Transcription Factor MyoD Research Articles

MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle.

In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem E-box to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of molecular clock amplitude as well as synergistic regulation of clock-controlled genes in skeletal muscle.

Abstract 17273: Long Non-Coding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Introduction: Long noncoding RNAs (lncRNAs), emerged as critical epigenetic regulators of transcriptome, play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction. In our work, we identified a novel lncRNA that regulates myogenesis. Hypothesis: LncRNA Ppp1r1b regulates myogenesis by modulating Histone 3 methylation Methods: After treated with antisense oligonucleotides (GapmeR) or siRNA against Ppp1r1b-LncRNA, real time PCR and Western blot analyses were performed to examine the expression of myogenic and sarcomere genes. Chromatin immunoprecipitation (CHIP) was used to comparatively analyze gene specific histone modification level. RNA pull-down was employed to identify the protein molecules that interact with Ppp1r1b-LncRNA. Results: By silencing Ppp1r1b-LncRNA with GapmeR, C2C12, a skeletal myoblast cell line, did not develop fully differentiated myotubes, but tend to remain in a quiescent mono-nucleated status. In vivo analysis of GapmeR injected neonatal mouse heart and in vitro siRNA silenced human skeletal myoblasts further confirmed the important role of Ppp1r1b-LncRNA on myogenesis. Members of the MyoD family of muscle-specific transcription factors (MyoD and myogenin) failed to be up-regulated during myogenic differentiation when treated with Ppp1r1b-LncRNA specific GapmeR or siRNA. Key proteins essential for establishing and maintaining normal skeletal muscle architecture, including Tcap and Dystropnin, are also suppressed in Ppp1r1b LncRNA- deficient heart. Analysis of histone modification levels at Myogenin, MyoD1 and Tcap in C2C12 cells revealed more histone tri-methylation at these myogenic and sarcomere structural genes compared to untreated cells. Additional lncRNA- protein complex isolation has further revealed insight into the biological roles of Ppp1r1b-LncRNA. Conclusions: Our results support the role of Ppp1r1b-LncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of multiple myogenic and sarcomere genes. In particular, the identification of EZH2 in pulled Pp1r1b LncRNA: protein complex implies that Polycomb repressive complex 2 (PRC2) is involved in Ppp1r1b-LncRNA modulated myoblast differentiation.

Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

Yes-associated protein (YAP) acts as a mechanotransducer in determining the cell fate of murine C2C12 mesenchymal precursors as investigated after stimulation with ultrasound. We applied Focused Low-Intensity Pulsed Ultrasound (FLIPUS) at a sound frequency of 3.6 MHz, 100 Hz pulse repetition frequency (PRF), 27.8% duty cycle (DC), and 44.5 mW/cm2 acoustic intensity ISATA for 5 minutes and evaluated early cellular responses. FLIPUS decreased the level of phosphorylated YAP on Serine 127, leading to higher levels of active YAP in the nucleus. This in turn enhanced the expression of YAP-target genes associated with actin nucleation and stabilization, cytokinesis, and cell cycle progression. FLIPUS enhanced proliferation of C2C12 cells, whereas silencing of YAP expression abolished the beneficial effects of ultrasound. The expression of the transcription factor MyoD, defining cellular myogenic differentiation, was inhibited by mechanical stimulation. This study shows that ultrasound exposure regulates YAP functioning, which in turn improves the cell proliferative potential, critical for tissue regeneration process.

Cis-regulatory determinants of MyoD function.

Muscle-specific transcription factor MyoD orchestrates the myogenic gene expression program by binding to short DNA motifs called E-boxes within myogenic cis-regulatory elements (CREs). Genome-wide analyses of MyoD cistrome by chromatin immnunoprecipitation sequencing shows that MyoD-bound CREs contain multiple E-boxes of various sequences. However, how E-box numbers, sequences and their spatial arrangement within CREs collectively regulate the binding affinity and transcriptional activity of MyoD remain largely unknown. Here, by an integrative analysis of MyoD cistrome combined with genome-wide analysis of key regulatory histones and gene expression data we show that the affinity landscape of MyoD is driven by multiple E-boxes, and that the overall binding affinity—and associated nucleosome positioning and epigenetic features of the CREs—crucially depend on the variant sequences and positioning of the E-boxes within the CREs. By comparative genomic analysis of single nucleotide polymorphism (SNPs) across publicly available data from 17 strains of laboratory mice, we show that variant sequences within the MyoD-bound motifs, but not their genome-wide counterparts, are under selection. At last, we show that the quantitative regulatory effect of MyoD binding on the nearby genes can, in part, be predicted by the motif composition of the CREs to which it binds. Taken together, our data suggest that motif numbers, sequences and their spatial arrangement within the myogenic CREs are important determinants of the cis-regulatory code of myogenic CREs.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

SummarySkeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

Identification of Epigenetic Regulators of DUX4-fl for Targeted Therapy of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes. We confirmed several of the strongest and most specific candidates (ASH1L, BRD2, KDM4C, and SMARCA5) in skeletal myocytes from two other unrelated FSHD1 patients, and we showed that knockdown led to reduced levels of DUX4-fl and DUX4-FL target genes, as well as altered chromatin at the D4Z4 locus. As a second mode of validation, targeting the CRISPR/dCas9-KRAB transcriptional repressor to the promoters of several candidates also led to reduced levels of DUX4-fl. Furthermore, these candidates can be repressed by different methods in skeletal myocytes without major effects on certain critical muscle genes. Our results demonstrate that expression of DUX4-fl is regulated by multiple epigenetic pathways, and they indicate viable, druggable candidates for therapeutic target development.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.

Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development

Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.

MYOD and HAND transcription factors have conserved recognition sites in mTOR promoter: insights from in silico analysis.

mTOR regulates multiple cellular processes that are critical for proper maintenance of cell growth and development. However, mechanisms and factors responsible for transcriptional regulation of mTOR are partially known. To identify different transcription factor binding sites in promoter region of mTOR, we performed in silico phylogenetic foot printing analysis of diverse set of human orthologs. Phylogenetic tree for the orthologs was generated to establish the evolutionary relationships among them. Conserved binding sites among the species were predicted by tool MEME. The predicted conserved sites were further analyzed for binding of transcription factors by MatInspector program. Predicted TFs were then integrated with known physical interactions and coexpression data to decipher the important transcriptional regulators of mTOR signaling. Our study suggests that motifs AGGCGGG (+ 15 to + 21) and GGCGGC (+ 60 to + 65) are highly conserved across the species and are recognition sequence for HAND and MYOD transcription factors, respectively. Also these two transcription factors show direct physical interaction in protein-protein interaction map, indicating their regulatory role on expression of mTOR for control of myogenesis. Our study provides novel clues on differential regulation of mTOR under diverse environmental conditions.

The Long Road to Making Muscle In Vitro.

The skeletal muscle lineage derives from the embryonic paraxial mesoderm (PM) which also gives rise to the axial skeleton, the dermis of the back, brown fat, meninges, and endothelial cells. Direct conversion was pioneered in skeletal muscle with overexpression of the transcription factor MyoD which can convert fibroblasts to a muscle fate. In contrast, directed differentiation of skeletal muscle from pluripotent cells (PC) in vitro has proven to be very difficult compared to other lineages and has only been achieved recently. Experimental strategies recapitulating myogenesis in vitro from mouse and human PC (ES/iPS) have now been reported and all rely on early activation of Wnt signaling at the epiblast stage. This leads to induction of neuromesodermal progenitors that can subsequently be induced to a PM fate and to skeletal muscle. These protocols can efficiently produce fetal muscle fibers and immature satellite cells. These new in vitro systems now open the possibility to better understand human myogenesis and to develop in vitro disease models as well as cell therapy approaches.

Dietary methionine restriction: Effects on glucose tolerance, lipid content and micro-RNA composition in the muscle of rainbow trout

Lean muscle mass plays an important role in overall health, as altered skeletal muscle metabolism can impact both the incidence and prevention of conditions related to metabolic health. Intriguingly, dietary methionine restriction (MR) has been shown to ameliorate this phenotype over time potentially through mechanisms related to changes in myogenic precursor cell (MPC) differentiation status. Recently the role of micro-RNAs (miRs) in regulating the expression of muscle specific transcription factors myoD and myogenin as well as signaling molecules involved in skeletal muscle differentiation has been reported in vitro. We performed an 8week feeding trial to determine if MR in vivo could alter miR abundance as well as change metabolic markers. Results show changes in muscle miR abundance for miR-133a at 4weeks with no significant difference seen in miR-210 or miR-206. After 8weeks of MR feeding fish demonstrated increased clearance of glucose, increased fat accumulation in the liver, and decreased fat accumulation in the muscle. These data demonstrate conservation of MR effects on fish metabolism, and suggest, for the first time, that miR-133a might play a role in tissue response to MR.

MiRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1

Skeletal muscle differentiation can be regulated by various transcription factors and non-coding RNAs. In our previous work, miR-223 is differentially expressed in the skeletal muscle of chicken with different growth rates, but its role, expression and action mechanism in muscle development still remains unknown. Here, we found that MYOD transcription factor can upregulate miR-223 expression by binding to an E-box region of the gga-miR-223 gene promoter during avian myoblast differentiation. IGF2 and ZEB1 are two target genes of miR-223. The target inhibition of miR-223 on IGF2 and ZEB1 are dynamic from proliferation to differentiation of myoblast. miR-223 inhibits IGF2 expression only in the proliferating myoblast, whereas it inhibits ZEB1 mainly in the differentiating myoblast. The inhibition of IGF2 by miR-223 resulted in the repression of myoblast proliferation. During myoblast differentiation, miR-223 would be upregulated owing to the promoting effect of MYOD, and the upregulation of miR-223 would inhibit ZEB1 to promote myoblast differentiation. These results not only demonstrated that the well-known muscle determination factor MYOD can promote myoblast differentiation by upregulate miR-223 transcription, but also identified that miR-223 can influence myoblast proliferation and differentiation by a dynamic manner regulates the expression of its target genes.

An isoflavone compound daidzein elicits myoblast differentiation and myotube growth

Preservation of muscle stem cell function is critical for muscle homeostasis and the regenerative capacity of stem cells declines with age, leading to muscle loss. Previously, daidzein, a natural isoflavone from Leguminosae, has been shown to prevent TNF-α-induced muscle atrophy. However, the molecular mechanisms of daidzein in these processes are currently unknown. In this study, we demonstrate a positive effect of black soybean extract (BS Ext) and daidzein on myoblast differentiation and myotube growth. Daidzein prevents dexamethasone-induced myotube atrophy through Akt activation. The promyogenic effect of BS Ext and daidzein is mediated through two promyogenic kinases, Akt and p38MAPK that in turn activates a key myogenic transcription factor MyoD. Additionally, BS Ext and daidzein promote myotube growth through Akt/mTOR/S6K pathway. Finally, daidzein augments MyoD-dependent myogenic conversion of mouse embryonic fibroblasts. Taken together, our data suggest that daidzein promotes myogenic differentiation and myotube hypertrophy through p38 and Akt/mTOR/S6K pathway.

The muscle regulatory transcription factor MyoD participates with p53 to directly increase the expression of the pro-apoptotic Bcl2 family member PUMA.

The muscle regulatory transcription factor MyoD is a master regulator of skeletal myoblast differentiation. We have previously reported that MyoD is also necessary for the elevated expression of the pro-apoptotic Bcl2 family member PUMA, and the ensuing apoptosis, that occurs in a subset of myoblasts induced to differentiate. Herein, we report the identification of a functional MyoD binding site within the extended PUMA promoter. In silico analysis of the murine PUMA extended promoter revealed three potential MyoD binding sites within 2kb of the transcription start site. Expression from a luciferase reporter construct containing this 2kb fragment was enhanced by activation of MyoD in both myoblasts and fibroblasts and diminished by silencing of MyoD in myoblasts. Experiments utilizing truncated versions of this promoter region revealed that the potential binding site at position - 857 was necessary for expression. Chromatin immunoprecipitation (ChIP) analysis confirmed binding of MyoD to the DNA region encompassing position - 857. The increase in MyoD binding to the PUMA promoter as a consequence of culture in differentiation media (DM) was comparable to the increase in MyoD binding at the myogenin promoter and was diminished in myoblasts silenced for MyoD expression. Finally, ChIP analysis using an antibody specific for the transcription factor p53 demonstrated that, in myoblasts silenced for MyoD expression, p53 binding to the PUMA promoter was diminished in response to culture in DM. These data indicate that MyoD plays a direct role in regulating PUMA expression and reveal functional consequences of MyoD expression on p53 mediated transcription of PUMA.

Incomplete MyoD-induced transdifferentiation is associated with chromatin remodeling deficiencies.

Our current understanding of cellular transdifferentiation systems is limited. It is oftentimes unknown, at a genome-wide scale, how much transdifferentiated cells differ quantitatively from both the starting cells and the target cells. Focusing on transdifferentiation of primary human skin fibroblasts by forced expression of myogenic transcription factor MyoD, we performed quantitative analyses of gene expression and chromatin accessibility profiles of transdifferentiated cells compared to fibroblasts and myoblasts. In this system, we find that while many of the early muscle marker genes are reprogrammed, global gene expression and accessibility changes are still incomplete when compared to myoblasts. In addition, we find evidence of epigenetic memory in the transdifferentiated cells, with reminiscent features of fibroblasts being visible both in chromatin accessibility and gene expression. Quantitative analyses revealed a continuum of changes in chromatin accessibility induced by MyoD, and a strong correlation between chromatin-remodeling deficiencies and incomplete gene expression reprogramming. Classification analyses identified genetic and epigenetic features that distinguish reprogrammed from non-reprogrammed sites, and suggested ways to potentially improve transdifferentiation efficiency. Our approach for combining gene expression, DNA accessibility, and protein–DNA binding data to quantify and characterize the efficiency of cellular transdifferentiation on a genome-wide scale can be applied to any transdifferentiation system.

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

Muscle-relevant genes marked by stable H3K4me2/3 profiles and enriched MyoD binding during myogenic differentiation.

Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.

PHRED-1 is a divergent neurexin-1 homolog that organizes muscle fibers and patterns organs during regeneration

Regeneration of body parts requires the replacement of multiple cell types. To dissect this complex process, we utilized planarian flatworms that are capable of regenerating any tissue after amputation. An RNAi screen for genes involved in regeneration of the pharynx identified a novel gene, Pharynx regeneration defective-1 (PHRED-1) as essential for normal pharynx regeneration. PHRED-1 is a predicted transmembrane protein containing EGF, Laminin G, and WD40 domains, is expressed in muscle, and has predicted homologs restricted to other lophotrochozoan species. Knockdown of PHRED-1 causes abnormal regeneration of muscle fibers in both the pharynx and body wall muscle. In addition to defects in muscle regeneration, knockdown of PHRED-1 or the bHLH transcription factor MyoD also causes defects in muscle and intestinal regeneration. Together, our data demonstrate that muscle plays a key role in restoring the structural integrity of closely associated organs, and in planarians it may form a scaffold that facilitates normal intestinal branching.

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient.

Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.