Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.
ABSTRACT: We previously reported that leukocyte specific ?2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for ?2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a ?2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and ?2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with ?2 integrin expressing myeloid cells.
Project description:BACKGROUND:Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. METHODS:We generated inducible satellite cell-specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2 . To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin-induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. RESULTS:Satellite cell-specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon-depleted myofibers exhibited 32 ± 9.6% of EdU-positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon-ablated myoblasts were positive for senescence-associated ?-galactosidase (SA-?-gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post-injury Day 4 revealed alterations in genes related to mitogen-activated protein kinase signalling (P < 8.29 e-5 ) and extracellular matrix (P < 2.65 e-24 ). Consistent with this, Cdon-depleted tibialis anterior muscles had reduced phosphorylated extracellular signal-regulated kinase (p-ERK) protein levels and expression of ERK targets, such as Fos (0.23-fold) and Egr1 (0.31-fold), relative to mock-treated control muscles (P < 0.001). Cdon-depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin ?1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24-/- myofibers, showed decreased Cdon levels (Cdon-positive cells in Zmpste24-/- = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin ?1 activation (weak or mislocalized integrin ?1 in Zmpste24-/- = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). CONCLUSIONS:Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.
Project description:Myogenic cell transplantation is an experimental approach for the treatment of myopathies. In this approach, transplanted cells need to fuse with pre-existing myofibers, form new myofibers, and generate new muscle precursor cells (MPCs). The last property was fully reported following myoblast transplantation in mice but remains poorly studied with human myoblasts. In this study, we provide evidence that the intramuscular transplantation of postnatal human myoblasts in immunodeficient mice generates donor-derived MPCs and specifically donor-derived satellite cells. In a first experiment, cells isolated from mouse muscles 1 month after the transplantation of human myoblasts proliferated in vitro as human myoblasts. These cells were retransplanted in mice and formed myofibers expressing human dystrophin. In a second experiment, we observed that inducing muscle regeneration 2 months following transplantation of human myoblasts led to myofiber regeneration by human-derived MPCs. In a third experiment, we detected by immunohistochemistry abundant human-derived satellite cells in mouse muscles 1 month after transplantation of postnatal human myoblasts. These human-derived satellite cells may correspond totally or partially to the human-derived MPCs evidenced in the first two experiments. Finally, we present evidence that donor-derived satellite cells may be produced in patients that received myoblast transplantation.
Project description:Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-?B and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy.
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.
Project description:The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair. We show that EphA7, a juxtacrine signaling receptor, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo. In EphA7-/- mice, hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exogenous EphA7 extracellular domain will rescue the null phenotype in vitro, and will also enhance commitment to differentiation in WT cells. We propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation.
Project description:Satellite cell proliferation is an essential step in proper skeletal muscle development and muscle regeneration. However, the mechanisms regulating satellite cell proliferation are relatively unknown compared to the knowledge associated with the differentiation of satellite cells. Moreover, it is still unclear whether overload muscle fiber hypertrophy is dependent on satellite cell proliferation. In general, cell proliferation is regulated by the activity of cell cycle regulators, such as cyclins and cyclin-dependent kinases (CDKs). Despite recent reports on the function of CDKs and CDK inhibitors in satellite cells, the physiological role of Cdk1 in satellite cell proliferation remains unknown. Herein, we demonstrate that Cdk1 regulates satellite cell proliferation, muscle regeneration, and muscle fiber hypertrophy. Cdk1 is highly expressed in myoblasts and is downregulated upon myoblast differentiation. Inhibition of CDK1 activity inhibits myoblast proliferation. Deletion of Cdk1 in satellite cells leads to inhibition of muscle recovery after muscle injury due to reduced satellite cell proliferation in vivo. Finally, we provide direct evidence that Cdk1 expression in satellite cells is essential for overload muscle fiber hypertrophy in vivo. Collectively, our results demonstrate that Cdk1 is essential for myoblast proliferation, muscle regeneration, and muscle fiber hypertrophy. These findings could help to develop treatments for refractory muscle injuries and muscle atrophy, such as sarcopenia.
Project description:In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.
Project description:Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.
Project description:Skeletal muscle possesses the ability to adapt its size in response to milieus, which is called plasticity. Overload like resistance training induces the increment of muscle mass called muscle hypertrophy. Muscle stem cells (also known as muscle stem cells) function to supply new nuclei for myofiber during the overload in muscle. Using compensatory hypertrophy in plantaris muscles, we isolated MuSCs from plantaris muscles 4 days after surgery. Control MuSCs were also prepared from sham plantaris muscles. Overall design: Control and overloaded satellite cells from Pax7-CreERT2:Rosa-YFP were used for RNA-seq analysis.
Project description:We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle.