Impaired Myogenic Differentiation Research Articles

Assessment of myogenic potency in patient-derived fibroblasts with c.1289-2A>G Desmin mutation

Abstract Objectives The ultra-rare DES c.1289-2A>G mutation, resulting in a 48-base pair insertion in the Desmin tail domain, is associated with late-onset MFM1 (myofibrillar myopathy-1; OMIM number; 601419) and exhibits distinctive pathological features. Despite sustained expression and cytoskeletal integrity, muscle biopsies reveal dystrophic characteristics through an unidentified mechanism. A deeper understanding of the molecular mechanisms underlying Desmin-related MFM1 could enhance our perspective and comprehension of the disease’s pathophysiology. In this study, we aimed to investigate the pathological phenotype by assessing the myogenic potency of MyoD-induced patient-derived fibroblasts. Methods Following the immortalization and myoconversion of unaffected and patient-derived fibroblast cells, we analyzed the myogenic potency of the mutant and control groups on day 5 post-differentiation. This analysis involved staining cells with MF20 antibody and DAPI after MyoD induction. Results Employing six parameters to quantify extra nuclei and myotube properties, we unveil impaired myogenic differentiation in c.1289-2A>G mutant cells, as evidenced by a compromised fusion index and distinctive myogenic features. In summary, our preliminary findings indicate phenotypic abnormalities and suggest an association between the DES c.1289-2A>G mutation and delayed maturation and MFM in affected individuals. Conclusions Our results indicate a significant involvement of Desmin in the myogenic maturation of muscle cells. Further investigation is required to understand the changes in the transcriptome during the myoconversion of patient-derived fibroblasts.

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

BackgroundRhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.ResultsWe demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.ConclusionsThe results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Induction of miR-665-3p Impairs the Differentiation of Myogenic Progenitor Cells by Regulating the TWF1-YAP1 Axis.

Actin dynamics are known to orchestrate various myogenic processes in progenitor cells. Twinfilin-1 (TWF1) is an actin-depolymerizing factor that plays a crucial role in the differentiation of myogenic progenitor cells. However, little is known about the mechanisms underlying the epigenetic regulation of TWF1 expression and impaired myogenic differentiation in the background of muscle wasting. This study investigated how miR-665-3p affects TWF1 expression, actin filaments' modulation, proliferation, and myogenic differentiation in progenitor cells. Palmitic acid, the most prevalent saturated fatty acid (SFA) in food, suppressed TWF1 expression and inhibited the myogenic differentiation of C2C12 cells while increasing the level of miR-665-3p expression. Interestingly, miR-665-3p inhibited TWF1 expression by targeting TWF1 3'UTR directly. In addition, miR-665-3p accumulated filamentous actin (F-actin) and enhanced the nuclear translocation of Yes-associated protein 1 (YAP1), consequently promoting cell cycle progression and proliferation. Furthermore, miR-665-3p suppressed the expressions of myogenic factors, i.e., MyoD, MyoG, and MyHC, and consequently impaired myoblast differentiation. In conclusion, this study suggests that SFA-inducible miR-665-3p suppresses TWF1 expression epigenetically and inhibits myogenic differentiation by facilitating myoblast proliferation via the F-actin/YAP1 axis.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.

Interleukin-11 (IL11) inhibits myogenic differentiation of C2C12 cells through activation of extracellular signal-regulated kinase (ERK)

Cancer-associated cachexia (CAC) is a multifactorial wasting syndrome characterized by loss of skeletal muscle. Interleukin-11 (IL11), one of the IL6 family cytokines, is highly expressed in various types of cancer including cancers frequently associated with cachexia. However, the impact of IL11 on muscle metabolism remains to be determined. Since one of the mechanisms of muscle wasting in cachexia is defective muscle regeneration due to impaired myogenic differentiation, we examined the effect of IL11 on the differentiation of C2C12 mouse myoblasts. Treatment of C2C12 cells with recombinant mouse IL11 resulted in decreased myotube formation. In addition, IL11 treatment reduced the protein and mRNA levels of myosin heavy chain (MHC), a marker of myogenic differentiation. Moreover, the levels of myogenic regulatory factors including myogenin and Mrf4 were significantly reduced by IL11 treatment. IL11 treatment increased the number of BrdU-positive cells and the level of phosphorylated retinoblastoma (Rb) protein, while the levels of p21Waf1 and p27Kip1 were reduced by IL11 treatment in differentiating C2C12 cells, suggesting that IL11 interferes with cell cycle exit during the early stages of myogenic differentiation. Consistent with this, IL11 treatment at the late stage of differentiation did not affect myotube formation and MHC expression. IL11 treatment resulted in an activation of ERK, STAT3, and AKT in differentiating C2C12 cells. However, only ERK inhibitors including PD98059 and U0126 were able to ameliorate the suppressive effect of IL11 on the expression of MHC and myogenin. Additionally, pretreatment with PD98059 and U0126 resulted in improved myotube formation and reduced BrdU staining in IL11-treated cells. Together, our results suggest that IL11 inhibits myogenic differentiation through delayed cell cycle exit in an ERK-dependent manner. To our knowledge, this study is the first to demonstrate an inhibitory role of IL11 in myogenic differentiation and identifies the previously unrecognized role of IL11 as a possible mediator of CAC.

Myogenetic Oligodeoxynucleotide Restores Differentiation and Reverses Inflammation of Myoblasts Aggravated by Cancer-Conditioned Medium

Cancer cachexia is characterized by irreversible muscle loss which is a critical factor in the prognosis of cancer patients. Myoblasts are myogenic precursor cells that are required to maintain skeletal muscle tissue. Previous studies reported that cancer-released factors deteriorate myoblast differentiation, which is one of the causes of cachexia-associated muscle wasting. We recently identified the myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes myogenesis. The present study investigated the effects of iSN04 on human myoblasts exposed to a conditioned medium (CM) of cancer cells. CM of colon cancer cell lines LoVo and HCT-116 significantly impaired myogenic differentiation and the myotube formation of human myoblasts by inducing the expression of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-α (TNF-α); however, the CM of the colon fibroblast cell line CCD-18Co did not. Intriguingly, iSN04 completely reversed the deterioration of myoblast differentiation by LoVo-CM by upregulating MyoD and myogenin, and downregulating myostatin, IL-1β, and TNF-α. TNF-α, of which a high level was produced in LoVo, alone inhibited myogenic differentiation and induced IL-1β, IL-6, and IL-8 transcriptions of myoblasts; however, pre-treatment with iSN04 reversed TNF-α-induced cachectic phenotypic features. The results indicate that iSN04 protects myoblasts against the effects of cancer-released factors and maintains their myogenic activity. This study provides a novel therapeutic strategy to prevent muscle loss associated with cancer cachexia.

An interplay between BRD4 and G9a regulates skeletal myogenesis.

Histone acetylation and methylation are epigenetic modifications that are dynamically regulated by chromatin modifiers to precisely regulate gene expression. However, the interplay by which histone modifications are synchronized to coordinate cellular differentiation is not fully understood. In this study, we demonstrate a relationship between BRD4, a reader of acetylation marks, and G9a, a writer of methylation marks in the regulation of myogenic differentiation. Using loss- and gain-of-function studies, as well as a pharmacological inhibition of its activity, we examined the mechanism by which BRD4 regulates myogenesis. Transcriptomic analysis using RNA sequencing revealed that a number of myogenic differentiation genes are downregulated in Brd4-depleted cells. Interestingly, some of these genes were upregulated upon G9a knockdown, indicating that BRD4 and G9a play opposing roles in the control of myogenic gene expression. Remarkably, the differentiation defect caused by Brd4 knockdown was rescued by inhibition of G9a methyltransferase activity. These findings demonstrate that the absence of BRD4 results in the upregulation of G9a activity and consequently impaired myogenic differentiation. Collectively, our study identifies an interdependence between BRD4 and G9a for the precise control of transcriptional outputs to regulate myogenesis.

Abstract 668: A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo

Abstract Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 668.

M6A Methylases Regulate Myoblast Proliferation, Apoptosis and Differentiation.

Simple SummaryN6-methyladenosine (m6A) is the most prevalent methylation modification in eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies found that m6A methylation plays a role in mammalian skeletal muscle development. Skeletal muscle is an important factor that regulates livestock muscle quality and maintains metabolic homeostasis, and skeletal myogenesis is regulated by a series of transcription factors. However, the role of m6A in bovine skeletal myogenesis is unclear. In this study, we examined the expression patterns of the m6A methylase genes METTL3, METTL14, WTAP, FTO and ALKBH5 in bovine skeletal muscle tissue and during myogenesis in myoblasts. Furthermore, we used bovine skeletal muscle myoblasts as the object of study to discover the regulatory role of these genes in the process of skeletal myogenesis in vitro. Our findings indicate that these five m6A methylases have pronounced and diverse functions in regulating bovine skeletal myoblast proliferation, apoptosis and myogenic differentiation, which can contribute to further understanding the roles of m6A in skeletal muscle development.N6-methyladenosine (m6A) plays an important role in regulating gene expression. Previous studies found that m6A methylation affects skeletal muscle development. However, the effect of m6A methylases on bovine skeletal myogenesis is still unclear. Here, we found that the expression of m6A demethylases (FTO and ALKBH5) was significantly higher in the longissimus dorsi muscle of adult cattle than in newborn cattle. In contrast, the expression of m6A methyltransferases (METTL3, METTL14 and WTAP) was reduced. The mRNA expression of all five genes was found to be increased during the myogenesis of myoblasts in vitro. Knockdown of FTO or METTL3 promoted myoblast proliferation, inhibited myoblast apoptosis and suppressed myogenic differentiation, whereas ALKBH5 knockdown had the opposite effect. METTL14 knockdown enhanced myoblast proliferation and impaired myogenic differentiation. WTAP knockdown attenuated proliferation and contributed to apoptosis but did not affect differentiation. Furthermore, the functional domains of these five m6A methylases are conserved across species. Our results suggest that m6A methylases are involved in regulating skeletal muscle development and that there may be a complex network of m6A methylation regulating skeletal myogenesis.

Palmitic Acid-Induced miR-429-3p Impairs Myoblast Differentiation by Downregulating CFL2.

MicroRNAs are known to play a critical role in skeletal myogenesis and maintenance, and cofilin-2 (CFL2) is necessary for actin cytoskeleton dynamics and myogenic differentiation. Nonetheless, target molecules and the modes of action of miRNAs, especially those responsible for the inhibitory mechanism on the myogenesis by saturated fatty acids (SFA) or obesity, still remain unclear. Here, we reported the role played by miR-429-3p on CFL2 expression, actin filament dynamics, myoblast proliferation, and myogenic differentiation in C2C12 cells. Palmitic acid (PA), the most abundant SFA in diet, inhibited the myogenic differentiation of myoblasts, accompanied by CFL2 reduction and miR-429-3p induction. Interestingly, miR-429-3p suppressed the expression of CFL2 by targeting the 3′UTR of CFL2 mRNA directly. Transfection of miR-429-3p mimic in myoblasts increased F-actin formation and augmented nuclear YAP level, thereby promoting cell cycle progression and myoblast proliferation. Moreover, miR-429-3p mimic drastically suppressed the expressions of myogenic factors, such as MyoD, MyoG, and MyHC, and impaired myogenic differentiation of C2C12 cells. Therefore, this study unveiled the crucial role of miR-429-3p in myogenic differentiation through the suppression of CFL2 and provided implications of SFA-induced miRNA in the regulation of actin dynamics and skeletal myogenesis.

Myogenetic Oligodeoxynucleotide (myoDN) Recovers the Differentiation of Skeletal Muscle Myoblasts Deteriorated by Diabetes Mellitus.

Skeletal muscle wasting in patients with diabetes mellitus (DM) is a complication of decreased muscle mass and strength, and is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle wasting. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8 (IL-8). iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat muscle wasting in patients with DM.

Propionate hampers differentiation and modifies histone propionylation and acetylation in skeletal muscle cells

Protein acylation via metabolic acyl-CoA intermediates provides a link between cellular metabolism and protein functionality. A process in which acetyl-CoA and acetylation are fine-tuned is during myogenic differentiation. However, the roles of other protein acylations remain unknown. Protein propionylation could be functionally relevant because propionyl-CoA can be derived from the catabolism of amino acids and fatty acids and was shown to decrease during muscle differentiation. We aimed to explore the potential role of protein propionylation in muscle differentiation, by mimicking a pathophysiological situation with high extracellular propionate which increases propionyl-CoA and protein propionylation, rendering it a model to study increased protein propionylation. Exposure to extracellular propionate, but not acetate, impaired myogenic differentiation in C2C12 cells and propionate exposure impaired myogenic differentiation in primary human muscle cells. Impaired differentiation was accompanied by an increase in histone propionylation as well as histone acetylation. Furthermore, chromatin immunoprecipitation showed increased histone propionylation at specific regulatory myogenic differentiation sites of the Myod gene. Intramuscular propionylcarnitine levels are higher in old compared to young males and females, possibly indicating increased propionyl-CoA levels with age. The findings suggest a role for propionylation and propionyl-CoA in regulation of muscle cell differentiation and ageing, possibly via alterations in histone acylation.

Enhancement of myogenic differentiation and inhibition of rhabdomyosarcoma progression by miR-28-3p and miR-193a-5p regulated by SNAIL

Rhabdomyosarcoma (RMS) is a soft tissue mesenchymal tumor that affects mostly children and adolescents. It originates from the impaired myogenic differentiation of stem cells or early progenitors. SNAIL, a transcription factor that regulates epithelial-to-mesenchymal transition in tumors of epithelial origin, is also a key regulator of RMS growth, progression, and myogenic differentiation. Here, we demonstrate that the SNAIL-dependent microRNAs (miRNAs) miR-28-3p and miR-193a-5p are crucial regulators of RMS growth, differentiation, and progression. miR-28-3p and miR-193a-5p diminished proliferation and arrested RMS cells in G0/G1 phase in vitro. They induced the myogenic differentiation of both RMS cells and human myoblasts by upregulating myogenic factors. Furthermore, miR-28-3p and miR-193a-5p inhibited migration in a scratch assay, adhesion to endothelial cells, chemotaxis, and invasion toward SDF-1 and HGF and regulated angiogenic capabilities of the cells. Overexpression of miR-28-3p and miR-193a-5p induced formation of fibrotic structures and abnormal blood vessels in RMS xenografts in immunodeficient mice in vivo. Simultaneous overexpression of both miRNAs diminished tumor growth after subcutaneous implantation and inhibited the engraftment of RMS cells into bone marrow after intravenous injection in vivo. To conclude, we discovered novel SNAIL-dependent miRNAs that may become new therapeutic targets in RMS in the future.

The phosphatidylcholine transfer protein StarD7 is important for myogenic differentiation in mouse myoblast C2C12 cells and human primary skeletal myoblasts

StarD7 is a phosphatidylcholine (PC)-specific lipid transfer protein essential for the maintenance of mitochondrial PC composition, morphogenesis, and respiration. Here, we studied the role of StarD7 in skeletal myoblast differentiation using mouse myoblast C2C12 cells and human primary myoblasts. Immunofluorescence and immuno-electron microscopy revealed that StarD7 was distributed in the cytosol, inner mitochondria space, and outer leaflet of the outer mitochondrial membrane in C2C12 cells. Unlike human kidney embryonic cell line HEK293 cells, the mitochondrial proteinase PARL was not involved in the processing and maturation of StarD7 in C2C12 cells. StarD7 was constantly expressed during myogenic differentiation of C2C12 cells. The siRNA-mediated knockdown of StarD7 in C2C12 cells and human primary myoblasts significantly impaired myogenic differentiation and reduced the expression of myomaker, myomerger and PGC-1α. The reduction in mitochondrial PC levels and oxygen consumption rates, decreased expression of myomaker, myomerger and PGC-1α, as well as impaired myogenic differentiation, were completely restored when the protein was reintroduced into StarD7-knockout C2C12 cells. These results suggest that StarD7 is important for skeletal myogenesis in mammals.

Effect of sarcolipin-mediated cell transdifferentiation in sarcopenia-associated skeletal muscle fibrosis

Fibrosis is a key pathological event during muscle aging that accelerates the development of sarcopenia. We show that sarcolipin (SLN) is highly expressed during aging, promotes intracellular calcium overload and participates in impaired myogenic differentiation. d-Galactose (D-gal) was used to induce senescence in C2C12 myoblasts. Conventional AAV-mediated SLN knockdown cells were used to study the role of SLN in muscle physiology and pathophysiology. C2C12 cells were treated with D-gal, which promoted fibrosis and SLN upregulation. The expression of TGF-β1 and α-SMA, which participate in myogenic transdifferentiation, were also elevated. C2C12 cells with reduced sarcolipin expression produced decreased amounts of collagen. Our study identified an unrecognized role of SLN in regulating myogenic transdifferentiation during aging-associated skeletal muscle cell fibrosis. Targeting SLN may be a novel therapeutic strategy to relieve sarcopenia-associated muscle fibrosis.

Altered dosage of noncoding RNAs expressed from the Dlk1‐Dio3 locus impairs skeletal muscle differentiation

Noncoding RNAs (ncRNAs) have emerged as key components of gene regulatory networks, yet their role in skeletal muscle diseases is still poorly understood. The maternally imprinted Dlk1‐Dio3 ncRNA locus has been shown to play an essential role in skeletal muscle development, regeneration, and metabolism. The diverse ncRNAs expressed from this locus, including over 50 microRNAs (miRNAs), several long noncoding RNAs (lncRNAs), and a cluster of C/D box snoRNAs, are thought to be transcribed as a ~200 kb polycistron initiating from the proximal promoter immediately upstream of the Gtl2 lncRNA, the most 5′ ncRNA in the locus. This project aimed to understand the importance of proper dosage of the Dlk1‐Dio3 ncRNAs in skeletal muscle differentiation. Toward this end, we utilized CRISPR‐Cas9 gene editing to generate deletions in the Gtl2 proximal promoter and the overlapping differentially methylated region (DMR) with the goal of modulating expression of the maternally expressed ncRNAs in C2C12 myoblasts, an established muscle cell line. This gene editing strategy yielded a clonal population of C2C12 myoblasts that harbored a deletion spanning a portion of the Gtl2 proximal promoter while leaving the Gtl2 promoter's TATA box intact. This CRISPR‐edited cell line showed a dramatic increase in the Dlk1‐Dio3 ncRNAs. As a result of Dlk1‐Dio3 ncRNA upregulation, this C2C12 cell line displays significantly impaired myogenic differentiation. The continued characterization of this novel C2C12 cell line will help elucidate the importance of maintaining proper dosage of the Dlk1‐Dio3 locus in skeletal muscle, which could serve as a therapeutic target for treating muscle diseases.Support or Funding InformationThis project was supported with funds from the Arnold &amp; Mabel Beckman Foundation and the Boston University Undergraduate Research Opportunities ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of the interferon type I response rather than autophagy contributes to myogenesis inhibition in congenital DM1 myoblasts

Congenital myotonic dystrophy type 1 (CDM1) is characterized by severe symptoms that affect patients from birth, with 40% mortality in the neonatal period and impaired skeletal muscle development. In this paper, we examined the relationship between autophagy and abnormal myogenic differentiation of CDM1 myoblasts. We investigated these pathological features at both ultrastructural and molecular levels, utilizing two CDM1 foetal myoblasts, CDM13 and CDM15, with 1800 and 3200 repeats, respectively. The congenital nature of these CDM1 myoblasts was confirmed by the high methylation level at the DMPK locus. Our results indicated that abnormal autophagy was independent of myogenic differentiation, as CDM13 myoblasts differentiated as well as control myoblasts but underwent autophagy like CDM15, displaying impaired differentiation. miRNA expression profiles revealed that CDM15 myoblasts failed to upregulate the complex network of myo-miRNAs under MYOD and MEF2A control, while this network was upregulated in CDM13 myoblasts. Interestingly, the abnormal differentiation of CDM15 myoblasts was associated with cellular stress accompanied by the induction of the interferon type 1 pathway (innate immune response). Indeed, inhibition of the interferon (IFN) type I pathway restores myogenic differentiation of CDM15 myoblasts, suggesting that the inappropriate activation of the innate immune response might contribute to impaired myogenic differentiation and severe muscle symptoms observed in some CDM1 patients. These findings open up the possibility of new therapeutic approaches to treat CDM1.

PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6-19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.

NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings

Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.