Quercetin induces a slow myofiber phenotype in engineered human skeletal muscle tissues.

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.

Piperine enhances contractile force in slow- and fast-twitch muscle.

Piperine has been shown to bind to myosin and shift the distribution of conformational states of myosin molecules from the super-relaxed state to the disordered relaxed state. However, little is known about the implications for muscle force production and potential underlying mechanisms. Muscle contractility experiments were performed using isolated muscles and single fibres from rats and mice. The dose-response effect of piperine on muscle force was assessed at several stimulation frequencies. The potentiation of muscle force was also tested in muscles fatigued by eccentric contractions. Potential mechanisms of force potentiation were assessed by measuring Ca2+ levels during stimulation in enzymatically dissociated muscle fibres, while myofibrillar Ca2+ sensitivity was assessed in chemically skinned muscle fibres. Piperine caused a dose-dependent increase in low-frequency force with no effect on high-frequency force in both slow- and fast-twitch muscle, with similar relative increases in twitch force, rate of force development and relaxation rate. The potentiating effect of piperine on low-frequency force was reversible, and piperine partially recovered low-frequency force in fatigued muscle. Piperine had no effect on myoplasmic free [Ca2+] levels in mouse muscle fibres, whereas piperine substantially augmented the force response to submaximal levels of [Ca2+] in rat MyHCII fibres and MyHCI fibres along with a minor increase in maximum Ca2+-activated force. Piperine enhances low-frequency force production in both fast- and slow-twitch muscle. The effects are reversible and can counteract muscle fatigue. The primary underlying mechanism appears to be an increase in Ca2+ sensitivity. KEY POINTS: Piperine is a plant alkaloid derived from black pepper. It is known to bind to skeletal muscle myosin and enhance resting ATP turnover but its effects on contractility are not well known. We showed for the first time a piperine-induced force potentiation that was pronounced during low-frequency electrical stimulation of isolated muscles. The effect of piperine was observed in both slow and fast muscle types, was reversible, and could counteract the force decrements observed after fatiguing muscle contractions. Piperine treatment caused an increase in myofibrillar Ca2+ sensitivity in chemically skinned muscle fibres, while we observed no effect on intracellular Ca2+ concentrations during electrical stimulation in enzymatically dissociated muscle fibres.

Azilsartan prevents muscle loss and fast- to slow-twitch muscle fiber shift in natural ageing sarcopenic rats.

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.

Arginine Regulates Skeletal Muscle Fiber Type Formation via mTOR Signaling Pathway.

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.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Effect of Porcine Whole Blood Protein Hydrolysate on Slow-Twitch Muscle Fiber Expression and Mitochondrial Biogenesis via the AMPK/SIRT1 Pathway.

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.

Eicosapentaenoic acid changes muscle transcriptome and intervenes in aging-related fiber type transition in male mice.

Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes are interrelated with each other and associated with systemic metabolism, the details of which, however, are largely unknown. Eicosapentaenoic acid (EPA) is a promising nutrient against sarcopenia and has multifaceted effects on systemic metabolism. In this study, we hypothesized that the aging process in skeletal muscle can be intervened by the administration of EPA. Seventy-five-week-old male mice were assigned to groups fed an EPA-deprived diet (EPA-) or an EPA-enriched diet with 1 wt% EPA (EPA+) for 12 wk. Twenty-four-week-old male mice fed with normal chow were also analyzed. At baseline, the grip strength of the aging mice was lower than that of the young mice. After 12 wk, EPA+ showed similar muscle mass but increased grip strength compared with EPA-. EPA+ displayed higher insulin sensitivity than EPA-. Immunohistochemistry and gene expression analysis of myosin heavy chains (MyHCs) revealed fast-to-slow fiber type transition in aging muscle, which was partially inhibited by EPA. RNA sequencing (RNA-Seq) analysis suggested that EPA supplementation exerts pathway-specific effects in skeletal muscle including the signatures of slow-to-fast fiber type transition. In conclusion, we revealed that aging skeletal muscle in male mice shows lower grip strength and fiber type changes, both of which can be inhibited by EPA supplementation irrespective of muscle mass alteration.NEW & NOTEWORTHY This study demonstrated that the early phenotype of skeletal muscle in aging male mice is characterized by muscle weakness with fast-to-slow fiber type transition, which could be ameliorated by feeding with EPA-enriched diet. EPA induced metabolic changes such as an increase in systemic insulin sensitivity and altered muscle transcriptome in the aging mice. These changes may be related to the fiber type transition and influence muscle quality.

Thyroid hormone receptor α in skeletal muscle is essential for T3-mediated increase in energy expenditure.

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole-body energy metabolism and body temperature has not been resolved. Here, we show that T3-induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα1 ) in skeletal muscle, but that T3-mediated elevation in body temperature is achieved in the absence of muscle-TRα1 . In slow-twitch soleus muscle, loss-of-function of TRα1 (TRαHSACre ) alters the fiber-type composition toward a more oxidative phenotype. The change in fiber-type composition, however, does not influence the running capacity or motivation to run. RNA-sequencing of soleus muscle from WT mice and TRαHSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα1 -linked control of whole-body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3-stimulated increase in whole-body energy expenditure.

An extract of Lycium barbarum mimics exercise to improve muscle endurance through increasing type IIa oxidative muscle fibers by activating ERRγ.

Lycium barbarum berry (gouqi, Goji, goji berry, or wolfberry), a traditional medicine and functional food, has a wide range of biological effects, including immuno-modulation, anti-aging, antitumor, neuro-protection, and hepato-protection. However, thus far, little is known about the traditional effects of L. barbarum on strengthening muscles. Therefore, this study focused on the effects of an extract of L. barbarum on skeletal muscles. First, the extract of L. barbarum significantly increased the mass of the tibial anterior muscle and gastrocnemius muscle and improved the average running distance of mice. Then, in vivo and in vitro experiments showed that the extract enhanced muscle endurance by increasing the proportion of type IIa oxidative muscle fibers and aerobic respiration. In an in-depth study of the molecular mechanism of these effects, we found that the extract upregulated the proportion of type IIa oxidative muscle fibers by activating ERRγ and that the PKA-CREB signaling pathway was involved in its activation. This study is the first to show that L. barbarum extract modulates skeletal muscle remodeling and has mimetic effects on skeletal muscles in a manner similar to exercise. It provides a scientific explanation based on modern biological technologies and concepts for the traditional function of L. barbarum in improving muscle fitness. This study lays a theoretical foundation for the application of L. barbarum in skeletal muscles as an exercise mimetic.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

Arginine promotes porcine type I muscle fibres formation through improvement of mitochondrial biogenesis.

The present study aimed to investigate whether arginine (Arg) promotes porcine type I muscle fibres formation via improving mitochondrial biogenesis. In the in vivo study, a total of sixty Duroc × Landrace × Yorkshire weaning piglets with an average body weight of 6·55 (sd 0·36) kg were randomly divided into four treatments and fed with a basal diet or a basal diet supplemented with 0·5, 1·0 and 1·5 % l-Arg, respectively, in a 4-week trial. Results showed that dietary supplementation of 1·0 % Arg significantly enhanced the activity of succinate dehydrogenase, up-regulated the protein expression of myosin heavy chain I (MyHC I) and increased the mRNA levels of MyHC I, troponin I1, C1 and T1 (Tnni1, Tnnc1 and Tnnt1) in longissimus dorsi muscle compared with the control group. In addition, ATPase staining analysis indicated that 1·0 % Arg supplementation significantly increased the number of type I muscle fibres and significantly decreased the number of type II muscle fibres. Furthermore, 1·0 % Arg supplementation significantly up-regulated PPAR-γ coactivator-1α (PGC-1α), sirtuin 1 and cytochrome c (Cytc) protein expressions, increased PGC-1α, nuclear respiratory factor 1 (NRF1), mitochondria transcription factor B1 (TFB1M), Cytc and ATP synthase subunit C1 (ATP5G) mRNA levels and increased mitochondrial DNA content. In the in vitro study, mitochondrial complex I inhibitor rotenone (Rot) was used. We found that Rot annulled Arg-induced type I muscle fibres formation. Together, our results provide for the first time the evidence that Arg promotes porcine type I muscle fibres formation through improvement of mitochondrial biogenesis.

Pterostilbene Enhances Endurance Capacity via Promoting Skeletal Muscle Adaptations to Exercise Training in Rats.

It has been demonstrated that skeletal muscle adaptions, including muscle fibers transition, angiogenesis, and mitochondrial biogenesis are involved in the regular exercise-induced improvement of endurance capacity and metabolic status. Herein, we investigated the effects of pterostilbene (PST) supplementation on skeletal muscle adaptations to exercise training in rats. Six-week-old male Sprague Dawley rats were randomly divided into a sedentary control group (Sed), an exercise training group (Ex), and exercise training combined with 50 mg/kg PST (Ex + PST) treatment group. After 4 weeks of intervention, an exhaustive running test was performed, and muscle fiber type transformation, angiogenesis, and mitochondrial content in the soleus muscle were measured. Additionally, the effects of PST on muscle fiber transformation, paracrine regulation of angiogenesis, and mitochondrial function were tested in vitro using C2C12 myotubes. In vivo study showed that exercise training resulted in significant increases in time-to-exhaustion, the proportion of slow-twitch fibers, muscular angiogenesis, and mitochondrial biogenesis in rats, and these effects induced by exercise training could be augmented by PST supplementation. Moreover, the in vitro study showed that PST treatment remarkably promoted slow-twitch fibers formation, angiogenic factor expression, and mitochondrial function in C2C12 myotubes. Collectively, our results suggest that PST promotes skeletal muscle adaptations to exercise training thereby enhancing the endurance capacity.

A myosin-based mechanism for stretch activation and its possible role revealed by varying phosphate concentration in fast and slow mouse skeletal muscle fibers.

Stretch activation (SA) is a delayed increase in force following a rapid muscle length increase. SA is best known for its role in asynchronous insect flight muscle, where it has replaced calcium's typical role of modulating muscle force levels during a contraction cycle. SA also occurs in mammalian skeletal muscle but has previously been thought to be too low in magnitude, relative to calcium-activated (CA) force, to be a significant contributor to force generation during locomotion. To test this supposition, we compared SA and CA force at different Pi concentrations (0-16 mM) in skinned mouse soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscle fibers. CA isometric force decreased similarly in both muscles with increasing Pi, as expected. SA force decreased with Pi in EDL (40%), leaving the SA to CA force ratio relatively constant across Pi concentrations (17-25%). In contrast, SA force increased in soleus (42%), causing a quadrupling of the SA to CA force ratio, from 11% at 0 mM Pi to 43% at 16 mM Pi, showing that SA is a significant force modulator in slow-twitch mammalian fibers. This modulation would be most prominent during prolonged muscle use, which increases Pi concentration and impairs calcium cycling. Based upon our previous Drosophila myosin isoform studies and this work, we propose that in slow-twitch fibers a rapid stretch in the presence of Pi reverses myosin's power stroke, enabling quick rebinding to actin and enhanced force production, while in fast-twitch fibers, stretch and Pi cause myosin to detach from actin.

Fiber type-selective exercise effects on AS160 phosphorylation.

Earlier research using muscle tissue demonstrated that postexercise elevation in insulin-stimulated glucose uptake (ISGU) occurs concomitant with greater insulin-stimulated Akt substrate of 160 kDa (AS160) phosphorylation (pAS160) on sites that regulate ISGU. Because skeletal muscle is a heterogeneous tissue, we previously isolated myofibers from rat epitrochlearis to assess fiber type-selective ISGU. Exercise induced greater ISGU in type I, IIA, IIB, and IIBX but not IIX fibers. This study tested if exercise effects on pAS160 correspond with previously published fiber type-selective exercise effects on ISGU. Rats were studied immediately postexercise (IPEX) or 3.5 h postexercise (3.5hPEX) with time-matched sedentary controls. Myofibers dissected from the IPEX experiment were analyzed for fiber type (myosin heavy chain isoform expression) and key phosphoproteins. Isolated muscles from the 3.5hPEX experiment were incubated with or without insulin. Myofibers (3.5hPEX) were analyzed for fiber type, key phosphoproteins, and GLUT4 protein abundance. We hypothesized that insulin-stimulated pAS160 at 3.5hPEX would exceed sedentary controls only in fiber types characterized by greater ISGU postexercise. Values for phosphorylation of AMP-activated kinase substrates (acetyl CoA carboxylaseSer79 and AS160Ser704) from IPEX muscles exceeded sedentary values in each fiber type, suggesting exercise recruitment of all fiber types. Values for pAS160Thr642 and pAS160Ser704 from insulin-stimulated muscles 3.5hPEX exceeded sedentary values for type I, IIA, IIB, and IIBX but not IIX fibers. GLUT4 abundance was unaltered 3.5hPEX in any fiber type. These results advanced understanding of exercise-induced insulin sensitization by providing compelling support for the hypothesis that enhanced insulin-stimulated phosphorylation of AS160 is linked to elevated ISGU postexercise at a fiber type-specific level independent of altered GLUT4 expression.

Skeletal muscle fiber type-selective effects of acute exercise on insulin-stimulated glucose uptake in insulin-resistant, high-fat-fed rats.

Insulin-stimulated glucose uptake (GU) by skeletal muscle is enhanced several hours after acute exercise in rats with normal or reduced insulin sensitivity. Skeletal muscle is composed of multiple fiber types, but exercise's effect on fiber type-specific insulin-stimulated GU in insulin-resistant muscle was previously unknown. Male rats were fed a high-fat diet (HFD; 2 wk) and were either sedentary (SED) or exercised (2-h exercise). Other, low-fat diet-fed (LFD) rats remained SED. Rats were studied immediately postexercise (IPEX) or 3 h postexercise (3hPEX). Epitrochlearis muscles from IPEX rats were incubated in 2-deoxy-[3H]glucose (2-[3H]DG) without insulin. Epitrochlearis muscles from 3hPEX rats were incubated with 2-[3H]DG ± 100 µU/ml insulin. After single fiber isolation, GU and fiber type were determined. Glycogen and lipid droplets (LDs) were assessed histochemically. GLUT4 abundance was determined by immunoblotting. In HFD-SED vs. LFD-SED rats, insulin-stimulated GU was decreased in type IIB, IIX, IIAX, and IIBX fibers. Insulin-independent GU IPEX was increased and glycogen content was decreased in all fiber types (types I, IIA, IIB, IIX, IIAX, and IIBX). Exercise by HFD-fed rats enhanced insulin-stimulated GU in all fiber types except type I. Single fiber analyses enabled discovery of striking fiber type-specific differences in HFD and exercise effects on insulin-stimulated GU. The fiber type-specific differences in insulin-stimulated GU postexercise in insulin-resistant muscle were not attributable to a lack of fiber recruitment, as indirectly evidenced by insulin-independent GU and glycogen IPEX, differences in multiple LD indexes, or altered GLUT4 abundance, implicating fiber type-selective differences in the cellular processes responsible for postexercise enhancement of insulin-mediated GLUT4 translocation.

Leucine promotes porcine myofibre type transformation from fast-twitch to slow-twitch through the protein kinase B (Akt)/forkhead box 1 signalling pathway and microRNA-27a.

Muscle fibre types can transform from slow-twitch (slow myosin heavy chain (MyHC)) to fast-twitch (fast MyHC) or vice versa. Leucine plays a vital effect in the development of skeletal muscle. However, the role of leucine in porcine myofibre type transformation and its mechanism are still unclear. In this study, effects of leucine and microRNA-27a (miR-27a) on the transformation of porcine myofibre type were investigated in vitro. We found that leucine increased slow MyHC protein level and decreased fast MyHC protein level, increased the levels of phospho-protein kinase B (Akt)/Akt and phospho-forkhead box 1 (FoxO1)/FoxO1 and decreased the FoxO1 protein level. However, blocking the Akt/FoxO1 signalling pathway by wortmannin attenuated the role of leucine in porcine myofibre type transformation. Over-expression of miR-27a decreased slow MyHC protein level and increased fast MyHC protein level, whereas inhibition of miR-27a had an opposite effect. We also found that expression of miR-27a was down-regulated following leucine treatment. Moreover, over-expression of miR-27a repressed transformation from fast MyHC to slow MyHC caused by leucine, suggesting that miR-27a is interdicted by leucine and then contributes to porcine muscle fibre type transformation. Our finding provided the first evidence that leucine promotes porcine myofibre type transformation from fast MyHC to slow MyHC via the Akt/FoxO1 signalling pathway and miR-27a.

Ca2+ release via two-pore channel type 2 (TPC2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos.

We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+ signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52-87nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

KEY POINTS: The Ca2+ -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross-sectional area of slow-twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. ABSTRACT: The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow-twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force-stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross-sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation.

Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility.

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.

MicroRNA-139-5p suppresses myosin heavy chain I and IIa expression via inhibition of the calcineurin/NFAT signaling pathway.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are widely involved in a variety of biological processes. Different skeletal muscle fiber type composition exhibits characteristic differences in functional properties and energy metabolism of skeletal muscle. However, the molecular mechanism by which miRNAs control the different type of muscle fiber formation is still not fully understood. In the present study, we characterized the role of microRNA-139-5p (miR-139-5p) in the regulation of myosin heavy chain (MyHC) isoform expression and its underlying mechanisms. Here we found that the expression of miR-139-5p was significantly higher in mouse slow-twitch muscle than in fast-twitch muscle. Overexpression of miR-139-5p downregulated the expression of MyHC I and MyHC IIa, whereas inhibition of miR-139-5p upregulated them. We also found that the levels of calcineurin (CaN), NFATc1, MEF2C and MCIP1.4, which are the components of CaN/NFAT signaling pathway that has shown to positively regulate slow fiber-selective gene expression, were notably inhibited by miR-139-5p overexpression. Furthermore, treatment of phenylephrine (PE), a α1-adrenoceptor agonist, abolished the inhibitory effect of miR-139-5p on MyHC I and MyHC IIa expression. Together, our findings indicated that the role of miR-139-5p in regulating the MyHC isoforms, especially MyHC I and MyHC IIa, may be achieved through inhibiting CaN/NFAT signaling pathway.