Skeletal myogenesis is required to maintain muscle mass and integrity, and impaired myogenesis is causally linked to the etiology of muscle wasting. Recently, it was shown that excessive uptake of saturated fatty acids (SFA) plays a significant role in the pathogenesis of muscle wasting. Although microRNA (miRNA) is implicated in the regulation of myogenesis, the molecular mechanism whereby SFA-induced miRNAs impair myogenic differentiation remains largely unknown. Here, we investigated the regulatory roles of miR-325-3p on CFL2 expression and myogenic differentiation in C2C12 myoblasts. PA impeded myogenic differentiation, concomitantly suppressed CFL2 and induced miR-325-3p. Dual-luciferase analysis revealed that miR-325-3p directly targets the 3'UTR of CFL2, thereby suppressing the expression of CFL2, a crucial factor for actin dynamics. Transfection with miR-325-3p mimic resulted in the accumulation of actin filaments (F-actin) and nuclear Yes-associated protein (YAP) in myoblasts and promoted myoblast proliferation and cell cycle progression. Consequently, miR-325-3p mimic significantly attenuated the expressions of myogenic factors and thereby impaired the myogenic differentiation of myoblasts. The roles of miR-325-3p on CFL2 expression, F-actin modulation, and myogenic differentiation suggest a novel miRNA-mediated regulatory mechanism of myogenesis and PA-inducible miR-325-3p may be a critical mediator between obesity and muscle wasting.

Actin cytoskeleton dynamics are essential regulatory processes in muscle development, growth, and regeneration due to their modulation of mechanotransduction, cell proliferation, differentiation, and morphological changes. Although the KN motif and ankyrin repeat domain-containing protein 1 (Kank1) plays a significant role in cell adhesion dynamics, actin polymerization, and cell proliferation in various cells, the functional significance of Kank1 during the myogenic differentiation of progenitor cells has not been explored. Here, we report that Kank1 acts as a critical regulator of the proliferation and differentiation of muscle progenitor cells. Kank1 was found to be expressed at a relatively high level in C2C12 myoblasts, and its expression was modulated during the differentiation. Depletion of Kank1 by siRNA (siKank1) increased the accumulation of filamentous actin (F-actin). Furthermore, it facilitated the nuclear localization of Yes-associated protein 1 (YAP1) by diminishing YAP1 phosphorylation in the cytoplasm, which activated the transcriptions of YAP1 target genes and promoted proliferation and cell cycle progression in myoblasts. Notably, depletion of Kank1 suppressed the protein expression of myogenic regulatory factors (i.e., MyoD and MyoG) and dramatically inhibited myoblast differentiation and myotube formation. Our results show that Kank1 is an essential regulator of actin dynamics, YAP1 activation, and cell proliferation and that its depletion impairs the myogenic differentiation of progenitor cells by promoting myoblast proliferation triggered by the F-actin-induced nuclear translocation of YAP1.

Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip's direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological "shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

Transforming Growth Factor β (TGF-β) is involved in fibrosis as well as the regulation of muscle mass, and contributes to the progressive pathology of muscle wasting disorders. However, little is known regarding the time-dependent signalling of TGF-β in myoblasts and myotubes, as well as how TGF-β affects collagen type I expression and the phenotypes of these cells. Here, we assessed effects of TGF-β on gene expression in C2C12 myoblasts and myotubes after 1, 3, 9, 24 and 48 h treatment. In myoblasts, various myogenic genes were repressed after 9, 24 and 48 h, while in myotubes only a reduction in Myh3 expression was observed. In both myoblasts and myotubes, TGF-β acutely induced the expression of a subset of genes involved in fibrosis, such as Ctgf and Fgf-2, which was subsequently followed by increased expression of Col1a1. Knockdown of Ctgf and Fgf-2 resulted in a lower Col1a1 expression level. Furthermore, the effects of TGF-β on myogenic and fibrotic gene expression were more pronounced than those of myostatin, and knockdown of TGF-β type I receptor Tgfbr1, but not receptor Acvr1b, resulted in a reduction in Ctgf and Col1a1 expression. These results indicate that, during muscle regeneration, TGF-β induces fibrosis via Tgfbr1 by stimulating the autocrine signalling of Ctgf and Fgf-2.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase (ADSL), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat, which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. The mechanisms of fish MSTN involved in muscle growth are not fully understood. In the present study, knockdown and overexpression of mstn-1 was performed in cultured Japanese flounder muscle cells to investigate the molecular function and the underlying mechanism of fish MSTN-1. Results showed that mstn-1 knockdown significantly induced cell proliferation and the mRNA expression of myogenic regulatory factors (MRFs), while overexpression of mstn-1 led to a significant decrease of cell proliferation and a suppression of the MRFs mRNA expression. The overexpression of mstn-1 also significantly increased the mRNA expression of ubiquitin-proteasomal pathway of proteolysis genes including muscle RING-finger protein 1 (murf-1) by 204.1% (p = 0.024) and muscle atrophy F-box protein (mafbx) by 165.7% (p = 0.011). However, mystn-1 overexpression inhibited the activation of mTOR signal pathway and the AKT/FoxO1 pathway through decreasing phosphorylation of AKT at Ser 473 by 56.0% (p = 0.001). Meanwhile, mystn-1 overexpression increased the dephosphorylation and nuclear localization of FoxO1 by 394.9% (p = 0.005). These results demonstrate that mstn-1 in Japanese flounder has the effects of inhibiting cell proliferation and growth, and the mTOR and AKT/FoxO1 pathways participated in these biological effects.

In this study, we investigated the effects and mechanisms of the pro-inflammatory cytokines IL-1β and TNF-α on the proliferation and commitment phases of myoblast differentiation. C2C12 mouse myoblast cells were cultured to reach a proliferated or committed status and were incubated with these cytokines for the evaluation of cell proliferation, cyclooxygenase 2 (COX-2) expression, release of prostaglandins (PGs) and myokines, and activation of myogenic regulatory factors (MRFs). We found that inhibition of the IL-6 receptor reduced IL-1β- and TNF-α-induced cell proliferation, and that the IL-1β effect also involved COX-2-derived PGs. Both cytokines modulated the release of the myokines myostatin, irisin, osteonectin, and IL-15. TNF-α and IL-6 reduced the activity of Pax7 in proliferated cells and reduced MyoD and myogenin activity at both proliferative and commitment stages. Otherwise, IL-1β increased myogenin activity only in committed cells. Our data reveal a key role of IL-6 and COX-2-derived PGs in IL-1β and TNF-α-induced myoblast proliferation and support the link between TNF-α and IL-6 and the activation of MRFs. We concluded that IL-1β and TNF-α induce similar effects at the initial stages of muscle regeneration but found critical differences between their effects with the progression of the process, bringing new insights into inflammatory signalling in skeletal muscle regeneration.