Skeletal Muscle-specific Genes Research Articles

Expression of the TGF-β Target Gene Mss51 Is Induced in Muscle From Tumor-Bearing Mice and Attenuated by Treatment With the AMPK Activator AICAR

Cachexia is a severe complication of cancer characterized by skeletal muscle atrophy and loss of function and is associated with a poor prognosis. AMPK is a cellular energy sensor that is upregulated in cancer cachexia. AMPK activation is well-established in promoting catabolic cell signaling in skeletal muscle, partly through the induction of muscle-specific ubiquitin ligases MAFBx and MuRF-1 expression. Despite AMPK’s acute catabolic effects, chronic treatment with the AMPK-activating drug 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been reported to preserve muscle function and metabolism in various muscle-wasting conditions, including cancer cachexia. Elevated TGF-β during cancer cachexia is known to disrupt mitochondrial homeostasis and lead to mitochondrial dysfunction. Mss51 is a skeletal muscle-specific mitochondrial gene that is upregulated through TGF-β signaling. Although the role of Mss51 in skeletal muscle cancer cachexia has not been studied as of yet, its expression is associated with mitochondrial and metabolic impairment. Therefore, we hypothesized that Mss-51 expression would be elevated in cachexic skeletal muscle and attenuated by chronic AMPK activation. To test this, we inoculated mice (n=4) with either Lewis Lung Carcinoma (LLC) cells or PBS. Mice were injected daily with 350 mg/kg AICAR or an equivalent volume of saline beginning seven days after inoculation. Twenty-four days after inoculation, the mice were euthanized, and tissues were harvested and weighed. RNA was isolated from the gastrocnemius-plantaris-soleus complex and analyzed by RT-PCR for Mss51, MuRF-1, and MAFBx expression. Protein expression for hexokinase II (HKII) was measured by western blotting. AICAR treatment did not attenuate muscle atrophy in tumor-bearing mice. Compared to the control, Mss-51 was elevated in LLC mice treated with saline and decreased in LLC mice treated with AICAR, consistent with the known effects of AMPK in blocking TGF-β signaling. Similarly, MuRF-1 and MAFBx tended to be elevated in LLC-saline. Still, this increase was attenuated in LLC-AICAR mice, though we were likely underpowered to detect significance in this measure. In conclusion, based on previous work, the rise in Mss51 in tumor-bearing mice may contribute to mitochondrial and metabolic dysfunction in cancer cachexia. The prevention of this increase by AICAR suggests that AMPK may benefit dysfunctional metabolism in the cachexic muscle through inhibition of Mss51. Funding for these experiments was from a BYU Widtsoe Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

Baf155 regulates skeletal muscle metabolism via HIF-1a signaling.

During exercise, skeletal muscle is exposed to a low oxygen condition, hypoxia. Under hypoxia, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is stabilized and induces expressions of its target genes regulating glycolytic metabolism. Here, using a skeletal muscle-specific gene ablation mouse model, we show that Brg1/Brm-associated factor 155 (Baf155), a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, is essential for HIF-1α signaling in skeletal muscle. Muscle-specific ablation of Baf155 increases oxidative metabolism by reducing HIF-1α function, which accompanies the decreased lactate production during exercise. Furthermore, the augmented oxidation leads to high intramuscular adenosine triphosphate (ATP) level and results in the enhancement of endurance exercise capacity. Mechanistically, our chromatin immunoprecipitation (ChIP) analysis reveals that Baf155 modulates DNA-binding activity of HIF-1α to the promoters of its target genes. In addition, for this regulatory function, Baf155 requires a phospho-signal transducer and activator of transcription 3 (pSTAT3), which forms a coactivator complex with HIF-1α, to activate HIF-1α signaling. Our findings reveal the crucial role of Baf155 in energy metabolism of skeletal muscle and the interaction between Baf155 and hypoxia signaling.

Engineered Circular RNA CircmiR‐29b Attenuates Muscle Atrophy by Sponging MiR‐29b

AbstractMuscle atrophy is commonly caused by various diseases but still lacks effective treatment in clinical practice. Here, an artificial circular RNA (circRNA) named circmiR‐29b, which is designed to be a molecular sponge for miR‐29b containing 12 imperfect bulged miR‐29b binding sites is constructed. CircmiR‐29b shows a favorable functional effect with respect to miR‐29b repression in a specific and drastic manner. CircmiR‐29b can protect against in vitro muscle atrophy induced by dexamethasone (Dex), angiotensin II (Ang II), and tumor necrosis factor alpha (TNF‐α). Besides, circmiR‐29b attenuates in vivo muscle atrophy induced by denervation, immobilization, and Ang II. More importantly, skeletal muscle specific gene therapy using circmiR‐29b is able to attenuate established muscle atrophy induced by spiral wire immobilization. This work has developed an engineered circmiR‐29b acting as an miR‐29b sponge, and offers a promising therapeutic tool to prevent muscle atrophy.

Contribution of Rare Genetic Variation to Disease Susceptibility in a Large Scandinavian Myositis Cohort.

Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs. Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune-related genes in a Scandinavian case-control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant- and gene-level enrichment analyses, was implemented to explore genotype-phenotype relations. Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle-specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants. Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.

LncEDCH1 improves mitochondrial function to reduce muscle atrophy by interacting with SERCA2

Skeletal muscle is a regulator of the body's energy expenditure and metabolism. Abnormal regulation of skeletal muscle-specific genes leads to various muscle diseases. Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in muscle growth and muscle atrophy. To explore the potential function of muscle-associated lncRNA, we analyzed our previous RNA-sequencing data and selected the lncRNA (LncEDCH1) as the research object. In this study, we report that LncEDCH1 is specifically enriched in skeletal muscle, and its transcriptional activity is positively regulated by transcription factor SP1. LncEDCH1 regulates myoblast proliferation and differentiation in vitro. In vivo, LncEDCH1 reduces intramuscular fat deposition, activates slow-twitch muscle phenotype, and inhibits muscle atrophy. Mechanistically, LncEDCH1 binds to sarcoplasmic/ER calcium ATPase 2 (SERCA2) protein to enhance SERCA2 protein stability and increase SERCA2 activity. Meanwhile, LncEDCH1 improves mitochondrial efficiency possibly through a SERCA2-mediated activation of the AMPK pathway. Our findings provide a strategy for using LncEDCH1 as an effective regulator for the treatment of muscle atrophy and energy metabolism.

Ablation of USP21 in skeletal muscle promotes oxidative fibre phenotype, inhibiting obesity and type 2 diabetes

BackgroundSkeletal muscle as a metabolic consumer determines systemic energy homeostasis by regulating myofibre type conversion and muscle mass control. Perturbation of the skeletal muscle metabolism elevates the risk of a variety of diseases including metabolic disorders. However, the regulatory pathways and molecules are not completely understood. The discovery of relevant responsible molecules and the associated network could be an attractive strategy to overcome diseases associated with muscle problems.MethodsAn initial screening using quantitative trait locus analysis enabled us to extract a set of genes including ubiquitin‐specific proteases21 (USP21) (r = 0.738; P = 0.004) as potential targets associated with fasting blood glucose content. Given tight regulation of the ubiquitination status of proteins in muscle, we focused on USP21 and generated whole‐body (KO) and skeletal muscle‐specific USP21 knockout (MKO) mice. Transcriptomics, proteomics, and lipidomics assays in combination with various in vivo and in vitro experiments were performed to understand the functions of USP21 and underlying mechanisms. A high‐fat diet (60%)‐fed mouse model and diabetic patient‐derived samples were utilized to assess the effects of USP21 on energy metabolism in skeletal muscle.ResultsUSP21 was highly expressed in both human and mouse skeletal muscle, and controlled skeletal muscle oxidative capacity and fuel consumption. USP21‐KO or USP21‐MKO significantly promoted oxidative fibre type changes (Δ36.6% or Δ47.2%), muscle mass increase (Δ13.8% to Δ22.8%), and energy expenditure through mitochondrial biogenesis, fatty acid oxidation, and UCP2/3 induction (P < 0.05 or P < 0.01). Consistently, cold exposure repressed USP21 expression in mouse skeletal muscle (Δ55.3%), whereas loss of USP21 increased thermogenesis (+1.37°C or +0.84°C; P < 0.01). Mechanistically, USP21 deubiquitinated DNA‐PKcs and ACLY, which led to AMPK inhibition. Consequently, USP21 ablation diminished diet‐induced obesity (WT vs. USP21‐KO, Δ8.02 g, 17.1%, P < 0.01; litter vs. USP21‐MKO, Δ3.48 g, 7.7%, P < 0.05) and insulin resistance. These findings were corroborated in a skeletal muscle‐specific gene KO mouse model. USP21 was induced in skeletal muscle of a diabetic patient (1.94‐fold), which was reciprocally changed to p‐AMPK (0.30‐fold).ConclusionsThe outcomes of this research provide novel information as to how USP21 in skeletal muscle contributes to systemic energy homeostasis, demonstrating USP21 as a key molecule in the regulation of myofibre type switch, muscle mass control, mitochondrial function, and heat generation and, thus, implicating the potential of this molecule and its downstream substrates network as targets for the treatment and/or prevention of muscle dysfunction and the associated metabolic diseases.

Impaired aerobic capacity and premature fatigue preceding muscle weakness in the skeletal muscle Tfam-knockout mouse model.

ABSTRACTMitochondrial diseases are genetic disorders that lead to impaired mitochondrial function, resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, deletion of the fast-twitch skeletal muscle-specific Tfam gene (Tfam KO) leads to a deficit in respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11- and 14-week-old Tfam KO mice, i.e. before and when mice are about to enter the terminal stage, respectively. Although force in the unfatigued state was reduced in Tfam KO mice compared to control littermates (wild type) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in Tfam KO muscle than in wild-type muscle at both ages, whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the Tfam KO mouse model represents a reliable model of lethal mitochondrial myopathy in which impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.

AUF1 gene transfer increases exercise performance and improves skeletal muscle deficit in adult mice

Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.

Acute Knockdown of MG29 in Mouse Muscle Cells Reveals Signaling Mechanisms Associated with Polyunsaturated Fatty Acid (PUFA) – Implications for Sarcopenia

Estimates of humans with sarcopenia (i.e., the age related loss of muscle loss and strength) ranges from 30% to over 70% depending on the age group, and specific population studied. Mitsugumin 29 (MG29), a muscle-specific protein, is a member of the synaptophysin-like protein family (SYPL2) that contain MAL and related proteins for vesicle trafficking and membrane link (MARVEL) domains. MG29 is important for the formation of the transverse-tubule (TT) system in skeletal muscles, efficient signaling between RyR1 and the store-operated Ca2+ entry (SOCE) machinery, and maintenance of lipid content of the sarcolemmal membrane. Our previous in vivo studies using a global knockout model of MG29 suggested a role of MG29 in the aging decline in muscle function. To avoid potential adaptive effects of the global absence of MG29, we also tested the siRNA-mediated acute knockdown of MG29 in mouse primary skeletal muscle cells, which led to extensive myogenic changes characterized by increased myogenic differentiation that led to abnormally thinner and longer myotubes. To examine the mechanistic basis of these changes, we measured changes in lipid signaling following knockdown of MG29. We collected both cell culture medium and cell homogenates 72 h after siRNA transfection, and applied a custom-targeted LC/MS lipidomics approach to quantify specific Lipid Signaling Mediators (LMs) in cell culture media and we used our customized skeletal muscle-specific RT-qPCR gene array to investigate specific genes showing alteration expression following acute MG29 knockdown. Compared with negative control siRNA treated cells, in MG29 siRNA treated cells, the cell culture media levels of arachidonoylethanolamine (AEA), oleoylethanolamide (OEA) increased significantly (p < 0.05) and the levels of prostaglandin D2 (PGD2), 9,10-DiHOME, 9-HOTrE, 9-HODE decreased significantly (p < 0.05). In addition, the PCR gene array revealed that in MG29 siRNA treated cells the expression of Cacna1s, RyR3, Btk and Sod3 increased 2.6 ± 0.48, 4.5 ± 0.89, 2.7 ± 0.51 and 6.5 ± 1.06 fold, respectively, while Fkbp1b and Ccl2 decreased 2.8 ± 0.38 and 7.3 ± 1.74 fold, respectively. These genes have function in calcium signaling and homeostasis and oxidative stress as well as inflammation and immune-regulation. We conclude that MG29 modulation of myogenic differentiation occurs via a complex signaling mechanism related to changes in LMs derived from polyunsaturated fatty acid (PUFA). This study further positions MG29 as an important regulator of signaling pathways associated with intracellular calcium homeostasis, oxidative stress, and inflammation, which emphasizes the role of MG29 as an orchestrator of alterations in skeletal muscle during sarcopenia.

Transcriptome profile analysis reveals KLHL30 as an essential regulator for myoblast differentiation

Skeletal muscle development is a sophisticated multistep process orchestrated by diverse myogenic transcription factors. Recent studies have suggested that Kelch-like genes play vital roles in muscle disease and myogenesis. However, it is still unclear how Kelch-like genes impact myoblast physiology. Here, through integrative analysis of the mRNA expression profile during chicken primary myoblast and C2C12 differentiation, many differentially expressed genes were found and suggested to be enriched in myoblast differentiation and muscle development. Interestingly, a little-known Kelch-like gene KLHL30 was screened as skeletal muscle-specific gene with essential roles in myogenic differentiation. Transcriptomic data and quantitative PCR analysis indicated that the expression of KLHL30 is upregulated under myoblast differentiation state. KLHL30 overexpression upregulated the protein expression of myogenic transcription factors (MYOD, MYOG, MEF2C) and induced myoblast differentiation and myotube formation, while knockdown of KLHL30 caused the opposite effect. Furthermore, KLHL30 was found to significantly decrease the numbers of cells in the S stage and thereby depress myoblast proliferation. Collectively, this study highlights that KLHL30 as a muscle-specific regulator plays essential roles in myoblast proliferation and differentiation.

UCHL1 regulates oxidative activity in skeletal muscle.

Ubiquitin C-terminal Hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally identified in neurons. Our recent study showed that UCHL1 was expressed in C2C12 myoblast cells and mouse skeletal muscle. Here we report that in mouse skeletal muscle, UCHL1 is primarily expressed in oxidative muscle fibers. Skeletal muscle specific gene knockout (smKO) of UCHL1 in mice reduced oxidative activity in skeletal muscle measured by SDH staining. The in situ muscle contraction test revealed that gastrocnemius muscle from UCHL1 smKO mice was more prone to fatigue in response to the repetitive stimulation. This data suggests that UCHL1 plays a role in maintenance of muscle oxidative metabolism. Moreover, UCHL1 smKO caused a significant reduction in key proteins that are involved in mitochondrial oxidative phosphorylation in soleus muscles, suggesting that UCHL1 may be involved in regulation of mitochondrial content and function. Immunostaining showed the co-localization of UCHL1 and mitochondrial marker VDAC in skeletal muscle. Mitochondrial fractionation assay revealed that, although UCHL1 was primarily present in the cytosolic fraction, a low level of UCHL1 protein was present in mitochondrial fraction. The level of phosphorylation of AMPKα, a master regulator of mitochondrial biogenesis, were unchanged in UCHL1 smKO muscle. On the other hand, immunoprecipitation from soleus muscle sample indicated the interaction between UCHL1 and HSP60, a chaperon protein that is involved in mitochondrial protein transport. There was a trend of downregulation of HSP60 in UCHL1 smKO muscle. Overall, our data suggests UCHL1 is a novel regulator of mitochondrial function and oxidative activity in skeletal muscle.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-β and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 μm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

DNA Methylation Reorganization of Skeletal Muscle-Specific Genes in Response to Gestational Obesity.

The goals were to investigate in umbilical cord tissue if gestational obesity: (1) was associated with changes in DNA methylation of skeletal muscle-specific genes; (2) could modulate the co-methylation interactions among these genes. Additionally, we assessed the associations between DNA methylation levels and infant’s variables at birth and at age 6. DNA methylation was measured in sixteen pregnant women [8-gestational obesity group; 8-control group] in umbilical cord using the Infinium Methylation EPIC Bead Chip microarray. Differentially methylated CpGs were identified with Beta Regression Models [false discovery rate (FDR) < 0.05 and an Odds Ratio > 1.5 or < 0.67]. DNA methylation interactions between CpGs of skeletal muscle-specific genes were studied using data from Pearson correlation matrices. In order to quantify the interactions within each network, the number of links was computed. This identification analysis reported 38 differential methylated CpGs within skeletal muscle-specific genes (comprising 4 categories: contractibility, structure, myokines, and myogenesis). Compared to control group, gestational obesity (1) promotes hypermethylation in highly methylated genes and hypomethylation in low methylated genes; (2) CpGs in regions close to transcription sites and with high CpG density are hypomethylated while regions distant to transcriptions sites and with low CpG density are hypermethylated; (3) diminishes the number of total interactions in the co-methylation network. Interestingly, the associations between infant’s fasting glucose at age 6 and MYL6, MYH11, TNNT3, TPM2, CXCL2, and NCAM1 were still relevant after correcting for multiple testing. In conclusion, our study showed a complex interaction between gestational obesity and the epigenetic status of muscle-specific genes in umbilical cord tissue. Additionally, gestational obesity may alter the functional co-methylation connectivity of CpG within skeletal muscle-specific genes interactions, our results revealing an extensive reorganization of methylation in response to maternal overweight. Finally, changes in methylation levels of skeletal muscle specific genes may have persistent effects on the offspring of mothers with gestational obesity.

Calcineurin Broadly Regulates the Initiation of Skeletal Muscle-Specific Gene Expression by Binding Target Promoters and Facilitating the Interaction of the SWI/SNF Chromatin Remodeling Enzyme.

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCβ) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

UCHL1 regulates muscle fibers and mTORC1 activity in skeletal muscle

AimsSkeletal muscle wasting is associated with many chronic diseases. Effective prevention and treatment of muscle wasting remain as a challenging task due to incomplete understanding of mechanisms by which muscle mass is maintained and regulated. This study investigated the functional role of Ubiquitin C-terminal hydrolase L1 (UCHL1) in skeletal muscle. Main methodsMice with skeletal muscle specific gene knockout of UCHL1 and C2C12 myoblast cells with UCHL1 knockdown were used. Muscle fiber types and size were measured using tissue or cell staining. The mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 activities were assessed with the phosphorylation of their downstream targets. Key findingsIn mouse skeletal muscle, UCHL1 was primarily expressed in slow twitch muscle fibers. Mice with skeletal muscle specific knockout (skmKO) of UCHL1 exhibited enlarged muscle fiber sizes in slow twitch soleus but not fast twitch extensor digitorum longus (EDL) muscle. Meanwhile, UCHL1 skmKO enhanced mTORC1 activity and reduced mTORC2 activity in soleus but not in EDL. Consistently, in C2C12 cells, UCHL1 knockdown increased the myotube size, enhanced mTORC1 activity, and reduced mTORC2 activities as compared with control cells. UCHL1 knockdown did not change the major proteins of mTOR complex but decreased the protein turnover of PRAS40, an inhibitory factor of mTORC1. SignificanceThese data revealed a novel function of UCHL1 in regulation of mTORC1 activity and skeletal muscle growth in slow twitch skeletal muscle. Given the upregulation of UCHL1 in denervation and spinal muscle atrophy, our finding advances understanding of regulators that are involved in muscle wasting.

Unique Skeletal Muscle Gene Expression Signature Induced by Acute Alcohol Intoxication in mice

High levels of alcohol intake lead to skeletal muscle myopathy and subsequent dysfunction both acutely and chronically. Our previous work has shown that a single dose of alcohol, as well as chronic intake, decreases rates of protein synthesis concurrent with suppression of the mTOR signaling pathway. Although evidence exists for the contribution of other anabolic and catabolic factors (myostatin, inflammation, insulin like growth factor, etc.), to the induction of alcoholic myopathy, no definitive mechanism(s) or pathway(s) has been identified. Therefore, the objective of this work was to identify novel pathways for future therapeutic targeting in the treatment of alcoholic myopathy via an unbiased screen of skeletal muscle specific genes differentially regulated by acute alcohol intoxication. Male, 12 week old, C57BL/6 mice were fasted overnight prior to receiving an intraperitoneal injection of 3g/kg BW alcohol or saline (Control group). The gastrocnemius/plantaris muscle complex was excised 1 hour post alcohol intoxication for isolation of RNA and preparation of cDNA prior to RNA deep sequencing. The data were mapped to the mouse genome (mm10, GRCm38) using TopHat 2 software. Using DESeq2 software, differentially expressed genes were determined based on an FDR‐adjusted p‐value of &lt;0.05. Under these parameters, 48 transcripts were identified in response to acute alcohol intoxication. Using the DAVID Bioinformatics Resource, 8 functional groups were recognized including Collagen, Extracellular matrix, Secreted, Hydroxylation, Cell Adhesion, Cytoplasm, Signal and Glycoprotein. Of the 48 genes, 15 were suppressed by alcohol intoxication including Regulator of Cell Cycle (Rgcc), previously shown to be upregulated in models of skeletal muscle hypertrophy and regrowth; Leiomodin2 (Lmod2) which is important in actin polymerization; and xin actin‐binding repeat containing 1 (Xirp1) which protects actin filaments from depolymerization and may be a marker of muscle damage. The remaining 32 genes were increased by acute alcohol intoxication including EMILIN1 which relies on the inhibition of TGF‐β; Aquaporin7 which is implicated in lipolysis and glycerol efflux via regulation of mTOR‐mediated leptin expression; and MAP3K14, also known as NF‐κB inducible kinase, indicating potential modulation of the NF‐κB pathway by acute alcohol. Of the differentially expressed gene transcripts, several do not appear to have a known functional role in skeletal muscle and will therefore provide novel targets for the further characterization of the effects of alcohol on skeletal muscle.Support or Funding InformationFunds provided by Florida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

BackgroundThe tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse.MethodsTo confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei.ResultsReverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells.ConclusionsThe HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle.

Transgenic overexpression of follistatin 2 in blunt snout bream results in increased muscle mass caused by hypertrophy

As a competitive binding protein, follistatin (FST) antagonizes myostatin (MSTN) function and promotes muscle growth. Overexpression of FST in transgenic mice results in dramatically increased muscle mass caused by hyperplasia and/or hypertrophy. Previously, we identified two functional divergent fst genes (gcfst1 and gcfst2) in grass carp (Ctenopharyngodon idella). The objectives of this study were to determine if gcfst genes have similar roles on promoting muscle growth in fish. We generated a heterozygous gcfst1- and gcfst2-transgenic F0 generation in blunt snout bream (Megalobrama amblycephala). We then produced a homozygous gynogenetic F1 generation, and eventually a homozygous transgenic F2 generation. Driven by the zebrafish skeletal muscle-specific gene Mylz2 promoter, high levels of gcfst1 or gcfst2 mRNAs were expressed in embryonic somites and adult muscles in homozygous gcfst1- or gcfst2-transgenic F2 blunt snout bream, respectively. In contrast to gcfst1-transgenic fish or wild-type controls, adult gcfst2-transgenic F2 blunt snout bream exhibited a double-muscling phenotype and marked increases in body height and thickness. Overexpression of gcfst2 caused significant fiber size enlargement in skeletal muscle in gcfst2-transgenic F2 blunt snout bream. Our findings suggest that enhanced muscle growth induced by gcfst2 overexpression in transgenic blunt snout bream is caused by hypertrophic muscle growth. This approach may be used to promote directional breeding in important aquaculture species such as blunt snout bream in the future. Statement of relevanceTo promote directional breeding in important aquaculture species blunt snout bream, our research papers presents novel data on fish breeding. Our article complies with the Policy Statement for submission of manuscripts to the Genetics Section.