The aligned structural environment in skeletal muscle is believed to be a crucial component in functional muscle regeneration. Myotube formation is increased on aligned biomaterials, but we do not ...fully understand the mechanisms that direct this enhanced fusion. Previous studies indicate that the α7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via α7β1 mediates the effect of alignment. To test this hypothesis, we took advantage of an in vitro model using random and aligned polydioxanone (PDO) matrices and C2C12 myoblasts. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of α7β1 signaling, we measured expression and production of α7, α5, and β1 and myoblast markers in wild type cells and in cells silenced for α7 and assessed effects of silencing on myogenic differentiation. Downstream signaling via ERK1/2 mitogen activated protein kinase (MAPK) was examined using a specific MEK1/2 inhibitor. Alignment increased mRNAs and protein for early (MyoD) and late (MyoG, MyHC) myoblast markers in comparison to non-aligned matrices, and these levels corresponded with increased α7 protein. α7-silencing reduced MyoG and MyHC protein in cells cultured on tissue culture polystyrene and aligned PDO matrices compared to wild type cells. Inhibition of ERK1/2 blocked effects of alignment. These data suggest that alignment regulates myogenic differentiation via α7β1 integrin signaling and ERK1/2 mediated gene expression.
Muscle regeneration in severe muscle injuries is complex, requiring a sequence of events to promote healing and not fibrosis. Aligned biomaterials that recapitulate muscle environments hold potential to facilitate regeneration, but it is important to understand cell-substrate signaling to form functional muscle. A critical component of muscle signaling is integrin α7β1, where mice lacking α7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of α7β1 signaling in myoblast differentiation on aligned biomaterials. α7-silenced myoblasts were found to regulate myogenic differentiation and demonstrate defective fusion. Our data shows reduced levels of myogenin and myosin heavy chain protein, while MyoD remains unchanged. These results support the hypothesis that α7β1 signaling plays a role in substrate-dependent tissue engineering strategies.
The myogenic regulatory factor MyoD has been implicated as a key regulator of myogenesis, and yet there is little information regarding its upstream regulators. We found that Deltex2 inhibits ...myogenic differentiation in vitro, and that skeletal muscle stem cells from Deltex2 knockout mice exhibit precocious myogenic differentiation and accelerated regeneration in response to injury. Intriguingly, Deltex2 inhibits myogenesis by suppressing MyoD transcription, and the Deltex2 knockout phenotype can be rescued by a loss-of-function allele for MyoD. In addition, we obtained evidence that Deltex2 regulates MyoD expression by promoting the enrichment of histone 3 modified by dimethylation at lysine 9 at a key regulatory region of the MyoD locus. The enrichment is attributed to a Deltex2 interacting protein, Jmjd1c, whose activity is directly inhibited by Deltex2 and whose expression is required for MyoD expression in vivo and in vitro. Finally, we find that Deltex2 causes Jmjd1c monoubiquitination and inhibits its demethylase activity. Mutation of the monoubiquitination site in Jmjd1c abolishes the inhibitory effect of Deltex2 on Jmjd1c demethylase activity. These results reveal a mechanism by which a member of the Deltex family of proteins can inhibit cellular differentiation, and demonstrate a role of Deltex in the epigenetic regulation of myogenesis.
Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in ...both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.
Display omitted
Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient ...mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.
Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are ...unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.
Display omitted
•Skeletal muscle growth is driven by stem cells that exhibit extensive clonal drift•The homeobox gene meox1 controls clonal drift during muscle growth•Meox1 directly inhibits ccnb1 to arrest stem cells in G2 phase•Artificially capturing cells in G2 arrest ectopically increases Meox1+ stem cells
Currie and colleagues reveal a dramatic clonal drift of muscle stem cells during tissue growth, which is regulated at the G2 phase of the cell cycle via Meox1-dependent control of ccnb1. This contrasts with G0/G1 phase control evident in adult stem cells, indicating divergent modes of stem cell regulation.
Context:
Immunosuppressive agents are associated with profound metabolic side effects including new-onset diabetes and dyslipidemia after organ transplantation.
Objective:
To investigate the effects ...of cyclosporine A (CsA) and tacrolimus on glucose uptake and insulin signaling in human adipocytes and their impact on the regulation of cellular trafficking of the glucose transporter 4 (GLUT4).
Design:
Isolated human adipocytes were incubated with therapeutic concentrations of either CsA or tacrolimus, and glucose uptake and expression of insulin signaling proteins were assessed. Furthermore, we studied effects of CsA and tacrolimus on the regulation of cellular trafficking of GLUT4 in differentiated human preadipocytes and L6 cells.
Results:
CsA and tacrolimus had a concentration-dependent inhibitory effect on basal and insulin-stimulated 14C-glucose uptake in adipocytes. Although phosphorylation at Tyr1146 of the insulin receptor was inhibited by tacrolimus, the phosphorylation and/or protein levels of the insulin signaling proteins IRS1/2, p85-PI3K, PKB, AS160, and mTORC1, as well as GLUT4 and GLUT1, were unchanged by CsA or tacrolimus. Furthermore, CsA and tacrolimus reduced the GLUT4 amount localized at the cell surface of differentiated human preadipocytes and L6 cells in the presence of insulin. This occurred by an increased rate of GLUT4 endocytosis, with no change in the exocytosis rate.
Conclusions:
These results suggest that therapeutic concentrations of CsA and tacrolimus can inhibit glucose uptake independent of insulin signaling by removing GLUT4 from the cell surface via an increased rate of endocytosis. This mechanism can contribute to the development of insulin resistance and diabetes associated with immunosuppressive therapy. In addition, it may provide novel pharmacological approaches for the treatment of diabetes.
Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing ...production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.
Display omitted
•Deactivated Cas9 (dCas9) impedes transcription of expanded microsatellite repeats•Repeat length, PAM sequence, and DNA strand influence extent of inhibition•dCas9 rescues molecular and cellular phenotypes in patient-derived cell lines•dCas9 rescues muscle phenotypes in a mouse model of myotonic dystrophy
Pinto, Saxena, et al. demonstrate that deactivated Cas9 efficiently blocks transcription of the expanded microsatellite repeats causing diseases such as myotonic dystrophy types 1 and 2 and C9orf72-ALS/FTD, thereby reversing toxic downstream molecular and cellular effects of repeat expression in vitro and in vivo.
Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; ...however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.
The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses ...were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.
MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress the expression of their target genes post-transcriptionally. MiRNAs participate in the regulation of a variety of biological ...processes, including development and diseases. However, the functional role and molecular mechanism by which miRNAs regulate skeletal muscle development and differentiation are not fully understood. In this report, we identified miR-23a as a key regulator of skeletal muscle differentiation. Using bioinformatics analyses, miR-23a is predicted to target multiple adult fast myosin heavy chain (Myh) genes, including Myh 1, 2 and 4. Luciferase reporter assays show that miR-23a directly targets the 3′ untranslated regions (UTRs) of these mRNAs. Interestingly, the expression level of mature miR-23a is inversely correlated with myogenic progression in mouse skeletal muscle. Both gain- and loss-of-function studies using C2C12 myoblasts demonstrate that miR-23a inhibits myogenic differentiation. These findings therefore reveal a novel role of miR-23a in regulating myogenic differentiation via inhibiting the expression of fast myosin heavy chain isoforms.
PDF
