Zfp423 Regulates Skeletal Muscle Regeneration and Proliferation.
ABSTRACT: Satellite cells (SCs) are skeletal muscle stem cells that proliferate in response to injury and provide myogenic precursors for growth and repair. Zfp423 is a transcriptional cofactor expressed in multiple immature cell populations, such as neuronal precursors, mesenchymal stem cells, and preadipocytes, where it regulates lineage allocation, proliferation, and differentiation. Here, we show that Zfp423 regulates myogenic progression during muscle regeneration. Zfp423 is undetectable in quiescent SCs but becomes expressed during SC activation. After expansion, Zfp423 is gradually downregulated as committed SCs terminally differentiate. Mice with satellite-cell-specific Zfp423 deletion exhibit severely impaired muscle regeneration following injury, with aberrant SC expansion, defective cell cycle exit, and failure to transition efficiently from the proliferative stage toward commitment. Consistent with a cell-autonomous role of Zfp423, shRNA-mediated knockdown of Zfp423 in myoblasts inhibits differentiation. Surprisingly, forced expression of Zfp423 in myoblasts induces differentiation into adipocytes and arrests myogenesis. Affinity purification of Zfp423 in myoblasts identified Satb2 as a nuclear partner of Zfp423 that cooperatively enhances Zfp423 transcriptional activity, which in turn affects myoblast differentiation. In conclusion, by controlling SC expansion and proliferation, Zfp423 is essential for muscle regeneration. Tight regulation of Zfp423 expression is essential for normal progression of muscle progenitors from proliferation to differentiation.
Project description:The remarkable regeneration capability of skeletal muscle depends on the coordinated proliferation and differentiation of satellite cells (SCs). The self-renewal of SCs is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in SCs in vivo remains largely unknown. Here, we demonstrate that SCs are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of SCs by maintaining their quiescence, increasing their self-renewal, and blocking their myogenic differentiation. HIF2A stabilization in SCs cultured under normoxia augments their engraftment potential in regenerative muscle. Conversely, HIF2A ablation leads to the depletion of SCs and their consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerates muscle regeneration by increasing SC proliferation and differentiation. Mechanistically, HIF2A induces the quiescence and self-renewal of SCs by binding the promoter of the Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in SCs and may be therapeutically targeted to improve muscle regeneration.
Project description:CCAAT/enhancer binding protein beta (C/EBP?), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBP? expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBP? can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBP?. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBP?, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1? (IL-1?), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBP? expression in myoblasts. High levels of C/EBP? expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBP? expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBP? in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBP? expression by IL-1? following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation.
Project description:Satellite cells (SCs) are stem cells that mediate skeletal muscle growth and regeneration. Here, we observe that adult quiescent SCs and their activated descendants expressed the homeodomain transcription factor Six1. Genetic disruption of Six1 specifically in adult SCs impaired myogenic cell differentiation, impaired myofiber repair during regeneration, and perturbed homeostasis of the stem cell niche, as indicated by an increase in SC self-renewal. Six1 regulated the expression of the myogenic regulatory factors MyoD and Myogenin, but not Myf5, which suggests that Six1 acts on divergent genetic networks in the embryo and in the adult. Moreover, we demonstrate that Six1 regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway during regeneration via direct control of Dusp6 transcription. Muscles lacking Dusp6 were able to regenerate properly but showed a marked increase in SC number after regeneration. We conclude that Six1 homeoproteins act as a rheostat system to ensure proper regeneration of the tissue and replenishment of the stem cell pool during the events that follow skeletal muscle trauma.
Project description:Myopathic changes are commonly described in hypothyroid and hyperthyroid patients, including muscular atrophy and weakness. Satellite cells (SCs) play a major role in skeletal muscle maintenance and regeneration after injury. A mouse model of resistance to thyroid hormone-TR?1PV demonstrated impaired skeletal muscle regeneration after injury with significant reduction of SCs, suggesting that exhaustion of the SC pool contributes to the impaired regeneration. To test this hypothesis, SC activation and proliferation were analyzed in vivo in response to skeletal muscle injury and during aging.SCs of TR?1PV male mice were analyzed four days after cardiotoxin-induced muscle injury, and they were compared to wild-type (WT) male animals. TR?-knockdown C2C12 myoblasts were injected into injured skeletal muscle, and four days after transplantation, the in vivo behavior was compared to control C2C12 myoblasts. Skeletal muscle regeneration was compared in younger and older TR?1PV and WT animals.The total number of SCs in skeletal muscle of TR?1PV mice was significantly lower than control, both before and shortly after muscle injury, with significant impairment of SC activation, consistent with SC pool exhaustion. TR?-knockdown myoblasts showed impaired in vivo proliferation and migration. TR?1PV mice had skeletal muscle loss and significant impairment in skeletal muscle regeneration with aging. This translated to a significant reduction of the SC pool with aging compared to WT mice.TR? plays an important role in the maintenance of the SC pool. Impaired skeletal muscle regeneration in TR?1PV mice is associated with insufficient SC activation and proliferation, as well as the progressive loss of the SC pool with aging. Regulation of the SC pool and SC proliferation provides a therapeutic target to enhance skeletal muscle regeneration and possibly slow age-associated sarcopenia.
Project description:The functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify SAM (Sugt1 asssociated muscle) lncRNA that is enriched in the proliferating myoblasts. Global deletion of SAM has no overt effect on mice but impairs adult muscle regeneration following acute damage; it also exacerbates the chronic injury-induced dystrophic phenotype in mdx mice. Consistently, inducible deletion of SAM in SCs leads to deficiency in muscle regeneration. Further examination reveals that SAM loss results in a cell-autonomous defect in the proliferative expansion of myoblasts. Mechanistically, we find SAM interacts and stabilizes Sugt1, a co-chaperon protein key to kinetochore assembly during cell division. Loss of SAM or Sugt1 both disrupts kinetochore assembly in mitotic cells due to the mislocalization of two components: Dsn1 and Hec1. Altogether, our findings identify SAM as a regulator of SC proliferation through facilitating Sugt1 mediated kinetochore assembly during cell division.
Project description:Cell differentiation status is defined by the gene expression profile, which is coordinately controlled by epigenetic mechanisms. Cell type-specific DNA methylation patterns are established by chromatin modifiers including de novo DNA methyltransferases, such as Dnmt3a and Dnmt3b. Since the discovery of the myogenic master gene MyoD, myogenic differentiation has been utilized as a model system to study tissue differentiation. Although knowledge about myogenic gene networks is accumulating, there is only a limited understanding of how DNA methylation controls the myogenic gene program. With an aim to elucidate the role of DNA methylation in muscle development and regeneration, we investigate the consequences of mutating Dnmt3a in muscle precursor cells in mice. Pax3 promoter-driven Dnmt3a-conditional knockout (cKO) mice exhibit decreased organ mass in the skeletal muscles, and attenuated regeneration after cardiotoxin-induced muscle injury. In addition, Dnmt3a-null satellite cells (SCs) exhibit a striking loss of proliferation in culture. Transcriptome analysis reveals dysregulated expression of p57Kip2, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKIs), in the Dnmt3a-KO SCs. Moreover, RNAi-mediated depletion of p57Kip2 replenishes the proliferation activity of the SCs, thus establishing a role for the Dnmt3a-p57Kip2 axis in the regulation of SC proliferation. Consistent with these findings, Dnmt3a-cKO muscles exhibit fewer Pax7+ SCs, which show increased expression of p57Kip2 protein. Thus, Dnmt3a is found to maintain muscle homeostasis by epigenetically regulating the proliferation of SCs through p57Kip2.
Project description:Long non-coding RNAs (lncRNAs) are family of gene regulators but the functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify SAM (Sugt1 associated muscle) lncRNA (also known as Snhg15, (small nucleolar RNA host gene 15) that is enriched in the proliferating myoblast cells and down-regulated as the cells progressed to differentiation. Gain- or loss- of function of SAM in muscle SCs alters myogenic proliferation and differentiation. Global deletion of SAM based on the KO-first strategy has no overt effect on mice but impairs adult muscle regeneration following acute damage; the loss also exacerbates the chronic injury induced dystrophic phenotype in the mdx mouse model. Consistently, inducible deletion of SAM in adult SCs leads to deficiency in acute injury-induced muscle regeneration. Further examination of SCs revealed that SAM loss results in cell autonomous defect in proliferative expansion of myoblasts. Mechanistically, we find SAM interacts and stabilizes Sugt1 (suppressor of G2 allele of SKP1) protein, a co-chaperon protein key to kinetochore assembly during cell division. Loss of SAM or Sugt1 both cause disruptive kinetochore assembly and microtubule attachment in mitotic cells due to mis-localization of key components Dsn1 and Hec1 proteins. Altogether, our findings identify SAM as a regulator of SC proliferation through facilitating Sugt1 mediated kinetochore assembly during cell division.
Project description:Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up-regulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors.We examined the histopathological stages, Pax7 SC content, and muscle-specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis.Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in the fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206.These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.
Project description:We isolated and cultured satellite cells (SCs) from the longissimus dorsi muscles of 1-day-old male Landrace and Lantang piglets to compare the SC differentiation capacity in the two breeds. Lantang piglets yielded more (P < 0.05) SCs per gram of muscle than Landrace piglets (5.2 ± 0.9×104 vs. 2.4 ± 0.2×104). Transcription of the differentiation markers myogenin and myosin heavy chain I (MyHC I) in the longissimus dorsi muscle was higher in Lantang than Landrace piglets (P < 0.05). Protein levels of myogenin (P < 0.05), MyHC I (P < 0.05), and myogenic regulatory factor 4 (P = 0.07) were higher in Lantang than Landrace piglet SCs after 72 h of differentiation. Creatine kinase activity was higher in Lantang than Landrace piglet SCs after 24, 48, and 72 h of differentiation (P < 0.05), and there was a greater fusion index in Landrace piglet SCs after 72 h of differentiation. In addition, phosphorylation of Akt, mTOR, S6K1, S6, and 4EBP1 was lower in Lantang than Landrace piglet SCs (P < 0.05). Thus differentiation was more extensive in Lantang than Landrace piglet SCs, but expression of the mTOR signaling pathway was lower in Lantang piglet SCs, suggesting mTOR signaling may inhibit myogenic differentiation. These findings reveal that mTOR signaling is a factor in myogenesis and imply that mTOR could potentially serve as an activator of myoblast differentiation and muscle regeneration.
Project description:Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and ?1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.