TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis.

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

Dual function of VGLL4 in muscle regeneration.

VGLL4 has previously been identified as a negative regulator of YAP. Here we show that VGLL4 regulates muscle regeneration in both YAP-dependent and YAP-independent manners at different stages. Knockout of VGLL4 in mice leads to smaller myofiber size and defective muscle contraction force. Furthermore, our studies reveal that knockout of VGLL4 results in increased muscle satellite cells proliferation and impaired myoblast differentiation, which ultimately leads to delayed muscle regeneration. Mechanistically, the results show that VGLL4 works as a conventional repressor of YAP at the proliferation stage of muscle regeneration. At the differentiation stage, VGLL4 acts as a co-activator of TEAD4 to promote MyoG transactivation and facilitate the initiation of differentiation in a YAP-independent manner. Moreover, VGLL4 stabilizes the protein-protein interactions between MyoD and TEAD4 to achieve efficient MyoG transactivation. Our findings define the dual roles of VGLL4 in regulating muscle regeneration at different stages and may open novel therapeutic perspectives for muscle regeneration.

Natural antisense transcript of MYOG regulates development and regeneration in skeletal muscle by shielding the binding sites of MicroRNAs of MYOG mRNA 3'UTR.

Natural antisense transcripts (NATs) are endogenous RNAs opposite to sense transcripts, and they can significantly contribute to regulating various biological processes through multiple epigenetic mechanisms. NATs can affect their sense transcripts to regulate the growth and development of skeletal muscle. Our analysis of third-generation full-length transcriptome sequencing data revealed that NATs represented a significant portion of the lncRNA, accounting for up to 30.19%-33.35%. The expression of NATs correlated with myoblast differentiation, and genes expressing NATs were mainly involved in RNA synthesis, protein transport, and cell cycle. We found a NAT of MYOG (MYOG-NAT) in the data. We found that the MYOG-NAT could promote the differentiation of myoblasts in vitro. Additionally, knockdown of MYOG-NAT in vivo led to muscle fiber atrophy and muscle regeneration retardation. Molecular biology experiments demonstrated that MYOG-NAT enhances the stability of MYOG mRNA by competing with miR-128-2-5p, miR-19a-5p, and miR-19b-5p for binding to MYOG mRNA 3'UTR. These findings suggest that MYOG-NAT plays a critical role in skeletal muscle development and provides insights into the post-transcriptional regulation of NATs.

The isolated effects of local cold application on proteolytic and myogenic signaling.

Post-exercise cooling studies reveal inhibitory effects on markers of skeletal muscle growth. However, the isolated effect of local cold application has not been adequately addressed. It is unclear if the local cold or the combination of local cold and exercise is driving negatively altered skeletal muscle gene expression. The purpose was to determine the effects of a 4 h local cold application to the vastus lateralis on the myogenic and proteolytic response. Participants (n = 12, 27 ± 6 years, 179 ± 9 cm, 82.8 ± 13.0 kg, 18.4 ± 7.1 %BF) rested with a thermal wrap placed on each leg with either circulating cold fluid (10 °C, COLD) or no fluid circulation (room temperature, RT). Muscle samples were collected to quantify mRNA (RT-qPCR) and proteins (Western Blot) associated with myogenesis and proteolysis. Temperatures in COLD were lower than RT at the skin (13.2 ± 1.0 °C vs. 34.8 ± 0.9 °C; p < 0.001) and intramuscularly (20.5 ± 1.3 °C vs. 35.6 ± 0.8 °C, p < 0.001). Myogenic-related mRNA, MYO-G and MYO-D1, were lower in COLD (p = 0.001, p < 0.001, respectively) whereas myogenic-mRNA, MYF6, was greater in COLD (p = 0.002). No other myogenic associated genes were different between COLD and RT (MSTN, p = 0.643; MEF2a, p = 0.424; MYF5, p = 0.523; RPS3, p = 0.589; RPL3-L, p = 0.688). Proteolytic-related mRNA was higher in COLD (FOXO3a, p < 0.001; Atrogin-1, p = 0.049; MURF-1, p < 0.001). The phosphorylation:total protein ratio for the translational repressor of muscle mass, 4E-BP1Thr37/46, was lower in COLD (p = 0.043), with no differences in mTORser2448 (p = 0.509) or p70S6K1Thr389 (p = 0.579). Isolated local cooling over 4 h exhibits inhibited myogenic and higher proteolytic skeletal muscle molecular response.

Phosphatase orphan 1 inhibits myoblast proliferation and promotes myogenic differentiation.

Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.

Myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Angiotensin II (Ang II), an important component of the renin-angiotensin system (RAS), plays a critical role in the pathogenesis of cardiovascular disorders. In addition, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been considered as a promising platform for studying personalized medicine for heart diseases. However, whether Ang II can induce the apoptosis of hiPSC-CMs is not known. METHODS: In this study, we treated hiPSC-CMs with different concentrations of Ang II [0 nM (vehicle as a control), 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 1 mM] for various time periods (24 h, 48 h, 6 days, and 10 days) and analyzed the viability and apoptosis of hiPSC-CMs. RESULTS: We found that treatment with 1 mM Ang II for 10 days reduced the viability of hiPSC-CMs by 41% (p = 2.073E-08) and increased apoptosis by 2.74-fold, compared to the control group (p = 6.248E-12). MYOG, which encodes the muscle-specific transcription factor myogenin, was also identified as an apoptosis-suppressor gene in Ang II-treated hiPSC-CMs. Ectopic MYOG expression decreased the apoptosis and increased the viability of Ang II-treated hiPSC-CMs. Further analysis of the RNA sequencing (RNA-seq) data illustrated that myogenin ameliorated Ang II-induced apoptosis of hiPSC-CMs by downregulating the expression of proinflammatory genes. CONCLUSION: Our findings suggest that Ang II induces the apoptosis of hiPSC-CMs and that myogenin attenuates Ang II-induced apoptosis.

Differences in the Role of HDACs 4 and 5 in the Modulation of Processes Regulating MAFbx and MuRF1 Expression during Muscle Unloading.

Unloading leads to skeletal muscle atrophy via the upregulation of MuRF-1 and MAFbx E3-ligases expression. Reportedly, histone deacetylases (HDACs) 4 and 5 may regulate the expression of MuRF1 and MAFbx. To examine the HDAC-dependent mechanisms involved in the control of E3-ubiquitin ligases expression at the early stages of muscle unloading we used HDACs 4 and 5 inhibitor LMK-235 and HDAC 4 inhibitor Tasqinimod (Tq). Male Wistar rats were divided into four groups (eight rats per group): nontreated control (C), three days of unloading/hindlimb suspension (HS) and three days HS with HDACs inhibitor LMK-235 (HSLMK) or Tq (HSTq). Treatment with LMK-235 diminished unloading-induced of MAFbx, myogenin (MYOG), ubiquitin and calpain-1 mRNA expression (p < 0.05). Tq administration had no effect on the expression of E3-ligases. The mRNA expression of MuRF1 and MAFbx was significantly increased in both HS and HSTq groups (1.5 and 4.0 folds, respectively; p < 0.05) when compared with the C group. It is concluded that during three days of muscle unloading: (1) the HDACs 4 and 5 participate in the regulation of MAFbx expression as well as the expression of MYOG, ubiquitin and calpain-1; (2) the inhibition of HDAC 4 has no effect on MAFbx expression. Therefore, HDAC 5 is perhaps more important for the regulation of MAFbx expression than HDAC 4.

WDR13 promotes the differentiation of bovine skeletal muscle-derived satellite cells by affecting PI3K/AKT signaling.

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet ß cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle-derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.

Transcription factor EGR1 promotes differentiation of bovine skeletal muscle satellite cells by regulating MyoG gene expression.

The transcription factor, early growth response 1 (EGR1), has important roles in various cell types in response to different stimuli. EGR1 is thought to be involved in differentiation of bovine skeletal muscle-derived satellite cells (MDSCs); however, the precise effects of EGR1 on differentiation of MDSCs and its mechanism of action remain unknown. In the present study, a time course of EGR1 expression and the effects of EGR1 on MDSC differentiation were determined. The results demonstrated that the expression of EGR1 mRNA and protein increased significantly in differentiating MDSCs relative to that in proliferating cells. Over-expression of the EGR1 gene in MDSCs promoted their differentiation and inhibited proliferation. Conversely, knock-down of EGR1 inhibited differentiation of MDSCs and promoted their proliferation, indicating that EGR1 promotes MDSC differentiation. Moreover, over-expression of EGR1 in MDSCs increased the expression of MyoG mRNA and protein, whereas its knock-down had the opposite effect. Furthermore, ChIP-PCR analyses demonstrated that EGR1 could bind directly to its putative binding site within the promoter region of MyoG, and determination of ERG1 subcellular localization in MDSCs demonstrated that it could relocate to the nucleus, indicating MyoG is likely an EGR1 target gene whose expression is positively regulated by this transcription factor. In conclusion, EGR1 can promote MDSC differentiation through positive regulation of MyoG gene expression.

Over-expression of DEC1 inhibits myogenic differentiation by modulating MyoG activity in bovine satellite cell.

Differentiated embryo chondrocyte 1 (DEC1), a member of basic-helix-loop-helix transcription factor Bhlhe40, also called stimulated by retinoic acid 13, STRA13, plays an important role in the regulation of adipogenesis, tumorigenesis, peripheral circadian output, response to hypoxia, and development of metabolic syndrome. Previous studies suggested that DEC1 was involved in skeletal muscle development; however, its precise role in myoblast differentiation has not been determined. In the present study, we showed that DEC1 expressed ubiquitously in different bovine tissues and was down-regulated in differentiated bovine satellite cells. Expression of muscle specific transcription factors (Myf5, MyoD, MyoG, and MHC) was significantly down-regulated when DEC1 was over-expressed by both CoCl2 -simulated hypoxia and Adenovirus-mediated transduction in bovine satellite cells. Consistent with that, promoter analyses via luciferase reporter assay also revealed that overexpression of bovine DEC1 could inhibit MyoG promoter activity. In conclusion, overexpression of DEC1 blocked myogenesis by inhibiting MyoG promoter activity in bovine. Our results provided a new mechanism for the muscle growth, which would contribute to increase cattle meat productivity.

Time course and side-by-side analysis of mesodermal, pre-myogenic, myogenic and differentiated cell markers in the chicken model for skeletal muscle formation.

The chicken is a well-established model for amniote (including human) skeletal muscle formation because the developmental anatomy of chicken skeletal muscle matches that of mammals. The accessibility of the chicken in the egg as well as the sequencing of its genome and novel molecular techniques have raised the profile of this model. Over the years, a number of regulatory and marker genes have been identified that are suited to monitor the progress of skeletal myogenesis both in wildtype and in experimental embryos. However, in the various studies, differing markers at different stages of development have been used. Moreover, contradictory results on the hierarchy of regulatory factors are now emerging, and clearly, factors need to be able to cooperate. Thus, a reference paper describing in detail and side-by-side the time course of marker gene expression during avian myogenesis is needed. We comparatively analysed onset and expression patterns of the key markers for the chicken immature paraxial mesoderm, for muscle-competent cells, for cells committed to myogenesis and for cells entering terminal differentiation. We performed this analysis from stages when the first paraxial mesoderm is being laid down to the stage when mesoderm formation comes to a conclusion. Our data show that, although the sequence of marker gene expression is the same at the various stages of development, the timing of the expression onset is quite different. Moreover, marker gene expression in myogenic cells being deployed from the dorsomedial and ventrolateral lips of the dermomyotome is different from those being deployed from the rostrocaudal lips, suggesting different molecular programs. Furthermore, expression of Myosin Heavy Chain genes is overlapping but different along the length of a myotube. Finally, Mef2c is the most likely partner of Mrf proteins, and, in contrast to the mouse and more alike frog and zebrafish fish, chicken Mrf4 is co-expressed with MyoG as cells enter terminal differentiation.

Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion.

The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3' UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.

Expression of Genes Related to Meat Productivity, Metabolic and Morphological Significance of Broiler Chickens with the Use of Nutritional Phytochemicals.

The study aimed to analyze gene expression linked to skeletal muscle growth and lipid metabolism in broiler chickens fed with plant extracts. Five groups of chickens were formed: four experimental groups and one control group. The diets of the experimental groups were supplemented with different plant extracts: chicory, St. John's wort, maral root, and creeping thyme, whereas the control group received feed without phytobiotic compounds. Weekly weighings were conducted (n = 36). The chickens were slaughtered at day 26 for tissue sampling of four birds from each group. Gene expression (MYOG, MSTN, FASN) related to muscle growth and fatty acid synthesis was analyzed using the ß-actin ACTB gene as a reference. Blood samples were taken at day 35 for biochemical analysis and anatomical dissection was performed. The study revealed that using plant extracts from chicory, thyme, and maral root increased MYOG gene activity by 4.21, 7.45, and 8.93 times, respectively. T. serpyllum extract boosted the MSTN gene by 10.93 times, impacting muscle growth regulation. FASN gene expression for fatty acid synthesis increased significantly by 18.22-184.12 times with plant extracts. The best results regarding meat productivity of chickens were obtained when using R. carthamoides extract. The results of the study will serve as a basis for further development of a phytocomposition designed to increase the meat productivity of broiler chickens in the production of environmentally safe poultry products.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.

MyoG-enhanced circGPD2 regulates chicken skeletal muscle development by targeting miR-203a.

Circular RNAs (circRNAs) are a subclass of RNA macromolecules that are reported to be involved in the regulation of skeletal muscle development. However, the functions and regulatory mechanisms of circRNAs in chicken myogenesis are still largely unclear. Here, we identified a novel circRNA, circGPD2, an RNA macromolecule with a calculated molecular weight of 215 kDa. We discovered that circGPD2 is a muscle-specific circRNA and is strongly expressed in the breast muscle of broilers by utilizing the comparison model of layers and broilers. Functional analysis revealed circGPD2 has a positive role in the proliferation and differentiation of myoblasts, and circGPD2 performs function through the release of the inhibition effect of miR-203a on c-JUN and MEF2C. Besides, the myogenic regulatory factor MyoG enhanced the expression of circGPD2 by targeting the E-box element on the GPD2 promoter. Importantly, lentivirus-mediated circGPD2 knockdown resulted in the breast muscle mass loss of the chicks. Overall, we revealed the crucial role of circGPD2 in chicken myogenesis in vitro and in vivo, and analyzed the upstream and downstream regulation mechanisms of circGPD2. Our study provides an attractive target for molecular marker-assisted breeding to improve the meat yield in the chicken meat industry.

Interaction of MyoD and MyoG with Myoz2 gene in bovine myoblast differentiation.

This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (-159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region -159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.

Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation.

In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.

Lnc-GD2H Promotes Proliferation by Forming a Feedback Loop With c-Myc and Enhances Differentiation Through Interacting With NACA to Upregulate Myog in C2C12 Myoblasts.

In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders.

Leucine Supplementation Decreases HDAC4 Expression and Nuclear Localization in Skeletal Muscle Fiber of Rats Submitted to Hindlimb Immobilization.

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells.

The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.