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Project description:Copper (Cu) is an essential trace element for diverse biological reactions such as respiration, neurotransmitter synthesis, oxidative stress and transcriptional regulation. If Cu homeostasis is disrupted, several pathological conditions can develop, leading to alterations of neuronal, cognitive, and muscular development. Known Cu+-binding transcription factors in mammalian cells are Atox1, Mtf1 and Sp1. We identified Crip2 as a novel Cu+-responsive transcriptional regulator that is required for the differentiation of primary myoblasts derived from mouse satellite cells. Functional characterization of CRISPR/Cas9-mediated deletion of Crip2 showed that myoblasts fail to differentiate and manifest a decrease in expression of the differentiation markers Myogenin and Myosin heavy chain. RNA-seq and CUT&RUN analysis were performed to understand the effect on Crip2 in the regulation of gene expression in proliferating and differentiating primary myoblasts stimulated with Cu.
2024-05-30 | GSE252158 | GEO
Project description:Copper (Cu) is an essential trace element for diverse biological reactions such as respiration, neurotransmitter synthesis, oxidative stress and transcriptional regulation. If Cu homeostasis is disrupted, several pathological conditions can develop, leading to alterations of neuronal, cognitive, and muscular development. Known Cu+-binding transcription factors in mammalian cells are Atox1, Mtf1 and Sp1. We identified Crip2 as a novel Cu+-responsive transcriptional regulator that is required for the differentiation of primary myoblasts derived from mouse satellite cells. Functional characterization of CRISPR/Cas9-mediated deletion of Crip2 showed that myoblasts fail to differentiate and manifest a decrease in expression of the differentiation markers Myogenin and Myosin heavy chain. RNA-seq and CUT&RUN analysis were performed to understand the effect on Crip2 in the regulation of gene expression in proliferating and differentiating primary myoblasts stimulated with Cu.
2024-05-30 | GSE252156 | GEO
Project description:Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation.
| PRJNA1057932 | ENA
Project description:Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation. [RNA-Seq]
| PRJNA1057942 | ENA
Project description:Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation. [CUT&Run]
| PRJNA1057943 | ENA
Project description:Skeletal muscle is the major site of postprandial peripheral glucose uptake, but in obesity-induced insulin-resistant states insulin-stimulated glucose disposal is markedly impaired. Despite the importance of skeletal muscle in regulating glucose homeostasis, the specific transcriptional changes associated with insulin-sensitive vs. -resistant states in muscle remain to be fully elucidated. Herein, using an RNA-seq approach we identified 20 genes differentially expressed in an insulin-resistant state in skeletal muscle, including cysteine- and glycine-rich protein 3 ( Csrp3), which was highly expressed in insulin-sensitive conditions but significantly reduced in the insulin-resistant state. CSRP3 has diverse functional roles including transcriptional regulation, signal transduction, and cytoskeletal organization, but its role in glucose homeostasis has yet to be explored. Thus, we investigated the role of CSRP3 in the development of obesity-induced insulin resistance in vivo. High-fat diet-fed CSRP3 knockout (KO) mice developed impaired glucose tolerance and insulin resistance as well as increased inflammation in skeletal muscle compared with wild-type (WT) mice. CSRP3-KO mice had significantly impaired insulin signaling, decreased GLUT4 translocation to the plasma membrane, and enhanced levels of phospho-PKC? in muscle, which all contributed to reduced insulin-stimulated glucose disposal in muscle in HFD-fed KO mice compared with WT mice. CSRP3 is a highly inducible protein and its expression is acutely increased after fasting. After 24h fasting, glucose tolerance was significantly improved in WT mice, but this effect was blunted in CSRP3-KO mice. In summary, we identify a novel role for Csrp3 expression in skeletal muscle in the development of obesity-induced insulin resistance.
| S-EPMC6139493 | biostudies-literature
Project description:BackgroundSkeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia.MethodsWe analyzed gene expression data from muscle of mice or human patients with diverse muscle pathologies and identified LMCD1 as a gene strongly associated with skeletal muscle function. We transiently expressed or silenced LMCD1 in mouse gastrocnemius muscle or in mouse primary muscle cells and determined muscle/cell size, targeted gene expression, kinase activity with kinase arrays, protein immunoblotting, and protein synthesis levels. To evaluate force, calcium handling, and fatigue, we transduced the flexor digitorum brevis muscle with a LMCD1-expressing adenovirus and measured specific force and sarcoplasmic reticulum Ca2+ release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling.ResultsHere, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease.ConclusionsOur gain- and loss-of-function studies show that LMCD1 controls protein synthesis, muscle fiber size, specific force, Ca2+ handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.
| S-EPMC6822430 | biostudies-literature
Project description:Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.
| S-EPMC1152326 | biostudies-other
Project description:BACKGROUND: In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers. RESULTS: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle. CONCLUSIONS: These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.
| S-EPMC2912822 | biostudies-other
Project description:Skeletal muscle differentiation is a precisely coordinated process, and the molecular mechanism regulating the process remains incompletely understood. Here we report the identification of serine/threonine kinase 40 (Stk40) as a novel positive regulator of skeletal myoblast differentiation in culture and fetal skeletal muscle formation in vivo We show that the expression level of Stk40 increases during skeletal muscle differentiation. Down-regulation and overexpression of Stk40 significantly decreases and increases myogenic differentiation of C2C12 myoblasts, respectively. In vivo, the number of myofibers and expression levels of myogenic markers are reduced in the fetal muscle of Stk40 knockout mice, indicating impaired fetal skeletal muscle formation. Mechanistically, Stk40 controls the protein level of histone deacetylase 5 (HDAC5) to maintain transcriptional activities of myocyte enhancer factor 2 (MEF2), a family of transcription factor important for skeletal myogenesis. Silencing of HDAC5 expression rescues the reduced myogenic gene expression caused by Stk40 deficiency. Together, our study reveals that Stk40 is required for fetal skeletal muscle development and provides molecular insights into the control of the HDAC5-MEF2 axis in skeletal myogenesis.
| S-EPMC5217693 | biostudies-literature