered insulin response remain unclear. The role of the CaN/NFAT pathway in regulating skeletal muscle hypertrophy is better described than its involvement in the pathogenesis of insulin resistance. Thus, there are opportunities for future research in that field. This review presents the role of CaN/NFAT signaling and suggests the relationship with insulin-resistant muscles., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD + level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD + is a possible approach for improving impaired cellular functions caused by aging or diseases., (© 2023. The Author(s).)

The purpose of this study was to evaluate the impact of resveratrol on slow-twitch muscle fiber expression in bovine myotubes. The results revealed that resveratrol enhanced slow myosin heavy chain (MyHC) and suppressed fast MyHC protein expression, accompanied by increased MyHC I/IIa and decreased MyHC IIx/IIb mRNA levels in bovine myotubes (P < 0.05). Resveratrol also enhanced the activities of succinic dehydrogenase (SDH), malate dehydrogenase (MDH) and the mitochondrial DNA (mtDNA) content, but reduced lactate dehydrogenase (LDH) activity (P < 0.05). Meanwhile, the protein and gene expression of AMPK, SIRT1 and PGC-1α were upregulated by resveratrol (P < 0.05). Furthermore, PGC-1α inhibitor SR-18292 could attenuate resveratrol-induced muscle fiber conversion from fast-twitch to slow-twitch. These results suggest that resveratrol might promote muscle fiber type transition from fast-twitch to slow-twitch through the AMPK/PGC-1α signaling pathway and mitochondrial biogenesis in bovine myotubes., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of financial competing interest or personal relationships., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Acute exercise induces changes in circulating proteins, which are known to alter metabolism and systemic energy balance. Skeletal muscle is a primary contributor to changes in the plasma proteome with acute exercise. An important consideration when assessing the endocrine function of muscle is the presence of different fiber types, which show distinct functional and metabolic properties and likely secrete different proteins. Similarly, adipokines are important regulators of systemic metabolism and have been shown to differ between depots. Given the health-promoting effects of exercise, we proposed that understanding depot-specific remodeling of protein secretion in muscle and adipose tissue would provide new insights into intertissue communication and uncover novel regulators of energy homeostasis. Here, we examined the effect of endurance exercise training on protein secretion from fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscle and visceral and subcutaneous adipose tissue. High-fat diet-fed mice were exercise trained for 6 wk, whereas a Control group remained sedentary. Secreted proteins from excised EDL and soleus muscle, inguinal, and epididymal adipose tissues were detected using mass spectrometry. We detected 575 and 784 secreted proteins from EDL and soleus muscle and 738 and 920 proteins from inguinal and epididymal adipose tissue, respectively. Of these, 331 proteins were secreted from all tissues, whereas secretion of many other proteins was tissue and depot specific. Exercise training led to substantial remodeling of protein secretion from EDL, whereas soleus showed only minor changes. Myokines released exclusively from EDL or soleus were associated with glycogen metabolism and cellular stress response, respectively. Adipokine secretion was completely refractory to exercise regulation in both adipose depots. This study provides an in-depth resource of protein secretion from muscle and adipose tissue, and its regulation following exercise training, and identifies distinct depot-specific secretion patterns that are related to the metabolic properties of the tissue of origin. NEW & NOTEWORTHY The present study examines the effects of exercise training on protein secretion from fast-twitch and slow-twitch muscle as well as visceral and subcutaneous adipose tissue of obese mice. Although exercise training leads to substantial remodeling of protein secretion from fast-twitch muscle, adipose tissue is completely refractory to exercise regulation.

Background: Betaglycan, also known as the TGFβ type III receptor (Tgfbr3), is a co-receptor that modulates TGFβ family signaling. Tgfbr3 is upregulated during C2C12 myoblast differentiation and expressed in mouse embryos myocytes., Results: To investigate tgfbr3 transcriptional regulation during zebrafish embryonic myogenesis, we cloned a 3.2 kb promoter fragment that drives reporter transcription during C2C12 myoblasts differentiation and in the Tg(tgfbr3:mCherry) transgenic zebrafish. We detect tgfbr3 protein and mCherry expression in the adaxial cells concomitantly with the onset of their radial migration to become slow-twitch muscle fibers in the Tg(tgfbr3:mCherry). Remarkably, this expression displays a measurable antero-posterior somitic gradient expression., Conclusions: tgfbr3 is transcriptionally regulated during somitic muscle development in zebrafish with an antero-posterior gradient expression that preferentially marks the adaxial cells and their descendants., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Vestigial-like family member 3 (VGLL3) is a cofactor for the TEA-domain transcription factor (TEAD) family. Although VGLL3 influences myogenic differentiation, its involvement in slow- and fast-twitch fiber specification remains unknown. In this study, we established a cell line stably overexpressing VGLL3 and analyzed effects of VGLL3 on the myogenic differentiation of murine myoblast C2C12 cells. We found that VGLL3 expression promotes slow-twitch muscle differentiation. Mechanistically, VGLL3 expression induced the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master transcriptional regulator of slow-twitch muscle development. We also found that VGLL3 proteins are degraded by the proteasome, which causes switching of TEAD cofactors from VGLL3 to Yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ). These results suggest that the balance between the two kinds of TEAD cofactors VGLL3 and YAP/TAZ controls muscle fiber-type specification., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The composition of fiber types within skeletal muscle impacts the tissue's physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber-type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light-stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and up-regulation of neuron receptor genes. We observed an up-regulation in the expression of fast myosin heavy-chain isoforms, which induced a shift towards a fast-twitch phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling., (© 2023 Hennig et al.)

Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle-specific deletion of Cdk4's target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber-specification that is of relevance to metabolic and muscular diseases.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle fiber type specification is changeable during muscle regeneration following cardiotoxin (CTX) injection; however, the mechanism of muscle fiber shift in regenerating muscle fibers remains unclear. Furthermore, it is unclear as to which factors determine skeletal muscle fiber types in regenerating muscle fibers. Previous studies showed that CTX-induced muscle damage resulted in a temporary hypoxic condition, indicating that hypoxia-inducible factor (HIF)-1α may be involved in muscle fiber type transition. Stabilization of HIF-1α has been shown to result in muscle fiber type transition toward slow-twitch phenotype through the calcineurin/nuclear factor activated T cell 1 (NFATc1) signaling pathway. Therefore, the aim of the present study was to determine whether the calcineurin/NFATc1 pathway is a key mediator of skeletal muscle fiber type transition during muscle regeneration. We found that CTX-induced muscle damage resulted in transient ischemia and HIF-1α expression in skeletal muscle. Additionally, it shifted the muscle fiber type proportion toward a slow-twitch phenotype in the soleus muscle (37.5% in the control muscle vs. 61.3% in the damaged muscle; p < 0.01) three weeks after muscle damage. Moreover, the NFATc1 protein levels increased in damaged muscle, and blockage of the calcineurin/NFATc1 signaling pathway by tacrolimus (FK-506) treatment substantially decreased the number of slow-twitch muscle fibers in the soleus muscle. This study demonstrated that CTX-induced muscle injury results in transient ischemia in hind limb muscle and stabilizes HIF-1α. Moreover, muscle damage increased oxidative phenotype muscle fibers through the calcineurin/NFATc1 signaling pathway during muscle regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle is highly developed after birth, consisting of glycolytic fast-twitch and oxidative slow-twitch fibers; however, the mechanisms of fiber-type-specific differentiation are poorly understood. Here, we found an unexpected role of mitochondrial fission in the differentiation of fast-twitch oxidative fibers. Depletion of the mitochondrial fission factor dynamin-related protein 1 (Drp1) in mouse skeletal muscle and cultured myotubes results in specific reduction of fast-twitch muscle fibers independent of respiratory function. Altered mitochondrial fission causes activation of the Akt/mammalian target of rapamycin (mTOR) pathway via mitochondrial accumulation of mTOR complex 2 (mTORC2), and rapamycin administration rescues the reduction of fast-twitch fibers in vivo and in vitro. Under Akt/mTOR activation, the mitochondria-related cytokine growth differentiation factor 15 is upregulated, which represses fast-twitch fiber differentiation. Our findings reveal a crucial role of mitochondrial dynamics in the activation of mTORC2 on mitochondria, resulting in the differentiation of muscle fibers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Purpose of the Research: Endurance training can modify signaling and gene expression pathways that play a pivotal role in determining the phenotype of the fibers. The present study aimed to investigate the effects of endurance training on the expression of some myomiRs and related genes in slow and fast twitch muscles., Methods: Twenty healthy male adult Wistar rats (281 ± 14 g) were randomized to either control (n = 10) or treated (n = 10). The treated group performed an endurance program for eight weeks (running on a treadmill for eight weeks, 50 min, 23 m/min). After the end of the training protocol, the slow (soleus) and fast (EDL) twitch muscles were removed to assess the miR-1, miR-133 expression, and hdac4, mef2c genes, and protein by real-time PCR and western blot, respectively., Results: The soleus muscle miR-1 expression and mef2c gene in the treated group were significantly lower compared control (p = 0.0001). In contrast, miR-133 and hdac4 gene expression of the soleus muscle of the treated group increased significantly (p = 0001), and the EDL miR-133 and mef2c expression of the treated group increased in the compared control group (p = 0.0001). The EDL MEF2c protein expression in the treated group significantly decreased compared to the control group, although the expression of EDL HDAC4 protein significantly increased (p = 0.0001)., Conclusions: Endurance training changes the expression of the miR-1, miR-133, and their predicted genes in slow and fast twitch muscles. Also, the rate of HDAC4 and MEF2c protein synthesis, which are upstream and downstream of these myomiRs, was affected by endurance training., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Introduction: Skeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of type 2 diabetes mellitus. Skeletal muscle is a heterogeneous tissue composed of different muscle fiber types that contribute distinctly to IR development. Glucose transport shows more protection in slow-twitch muscles than in fast-twitch muscles during IR development, while the mechanisms involved remain unclear. Therefore, we investigated the role of the mitochondrial unfolded protein response (UPRmt) in the distinct resistance of two types of muscle in IR., Methods: Male Wistar rats were divided into high-fat diet (HFD) feeding and control groups. We measured glucose transport, mitochondrial respiration, UPRmt and histone methylation modification of UPRmt-related proteins to examine the UPRmt in the slow fiber-enriched soleus (Sol) and fast fiber-enriched tibialis anterior (TA) under HFD conditions., Results: Our results indicate that 18 weeks of HFD can cause systemic IR, while the disturbance of Glut4-dependent glucose transport only occurred in fast-twitch muscle. The expression levels of UPRmt markers, including ATF5, HSP60 and ClpP, and the UPRmt-related mitokine MOTS-c were significantly higher in slow-twitch muscle than in fast-twitch muscle under HFD conditions. Mitochondrial respiratory function is maintained only in slow-twitch muscle. Additionally, in the Sol, histone methylation at the ATF5 promoter region was significantly higher than that in the TA after HFD feeding., Conclusion: The expression of proteins involved in glucose transport in slow-twitch muscle remains almost unaltered after HFD intervention, whereas a significant decline of these proteins was observed in fast-twitch muscle. Specific activation of the UPRmt in slow-twitch muscle, accompanied by higher mitochondrial respiratory function and MOTS-c expression, may contribute to the higher resistance to HFD in slow-twitch muscle. Notably, the different histone modifications of UPRmt regulators may underlie the specific activation of the UPRmt in different muscle types. However, future work applying genetic or pharmacological approaches should further uncover the relationship between the UPRmt and insulin resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Li, Zhang, Song, Li, Wang, Bo and Zhang.)

Skeletal muscles support the stability and mobility of the skeleton but differ in biomechanical properties and physiological functions. The intrinsic factors that regulate muscle-specific characteristics are poorly understood. To study these, we constructed a large atlas of RNA-seq profiles from six leg muscles and two locations from one muscle, using biopsies from 20 healthy young males. We identified differential expression patterns and cellular composition across the seven tissues using three bioinformatics approaches confirmed by large-scale newly developed quantitative immune-histology procedures. With all three procedures, the muscle samples clustered into three groups congruent with their anatomical location. Concomitant with genes marking oxidative metabolism, genes marking fast- or slow-twitch myofibers differed between the three groups. The groups of muscles with higher expression of slow-twitch genes were enriched in endothelial cells and showed higher capillary content. In addition, expression profiles of Homeobox ( HOX ) transcription factors differed between the three groups and were confirmed by spatial RNA hybridization. We created an open-source graphical interface to explore and visualize the leg muscle atlas (https://tabbassidaloii.shinyapps.io/muscleAtlasShinyApp/). Our study reveals the molecular specialization of human leg muscles, and provides a novel resource to study muscle-specific molecular features, which could be linked with (patho)physiological processes., Competing Interests: TA, Se, LV, TV, DC, HM, DM, Ev, P', HK, VR No competing interests declared, (© 2023, Abbassi-Daloii et al.)

The conversion of skeletal muscle fiber from fast-twitch to slow-twitch is crucial for sustained contractile and stretchable events, energy homeostasis, and anti-fatigue ability. The purpose of our study was to explore the mechanism and effects of garcinol on the regulation of skeletal muscle fiber type transformation. Forty 21-day-old male C57/BL6J mice (n = 10/diet) were fed a control diet or a control diet plus garcinol at 100 mg/kg (Low Gar), 300 mg/kg (Mid Gar), or 500 mg/kg (High Gar) for 12 weeks. The tibialis anterior (TA) and soleus muscles were collected for protein and immunoprecipitation analyses. Dietary garcinol significantly downregulated ( p < 0.05) fast myosin heavy chain (MyHC) expression and upregulated ( p < 0.05) slow MyHC expression in the TA and soleus muscles. Garcinol significantly increased ( p < 0.05) the activity of peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) and markedly decreased ( p < 0.05) the acetylation of PGC-1α. In vitro and in vivo experiments showed that garcinol decreased ( p < 0.05) lactate dehydrogenase activity and increased ( p < 0.05) the activities of malate dehydrogenase and succinic dehydrogenase. In addition, the results of C2C12 myotubes showed that garcinol treatment increased ( p < 0.05) the transformation of glycolytic muscle fiber to oxidative muscle fiber by 45.9%. Garcinol treatment and p300 interference reduced ( p < 0.05) the expression of fast MyHC but increased ( p < 0.05) the expression of slow MyHC in vitro. Moreover, the acetylation of PGC-1α was significantly decreased ( p < 0.05). Garcinol promotes the transformation of skeletal muscle fibers from the fast-glycolytic type to the slow-oxidative type through the p300/PGC-1α signaling pathway in C2C12 myotubes.

Sarcopenia is distinct from normal muscle atrophy in that it is closely related to a shift in the muscle fiber type. Deficiency of the anabolic action of androgen on skeletal muscles is associated with sarcopenia; however, the function of the androgen receptor (AR) pathway in sarcopenia remains poorly understood. We generated a mouse model (fast-twitch muscle-specific AR knockout [fmARKO] mice) in which the AR was selectively deleted in the fast-twitch muscle fibers. In young male mice, the deletion caused no change in muscle mass, but it reduced muscle strength and fatigue resistance and induced a shift in the soleus muscles from fast-twitch fibers to slow-twitch fibers (14% increase, P = 0.02). After middle age, with the control mice, the male fmARKO mice showed much less muscle function, accompanied by lower hindlimb muscle mass; this phenotype was similar to the progression of sarcopenia. The bone mineral density of the femur was significantly reduced in the fmARKO mice, indicating possible osteosarcopenia. Microarray and gene ontology analyses revealed that in male fmARKO mice, there was downregulation of polyamine biosynthesis-related geneswhich was confirmed by liquid chromatography-tandem mass spectrometry assay and the primary cultured myofibers. None of the AR deletion-related phenotypes were observed in female fmARKO mice. Our findings showed that the AR pathway had essential muscle type- and sex-specific roles in the differentiation toward fast-twitch fibers and in the maintenance of muscle composition and function. The AR in fast-twitch muscles was the dominant regulator of muscle fiber-type composition and muscle function, including the muscle-bone relationship.

The aim of this study was to explore the effect of dietary L-theanine (LT) supplementation on skeletal muscle fiber type transformation in weaning piglets. Our data showed that LT significantly increased the slow-twitch fiber-related genes expression and the percentage of slow oxidative fiber, and decreased the MyHC IIb mRNA expression and the percentage of fast glycolytic fiber. In addition, LT significantly increased the succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities and increased the LDH activities. In addition, LT significantly affected mitochondrial biogenesis and function and antioxidative related genes expression, and increased the protein expression of p-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear factor E2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) and decreased the Keap1 protein levels. Furthermore, our data indicated that LT significantly increased the mRNA and protein expression of prospero-related homeobox 1 (Prox1), calcineurin A (CnA), and NFATc1, suggesting that dietary LT supplementation promoted skeletal muscle fiber transition from types II to I might be via activation of calcineurin signaling pathway. Taken together, these findings suggested that LT promoted the transformation of muscle fiber types from slow oxidative to fast glycolytic by increasing antioxidant capacity and improving mitochondrial biogenesis and function and activation of calcineurin signaling pathway.

The skeletal muscle fiber profile is closely related to livestock meat quality. However, the molecular mechanisms determining muscle fiber types in donkeys are not completely understood. In this study, we selected the psoas major muscle (PM; mainly composed of oxidative-type muscle fibers) and biceps femoris muscle (BF; mainly composed of glycolytic-type muscle fibers) and systematically compared their mRNA and microRNA transcriptomes via RNA-seq. We identified a total of 2881 differentially expressed genes (DEGs) and 21 known differentially expressed miRNAs (DEmiRs). Furthermore, functional enrichment analysis showed that the DEGs were mainly involved in energy metabolism and actin cytoskeleton regulation. The glycolysis/gluconeogenesis pathway (including up-regulated genes such as PKM , LDHA , PGK1 and ALDOA ) was more highly enriched in BF, whereas the oxidative phosphorylation pathway and cardiac muscle contraction (including down-regulated genes such as LDHB , ATP2A2 , myosin-7 (MYH7) , TNNC1 , TPM3 and TNNI1 ) was more enriched in PM. Additionally, we identified several candidate miRNA-mRNA pairs that might regulate muscle fiber types using the integrated miRNA-mRNA analysis. Combined with the results of protein-protein interaction (PPI) analysis, some interesting DEGs (including ACTN3 , TNNT3 , TPM2 , TNNC2 , PKM , TNNC1 and TNNI1 ) might be potential candidate target genes involved in the miRNA-mediated regulation of the myofibril composition. This study is the first to indicate that DEmiRs, especially eca-miR-193a-5p and eca-miR-370, and potential candidate target genes that are mainly involved in actin binding (e.g., ACTN3 , TNNT3 and TNNC1 ) and the glycolysis/gluconeogenesis pathways (e.g., PKM ) might coregulate the myofibril composition in donkeys. This study may provide useful information for improving meat quality traits in Dezhou donkeys.

Acute respiratory distress syndrome is associated with skeletal muscle compromise, which decreases survival and impairs functional capacity. A comparative analysis of peripheral and respiratory muscles' atrophy and dysfunction in acute lung injury (ALI) has not been performed. We aimed to evaluate diaphragmatic and peripheral muscle mass and contractility in an ALI animal model. ALI was induced in C57BL/6 mice by intratracheal lipopolysaccharides instillation. Muscle mass and in vitro contractility were evaluated at different time points in hindlimb soleus (slow-twitch) and extensor digitorum longus (EDL, fast-twitch), as well as in the main respiratory muscle diaphragm. Myogenic precursor satellite cell-specific transcription factor Pax7 expression was determined by Western blot. Lung injury was associated with atrophy of the three studied muscles, although it was more pronounced and persistent in the diaphragm. Specific contractility was reduced during lung injury in EDL muscle but restored by the time lung injury has resolved. Specific force was not affected in soleus and diaphragm. A persistent increase in Pax7 expression was detected in diaphragm and EDL muscles after induction of ALI, but not in soleus muscle. Different peripheral and respiratory skeletal muscles are distinctly affected during the course of ALI. Each of the studied muscles presented a unique pattern in terms of atrophy development, contractile dysfunction and Pax7 expression., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Rapid oscillations in cytosolic calcium (Ca 2+ ) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca 2+ , which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca 2+ ATPase (SERCA) to sequester Ca 2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency ( P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca 2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue. NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.

The aim of this study was to investigate the effect and mechanism of L-theanine (LT) on muscle fiber type transformation in C2C12 myotubes. Our data showed that LT exhibited significantly higher slow oxidative muscle fiber expression and lower glycolytic fibers expression. In addition, LT significantly increased the activities of malate dehydrogenase (MDH) and succinic dehydrogenase (SDH), and decreased lactate dehydrogenase (LDH) activity, the calcineurin (CaN) activity and the protein expressions of nuclear factor of activated T cell 1 (NFATc1), prospero-related homeobox1 (prox1), and calcineurin A (CnA) were significantly increased. However, inhibition of CaN activity by cyclosporine A (CsA) abolished LT-induced increase of slow oxidative muscle fiber expression and decrease of glycolytic fibers expression. Moreover, inhibition of prox1 expression by prox1-siRNA disrupted LT-induced activation of CaN signaling pathway and muscle fiber type transformation. Taken together, these results indicated that LT could promote skeletal muscle fiber type transformation from type II to type I via activation of prox1/CaN signaling pathway., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Skeletal muscles are made up of various muscle fiber type including slow and fast-twitch fibers. Because each muscle fiber has its own physiological characteristics, the effects of aging and exercise vary depending on the type of muscle fiber. We used bioinformatics screening techniques such as differentially expressed gene analysis, gene ontology analysis and gene set enrichment analysis, to try to understand the genetic differences between muscle fiber types. The experiment and gene expression profiling in this study used the soleus (SOL, slow-twitch muscle) and gastrocnemius (GAS, fast-twitch muscle). According to our findings, fatty acid metabolism is significantly up-regulated in SOL compared to GAS, whereas the glucose metabolism pathway is significantly down-regulated in SOL compared to GAS. Furthermore, apoptosis and myogenesis patterns differ between SOL and GAS. SOL did not show differences in apoptosis due to the aging effect, but apoptosis in GAS was significantly up-regulated with age. Apoptosis in GAS of old groups is significantly reduced after 4 weeks of aerobic exercise, but no such finding was found in SOL. In terms of myogenesis, exercise intervention up-regulated this process in GAS of old groups but not in SOL. Taken together, muscle fiber type significantly interacts with aging and exercise. Despite the importance of the interaction between these factors, large-scale gene expression data has rarely been studied. We hope to contribute to a better understanding of the relationship between muscle fiber type, aging and exercise at the molecular level.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Aim: To develop a method for direct measurement of the fluorescent d-glucose analogue 2-NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d-glucose transport under basal conditions., Methods: A novel method is described for measuring free 2-NBDG transport across plasma membrane of single rat muscle fibres at rest. The 2-NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast-twitch and slow-twitch fibres, indicating that the 2-NBDG transport is GLUT-mediated. Fibres were identified as fast-twitch or slow-twitch based on the differential sensitivity of their contractile apparatus to Sr 2+ ., Results: The time course of 2-NBDG uptake in the presence of 50 µM 2-NBDG follows a one-phase exponential plateau curve and is faster in fast-twitch (rate constant 0.053 ± 0.0024 s -1 ) than in slow-twitch fibres (rate constant 0.031 ± 0.0021 s -1 ). The rate constants were markedly reduced in the presence of 20 mM d-glucose to 0.0082 ± 0.0004 s -1 and 0.0056 ± 0.0002 s -1 in fast-twitch and slow-twitch fibres respectively. 2-NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2-NBDG in muscle fibres at rest. The parameters describing the transport kinetics for both 2-NBDG and d-glucose (dissociation constants, Michaelis-Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2-NBDG., Conclusion: Free 2-NBDG and d-glucose transport across the plasma membrane of single rat muscle fibres at rest is fast, conclusively showing that the rate-limiting step in d-glucose uptake in skeletal muscle is not necessarily the GLUT-mediated transport of d-glucose., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

In this study, our aim is to investigate the effect of dimer procyanidin B2 [epicatechin-(4β-8)-epicatechin] (PB2) on porcine skeletal myofiber gene expression in vitro . Our data showed PB2 promoted the protein expression of slow myosin heavy chain (MyHC) in porcine myotubes, concomitant with the increases in mRNA levels of MyHC I , MyHC IIa and Tnni1 . We also found PB2 activated AMPK signaling in porcine myotubes. NRF1 and CaMKKβ that are two important upstream factors of AMPK, and Sirt1 and PGC-1α that are two major downstream factors of AMPK, were also up-regulated by PB2. The mechanism study showed the effect of PB2 on slow-twitch myofiber gene expression was abolished by AMPK inhibitor compound C or by AMPKα1 siRNA. Together, we found PB2 induced porcine skeletal slow-twitch myofiber gene expression by AMPK signaling pathway.

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s -1 ) and super-relaxed states (SRX, 0.005 s -1 ) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969-1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C-30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs., Competing Interests: Conflict of interest This article reflects only the authors' view. The European Commission is not responsible for any use that may be made of the information it contains., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian skeletal muscle contains heterogenous myofibers with different contractile and metabolic properties that sustain muscle mass and endurance capacity. The transcriptional regulators that govern these myofiber gene programs have been elucidated. However, the hormonal cues that direct the specification of myofiber types and muscle endurance remain largely unknown. Here, we uncover the secreted factor Tsukushi (TSK) as an extracellular signal that is required for maintaining muscle mass, strength, and endurance capacity and that contributes to muscle regeneration. Mice lacking TSK exhibited reduced grip strength and impaired exercise capacity. Muscle transcriptomic analysis revealed that TSK deficiency results in a remarkably selective impairment in the expression of myofibrillar genes, characteristic of slow-twitch muscle fibers, that is associated with abnormal neuromuscular junction formation. AAV-mediated overexpression of TSK failed to rescue these myofiber defects in adult mice, suggesting that the effects of TSK on myofibers are likely restricted to certain developmental stages. Finally, mice lacking TSK exhibited diminished muscle regeneration following cardiotoxin-induced muscle injury. These findings support a crucial role of TSK as a hormonal cue in the regulation of contractile gene expression, endurance capacity, and muscle regeneration.

Skeletal muscle is a heterogeneous tissue composed of a variety of functionally different fiber types. Slow-twitch type I muscle fibers are rich with mitochondria, and mitochondrial biogenesis promotes a shift towards more slow fibers. Leucine, a branched-chain amino acid (BCAA), regulates slow-twitch muscle fiber expression and mitochondrial function. The BCAA content is increased in porcine whole-blood protein hydrolysates (PWBPH) but the effect of PWBPH on muscle fiber type conversion is unknown. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased time to exhaustion in the forced swimming test and the mass of the quadriceps femoris muscle but decreased the levels of blood markers of exercise-induced fatigue. PWBPH also promoted fast-twitch to slow-twitch muscle fiber conversion, elevated the levels of mitochondrial biogenesis markers (SIRT1, p-AMPK, PGC-1α, NRF1 and TFAM) and increased succinate dehydrogenase and malate dehydrogenase activities in ICR mice. Similarly, PWBPH induced markers of slow-twitch muscle fibers and mitochondrial biogenesis in C2C12 myotubes. Moreover, AMPK and SIRT1 inhibition blocked the PWBPH-induced muscle fiber type conversion in C2C12 myotubes. These results indicate that PWBPH enhances exercise performance by promoting slow-twitch muscle fiber expression and mitochondrial function via the AMPK/SIRT1 signaling pathway.

BDNF (brain-derived neurotrophic factor) is present in skeletal muscle, controlling muscular metabolism, strength and regeneration processes. However, there is no consensus on BDNF cellular source. Furthermore, while endothelial tissue expresses BDNF in large amount, whether endothelial cells inside muscle expressed BDNF has never been explored. The aim of the present study was to provide a comprehensive analysis of BDNF localization in rat skeletal muscle. Cellular localization of BDNF and activated Tropomyosin-related kinase B (TrkB) receptors was studied by immunohistochemical analysis on soleus (SOL) and gastrocnemius (GAS). BDNF and activated TrkB levels were also measured in muscle homogenates using Western blot analysis and/or Elisa tests. The results revealed BDNF immunostaining in all cell types examined with a prominent staining in endothelial cells and a stronger staining in type II than type I muscular fibers. Endothelial cells but not other cells displayed easily detectable activated TrkB receptor expression. Levels of BDNF and activated TrkB receptors were higher in SOL than GAS. In conclusion, endothelial cells are an important and still unexplored source of BDNF present in skeletal muscle. Endothelial BDNF expression likely explains why oxidative muscle exhibits higher BDNF levels than glycolytic muscle despite higher the BDNF expression by type II fibers., (© 2022. The Author(s).)