Mammalian Skeletal Muscle Research Articles

Splicing regulation of GFPT1 muscle-specific isoform and its roles in glucose metabolisms and neuromuscular junction

Glutamine:fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP). A 54-bp exon 9 of GFPT1 is specifically included in skeletal and cardiac muscles to generate a long isoform of GFPT1 (GFPT1-L). We showed that SRSF1 and Rbfox1/2 cooperatively enhance, and hnRNP H/F suppresses, the inclusion of human GFPT1 exon 9 by modulating recruitment of U1 snRNP. Knockout (KO) of GFPT1-L in skeletal muscle markedly increased the amounts of GFPT1 and UDP-HexNAc, which subsequently suppressed the glycolytic pathway. Aged KO mice showed impaired insulin-mediated glucose uptake, as well as muscle weakness and fatigue likely due to abnormal formation and maintenance of the neuromuscular junction. Taken together, GFPT1-L is likely to be acquired in evolution in mammalian striated muscles to attenuate the HBP for efficient glycolytic energy production, insulin-mediated glucose uptake, and the formation and maintenance of the neuromuscular junction.

Matching Mechanics and Energetics of Muscle Contraction Suggests Unconventional Chemomechanical Coupling during the Actin-Myosin Interaction.

The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between mechanical and biochemical steps in the myosin-actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin-actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.

Slow or fast: Implications of myofibre type and associated differences for manifestation of neuromuscular disorders.

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow- to fast-twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow-twitch soleus and fast-twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre-type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders.

Analysis of <i>BMP2</i> gene polymorphism of bone morphogenetic protein-2 in reindeer

The BMP2 gene of reindeer was study by Sanger sequencing. The BMP2 gene is involved in the formation of bone tissue and skeletal muscle in mammals. The BMP2 gene is associated with an increase in height, size, and body weight in productive animals. For the first time, the nucleotidesequence of the coding and regulatory regions of the BMP2 gene was determined in populations of wild and domestic reindeer.As a result, a segment of the reindeer BMP2 candidate gene was sequence, including the first untranslated and second exons. Loci with low (exon1) and high (exon2) degree of polymorphism identified.Single nucleotide mismatches with the reference genome (white-tailed deer (Odocoileus virginianus texanus) genome) identified, which can be used for phylogenetic analysis of the evolution of orthologue genes, as well as for genetic identification of species. Twelve polymorphic loci of two types were identified: in/del and SNP. The most significant substitutions were also determined in accordance with the change in the amino acid composition of the protein and the chemical properties of the replaced amino acids. Calculations were made of the occurrence of genotypes and alleles according to the identified variants of genetic polymorphism in the analyzed populations of reindeer (Nenets and Evenk breeds, wild reindeer).The object of research was wild and domestic reindeer on the farms of the Taimyr Peninsula and Evenkia. The material for the study was DNA isolated by the classical phenol method from the blood of domestic reindeer of two breeds - Nenets (n=20) and Evenki (n=20), as well as from tissue samples of wild reindeer (n=20).The aim of the study was to search for and select potential variants of genetic polymorphism in the BMP2 gene as molecular genetic markers that can be recommend for use in the practice of reindeer breeding to increase meat productivity.For the first time, the nucleotide sequence of significant regions of the reindeer BMP2 gene was determined. The polymorphic loci in the BMP2 gene identified as a result of the studies had differences in the frequency of occurrence of alleles of mutant and wild types in the studied samples of domestic reindeer of the Nenets and Evenk breeds, as well as in the sample of wild reindeer. Differences in occurrence indicate the effect of selection factors on the region of the BMP2 gene. The revealed genetic variability of the studied region in reindeer breeds contrasting in productivity is a preliminary confirmation of the influence of genetic polymorphism in the BMP2 gene on the variability of phenotypes in terms of height and weight indicators. Indicators of growth and live weight are key to the formation of the meat productivity of reindeer. The results of studies of the effect of BMP2 gene polymorphism on growth and weight parameters in other animal species suggest that some of the identified variants of genetic polymorphism in the studied region of the BMP2 gene may turn out to be casual mutations that can affect the formation of quantitative traits in domestic reindeer.

Effects of Leucine Ingestion and Contraction on the Sestrin/GATOR2 Pathway and mTORC1 Activation in Rat Fast-Twitch muscle

BackgroundLeucine activates the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in mammalian skeletal muscle. Recent studies have shown that Sestrin, a leucine sensor, might play a role in this process. However, it remains unknown whether Sestrin dissociates from GATOR2 in a dose- and time-dependent manner and whether an acute bout of muscle contraction augments this dissociation. ObjectiveThis study aimed to examine the effects of leucine ingestion and muscle contraction on the interaction between Sestrin1/2 and GATOR2 and on mTORC1 activation. MethodsMale Wistar rats were randomly assigned to control (C), leucine 3 (L3), or leucine 10 (L10) groups. Intact gastrocnemius muscles were subjected to 30 repetitive unilateral contractions. The L3 and L10 groups were then orally administered 3 and 10 mmol/kg body weight of L-leucine 2 h after the end of the contractions, respectively. Blood and muscle samples were collected 30, 60, or 120 min after the administration. ResultsThe blood and muscle leucine concentrations increased in a dose-dependent manner. The ratio of phosphorylated ribosomal protein S6 kinase (S6K) to total S6K (which indicates mTORC1 signaling activation) was markedly increased by muscle contraction and increased in a dose-dependent manner only in rested muscle. Leucine ingestion but not muscle contraction increased Sestrin1 dissociation from GATOR2 and Sestrin2 association with GATOR2. A negative relationship was observed between the blood and muscle leucine concentrations and the Sestrin1 association with GATOR2. ConclusionsThe results suggest that Sestrin1, but not Sestrin2, regulates leucine-related mTORC1 activation via its dissociation from GATOR2 and that acute exercise-induced mTORC1 activation involves pathways other than the leucine-related Sestrin1/GATOR2 pathway.

Skeletal muscle growth activity of Olive Flounder (Paralichthys olivaceus) meat digest

Mammalian skeletal muscles play a pivotal role in the body and are mainly responsible for movement, metabolism, and energy consumption. Proteins that regulate muscle hypertrophy and atrophy are involved in muscle growth. A sufficient intake of dietary protein is essential for maintaining good health during normal growth and aging. Olive flounder (Paralichthys olivaceus) is one of the most common flatfish species in the South Korean aquaculture industry, and its consumption has been associated with various health benefits, such as a lower risk of cancers, heart failure, and chronic diseases. The present study attempted to determine the effect on muscle growth of flounder meat digest prepared using an in vitro gastric model. The capacity of digested flounder meat to enhance muscle growth was evaluated by examining glycogen levels and creatine kinase activity during muscle differentiation. The expression of muscle growth regulatory proteins was also evaluated. Treatment with flounder meat digest significantly and dose-dependently increased glycogen levels and creatine kinase activity in C2C12 cells. Moreover, proteins that are responsible for muscle hypertrophy and atrophy were significantly upregulated and downregulated, respectively. These results were confirmed using western blotting and immunofluorescence assays. The results of this study suggest that digested flounder meat has great potential for skeletal muscle growth and can be used as a functional food ingredient.

New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4.

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing - comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.

Ryanodine receptor 3 impacts on the expression of aminopeptidase in mammalian skeletal muscle cells during myogenic differentiation

In mammalian skeletal muscle, ryanodine receptor (Ryr) expresses in the membrane of sarcoplasmic reticulum (SR), and is known as Ca2+-releasing channel. Ryr in mammalian skeletal muscle consists of 2 isoforms, Ryr1 and Ryr3. Ryr1 is coupled with dihydropyridine receptors (DHPRs) in transverse tubules, and Ca2+ release is a key step for the excitation-contraction coupling (E-C coupling). However, a physiological role of ryr3 is still unclear. Recently, we confirmed that Ryr3 knockdown induces abnormal myotube formation of C2C12 cells. Further, it is reported that the abnormal shape of C2C12 myotubes were observed by the knockdown of puromycin-sensitive aminopeptidase, a selective hydrolyze of amino acid residue from the N-terminus of peptide and proteins. In the present study, we investigated that the molecular mechanisms for the abnormal myotube formation of C2C12 cells following the knockdown of Ryr3. Aggregation of phalloidin-stained actin fibers were observed in Ryr3-knockdown myotubes. On the other hand, a significantly decrease in the mRNA expression level of puromycin-sensitive aminopeptidase and endoplasmic reticulum aminopeptidase 1 was observed in Ryr3-knockdown C2C12 cells (p<0.05). Therefore, Ryr3 may play a crucial role in the quality control of proteins and peptides during myogenic differentiation via the up-regulation of puromycin-sensitive aminopeptidase and endoplasmic reticulum aminopeptidase 1. This study was partially supported by JSPS KAKENHI (18H03160, K.G.; 19K22825, K.G.; 19KK0254, K.G., S.Y., T.E.; 22H03474, K.G., T.E.; 22K19722, K.G., T.E.; 22H03319, K.G., T.E.; 22K18413, K.G., T.E.), a grant from Graduate School of Health Science, Toyohashi SOZO University (K.G.), and a research grant from Toyohashi SOZO university (K.G.). All authors declare that they have no conflicts of interest. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Muscle fusogens go viral for gene delivery to skeletal muscle

Viruses and multinucleated cells rely on fusogens to facilitate the fusion of their membranes. In this issue of Cell, Millay and colleagues demonstrate that replacing viral fusogens with mammalian skeletal muscle fusogens leads to the specific transduction of skeletal muscle and the ability to deliver gene therapy constructs in a therapeutically relevant muscle disease.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver μDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle.

The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.

No Effect of Acute Balenine Supplementation on Maximal and Submaximal Exercise Performance in Recreational Cyclists.

Carnosine (β-alanyl-L-histidine) and its methylated analogues anserine and balenine are highly concentrated endogenous dipeptides in mammalian skeletal muscle that are implicated in exercise performance. Balenine has a much better bioavailability and stability in human circulation upon acute ingestion, compared to carnosine and anserine. Therefore, ergogenic effects observed with acute carnosine and anserine supplementation may be even more pronounced with balenine. This study investigated whether acute balenine supplementation improves physical performance in four maximal and submaximal exercise modalities. A total of 20 healthy, active volunteers (14 males; six females) performed cycling sprints, maximal isometric contractions, a 4-km TT and 20-km TT following either preexercise placebo or 10mg/kg of balenine ingestion. Physical, as well as mental performance, along with acid-base balance and glucose concentration were assessed. Balenine was unable to augment peak power (p = .3553), peak torque (p = .3169), time to complete the 4km (p = .8566), nor 20km time trial (p = .2660). None of the performances were correlated with plasma balenine or CN1 enzyme activity. In addition, no effect on pH, bicarbonate, and lactate was observed. Also, the supplement did not affect mental performance. In contrast, glucose remained higher during and after the 20km time trial following balenine ingestion. In conclusion, these results overall indicate that the functionality of balenine does not fully resemble that of carnosine and anserine, since it was unable to elicit performance improvements with similar and even higher plasma concentrations.

Transcriptional and open chromatin analysis of bovine skeletal muscle development by single-cell sequencing.

Skeletal muscle is a complex heterogeneous tissue and characterizing its cellular heterogeneity and transcriptional and epigenetic signatures are important for understanding the details of its ontogeny. In our study, we applied scRNA-seq and scATAC-seq to investigate the cell types, molecular features, transcriptional and epigenetic regulation, and patterns of developing bovine skeletal muscle from gestational, lactational and adult stages. Detailed molecular analyses were used to dissect cellular heterogeneity, and we deduced the differentiation trajectory of myogenic cells and uncovered their dynamic gene expression profiles. SCENIC analysis was performed to demonstrate key regulons during cell fate decisions. We explored the future expression states of these heterogeneous cells by RNA velocity analysis and found extensive networks of intercellular communication using the toolkit CellChat. Moreover, the transcriptomic and chromatin accessibility modalities were confirmed to be highly concordant, and integrative analysis of chromatin accessibility and gene expression revealed key transcriptional regulators acting during myogenesis. In bovine skeletal muscle, by scRNA-seq and scATAC-seq analysis, different cell types such as adipocytes, endothelial cells, fibroblasts, lymphocytes, monocytes, pericyte cells and eight skeletal myogenic subpopulations were identified at the three developmental stages. The pseudotime trajectory exhibited a distinct sequential ordering for these myogenic subpopulations and eight distinct gene clusters were observed according to their expression pattern. Moreover, specifically expressed TFs (such as MSC, MYF5, MYOD1, FOXP3, ESRRA, BACH1, SIX2 and ATF4) associated with muscle development were predicted, and likely future transcriptional states of individual cells and the developmental dynamics of differentiation among neighbouring cells were predicted. CellChat analysis on the scRNA-seq data set then classified many ligand-receptor pairs among these cell clusters, which were further categorized into significant signalling pathways, including BMP, IGF, WNT, MSTN, ANGPTL, TGFB, TNF, VEGF and FGF. Finally, scRNA-seq and scATAC-seq results were successfully integrated to reveal a series of specifically expressed TFs that are likely to be candidates for the promotion of cell fate transition during bovine skeletal muscle development. Overall, our results outline a single-cell dynamic chromatin/transcriptional landscape for normal bovine skeletal muscle development; these provide an important resource for understanding the structure and function of mammalian skeletal muscle, which will promote research into its biology.

The influence of temperature on contractile properties of motor units in rat medial gastrocnemius

The effects of hypothermia and hyperthermia on mammalian skeletal muscle function have previously been reported. However, their effects on the contractile properties of different motor unit (MU) types were not described. This study aimed to explore the effect of temperature on contractile properties of MUs in rat medial gastrocnemius kept at 25°C (hypothermia), 37°C (normothermia), and 41°C (hyperthermia). Hypothermia prolonged the twitch time parameters of all MU types, shifting the steep part of the force-frequency curve towards lower frequencies and increasing its steepness. In addition, it reduced the rate of force development but not the twitch and tetanus forces of slow-twitch (S) MUs. Moreover, it reduced the tetanic force of fast-twitch fatigable (FF) MUs and increased the twitch force of fast-twitch fatigue-resistant (FR) MUs. In contrast, hyperthermia had opposite effects on twitch time properties and the force-frequency relationship. The twitch-to-tetanus ratio decreased for FF and FR MUs, and the steep part of the force-frequency curve shifted towards higher frequencies and decreased in steepness. Our findings indicate that FF MUs are the most sensitive and S MUs are the least sensitive to temperature. Furthermore, force control processes involving changes in motoneuronal firing frequency were radically modified for fast MUs, especially FF MUs.

Muscle length alters activation kinetics in fast-twitch edl muscle and relaxation kinetics in slow-twitch soleus muscle.

Mammalian skeletal muscles typically comprise mixtures of slow and fast-twitch fiber types. By studying muscles that homogenously express fast-twitch (EDL) and slow-twitch soleus (SOL) fibers, we can compare the activation and relaxation kinetics in fast and slow muscle fibers in intact and permeabilized muscle preparations at different sarcomere lengths (SL). Intact EDL muscle at optimal length (L0) had significantly longer times (p<0.05) to 50% (½ time) activation at 38.02 ± 2.26 ms (t1/2ACT), compared to 95% L0 and 90% L0 (33.65±1.86 and 31.31±1.61 respectfully). There were, however, no significant differences in activation ½ times of intact SOL muscle as muscle length increases. The relaxation ½ times are indistinguishable for intact EDL muscle at different muscle lengths, while the relaxation ½ times for intact SOL muscle increased as the muscle lengthen increased. To deconvolute the contribution of calcium transients and intrinsic myofilament kinetics to the length dependent activation and relaxation kinetics seen in intact muscle, we next investigate the kinetics of skinned myofibrils at different sarcomere lengths. Rate constant values for initial myofibril activation for EDL preparations showed significant (p<0.05) length dependence, decreasing from 7.25±0.47 s−1 to 5.97±0.44 s−1, 4.99±0.477 s−1, and 3.98±0.41 s−1 at 2.2, 2.5, 2.75, and 2.90 µm respectively). In contrast, SOL myofibrils had minimal length dependence, with no significant changes in activation rate constants. Fast relaxation rates for soleus muscle increased from 11.51±0.91 s−1 to 13.26±0.86 s−1, 15.13±0.99 s−1, and 16.34±1.02 s−1 (at 2.2, 2.5, 2.75, and 2.90 μm respectively) while EDL preparations showed no change. The results suggest that activation kinetics in EDL muscle and relaxation kinetics in SOL are length dependent and are intrinsic properties of the myofilaments.

The m6A reader YTHDC1 regulates muscle stem cell proliferation via PI4K-Akt-mTOR signalling.

Muscle stem cells are required for the homeostasis and regeneration of mammalian skeletal muscles. It has been reported that RNA N6-methyladenosine (m6A) modifications play a pivotal role in muscle development and regeneration. Nevertheless, we know little about which m6A reader regulates mammalian muscle stem cells. Here, we discovered that the m6A reader Ythdc1 is indispensable for mouse skeletal muscle regeneration and proliferation of muscle stem cells. In the absence of Ythdc1, Muscle stem cells in adult mice are unable to exit from quiescence. Mechanistically, Ythdc1 binds to m6A-modified Pi4k2a and Pi4kb mRNAs to regulate their alternative splicing and thus PI4K-Akt-mTOR signalling. Ythdc1-null muscle stem cells show a deficiency in phosphatidylinositol (PI) 3,4,5-trisphosphate, phospho-Akt and phospho-S6, which correlates with a failure in exit from quiescence. Our findings connect dynamic RNA methylation to the regulation of PI4K-Akt-mTOR signalling during stem cell proliferation and adult tissue regeneration.

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

Targeting the DP2 receptor alleviates muscle atrophy and diet-induced obesity in mice through oxidative myofiber transition.

Mammalian skeletal muscles consist of two main fibre types: slow-twitch (type I, oxidative) and fast-twitch (type IIa, fast oxidative; type IIb/IIx, fast glycolytic). Muscle fibre composition switch is closely associated with chronic diseases such as muscle atrophy, obesity, type II diabetes and athletic performance. Prostaglandin D2 (PGD2 ) is a bioactive lipid derived from arachidonic acid that aggravates muscle damage and wasting during muscle atrophy. This study aimed to investigate the precise mechanisms underlying PGD2 -mediated muscle homeostasis and myogenesis. Skeletal muscle-specific PGD2 receptor DP2-deficient mice (DP2fl/fl HSACre ) and their littermate controls (DP2fl/fl ) were subjected to exhaustive exercise and fed a high-fat diet (HFD). X-linked muscular dystrophy (MDX) mice and HFD-challenged mice were treated with the selective DP2 inhibitor CAY10471. Exercise tolerance, body weight, glycometabolism and skeletal muscle fibre composition were measured to determine the role of the skeletal muscle PGD2 /DP2 signalling axis in obesity and muscle disorders. Multiple genetic and pharmacological approaches were also used to investigate the intracellular signalling cascades underlying the PGD2 /DP2-mediated skeletal muscle fibre transition. PGD2 generation and DP2 expression were significantly upregulated in the hindlimb muscles of HFD-fed mice (P<0.05 or P<0.01 vs. normal chow diet). Compared with DP2fl/fl mice, DP2fl/fl HSACre mice exhibited remarkable glycolytic-to-oxidative fibre-type transition in hindlimb muscles and were fatigue resistant during endurance exercise (154.9±6.0 vs. 124.2±8.1min, P<0.05). DP2fl/fl HSACre mice fed an HFD showed less weight gain (P<0.05) and hepatic lipid accumulation (P<0.01), reduced insulin resistance and enhanced energy expenditure (P<0.05) compared with DP2fl/fl mice. Mechanistically, DP2 deletion promoted the nuclear translocation of nuclear factor of activated T cells 1 (NFATc1) by suppressing RhoA/Rho-associated kinase 2 (ROCK2) signalling, which led to enhanced oxidative fibre-specific gene transcription in muscle cells. Treatment with CAY10471 enhanced NFATc1 activity in the skeletal muscles and ameliorated HFD-induced obesity (P<0.05 vs. saline) and insulin resistance in mice. CAY10471 also enhanced exercise tolerance in MDX mice (100.8±8.0 vs. 68.9±11.1min, P<0.05 vs. saline) by increasing the oxidative fibre-type ratio in the muscles (45.1±2.3% vs. 32.3±2.6%, P<0.05 vs. saline). DP2 activation suppresses oxidative fibre transition via RhoA/ROCK2/NFATc1 signalling. The inhibition of DP2 may be a potential therapeutic approach against obesity and muscle disorders.

Disrupted T-tubular network accounts for asynchronous calcium release in MTM1-deficient skeletal muscle.

In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage-sensor dihydropyridine receptors. Deficiency in 3-phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T-tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1-deficient mice were compared with the results from a model where propagation of the depolarization along the T-tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T-tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium-induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+ -triggered opening of RyRs provides an alleviating support to the diseased process. KEY POINTS: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1. Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease-associated muscle function failure. Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets. The mathematical modelling approach used in the present work proves that the disease-associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation-contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure.

Ultrastructural and kinetic evidence support that thick filaments slide through the Z-disc.

How myofilaments operate at short mammalian skeletal muscle lengths is unknown. A common assumption is that thick (myosin-containing) filaments get compressed at the Z-disc. We provide ultrastructural evidence of sarcomeres contracting down to 0.44 µm-approximately a quarter of thick filament resting length-in long-lasting contractions while apparently keeping a regular, parallel thick filament arrangement. Sarcomeres produced force at such extremely short lengths. Furthermore, sarcomeres adopted a bimodal length distribution with both modes below lengths where sarcomeres are expected to generate force in classic force-length measurements. Mammalian fibres did not restore resting length but remained short after deactivation, as previously reported for amphibian fibres, and showed increased forces during passive re-elongation. These findings are incompatible with viscoelastic thick filament compression but agree with predictions of a model incorporating thick filament sliding through the Z-disc. This more coherent picture of mechanical mammalian skeletal fibre functioning opens new perspectives on muscle physiology.