Myoblast Apoptosis Research Articles

Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through Ca2+ overloading.

Mechanical stretch exerted pro-apoptotic effect on myoblasts, the mechanism of which is currently unknown. Intracellular Ca2+ accumulation has been implicated in stretch-induced apoptosis. calreticulin (CRT) and plasma membrane Ca2+ transporting ATPase 1 (PMCA1) are two critical components of Ca2+ signaling system participating in intracellular Ca2+ homeostasis. In this study, we explored the contribution of CRT and PMCA1 in mediating stretch-induced Ca2+ accumulation and apoptosis of myoblasts. Stretching stimuli elevated level of CRT while inhibited activity of PMCA1. Moreover, there were bidirectional regulations between CRT and PMCA1, which formed the positive feedback loop leading to continuous increment of CRT level and repression of PMCA1 activity, in stretched myoblasts. Specifically, increased CRT level inhibited PMCA1 activity via suppressing Calmodulin (CaM), while reduced PMCA1 activity promoted CRT expression through activating p38MAPK pathway. Thus, the CRT-CaM-PMCA1 and PMCA1-p38MAPK-CRT pathways constituted a close cycle comprising CRT, PMCA1, CaM and p38MAPK. Inhibition of both CaM and p38MAPK affected the other three factors in stretched myoblasts. Circulation of the vicious cycle resulted in escalated Ca2+ overloading in myoblasts under continuous stretching stimuli. CRT knock-down, PMCA1 overexpression, and p38MAPK inhibition all attenuated the raised intracellular Ca2+ level and ameliorated myoblast apoptosis in the stretching environment. Conversely, CRT overexpression, PMCA1 knock-down, and CaM inhibition all aggravated stretch-induced Ca2+ overloading and myoblast apoptosis. A positive feedback loop between CRT and PMCA1 was activated in stretched myoblasts, which contributed to intracellular Ca2+ accumulation and resultant myoblast apoptosis.

VMP1 Regulated by chi-miR-124a Effects Goat Myoblast Proliferation, Autophagy, and Apoptosis through the PI3K/ULK1/mTOR Signaling Pathway.

The production of goat meat is determined by the growth speed of muscle fibers, and the autophagy and apoptosis of myoblast cells is a crucial process in the growth of muscle fibers. The rapid growth of muscle fibers occurs from one month old to nine months old in goats; however, the mechanisms of myoblast cells’ autophagy and apoptosis in this process are still unknown. To identify candidate genes and signaling pathway mechanisms involved in myoblast apoptosis and autophagy, we compared the expression characteristics of longissimus dorsi tissues from Wu’an goats—a native goat breed of China—at 1 month old (mon1 group) and 9 months old (mon9 group). Herein, a total of 182 differentially expressed mRNAs (DEGs) in the mon1 vs. mon9 comparison, along with the KEGG enrichments, showed that the PI3K-Akt pathway associated with autophagy and apoptosis was significantly enriched. Among these DEGs, expression of vacuole membrane protein 1 (VMP1)—a key gene for the PI3K-Akt pathway—was significantly upregulated in the older goats relative to the 1-month-old goats. We demonstrated that VMP1 promotes the proliferation and autophagy of myoblasts, and inhibits their apoptosis. The integration analysis of miRNA–mRNA showed that miR-124a was a regulator of VMP1 in muscle tissue, and overexpression and inhibition of miR-124a suppressed the proliferation and autophagy of myoblasts. The PI3K/Akt/mTOR pathway was an important pathway for cell autophagy. Additionally, the activator of the PI3K/Akt/mTOR pathway, the expression of VMP1, and ULK1 were higher than the negative control, and the expression of mTOR was depressed. The expression of VMP1, ULK1, and mTOR was the opposite when the inhibitor was added to the myoblasts. These results show that the PI3K/Akt/mTOR pathway promoted the expression of VMP1 and ULK1. By using adenovirus-mediated apoptosis and proliferation assays, we found that that miR-124a inhibits myoblast proliferation and autophagy, and promotes their apoptosis by targeting VMP1. In conclusion, our results indicated that VMP1 was highly expressed in the LD muscle tissues of nine-month-old goats, and that it was regulated by miR-124a to inhibit myoblast cells’ apoptosis through the PI3K/Akt/mTOR pathway, and to promote proliferation and autophagy. These findings contribute to the understanding of the molecular mechanisms involved in myoblast proliferation, autophagy, and apoptosis.

Regulation of Non-Coding RNA in the Growth and Development of Skeletal Muscle in Domestic Chickens.

Chicken is the most widely consumed meat product worldwide and is a high-quality source of protein for humans. The skeletal muscle, which accounts for the majority of chicken products and contains the most valuable components, is tightly correlated to meat product yield and quality. In domestic chickens, skeletal muscle growth is regulated by a complex network of molecules that includes some non-coding RNAs (ncRNAs). As a regulator of muscle growth and development, ncRNAs play a significant function in the development of skeletal muscle in domestic chickens. Recent advances in sequencing technology have contributed to the identification and characterization of more ncRNAs (mainly microRNAs (miRNAs), long non-coding RNAs (LncRNAs), and circular RNAs (CircRNAs)) involved in the development of domestic chicken skeletal muscle, where they are widely involved in proliferation, differentiation, fusion, and apoptosis of myoblasts and satellite cells, and the specification of muscle fiber type. In this review, we summarize the ncRNAs involved in the skeletal muscle growth and development of domestic chickens and discuss the potential limitations and challenges. It will provide a theoretical foundation for future comprehensive studies on ncRNA participation in the regulation of skeletal muscle growth and development in domestic chickens.

MicroRNA profiling reveals miR-145-5p inhibits goat myoblast differentiation by targeting the coding domain sequence of USP13.

MicroRNAs (miRNAs) are evolutionarily conserved endogenous small non-coding RNAs that play critical roles in skeletal muscle development. In this study, we identified putative miRNAs that were differentially expressed in the longissimus dorsi muscle between fetus (75days of pregnancy) and lamb (1day of age). We detected 1208miRNAs, 313 of which were differentially expressed. In particular, we found that miR-145-5p was differentially and highly expressed in lamb skeletal muscle. In addition, our results demonstrated that overexpression of miR-145-5p inhibited the differentiation and apoptosis of goat primary myoblasts (GPMs), whereas knockdown of miR-145-5p had the opposite effect. The coding domain sequence (CDS) of ubiquitin-specific peptidase 13 (USP13) was predicted and validated as a target of miR-145-5p. We also demonstrated that the influence as a key regulator of GPMs differentiation is primarily mediated by targeting and inhibiting USP13. Taken together, these results revealed a novel pathway in skeletal muscle development in which miR-145-5p targets CDS region of USP13 to regulate differentiation and apoptosis of GPMs.

Ablation of PRAK exacerbates the mitochondria dysfunction in C2C12 myoblasts exposed to palmitate stress

Backgroundp38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating survival and metabolism. However, the role of PRAK in the regulation of palmitate‐induced metabolic stress remains unknown. We examined whether deletion of PRAK would suppress cell survival and mitochondrial function and whether PRAK‐induced detrimental effect was mitigated by irisin in C2C12 myoblasts in response to palmitate (PA) stress.MethodsPRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas‐9 genome editing technology. Both wild type and PRAK‐/‐ C2C12 cells were exposed to palmitate at the concentration of 200 mmol/L for 48 h to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK‐/‐cells was determined to explore its relationship with RPAK in response to palmitate stress. Cell viability, ATP product, glucose uptake, mitochondrial membrane potential and mitochondrial swelling were assessed.ResultsPRAK knockout decreased C2C12 viability in response to PA stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to PA treatment, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential and increasing mitochondrial swelling. However, PRAK knockout induced an impairment of mitochondrial and cell damage in C2C12 cells exposed to PA stress were rescued by treatment of irisin (10ng/ml).ConclusionOur finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function and glucose uptake in C2C12 cells exposed to palmitate induced metabolic stress.

Pleomorphic adenoma gene 1 (PLAG1) promotes proliferation and inhibits apoptosis of bovine primary myoblasts through the PI3K-Akt signaling pathway.

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in various cellular processes in organismal growth and development. However, its role in muscle function is unclear. This work investigated the roles of PLAG1 in muscle development and explored its regulatory mechanisms. The PLAG1 was proved to promote the proliferation of bovine primary myoblasts using the cell counting kit 8 (CCK-8) assay (P < 0.001), 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay (P = 0.005), quantitative real-time polymerase chain reaction (qRT-PCR) (P = 0.028), western blot, and flow cytometry (P < 0.05), and to inhibit apoptosis of bovine primary myoblasts using qRT-PCR (P = 0.038), western blot, and flow cytometry (P < 0.001). Chromatin immunoprecipitation sequencing (ChIP-seq) and western blot showed PLAG1 upregulated phosphorylated (p)-PI3K, PI3K, p-Akt, Akt, Cyclin D1, and CDK2 and inhibited the expression of p21 and p27 to enhance myoblast proliferation, and increased expression of Bcl-2, and Bcl-xL to inhibit apoptosis. Additionally, PLAG1 was identified as a target of miR-1 using dual-luciferase assay (P < 0.001), qRT-PCR (P < 0.001), and western blot. Furthermore, miR-1 might be a potential mediator of the positive feedback regulation relationship between PLAG1 and the PI3K-Akt signaling pathway.

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.

Transcriptome sequencing to explore the effect of miR-214 on chicken primary myoblasts

MicroRNAs are involved in a series of biological processes, such as proliferation, differentiation and apoptosis of primary myoblasts. The research group found that miR-214 is highly expressed in chicken primary myoblasts (CPMs), so we used miR-214 as a starting point to explore the biological function of miR-214 in skeletal muscle growth and development. In this experiment, CPMs were cultured in vitro; miR-214 was overexpressed in CPMs; and cell samples were collected for subsequent transcriptome sequencing (RNA-seq). After miR-214 overexpression, we identified 97 differentially expressed genes (DEGs), of which 21 DEGs were up-regulated and 76 DEGs were down-regulated. After bioinformatics analysis, these DEGs were found to be significantly enriched in myofibrils, muscle system processes, myofibril assembly and other biological processes related to muscle development. The significantly enriched KEGGs include focal adhesion and type II diabetes mellitus. The protein network of DEGs was drawn by STRING and Cytoscape software, and 5 DEGs were randomly selected to verify the sequencing results by real-time fluorescence quantification. CAV3 is not only an important node protein in the protein network but also a member of the focal adhesion signaling pathway. It is speculated that miR-214 may regulate muscle development through the focal adhesion signaling pathway.

Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast.

Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

Circular RNA circUSP13 sponges miR-29c to promote differentiation and inhibit apoptosis of goat myoblasts by targeting IGF1.

Circular RNAs (circRNAs) are an indispensable element of post-transcriptional gene regulation, influencing a variety of biological processes including myogenic differentiation; however, little is known about the function of circRNA in goat myogenic differentiation. Using RNA-sequencing data from our laboratory, we explored the influences of circUSP13, as a candidate circRNA, on myoblast differentiation since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). In in vitro experiments, circUSP13 significantly promoted differentiation and inhibited apoptosis in goat primary myoblasts. Mechanistically, circUSP13localized with miR-29c in the cytoplasm of goat myoblasts to regulate IGF1 expression. We further demonstrated that circUSP13sponges miR-29c, promoting IGF1 expression that upregulated the expression of MyoG and MyHC. Thus, our results identified circUSP13 as a molecular marker for breeding programs of mutton production, as well as the circUSP13-miR-29c-IGF1 axis as a potential therapeutic target for combating muscle wasting.

Hyperglycemia induced C2C12 myoblast cell cycle arrest and skeletal muscle atrophy by modulating sirtuins gene expression in rats.

Diabetes is characterized by high blood glucose level termed hyperglycemia affecting skeletal muscle structure and function by an unclear molecular mechanism. This study aimed to investigate the effect and underlying mechanism(s) of hyperglycemia on skeletal muscle both in vitro and in vivo. Treatment with hyperglycemic condition (25 mM) for 48 h significantly inhibited C2C12 myoblast proliferation detected by MTT assay whilst flow cytometry revealed an interruption of the cell cycle at subG1 and G2/M phases. An exposure to hyperglycemic condition significantly decreased the myosin heavy chain (MHC) protein expression in the differentiated myotube and tibialis anterior (TA) muscle of Wistar rats. In addition, the muscle cross-section area (MCA) of TA muscle in diabetic rats were significantly decreased compared to the non-diabetic control. Western blotting analysis of C2C12 myoblasts and differentiated myotubes revealed the increased expressions of cleaved-caspase-9 and cleaved-caspase-3, but not cleaved-caspase-8. Of note, these caspases in the TA muscles were not changed under hyperglycemic condition. Quantitative real-time polymerase chain reaction (qRT-PCR) of the hyperglycemic myoblasts and TA muscles revealed modulation of the gene expression of sirtuins (SIRTs). In C2C12 myoblasts, the expressions of SIRT1, SIRT2 and SIRT4 were upregulated whilst SIRT7 was downregulated. Meanwhile, the expressions of SIRT1, SIRT2 in TA muscles were upregulated whilst SIRT4 was downregulated. Taken together, this study showed that hyperglycemia induced cell cycle arrest and apoptosis in myoblasts, and protein degradation and atrophy in skeletal muscle most likely via modulation of SIRTs gene expression.

Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK−/− C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK−/− cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

The circular RNA circCPE regulates myoblast development by sponging miR-138

BackgroundSkeletal muscle development, a long-term and complex process, is controlled by a set of the myogenic genes. Circular RNAs (circRNAs), a class of noncoding RNA, have been shown to regulate various biological processes. Recent studies indicate circRNAs may be involved in myogenesis, but the role and regulatory mechanism of circRNAs in myogenesis is largely unknown. In the present study, circCPE was firstly found to promote the bovine myoblast proliferation and inhibit cell apoptosis and differentiation by influencing the expression of FOXC1 in a miR138-mediated manner. And in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration.ResultsWe identified a novel circular RNA circCPE by analyzing circRNAs sequencing data of bovine muscle tissue. Sequencing verification, RNase R treatment and Actinomycin D treatment confirmed the circular nature of circCPE in bovine muscle. Functional assays showed that overexpression of circCPE could inhibit bovine myoblast apoptosis and differentiation, as well as facilitate cell proliferation. Moreover, in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration. In consideration of circRNA action as miRNAs sponge, we found that circCPE harbors miR-138 binding sites and absorbed miR-138. Mechanistically, the rescue experiments showed that the overexpression of circCPE can counteract the inhibitory effect of miR-138 on the cell proliferation and the accelerated effects on the differentiation and apoptosis. Subsequently, we found that circCPE sequester the inhibitory effect of miR-138 on FOXC1 so as to involve in myogenesis.ConclusionsCollectively, we constructed a novel circCPE/miR-138/FOXC1 regulatory network in bovine myogenesis, which further provide stronger evidence that circRNA involved in muscle development acting as miRNA sponge.

MiR-7 regulates the apoptosis of chicken primary myoblasts through the KLF4 gene

ABSTRACT 1. MicroRNAs (miRNAs) play a vital role in the proliferation, differentiation, and apoptosis of myoblasts. However, the effect of miR-7 on the apoptosis of chicken primary myoblasts (CPMs) and its mechanism is still unclear. 2. In this study, the expression of apoptosis marker genes (RAF1, Caspase3, Caspase9, Cytc, Fas) in CPMs was significantly increased after transfection of miR-7 mimic. The expression of the apoptosis marker genes in CPMs was significantly reduced after transfection with miR-7 inhibitor. Flow cytometry showed that the late apoptosis rate of the mimic group was significantly higher than the negative control (NC). The viable cells of the mimic group were significantly lower than the NC. In contrast, inhibition of miR-7 had the opposite effect. 3. The dual-luciferase assay showed that the KLF4 was a target gene of miR-7. The rescue experiment showed that the KLF4 gene could attenuate the effect of miR-7 on the expression of apoptosis marker genes in CPMs. 4. Determination of the function the KLF4 gene showed that the expression of the apoptosis marker genes in CPMs decreased significantly compared with the NC after its overexpression. Inhibition of KLF4 gene had the opposite effect. Flow cytometry showed that overexpression of the KLF4 gene inhibited early apoptosis of myoblasts (P ≤ 0.01), while interference with the KLF4 gene could promote early apoptosis of myoblasts (P ≤ 0.001). 5. The results demonstrated, for the first time, that miR-7 promotes apoptosis in chicken primary myoblasts by regulating the expression of the KLF4 gene.

2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways

SUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.

Downregulated hypoxia-inducible factor 1α improves myoblast differentiation under hypoxic condition in mouse genioglossus.

The treatment of obstructive sleep apnea-hypopnea syndrome targets the narrow anatomic structure of the upper airway (UA) and lacks an effective therapy for UA dilator muscle dysfunction. Long-term hypoxia can cause damage to UA dilator muscles and trigger a vicious cycle. We previously confirmed that hypoxia-inducible factor 1α (HIF-1α) upregulation mediates muscle fatigue in hypoxia condition, but the underlying mechanism remains to be determined. The present study investigated the intrinsic mechanisms and related pathways of HIF-1α that affect myoblast differentiation, with an aim to search for compounds that have protective effects in hypoxic condition. Differentiation of myoblasts was induced under hypoxia, and we found that hypoxia significantly inhibits the differentiation of myoblasts, damages the ultrastructure of mitochondria, and reduces the expression of myogenin, PGC-1β and pAMPKα1. HIF-1α has a negative regulation effect on AMPK. Downregulation of HIF-1α increases the expression of the abovementioned proteins, promotes the differentiation of myoblasts, and protects mitochondrial integrity. In addition, mitochondrial biogenesis occurs during myogenic differentiation. Inhibition of the AMPK pathway inhibits mitochondrial biogenesis, decreases the level of PGC-1β, and increases apoptosis. Resveratrol dimer can reverse the mitochondrial damage induced by AMPK pathway inhibition and decrease myoblast apoptosis. Our results provided a regulatory mechanism for hypoxic injury in genioglossus which may contribute to the pathogenesis and treatment of OSAHS.

The Regulation Mechanism of AMPK/FOXO3 Signal Pathway in the Apoptosis and Differentiation of Duck Myoblasts

The aim of this study was to investigate the effect of AMPK/FOXO3 signal pathway on duck myoblasts apoptosis and differentiation the possible mechanisms. Dorsomorphin 2HCl (DOR) and AICAR were used to inhibited and activated AMPK expression, respectively. And then, the myoblasts apoptosis and differentiation were established by dexamethasone (DEX) and 2% pregnant mare serum gonadotropin (PMSG) induction. In the present work, DEX succeed promoting the apoptosis of duck myoblasts and the expression of marker genes Caspase 3, LC3 and Caspase 7. Further, the mRNA expression of AMPKα1 and FOXO3 and the protein expression of p-AMPK and FOXO3 significantly increased in the 1000 and 10 000 ng/mL group but AMPKα2 only in 10 000 ng/mL group. However, the positive effects of DEX on apoptosis of duck myoblasts could be inhibited by DOR. We found that the activator of AMPK/FOXO3 pathway could increase the expression of Caspase 3, LC3 and Fas and promoted the G0/G1 phase arrest. On the other hand, the AMPK/FOXO3 signal pathway could also be activated by 2% PMSG. Similarity, the expression increased of MyHC was induced by 2% PMSG could also be inhibited by DOR. Corresponding, the activation of AMPK/FOXO3 signal pathway significantly increased the expression of MyHC. Therefore, our results indicated that the activation of AMPK/FOXO3 signal pathway could promote the duck myoblasts apoptosis and differentiation.

Pharmacological Pre- and Postconditioning With Levosimendan Protect H9c2 Cardiomyoblasts From Anoxia/Reoxygenation-induced Cell Death via PI3K/Akt Signaling.

The calcium sensitizer levosimendan is indicated for the hemodynamic stabilization of patients with acutely decompensated heart failure and has been shown to be protective against reperfusion injury after myocardial infarction. However, affected forms of cell death and underlying signaling pathways remain controversial. Therefore, the aim of this study was to examine the influence of levosimendan preconditioning and postconditioning on anoxia/reoxygenation-induced apoptosis, necrosis, and autophagy in H9c2 myoblasts. To mimic conditions of myocardial ischemia/reperfusion, rat cardiac H9c2 myoblasts were exposed to anoxia/starvation, followed by reoxygenation/refeeding. Apoptosis, necrosis, autophagy, cell viability, survival signaling, and mitochondrial permeability transition pore (mPTP) opening were measured. Both, pharmacological preconditioning and postconditioning with levosimendan were capable to reduce apoptosis as well as necrosis in stressed H9c2 cells. However, preconditioning showed to have the stronger impact compared with postconditioning. Moreover, levosimendan preconditioning increased autophagy, suggesting enhanced repair processes initiated by the early presence of the drug. Underlying mechanisms differ between both interventions: Although both are associated with PI3/Akt activation and reduced mPTP opening, only postconditioning but not preconditioning depended on mKATP activation. This variation might indicate that a pharmacological treatment after the onset of reoxygenation at least in part directly addresses mitochondrial structures for protection. In conclusion, we demonstrate that both pharmacological preconditioning and postconditioning with levosimendan protect anoxia/reoxygenation-stressed cells but differ in the underlying mechanisms. These results are decisive to obtain more insights into the beneficial effects of levosimendan in the treatment of reperfusion-mediated damage.

Lmod3 promotes myoblast differentiation and proliferation via the AKT and ERK pathways

Mutations in the Lmod3 gene have been identified as a genetic cause of nemaline myopathy. However, the mechanism underlying this disease and the function of Lmod3 remain largely unknown. In this study, we found that Lmod3 knockdown in C2C12 cells impaired myoblast differentiation, whereas enforced Lmod3 expression enhanced such differentiation. We also discovered that myoblast proliferation was promoted by Lmod3 overexpression but impeded by its knockdown. Additionally, knockdown of Lmod3 led to apoptosis in myoblasts. Concurrently, forced Lmod3 expression in C2C12 cells contributed to activation of the AKT and ERK pathways during myoblast differentiation and proliferation, respectively. Conversely, knockdown of Lmod3 in C2C12 cells produced the opposite results. Furthermore, administration of IGF-1, a booster of both AKT and ERK pathways, partially rescued the inhibitory effect of Lmod3 knockdown on both differentiation and proliferation of C2C12 cells. These results suggest that Lmod3 promotes differentiation and proliferation of myoblasts through the AKT and ERK pathways, respectively.

Indole-6-carboxaldehyde prevents oxidative stress-induced mitochondrial dysfunction, DNA damage and apoptosis in C2C12 skeletal myoblasts by regulating the ROS-AMPK signaling pathway

Indole-6-carboxaldehyde (I6CA), a natural indole derivative derived from the brown algae Sargassum thunbergii (Mertens) Kuntze, is known to have several pharmacological activities. However, the antioxidant effects of I6CA have not been identified. The study aimed to investigate the protective effect of I6CA and its underlying mechanism against oxidative stress-induced damage in C2C12 mouse skeletal myoblasts. The findings revealed that pretreatment with I6CA protected hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage through blockage of intracellular reactive oxygen species (ROS) generation. I6CA also significantly suppressed C2C12 cells against H2O2-induced apoptosis by preventing loss of mitochondrial membrane potential and cytosolic release of cytochrome c, decreasing the rate of Bax/Bcl-2 expression and reducing the activity of caspases. In addition, I6CA markedly attenuated the decrease in ATP content induced by H2O2 and restored H2O2-induced activation of AMP-activated protein kinase (AMPK). However, the cytoprotective effects of I6CA against H2O2 were eliminated by compound C, a specific AMPK signaling blocker. The current results indicate that I6CA was able to protect C2C12 myoblast DNA damage and apoptosis from oxidative stress by at least preserving mitochondrial homeostasis mediated through the ROS-AMPK signaling pathway.