ABSTRACT: Myo-satellite cells regenerate and differentiate into skeletal muscle (SM) after acute or chronic injury. Changes in the redox milieu towards the oxidative arm at the wound site are known to compromise SM regeneration. Recently, we reported that abrogation of Nrf2/antioxidant signaling promotes oxidative stress and impairs SM regeneration in C57/Bl6 mice. Here, we investigated whether the activation of intracellular Nrf2 signaling favors antioxidant transcription and promotes myoblast differentiation. Satellite cell-like C2C12 myoblasts were treated with sulforaphane (SF; 1.0 & 5.0 ?M) to activate Nrf2/antioxidant signaling during proliferation and differentiation (i.e. formation of myotubes/myofibers). SF-mediated Nrf2 activation resulted in increased expression of Nrf2-antioxidants (e.g. GCLC and G6PD) and augmented the production of reduced glutathione (GSH) leading to a reductive redox state. Surprisingly, this resulted in significant inhibition of myoblast differentiation, as observed from morphological changes and reduced expression of MyoD, Pax7, and Myh2, due to reductive stress (RS). Furthermore, supplementation of N-acetyl-cysteine (NAC) or GSH-ester or genetic knock-down of Keap1 (using siRNA) also resulted in RS-driven inhibition of differentiation. Interestingly, withdrawing Nrf2 activation rescued differentiation potential and formation of myotubes/myofibers from C2C12 myoblasts. Thus, abrogation of physiological ROS signaling through over-activation of Nrf2 (i.e. RS) and developing RS hampers differentiation of muscle satellite cells.
Project description:Skeletal muscle (SM) regeneration after injury is impaired by excessive inflammation. Particularly, the inflammatory cytokine tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent inducer of skeletal muscle wasting and fibrosis. In this study we investigated the role of Nrf2, a major regulator of oxidative stress defence, in SM ischemia/reperfusion (I/R) injury and TWEAK induced atrophy. We explored the time-dependent expression of TWEAK after I/R in SM of Nrf2-wildtype (WT) and knockout (KO) mice. Nrf2-KO mice expressed significant higher levels of TWEAK as compared to WT mice. Consequently, Nrf2-KO mice present an insufficient regeneration as compared to Nrf2-WT mice. Moreover, TWEAK stimulation activates Nrf2 in the mouse myoblast cell line C2C12. This Nrf2 activation inhibits TWEAK induced atrophy in C2C12 differentiated myotubes. In summary, we show that Nrf2 protects SM from TWEAK-induced cell death in vitro and that Nrf2-deficient mice therefore have poorer muscle regeneration.
Project description:Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKC? during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKC?-HMGA1 signaling axis in myoblast differentiation and regeneration processes.PKC? expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKC? and HMGA1 in myogenic differentiation.PKC? expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKC? blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKC? accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKC? impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration.This work identifies the PKC?-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.
Project description:Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.
Project description:BACKGROUND: The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors. RESULTS: Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation. CONCLUSION: Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.
Project description:AIM:The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion. METHODS AND RESULTS:By using immunoassaying and western blot analyses, we found that β1 and β2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of β2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a β-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased β1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-β2-AdR levels while decreasing β2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a. CONCLUSION:Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced β2-AdR and increased β1-AdR levels.
Project description:Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3' untranslated region (3' UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3' UTR of c-Myb was also important because the expression of c-Myb coding region with its 3' UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3' UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.
Project description:Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.
Project description:Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and beta-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.
Project description:Pax3 is an essential myogenic regulator of fetal and embryonic development, but its role in postnatal myogenesis remains a topic of debate. We show that constitutive expression of Pax3 in postnatal, juvenile mouse skeletal muscle stem cells, a subset of the heterogeneous satellite cell pool highly enriched for myogenic activity, potently induces differentiation. This differentiation-promoting activity stands in contrast to the differentiation-inhibiting effects of Pax3 in the commonly used mouse myoblast cell line C2C12. Pax3 mRNA levels in distinct muscles correlate with the rate of myogenic differentiation of their muscle stem cells. Although Pax3 controls embryonic myogenesis through regulation of the canonical myogenic regulatory factors (MRFs) Myf-5, MyoD, myogenin and Mrf4, we find that in postnatal muscle stem cells, ectopic Pax3 expression fails to induce expression of any of these factors. Unexpectedly, overexpression of neither Myf-5 nor myogenin is sufficient to induce differentiation of juvenile stem cells; and knockdown of Myf-5, rather than inhibiting differentiation, promotes it. Taken together, our results suggest that there are distinct myogenic regulatory pathways that control the embryonic development, juvenile myogenesis and adult regeneration of skeletal myofibers.
Project description:Adult skeletal muscles are comprised of multinuclear muscle cells called myofibers. During skeletal muscle development and regeneration, mononuclear progenitor cells (myoblasts) fuse to form multinuclear myotubes, which mature and become myofibers. The molecular events mediating myoblast fusion are not fully understood. Here we report that Shisa2, an endoplasmic reticulum (ER) localized protein, regulates the fusion of muscle satellite cell-derived primary myoblasts. Shisa2 expression is repressed by Notch signaling, elevated in activated compared to quiescent satellite cells, and further upregulated during myogenic differentiation. Knockdown of Shisa2 inhibits the fusion of myoblasts without affecting proliferation. Conversely, Shisa2 overexpression in proliferating myoblasts inhibits their proliferation but promotes premature fusion. Interestingly, Shisa2-overexpressing nascent myotubes actively recruit myoblasts to fuse with. At the molecular level, Rac1/Cdc42-mediated cytoskeletal F-actin remodeling is required for Shisa2 to promote myoblast fusion. These results provide a novel mechanism through which an ER protein regulates myogenesis.