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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.)

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.)

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.)

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.)

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 fusion occurs during development, growth, and regeneration. To investigate how muscle fusion compares among different muscle cell types and developmental stages, we studied muscle cell fusion over time in wild-type, myomaker (mymk), and jam2a mutant zebrafish. Using live imaging, we show that embryonic myoblast elongation and fusion correlate tightly with slow muscle cell migration. In wild-type embryos, only fast muscle fibers are multinucleate, consistent with previous work showing that the cell fusion regulator gene mymk is specifically expressed throughout the embryonic fast muscle domain. However, by 3 weeks post-fertilization, slow muscle fibers also become multinucleate. At this late-larval stage, mymk is not expressed in muscle fibers, but is expressed in small cells near muscle fibers. Although previous work showed that both mymk and jam2a are required for embryonic fast muscle cell fusion, we observe that muscle force and function is almost normal in mymk and jam2a mutant embryos, despite the lack of fast muscle multinucleation. We show that genetic requirements change post-embryonically, with jam2a becoming much less important by late-larval stages and mymk now required for muscle fusion and growth in both fast and slow muscle cell types. Correspondingly, adult mymk mutants perform poorly in sprint and endurance tests compared to wild-type and jam2a mutants. We show that adult mymk mutant muscle contains small mononucleate myofibers with average myonuclear domain size equivalent to that in wild type adults. The mymk mutant fibers have decreased Laminin expression and increased numbers of Pax7-positive cells, suggesting that impaired fiber growth and active regeneration contribute to the muscle phenotype. Our findings identify several aspects of muscle fusion that change with time in slow and fast fibers as zebrafish develop beyond embryonic stages., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Skeletal muscle is the largest organ system in the mammalian body and plays a key role in locomotion of horses. Fast and slow muscle fibers have different abilities and functions to adapt to exercises. To investigate the RNA and miRNA expression profiles in the muscles with different muscle fiber compositions on Mongolian horses. We examined the muscle fiber type population and produced deep RNA sequencing for different parts of skeletal muscles. And chose two of them with the highest difference in fast and slow muscle fiber population (splenius and gluteus medius) for comparing the gene expression profile of slow and fast muscle fiber types. We identified a total of 275 differentially expressed genes (DEGs), and 11 differentially expressed miRNAs (DEmiRs). In addition, target gene prediction and alternative splicing analysis were also performed. Significant correlations were found between the differentially expressed gene, miRNAs, and alternative splicing events. The result indicated that differentially expressed muscle-specific genes and target genes of miRNAs might co-regulating the performance of slow and fast muscle fiber types in Mongolian horses., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim This study sought to investigate the effects of two different maternal high-fat diets, during gestation and lactation, on the morphology of the skeletal muscle of the adult offspring rats., Methods: Female Wistar rats were fed Control (C) or High-fat/high-caloric (HH) or High-fat/isocaloric (HI) diet during gestation and lactation. The somatic growth of the offspring was measured throughout lactation. Glucose and insulin tolerance tests were performed at PND61 and PND65, respectively. At PND70, soleus and extensor digitorum longus (EDL) muscles were removed for myofibrillar ATPase staining analysis., Key Findings: HH pups were heavier and longer at weaning but presented same body weight at PND70. No difference among groups in glucose or insulin tolerance tests was observed. In the soleus muscle, HH offspring showed increased proportion and size of type 1 fibres and reduced proportion and number with increased size of type 2A fibres. In EDL muscle, there was no difference in proportion and number of fibres. HH and HI animals presented reduced type 1 and 2A fibres size while HH animals presented increased type 2B fibres size, in EDL muscle., Significance: Maternal HH diet promoted a more oxidative profile in soleus muscle. Though, maternal high-fat/isocaloric diet influenced only fibres size. Glycolytic muscle is more resistant to maternal diet influence. These results emphasize the importance of maternal diet during the critical period of development on muscle morphology of the offspring., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Emerging evidence indicates that skeletal muscle possesses endocrine function to secret myokines. Interleukin 6 (IL-6) is a well-characterized myokine that is involved in regulation of metabolism and muscle function. Metabolism type and contractile dynamics vary in different muscle types. It is not clear, however, if IL-6 secretion differs in different muscle types. In this study, we first established an ex vivo approach to test the inducible muscle secretion. Freshly isolated muscles were incubated in Krebs solution at 37 °C with oxygen supply. Secreted IL-6 in the incubation media was measure using Western blot and ELISA assay. We first confirmed that the IL-6 release was inducible by treating the incubated muscle with a cytokine stimulant. We demonstrated that physiological temperature (37 °C) and O 2 supply were essential for the induction of IL-6 release from the incubated muscle, suggesting it is a controlled secretion rather than a spontaneous leak. Using this approach, we found that IL-6 release was only inducible from soleus muscle but not EDL muscle. We further showed that IL-6 protein level was higher in slow oxidative muscle fibers. Moreover, we showed that EDL, although lacks of IL-6 release, surely has inducible secretory function that had different secretory pattern from soleus., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Existing evidence emphasize the role of mitochondrial dysfunction in sarcopenia which is revealed as loss of skeletal muscle mass and neuromuscular junction remodeling. We assessed the effect of low-intensity aerobic training along with blood flow restriction on muscle hypertrophy index, muscle-specific kinase (MuSK), a pivotal protein of the neuromuscular junction and Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) in aged male rats., Main Methods: Animals groups were control (CTL), sham (Sh), leg blood flow restriction (BFR), exercise (Ex), sham + exercise (Sh + Ex), and BFR plus exercise (BFR + Ex) groups. The exercise groups were trained with low intensity exercise for 10 weeks. 48 h after the last training session, animals were sacrificed under anesthesia. Soleus and EDL muscles were isolated, hypertrophy index was estimated and MuSK and PGC-1α were measured by western blot method., Key Findings: Hypertrophy index enhanced in soleus and Extensor digitorum longus (EDL) muscles of BFR + Ex group (P < 0.01 versus CTL and Sh groups, and P < 0.001 versus other groups). The MuSK protein of soleus and EDL muscles increased in BFR + Ex group (P < 0.01 and P < 0.001, respectively) in comparison with CTL and Sh groups. In BFR + Ex group, the PGC-1α protein increased in both soleus and EDL (P < 0.001 compared to other groups). Also the PGC-1α of soleus muscle was higher in Ex and Sh + Ex groups versus CTL and Sh groups (P < 0.05)., Significance: Findings suggest that low endurance exercise plus BFR improves the MuSK and hypertrophy index of both slow and fast muscles of elderly rats probably through the rise of PGC-1α expression., (Copyright © 2018. Published by Elsevier Inc.)

Fast twitch muscle and slow twitch muscle are two important organs of Takifugu rubripes. Both tissues are of ectodermic origin, and the differences between the two muscle fibers reflect the differences in their myofibril protein composition and molecular structure. In order to identify and characterize the gene expression profile in the two muscle fibers of T. rubripes, we generated 54 million and 44 million clean reads from the fast twitch muscle and slow twitch muscle, respectively, using RNA-Seq and identified a total of 580 fast-muscle-specific genes, 1533 slow-muscle-specific genes and 11,806 genes expressed by both muscles. Comparative transcriptome analysis of fast and slow twitch muscles allowed the identification of 1508 differentially expressed genes, of which 34 myosin and 30 ubiquitin family genes were determined. These differentially expressed genes (DEGs) were also analyzed by Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. In addition, alternative splicing analysis was also performed. The generation of larger-scale transcriptomic data presented in this work would enrich the genetic resources of Takifugu rubripes, which could be valuable to comparative studies of muscles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Obesity is often accompanied by decreases in the proportion of skeletal muscle slow-twitch fibers and insulin sensitivity. Increased plasma non-esterified fatty acids (NEFA) levels are responsible for obesity-associated insulin resistance. Palmitate, one of the most elevated plasma NEFA in obesity, has been recognized as the principle inducer of insulin resistance. The present study showed that increased plasma NEFA levels were negatively linked to slow-twitch fiber proportion and insulin sensitivity, while slow-twitch fiber proportion was positively correlated to insulin sensitivity in high fat diet (HFD)-fed and ob/ob mice. Dihydromyricetin (DHM) intervention increased slow-twitch fiber proportion and improved insulin resistance. In cultured C2C12 myotubes, palmitate treatment resulted in decrease of slow-twitch fiber specific Myh7 expression and insulin resistance, concomitant with folliculin (FLCN) and folliculin-interacting protein 1 (FNIP1) expression increase, AMP-activated protein kinase (AMPK) inactivation and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression decrease. Those palmitate-induced effects could be blocked by knock-down of FLCN expression or DHM intervention. Meanwhile, the protective effects of DHM were alleviated by over-expression of FLCN. In addition, the changes in AMPK activity and expression of FLCN and FNIP1 in vivo were consistent with those occurring in vitro. These findings suggest that DHM treatment prevents palmitate-induced slow-twitch fibers decrease partially via FLCN-FNIP1-AMPK pathway thereby improving insulin resistance in obesity., (Copyright © 2017. Published by Elsevier B.V.)

We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca 2+ 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 tpcn2 dhkz1a 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 IP 3 /BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP 3 R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca 2+ 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 Ca 2+ release via TPC2 might trigger the generation of more global Ca 2+ release from the SR via Ca 2+ -induced Ca 2+ release., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During skeletal muscle development, myocytes aggregate and fuse to form multinucleated muscle fibers. Inhibition of myocyte fusion is thought to significantly derail the differentiation of functional muscle fibers. Despite the purported importance of fusion in myogenesis, in vivo studies of this process in vertebrates are rather limited. Myomaker, a multipass transmembrane protein, has been shown to be the first muscle-specific fusion protein essential for myocyte fusion in the mouse. We have generated loss-of-function alleles in zebrafish myomaker, and found that fusion of myocytes into syncytial fast-twitch muscles was significantly compromised. However, mutant myocytes could be recruited to fuse with wild-type myocytes in chimeric embryos, albeit rather inefficiently. Conversely, overexpression of Myomaker was sufficient to induce hyperfusion among fast-twitch myocytes, and it also induced fusion among slow-twitch myocytes that are normally fusion-incompetent. In line with this, Myomaker overexpression also triggered fusion in another myocyte fusion mutant compromised in the function of the junctional cell adhesion molecule, Jam2a. We also provide evidence that Rac, a regulator of actin cytoskeleton, requires Myomaker activity to induce fusion, and that an approximately 3kb of myomaker promoter sequence, with multiple E-box motifs, is sufficient to direct expression within the fast-twitch muscle lineage. Taken together, our findings underscore a conserved role for Myomaker in vertebrate myocyte fusion. Strikingly, and in contrast to the mouse, homozygous myomaker mutants are viable and do not exhibit discernible locomotory defects. Thus, in the zebrafish, myocyte fusion is not an absolute requirement for skeletal muscle morphogenesis and function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: Acute heat stress negatively impacts both human health and livestock production. In order to characterize the skeletal muscle cellular response to acute heat stress, the muscle sarcoplasmic proteome was analyzed via 2-D DIGE. Pigs (n=8 per treatment) were exposed to one of the three treatments for 12 h: heat stress (HS; 37 °C), thermal neutral (TN; 21°C), or TN while pair-fed (PFTN; 21 °C, feed limited based on HS group consumption). After euthanasia, the semitendinosus muscle was excised, separated into predominately red (RST) and white (WST) fiber type portions, and sarcoplasmic proteins were extracted. Spots determined in 2D-DIGE to be different due to HS were identified using ESI-MS or LC-MS/MS. Several proteins involved in glycolysis, glycogenesis, and glycogenolysis were increased or modified, indicating enhanced glycolytic capacity in response to HS. In the WST, HS decreased abundance of tubulins and soluble actin and increased phosphorylated cofilin 2 abundance, indicating a loss of microtubule structure and a likely increase in stable actin microfilaments. HS increased manganese superoxide dismutase abundance, but decreased peroxiredoxin 2 abundance, indicating an antioxidant response to HS. The proteomic response to HS suggests marked cellular changes in carbohydrate metabolism, structure, and antioxidant machinery in skeletal muscle., Significance: This paper examines the proteome response of skeletal muscle to acute (short duration, high intensity) heat stress (HS). Defining changes in the sarcoplasm proteome increases our understanding of the mechanisms of how muscle responds to HS. Moreover, demonstration of a fiber type differential response to HS illustrates the dynamic nature of muscle. The experimental design of the experiment allows for the differentiation between the true effects of HS and HS-induced hypophagia. Data such as these will provide the foundation for developing future mitigating solutions and preventative therapies to reduce the detrimental effects of acute heat stress on muscle function and metabolism., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5'-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr(172) phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-D-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species. The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Muscle wasting occurs in a variety of conditions, including both genetic diseases, such as muscular dystrophies, and acquired disorders, ranging from muscle disuse to cancer cachexia, from heart failure to aging sarcopenia. In most of these conditions, the loss of muscle tissue is not homogeneous, but involves specific muscle groups, for example Duchenne muscular dystrophy affects most body muscles but spares extraocular muscles, and other dystrophies affect selectively proximal or distal limb muscles. In addition, muscle atrophy can affect specific fiber types, involving predominantly slow type 1 or fast type 2 muscle fibers, and is frequently accompanied by a slow-to-fast or fast-to-slow fiber type shift. For example, muscle disuse, such as spinal cord injury, causes type 1 fiber atrophy with a slow-to-fast fiber type shift, whereas cancer cachexia leads to preferential atrophy of type 2 fibers with a fast-to-slow fiber type shift. The identification of the signaling pathways responsible for the differential response of muscles types and fiber types can lead to a better understanding of the pathogenesis of muscle wasting and to the design of therapeutic interventions appropriate for the specific disorders. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Vertebrate skeletal muscle is composed of distinct types of fibre that are functionally adapted through differences in their physiological and metabolic properties. An understanding of the molecular basis of fibre-type specification is of relevance to human health and fitness. The zebrafish provides an attractive model for investigating fibre type specification; not only are their rapidly developing embryos optically transparent, but in contrast to amniotes, the embryonic myotome shows a discrete temporal and spatial separation of fibre type ontogeny that simplifies its analysis. Here we review the current state of understanding of muscle fibre type specification and differentiation during embryonic development of the zebrafish, with a particular focus on the roles of the Prdm1a and Sox6 transcription factors, and consider the relevance of these findings to higher vertebrate muscle biology., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Erythropoietin activity, required for erythropoiesis, is not restricted to the erythroid lineage. In light of reports on the metabolic effects of erythropoietin, we examined the effect of erythropoietin signaling on skeletal muscle fiber type development. Skeletal muscles that are rich in slow twitch fibers are associated with increased mitochondrial oxidative activity and corresponding expression of related genes compared to muscle rich in fast twitch fibers. Although erythropoietin receptor is expressed on muscle progenitor/precursor cells and is down regulated in mature muscle fibers, we found that skeletal muscles from mice with high erythropoietin production in vivo exhibit an increase in the proportion of slow twitch myofibers and increased mitochondrial activity. In comparison, skeletal muscle from wild type mice and mice with erythropoietin activity restricted to erythroid tissue have fewer slow twitch myofibers and reduced mitochondrial activity. PGC-1α activates mitochondrial oxidative metabolism and converts the fast myofibers to slow myofibers when overexpressed in skeletal muscle and PGC-1α was elevated by 2-fold in mice with high erythropoietin. In vitro erythropoietin treatment of primary skeletal myoblasts increased mitochondrial biogenesis gene expression including PGC-1α by 2.6-fold, CytC by 2-fold, oxygen consumption rate by 2-fold, and citrate synthase activity by 58%. Erythropoietin also increases AMPK, which induces PGC-1α and stimulates slow oxidative fiber formation. These data suggest that erythropoietin contributes to skeletal muscle fiber programming and metabolism, and increases PGC-1α and AMPK activity during muscle development directly to affect the proportion of slow/fast twitch myofibers in mature skeletal muscle., (Published by Elsevier Ltd.)

The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a's synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In rat fast muscles, collagen Q (ColQ) expression is restricted to the neuromuscular junctions. In contrast, it is high also extrajunctionally in the slow soleus muscles. Fast muscles activated by chronic low-frequency electrical stimulation, similar to neural activation of the soleus muscles, did not increase their extrajunctional expression of ColQ. We assumed that the myogenic stem cells (satellite cells) in fast and slow muscles were intrinsically different in regard to the capacity that they convey to their respective muscle fibers to increase the extrajunctional ColQ expression upon innervation. ColQ mRNA levels were determined by quantitative real-time PCR. Extensive neural suppression of the extrajunctional ColQ expression in regenerating fast muscles during maturation is a very slow process requiring 30-60 days. If the immature regenerating fast EDL muscles were indirectly or directly electrically stimulated immediately after innervation by chronic low-frequency impulse pattern for 8 days, no significant increase of the extrajunctional ColQ mRNA levels was observed in stimulated regenerates in comparison to non-stimulated ones. In contrast, the extrajunctional ColQ mRNA levels in the regenerates of the soleus muscles, trans-innervated by the EDL nerve at the time of muscle injury, increased 4- to 5-fold after 8 days of the same chronic low-frequency electrical stimulation in comparison to those in the stimulated EDL regenerates. Since both fast and slow muscles completely regenerated only from their own myogenic stem cells and were innervated by the same nerve and later activated by the same tonic pattern of impulses, these results demonstrated that the mechanism causing incapacity of regenerating fast muscles to increase their extrajunctional ColQ expression upon tonic activation is encoded in their satellite cells, which in this respect differ from those in the slow muscles., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In the present study, we tested the hypothesis that niacin increases the oxidative capacity of muscle by increasing the oxidative type I muscle fiber content. Twenty-four obese Zucker rats were assigned to 2 groups of 12 rats that were fed either a control diet (O group) or a diet supplemented with 750 mg/kg diet niacin (O+N group) for 4 wk. In addition, one group of lean rats (L group) was included in the experiment and fed the control diet for 4 wk. Plasma and liver concentrations of TG were markedly greater in obese groups than in the L group but markedly lower in the O+N group than in the O group (P < 0.05). Rats of the O+N group had a higher percentage of oxidative type I fibers and higher mRNA levels of genes encoding regulators of muscle fiber composition (Ppard, Ppargc1a, Ppargc1b), angiogenic factors (Vegfa, Vegfb), and genes involved in fatty acid utilization (Cpt1b, Slc25a20, Slc22a4, Slc22a5, Slc27a1) and oxidative phosphorylation (Cox4i1, Cox6a2) and a higher activity of the mitochondrial oxidative enzyme succinate dehydrogenase in muscle than rats of the O and L groups (P < 0.05). These niacin-induced changes in muscle metabolic phenotype are indicative of an increased capacity of muscle for oxidative utilization of fatty acids and are likely mediated by the upregulation of Ppard, Ppargc1a, and Ppargc1b, which are key regulators of muscle fiber composition, mitochondrial biogenesis, angiogenesis, and genes involved in fatty acid catabolism and oxidative phosphorylation. The increased utilization of fatty acids by muscle might contribute to the strong TG-lowering effect of niacin treatment.

Fibre type percentage and changes in textural parameters, sensory properties as well as mean fibre cross sectional area (CSA), fibre shape, endomysium and perimysium thickness of wild boar and deer longissimus (L) muscle subjected to ageing with kefir, dry red wine, lemon and pineapple juice marinades for 4 days were studied. Among the non-marinated and non-aged samples of muscles it was found that wild boar meat with its higher percentage of red fibres, higher CSA, thicker connective tissue as compared with deer meat, was harder, more springy and stringy. Muscles ageing, regardless of methods, resulted in a decrease in both the CSA and thickness of the connective tissue, and improve in fibre shape. As a consequence ageing caused a reduction in hardness, cohesiveness, springiness, and stringiness as well as in augmentation of tenderness, juiciness and general attractiveness of the muscles studied. As demonstrated by obtained data, regardless of ageing methods, deer L muscle contained more white fibres compared to wild boar muscle, were more susceptible to tenderization. The highest structural and textural changes, but the worst general attractiveness was found in muscles marinated with pineapple juice addition. Insignificantly lower changes in both quality traits were found in muscles aged with kefir marinade which at the same time were characterized by the high tenderness, the highest juiciness and general attractiveness., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Skeletal muscles in the limb and body trunk are composed of heterogeneous myofibers expressing different isoforms of myosin heavy chain (Myh), including type I (slow, Myh7), IIA (intermediate, Myh2), IIX (fast, Myh1), and IIB (very fast, Myh4). While the contraction force and speed of a muscle are known to be determined by the relative abundance of myofibers expressing each Myh isoform, it is unclear how specific combinations of myofiber types are formed and regulated at the cellular and molecular level. We report here that myostatin (Mstn) positively regulates slow but negatively regulates fast Myh isoforms. Mstn was expressed at higher levels in the fast muscle myoblasts and myofibers than in the slow muscle counterparts. Interestingly, Mstn knockout led to a shift of Myh towards faster isoforms, suggesting an inhibitory role of Mstn in fast Myh expression. Consistently, when induced to differentiate, Mstn null myoblasts formed myotubes preferentially expressing fast Myh. Conversely, treatment of myoblasts with a recombinant Mstn protein upregulated Myh7 but downregulated Myh4 gene expression in newly formed myotubes. Importantly, both Mstn antibody and soluble activin type 2B receptor inhibited slow Myh7 and promoted fast Myh4 expression, indicating that myostatin acts through canonical activin receptor to regulate the expression of Myh genes. These results demonstrate a role of myostatin in the specification of myofiber types during myogenic differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The purpose of this study was to compare parameters associated with pork quality, muscle fiber, and eating quality among various breeds, and to examine if differences in eating quality were associated to pork quality and muscle fiber characteristics. For carcass and pork quality, although there were significant differences among breeds, the values of parameters in all pigs were assigned a normal quality class, a likely outcome of the similarity in the area percentage of type I and IIB fibers. For eating quality, pork loins from Berkshire pigs were more tender and full of pork flavor than Landrace and Yorkshire pigs. Except juiciness and mouth coating, over 20% of the variability in the eating quality parameters can be explained by pork quality traits and muscle fiber characteristics using multiple regression analysis. Furthermore, differences in muscle pH(24h), cooking loss, shear force, and NPPC marbling score could explain a large proportion of variation in eating quality parameters associated with the texture of pork., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aims: Glucocorticoids bind to the glucocorticoid receptor (GR) and increase catabolism of muscle proteins via the ubiquitin-proteasome pathway. Activation of β(2)-adrenergic receptor (β(2)-AR) in skeletal muscle has been shown to induce muscle hypertrophy by promoting muscle protein synthesis and/or attenuating protein degradation. The aim of this study was to investigate the correlation between disuse-induced muscle atrophy, and expression of GR and β(2)-AR., Methods: Rats were subjected to casted-immobilization (knee and foot arthrodesis), a model for muscle disuse, for 10 days. Fast-twitch (extensor digitorum longus: EDL) and slow-twitch (soleus: SOL) muscles were isolated and subsequently used for analysis. The expression of GR and β(2)-AR was analyzed by real-time RT-PCR and western blotting. In addition, we analyzed plasma catecholamine and corticosterone concentrations by ELISA., Key Findings: Casted-immobilization-induced muscle atrophy was much greater in the SOL muscle than in the EDL muscle. Casted-immobilization decreased the expression of GR mRNA and protein in the SOL muscle but not in the EDL muscle. Although the expression of β(2)-AR protein in the cytosol and membrane-rich fractions was not changed by casted-immobilization in either muscle, casted-immobilization decreased the expression of β(2)-AR mRNA in the SOL muscle. Plasma catecholamine and corticosterone concentrations, however, were largely unaffected by casted-immobilization during the experimental period., Significance: This study provides evidence that casted-immobilization-induced muscle disuse downregulates GR expression in slow-twitch muscle. These results suggest that muscle disuse suppresses glucocorticoid signals, such as muscle protein breakdown and transcription of the β(2)-AR gene, via downregulation of GR expression in slow-twitch muscle., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A novel approach was applied in this study to directly evaluate the effect of muscle fiber type on postmortem protein degradation. Porcine muscle fibers were isolated from longissimus muscle at day 1, 3, and 6 postmortem. Fibers were sorted by immunochemical myosin heavy chain isoform typing. Western blot analysis of fibers pooled separately into type I or IIb showed that the relative amounts of 39- and 50-kDa desmin degradation fragments at day 6, and 28- to 31-kDa fragments of troponin T fast type isoform (fTnT) at day 1 and 6 postmortem were higher in type IIb than in type I fibers. At day 6 troponin T slow type isoform (sTnT) was less degraded than fTnT in type I fibers. These results indicated greater rate and extent of proteolysis in type IIb than in type I fibers and higher susceptibility of fTnT to proteolysis than that of sTnT isoform., (Copyright © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

To investigate the relationships between muscle fiber characteristics and meat quality traits of Korean native cattle, Hanwoo, Longissimus dorsi (LD), Psoas major (PM) and Semimembranosus (SM) muscles obtained from 18 Hanwoo steers and the muscle fiber characteristics were measured by histochemical analysis. Fiber number, area percentages and density of type IIA and IIB were lower in SM muscle, but higher in PM muscle than other muscles. LD muscle had higher pH(24h), L* value and fat content whereas SM muscle had lower L* value and fat content. The lowest WBSF with longer sarcomere length was observed in PM muscle, while SM muscle showed the highest WBSF with shorter sarcomere length. Consequently, the percentage of type I and IIB were highly correlated with meat quality traits and inversely correlated with fat content, L* value and WBSF. Fiber number and area percentage of type I had a positive correlation with fat content and L* value and a negative correlation with WBSF. These results suggest that Hanwoo steers had high marbling, more lightness and tenderness when the percentage of type I was high and the percentage of type IIB was low in muscle., (Copyright (c) 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

There is a major difference between fast and slow rat muscles in regard to acetylcholinesterase (AChE) expression in their extrajunctional regions: the activity of the asymmetric forms of AChE (A(8) and A(12)) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the nearly complete suppression of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. This difference is partly caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the levels of mRNA of ColQ. Whereas the changes of the levels of ColQ mRNA in slow muscles, observed in response to different electrical stimulation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible in this respect. Calcineurin signaling pathway in slow muscle fibers, activated by high average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, maintains high mRNA levels of ColQ in the extrajunctional regions of the slow soleus muscles. A different, calcineurin-independent regulatory pathway is responsible for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers. Immature rat muscle fibers, both fast and slow, however, display relatively high levels of the A forms of AChE and ColQ mRNA during the early postnatal period. Four days after birth, ColQ mRNA levels are already 2-fold higher in slow than in fast muscle fibers. Muscle regeneration after injury is a repetition of its ontogenetic development, originating from the muscle satellite cells. The extrajunctional levels of ColQ mRNA in non-innervated regenerating fast and slow muscles, however, are not significantly different, but they become about 2- to 3-fold higher in the regenerating soleus than in the fast STM already after several days of innervation by their respective nerves. We are currently testing a hypothesis that intrinsic differences exist between fast and slow muscle fibers in regard to their capacity to express ColQ extrajunctionally, and that these differences may originate in the stem cells of these muscle fibers., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2(-/-)-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2(-/-)-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2(-/-)-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2(-/-)-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2(-/-)-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2(-/-)-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2(-/-)-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2(-/-)-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2(-/-)-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2(-/-)-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling., (Copyright © 2010 International Society of Matrix Biology. All rights reserved.)

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Menopause, the age-related loss of ovarian hormone production, promotes increased adiposity and associated metabolic pathology, but molecular mechanisms remain unclear. We previously reported that estrogen increases skeletal muscle PPARdelta expression in vivo, and transgenic mice overexpressing muscle-specific PPARdelta are reportedly protected from diet-induced obesity. We thus hypothesized that obesity observed in ovariectomized mice, a model of menopause, may result in part from abrogated expression of muscle PPARdelta and/or downstream mediators such as FoxO1. To test this hypothesis, we ovariectomized (OVX) or sham-ovariectomized (SHM) 10-week old female C57Bl/6J mice, and subsequently harvested quadriceps muscles 12weeks later for gene expression studies. Compared to SHM, muscle from OVX mice displayed significantly decreased expression of PPARdelta (3.4-fold), FoxO1 (4.5-fold), PDK-4 (2.3-fold), and UCP-2 (1.8-fold). Consistent with studies indicating PPARdelta and FoxO1 regulate muscle fiber type, we observed dramatic OVX-specific decreases in slow isoforms of the contractile proteins myosin light chain (11.1-fold) and troponin C (11.8-fold). In addition, muscles from OVX mice expressed 57% less myogenin (drives type I fiber formation), 2-fold more MyoD (drives type II fiber formation), and 1.6-fold less musclin (produced exclusively by type II fibers) than SHM, collectively suggesting a shift towards less type I oxidative fibers. Finally, and consistent with changes in PPARdelta and FoxO1 activity, we observed decreased expression of atrogin-1 (2.3-fold) and MuRF-1 (1.9-fold) in OVX mice. In conclusion, muscles from ovariectomized mice display decreased PPARdelta and FoxO1 expression, abrogated expression of downstream targets involved in lipid and protein metabolism, and gene expression profiles indicating less type I oxidative fibers., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Although tight quantitative control of gene expression is required to ensure that organs and tissues function correctly, the transcriptional mechanisms underlying this process still remain poorly understood. Here, we describe novel and evolutionary conserved secondary enhancers that are needed for the regulation of the expression of Troponin I genes. Secondary enhancers are silent when tested individually in electroporated muscles but interact with the primary enhancers and are required to precisely control the appropriate timing, the tissue and fibre specificity, and the quantitative expression of these genes during muscle differentiation. Synergism is completely dependent of the fully conserved MEF2 site present on the primary enhancers core of skeletal muscle Troponin I genes. Thus, while each of these paired enhancers has a different function, the concerted action of both is crucial to recapitulate endogenous gene expression. Through comparative genomics, we predict that this mechanism has also arisen in other mammalian muscle genes. Our results reveal the existence of a novel mechanism, conserved from flies to mammals, to fine-tune gene expression in each muscle and probably other tissues.

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using alpha-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.

Skeletal muscles of vertebrates are typically composed of slow- and fast-twitch fibers that differ in their morphology, gene expression profiles, contraction speeds, metabolic properties and patterns of innervation. During myogenesis, how muscle precursors are induced to mature into distinct slow- or fast-twitch fiber-types is inadequately understood. We have previously shown that within the somites of the zebrafish embryo, the activity of the zinc finger and SET domain-containing transcriptional regulator Blimp1 is essential for the specification of slow muscle fibers. Here, we have investigated the mechanism by which Blimp1 programs myoblasts to adopt the slow-twitch fiber fate. In slow myoblasts, expression of the Blimp1 protein is transient, and precedes the expression of slow muscle-specific differentiation genes. We demonstrate that the competence of somitic myoblasts to commit to the slow lineage in response to Blimp1 changes as a function of developmental time. Furthermore, we provide evidence that mammalian Blimp1 can recapitulate the slow myogenic program in zebrafish, suggesting that zebrafish Blimp1 can recognize the same consensus DNA sequence that is bound by the mammalian protein. Finally, we show that zebrafish Blimp1 can repress the expression of fast muscle-specific myosin light chain, mylz2, through direct binding near the promoter of this gene, indicating that an important function of the transcriptional activity of Blimp1 in slow muscle development is the suppression of fast muscle-specific gene expression. Taken together, these findings provide new insights into the molecular basis of vertebrate muscle fiber-type specification, and underscore Blimp1 as the central determinant of this process.

Human MusTRD1alpha1 was isolated as a result of its ability to bind a critical element within the Troponin I slow upstream enhancer (TnIslow USE) and was predicted to be a regulator of slow fiber-specific genes. To test this hypothesis in vivo, we generated transgenic mice expressing hMusTRD1alpha1 in skeletal muscle. Adult transgenic mice show a complete loss of slow fibers and a concomitant replacement by fast IIA fibers, resulting in postural muscle weakness. However, developmental analysis demonstrates that transgene expression has no impact on embryonic patterning of slow fibers but causes a gradual postnatal slow to fast fiber conversion. This conversion was underpinned by a demonstrable repression of many slow fiber-specific genes, whereas fast fiber-specific gene expression was either unchanged or enhanced. These data are consistent with our initial predictions for hMusTRD1alpha1 and suggest that slow fiber genes contain a specific common regulatory element that can be targeted by MusTRD proteins.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) and butyrylcholinesterase at vertebrate neuromuscular junctions, which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow- and fast-twitch muscles in mammals, respectively. Both ColQ transcripts are derived from a single COLQ gene. Transcripts encoding ColQ increased during myogenic differentiation of C2C12 cells; the increase was in parallel with AChE catalytic subunit. Quantitative PCR analysis indicated that the increase during the myotube formation was due to the up regulation of ColQ-1 transcript instead of ColQ-1a. In order to reveal the regulatory mechanism of ColQ transcripts, two distinct promoters, pColQ-1 and pColQ-1a, were isolated from human COLQ gene. The ColQ promoters showed a muscle fiber type-specific expression pattern, and which was in line with the expression of endogenous transcript. After in vivo DNA transfection, pColQ-1 showed strong activity in slow-twitch muscle (e.g. soleus), while pColQ-1a was preferably expressed in fast-twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts was regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These results explain the specific expression patterns of collagen-tailed AChE in slow and fast muscle fibers.

The expression pattern of the RyR3 isoform of Ca2+ release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.

Objective: To better understand the association between skeletal muscle and exercise intolerance in peripheral arterial disease (PAD), we assessed treadmill-walking performance and gastrocnemius muscle phenotype in healthy control subjects and in patients with PAD. We hypothesized that gastrocnemius muscle characteristics would be altered in PAD compared with control subjects and that exercise tolerance in patients PAD would be related to muscle phenotype., Methods: Sixteen patients with PAD and intermittent claudication and 13 healthy controls of the same age participated. Each subject completed a graded treadmill-walking test and underwent a resting muscle biopsy. Muscle biopsy samples were obtained from the medial gastrocnemius muscle of the most ischemic limb in PAD and a limb chosen at random in controls. Samples were analyzed for fiber type and cross-sectional area, capillary-to-fiber ratio, the number of capillaries in contact with each fiber type, and the optical density of glycogen within each fiber by using histochemical procedures. Total muscle glycogen content was determined biochemically., Results: Exercise capacity measured on the incremental walking test in the PAD group was only 30% to 40% of that observed in controls. The PAD group had a lower proportion of type I muscle fibers (P < .05), fewer capillaries per muscle fiber (P < .05), and tended to have smaller fiber areas (P = .08). The relative area of type I fibers, the capillary-to-fiber ratio, capillary contacts with type I and IIa fibers, and the optical density of glycogen in type I fibers were all positively correlated with exercise tolerance in the PAD group (P < .05) but not controls., Conclusions: These data suggest that muscle phenotype is altered in PAD and that such alterations are associated with the exercise intolerance in these patients. In light of these findings, therapies such as resistance training or electrical stimulation that target skeletal muscle in PAD may prove beneficial, and further investigation of such therapies is warranted.

Acute toxic effects of acetylcholinesterase (AChE) inhibitors on skeletal muscles are thought to involve oxidative stress with increased generation of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). Muscle hyperactivity with its increased oxygen and energy consumption appear to be the primary cause of oxidative stress. The present investigation was therefore undertaken to establish the normal levels of F(2)-isoprostanes (F(2)-IsoPs, specific markers of ROS/oxidative stress), citrulline (determinant of NO/NOS and marker of RNS), and high-energy phosphates (HEP: adenosine triphosphate, ATP and phosphocreatine, PCr) in slow (soleus) and fast (extensor digitorum longus, EDL) muscles of rats. In addition, we aimed to determine if memantine HCl (MEM), in combination with atropine sulfate (ATS), prevents carbofuran-induced changes in markers of oxidative stress. Control values were not significantly different for F(2)-IsoPs (1.142 +/- 0.027 and 1.177 +/- 0.092 ng/g) and citrulline (469.7 +/- 31.8 and 417.8 +/- 18.5 nmol/g) in soleus and EDL muscles, while the values were different for HEP (ATP, 3.66 +/- 0.11 and 5.85 +/- 0.14 micromol/g; PCr, 7.91 +/- 0.26 and 13.14 +/- 0.31 micromol/g). Rats acutely intoxicated with carbofuran (1.5 mg/kg, s.c.) showed the signs of maximal toxicity including muscle hyperactivity within 60 min of exposure. At this time, F(2)-IsoPs (177 and 153%) and citrulline (267 and 304%) levels were significantly increased, while ATP (46 and 43%) and PCr (44 and 46%) levels were decreased in soleus and EDL, respectively. Rats pretreated with MEM (18 mg/kg, s.c.) and ATS (16 mg/kg, s.c.), 60 and 15 min prior to carbofuran, respectively, showed no signs of toxicity. MEM in combination with ATS protected muscles from carbofuran-induced hyperactivity and attenuated increases in F(2)-IsoPs and citrulline, and depletion of HEP. Carbofuran-induced changes and protection by MEM and ATS were of similar magnitude in both muscles. These findings indicate that carbofuran-induced muscle hyperactivity produces oxidative stress as measured by increased ROS and RNS generation, and HEP depletion. MEM and ATS prevent the carbofuran-induced chain of events involved in oxidative stress.

Aim: The aim of the study was to investigate the impact of creatine feeding (5 g kg(-1) body weight day(-1)) upon the deleterious adaptations in skeletal muscle induced by immobilization., Methods: Male Wistar rats were submitted to hind limb immobilization together with three dietary manipulations: control, supplemented with creatine for 7 days (along with immobilization) and supplemented with creatine for 14 days (7 days before immobilization and together with immobilization). Muscle weight (wet/dry) was determined in the soleus (SOL) and gastrocnemius (GAS). The analysis of lean mass was performed by DEXA and myosin heavy chain (MHC) distribution by SDS-PAGE., Results: After 14 days of creatine loading, immobilized SOL and GAS total creatine content were increased by 25% and 18%, respectively. Regardless of dietary manipulation, the immobilization protocol induced a decrease in the weight of SOL and GAS (P < 0.001). However, creatine feeding for 14 days minimized mass loss in the SOL and GAS (P < 0.05). Our findings also indicate that creatine supplementation maximizes the expected slow-to-fast MHC shift driven by immobilization (P < 0.05)., Conclusions: Previous creatine supplementation attenuates muscle wasting induced by immobilization. This effect is associated with the increment of intramuscular creatine content.