Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration

When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest^+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest^+/-; DMD-null mice, decreased muscle growth was observed in Mest^+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest^+/-; DMD-null mice compared to DMD-null mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

Heparan sulfate controls skeletal muscle differentiation and motor functions

BackgroundHeparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear.MethodsWe examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system.ResultsCRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl₂ injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells.SignificanceThese results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.     

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr^−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.     

Collagen XV, a novel factor in zebrafish notochord differentiation and muscle development

Muscle cells are surrounded by extracellular matrix, the components of which play an important role in signalling mechanisms involved in their development. In mice, loss of collagen XV, a component of basement membranes expressed primarily in skeletal muscles, results in a mild skeletal myopathy. We have determined the complete zebrafish collagen XV primary sequence and analysed its expression and function in embryogenesis. During the segmentation period, expression of the Col15a1 gene is mainly found in the notochord and its protein product is deposited exclusively in the peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1 causes defects in notochord differentiation and in fast and slow muscle formation as shown by persistence of axial mesodermal marker gene expression, disorganization of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome. In addition, the number of medial fast-twitch muscle fibers was substantially increased, suggesting that the signalling by notochord derived Hh proteins is enhanced by loss of collagen XV. Consistent with this, there is a concomitant expansion of patched-1 expression in the myotome of morphant embryos. Together, these results indicate that collagen XV is required for notochord differentiation and muscle development in the zebrafish embryo and that it interplays with Shh signalling.     

Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

Calpains are Ca²⁺-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6''s in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

GdX/UBL4A null mice exhibit mild kyphosis and scoliosis accompanied by dysregulation of osteoblastogenesis and chondrogenesis

GdX, also named ubiquitin‐like protein 4A, is a ubiquitin‐domain protein characterized by a ubiquitin‐like domain that regulates the movement of misfolded proteins from the endoplasmic reticulum membrane to proteasome. However, its function in skeletal biology remains unclear. Here, we report that GdX plays a crucial role in skeletal development as mice lacking GdX exhibit skeletal dysplasias, mild kyphosis, and scoliosis. During embryonic stage, GdX knockout mice display decreased bone mineral density and trabecular bone accompanied by delayed osteogenic formation. GdX knockout mice also have blended spine and small body size. At the molecular level, GdX knockout mice showed perturbed expression of osteogenesis‐related genes and cartilage developmental genes, indicative of altered differentiation of mesenchymal cell lineage. Collectively, our results uncovered GdX as a novel regulator in bone development and a potential candidate gene for skeletal dysplasias.     

Control of organization and function of muscle and tendon by thrombospondin-4

Thrombospondins (TSPs) are multifunctional proteins that are deposited in the extracellular matrix where they directly affect the function of vascular and other cell types. TSP-4, one of the 5 TSP family members, is expressed abundantly in tendon and muscle. We have examined the effect of TSP-4 deficiency on tendon collagen and skeletal muscle morphology and function.In Thbs4^−/− mice, tendon collagen fibrils are significantly larger than in wild-type mice, and there is no compensatory over-expression of TSP-3 and TSP-5, the two TSPs most highly homologous to TSP-4, in the deficient mice. TSP-4 is expressed in skeletal muscle, and higher levels of TSP-4 protein are associated with the microvasculature of red skeletal muscle with high oxidative metabolism. Lack of TSP-4 in medial soleus, red skeletal muscle with predominant oxidative metabolism, is associated with decreased levels of several specific glycosaminoglycan modifications, decreased expression of a TGFβ receptor beta-glycan, decreased activity of lipoprotein lipase, which associates with vascular cell surfaces by binding to glycosaminoglycans, and decreased uptake of VLDL. The soleus muscle is smaller and hind- and fore-limb grip strength is reduced in Thbs4^−/− mice compared to wild-type mice. These observations suggest that TSP-4 regulates the composition of the ECM at major sites of its deposition, tendon and muscle, and the absence of TSP-4 alters the organization, composition and physiological functions of these tissues.     

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.     

SMAD7, an antagonist of TGF-beta signaling,is a candidate of prenatal skeletal muscle development and weaning weight in pigs

SMAD7 promotes and enhances skeletal muscle differentiation by inhibiting transforming growth factor beta (TGF-β)/activin signaling and bone morphogenetic protein (BMP) pathways. However, its function, the mechanism regulating its translation, and its association with production meat traits remain unclear in pigs. In this study, we explored SMAD7 gene spatio-temporal and tissue distribution, conducted a single nucleotide polymorphism association analysis, and examined regulation of its expression during skeletal muscle development. We found that SMAD7 was positively related to TGF-β pathway genes and mainly expressed in prenatal developing muscle, and dual luciferase and western blot assays demonstrated that SMAD7 expression was regulated by miRNA-21 at the protein level via inhibition of mRNA translation. Finally, the association analysis showed that a single nucleotide mutation (Exon 4_28816;C/A) was significantly associated with the weaning weight of piglets among Yorkshire pigs. These data indicate that SMAD7 plays a potentially important role in mammalian prenatal skeletal muscle development and is a candidate gene for promoting greater weaning weight in pig breeding.