Single nucleotide polymorphisms in the MRFs gene family associated with growth in Nile tilapia.

BACKGROUND: Muscle occupies most of the fish body, promoting the proliferation of fish muscle fibers can facilitate rapid growth and increase the body weight of fish. Some studiesSeveral previous suggest that Myogenic regulatory factors (MRFs) play an important role in the growth of fish. OBJECTIVE: To investigate the association between the polymorphism of MRFs gene family and growth traits in Nile tilapia (Oreochromis niloticus), get more molecular markers for growth. METHODS: Amplified the Nile tilapia MRFs family gene, including Myogenic determination 1 (Myod1), Myogenic determination 2 (Myod2), Myogenin (Myog), Myogenic factor 5 (Myf5), and Myogenic factor 6 (Myf6), single nucleotide polymorphism (SNP) were screened by Sanger sequencing. RESULTS: A total of 16 SNP loci were screened, including six for Myf5, six for Myf6, one for Myog, one for Myod1 and two for Myod2. The growth traits were analyzed in relation to these 16 SNP loci, and the results indicated significant associations between all 16 SNP loci and the growth traits (P < 0.05). The linkage disequilibrium analysis revealed that D1 and D2 diplotypes of Myf5 gene, E1, E2, E3 and E4 of Myf6 gene, and F1 diplotype of Myod2 gene were significantly associated with superior growth traits. CONCLUSION: There were 6, 6, 1, 1 and 2 growth-related molecular markers in Myf5, Myf6, Myog, Myod1 and Myod2 genes, respectively, which could be applied to the breeding of Nile tilapia.

Heat shock protein 27 regulates myogenic and self-renewal potential of bovine satellite cells under heat stress.

While satellite cells play a key role in the hypertrophy, repair, and regeneration of skeletal muscles, their response to heat exposure remains poorly understood, particularly in beef cattle. This study aimed to investigate the changes in the transcriptome, proteome, and proliferation capability of bovine satellite cells in response to different levels of heat stress (HS) and exposure times. Satellite cells were isolated from 3-mo-old Holstein bulls (body weight: 77.10 ± 2.02 kg) and subjected to incubation under various temperature conditions: 1) control (38 °C; CON), 2) moderate (39.5 °C; MHS), and extreme (41 °C; EHS) for different durations ranging from 0 to 48 h. Following 3 h of exposure to extreme heat (EHS), satellite cells exhibited significantly increased gene expression and protein abundance of heat shock proteins (HSPs; HSP70, HSP90, HSP20) and paired box gene 7 (Pax7; P < 0.05). HSP27 expression peaked at 3 h of EHS and remained elevated until 24 h of exposure (P < 0.05). In contrast, the expression of myogenic factor 5 (Myf5) and paired box gene 3 (Pax3) was decreased by EHS compared to the control at 3 h of exposure (P < 0.05). Notably, the introduction of HSP27 small interference RNA (siRNA) transfection restored Myf5 expression to control levels, suggesting an association between HSP27 and Myf5 in regulating the self-renewal properties of satellite cells upon heat exposure. Immunoprecipitation experiments further confirmed the direct binding of HSP27 to Myf5, supporting its role as a molecular chaperone for Myf5. Protein-protein docking algorithms predicted a high probability of HSP27-Myf5 interaction as well. These findings indicate that extreme heat exposure intrinsically promotes the accumulation of HSPs and modulates the early myogenic regulatory factors in satellite cells. Moreover, HSP27 acts as a molecular chaperone by binding to Myf5, thereby regulating the division or differentiation of satellite cells in response to HS. The results of this study provide a better understanding of muscle physiology in heat-stressed cells, while unraveling the intricate molecular mechanisms that underlie the HS response in satellite cells.

This study aimed to elucidate the response of bovine satellite cells to heat exposure. Satellite cells were isolated from Holstein bulls and subjected to varying temperatures. Transcriptional, proteomic, and proliferative changes were assessed. Following extreme heat exposure, cells exhibited upregulated expression of heat shock proteins (HSPs; HSP70, HSP90, HSP20) and paired box gene 7 (Pax7). Conversely, the expression of myogenic factor 5 (Myf5) and paired box gene 3 (Pax3), key regulators of myogenesis, decreased under conditions of extreme heat. Notably, downregulation of HSP27 expression using siRNA restored Myf5 expression to normal levels, implying an association between HSP27 and Myf5 in the modulation of satellite cell properties during heat exposure. Our results validated the direct binding of HSP27 to Myf5, substantiating its role as a molecular chaperone. These findings underscore the elevation of HSPs, and alteration of early myogenic regulatory factors implicated in muscle development upon exposure to extreme heat. HSP27 functions as a molecular chaperone by engaging with Myf5, thereby influencing the division or differentiation of satellite cells during heat stress (HS). This study contributes to the advancement of our comprehension regarding the muscular physiology of heat-stressed animals, while clarifying the intricate molecular mechanisms governing the response of satellite cells to HS.

Exploring the Temporal Correlation of Sarcopenia with Bone Mineral Density and the Effects of Osteoblast-Derived Exosomes on Myoblasts through an Oxidative Stress-Related Gene.

Sarcopenia is an age-related accelerated loss of muscle strength and mass. Bone and muscle are closely related as they are physically adjacent, and bone can influence muscle. However, the temporal association between bone mineral density (BMD) and muscle mass in different regions of the body after adjustment for potential indicators and the mechanisms by which bone influences muscle in sarcopenia remain unclear. Therefore, this study aimed to explore the temporal association between muscle mass and BMD in different regions of the body and mechanisms by which bone regulates muscle in sarcopenia. Here, cross-lagged models were utilized to analyze the temporal association between BMD and muscle mass. We found that low-density lipoprotein (LDL-C) positively predicted appendicular lean mass. Mean whole-body BMD (WBTOT BMD), lumbar spine BMD (LS BMD), and pelvic BMD (PELV BMD) temporally and positively predicted appendicular lean mass, and appendicular lean mass temporally and positively predicted WBTOT BMD, LS BMD, and PELV BMD. Moreover, this study revealed that primary mice femur osteoblasts, but not primary mice skull osteoblasts, induced differentiation of C2C12 myoblasts through exosomes. Furthermore, the level of long noncoding RNA (lncRNA) taurine upregulated 1 (TUG1) was decreased, and the level of lncRNA differentiation antagonizing nonprotein coding RNA (DANCR) was increased in skull osteoblast-derived exosomes, the opposite of femur osteoblast-secreted exosomes. In addition, lncRNA TUG1 enhanced and lncRNA DANCR suppressed the differentiation of myoblasts through regulating the transcription of oxidative stress-related myogenin (Myog) gene by modifying the binding of myogenic factor 5 (Myf5) to the Myog gene promoter via affecting the nuclear translocation of Myf5. The results of the present study may provide novel diagnostic biomarkers and therapeutic targets for sarcopenia.

What snakes and caecilians have in common? Molecular interaction units and the independent origins of similar morphotypes in Tetrapoda.

Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.

Myogenic regulatory factors Myod and Myf5 are required for dorsal aorta formation and angiogenic sprouting.

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.

Characterization of myogenic regulatory factors, myod and myf5 from Megalobrama amblycephala and the effect of lipopolysaccharide on satellite cells in skeletal muscle.

Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala, an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod, myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection.

miR-9-5p promotes myogenic differentiation via the Dlx3/Myf5 axis.

MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3'UTR of Dlx3 and downregulated Dlx3 expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.

Assessing Myf5 and Lbx1 contribution to carapace development by reproducing their turtle-specific signatures in mouse embryos.

BACKGROUND: The turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF. RESULTS: We investigated these turtle-specific expression differences using genetic approaches in mouse embryos. At mid-gestation, mouse embryos producing Myf5 transcripts lacking exon 2 replicated some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs. CONCLUSIONS: Turtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.

Single-Cell RNA Sequencing Reveals Heterogeneity of Myf5-Derived Cells and Altered Myogenic Fate in the Absence of SRSF2.

Splicing factor SRSF2 acts as a critical regulator for cell survival, however, it remains unknown whether SRSF2 is involved in myoblast proliferation and myogenesis. Here, knockdown of SRSF2 in myoblasts causes high rates of apoptosis and defective differentiation. Combined conditional knockout and lineage tracing approaches show that Myf5-cre mice lacking SRSF2 die immediately at birth and exhibit a complete absence of mature myofibers. Mutant Myf5-derived cells (tdtomato-positive cells) are randomly scattered in the myogenic and non-myogenic regions, indicating loss of the community effect required for skeletal muscle differentiation. Single-cell RNA-sequencing reveals high heterogeneity of myf5-derived cells and non-myogenic cells are significantly increased at the expense of skeletal muscle cells in the absence of SRSF2, reflecting altered cell fate. SRSF2 is demonstrated to regulate the entry of Myf5 cells into the myogenic program and ensures their survival by preventing precocious differentiation and apoptosis. In summary, SRSF2 functions as an essential regulator for Myf5-derived cells to respond correctly to positional cues and to adopt their myogenic fate.

Novel Single Nucleotide Polymorphisms and Haplotype of MYF5 Gene Are Associated with Body Measurements and Ultrasound Traits in Grassland Short-Tailed Sheep.

Myogenic factor 5 plays active roles in the regulation of myogenesis. The aim of this study is to expose the genetic variants of the MYF5 and its association with growth performance and ultrasound traits in grassland short-tailed sheep (GSTS) in China. The combination technique of sequencing and SNaPshot revealed seven SNPs in ovine MYF5 from 533 adult individuals (male 103 and female 430), four of which are novel ones located at g.6838G > A, g.6989 G > T, g.7117 C > A in the promoter region and g.9471 T > G in the second intron, respectively. Genetic diversity indexes showed the seven SNPs in low or intermediate level, but each of them conformed HWE (p > 0.05) in genotypic frequencies. Association analysis indicated that g.6838G > A, g.7117 C > A, g.8371 T > C, g.9471 T > G, and g.10044 C > T had significant effects on growth performance and ultrasound traits. The diplotypes of H1H3 and H2H3 had higher body weight and greater body size, and haplotype H3 had better performance on meat production than the others. In addition, the dual-luciferase reporter assay showed that there are two active regions in the MYF5 promoter located at −1799~−1197 bp and −514~−241 bp, respectively, but g.6838G > A and g.7117 C > A were out of the region, suggesting these two SNPs influence the phenotype by other pathway. The results suggest that the MYF5 gene might be applied as a promising candidate of functional genetic marker in GSTS breeding.

TLE4 regulates muscle stem cell quiescence and skeletal muscle differentiation.

Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111 kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.

Association of GH, STAT5A, MYF5 gene polymorphisms with milk somatic cell count, EC and pH levels of Holstein dairy cattle.

This study was conducted to ivnestigate the associations of GH-AluI, STAT5A-AvaI and MYF5-TaqI gene polymorphisms with milk somatic cell count (SCC), electrical conductivity (EC) and pH levels in Holstein dairy cows. For this purpose, 167 blood and 1670 milk samples of 167 Holstein cows in their 2nd lactation were used. There were significant relationships between GH-AluI genotypes and milk EC (p < 0.001) and between STAT5A-AvaI genotypes and milk EC (p = 0.007), but there were not any significant relationships between MYF5 gene polymorphism and the investigated traits (p > 0.05). The greatest EC values were observed in GH-AluI-LV and STAT5A-AvaI-TT-genotyped individuals. Just because of association of EC with mastitis, it was concluded that present GH-AluI and STAT5A-AvaI polymorphisms could be used in further studies to be conducted to improve mastitis resistance and milk quality traits of Holstein dairy cows.

Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat.

Brown adipocytes share the same developmental origin with skeletal muscle. Here we find that a brown adipocyte-to-myocyte remodeling also exists in mature brown adipocytes, and is induced by prolonged high fat diet (HFD) feeding, leading to brown fat dysfunction. This process is regulated by the interaction of epigenetic pathways involving histone and DNA methylation. In mature brown adipocytes, the histone demethylase UTX maintains persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high Prdm16 expression. PRDM16 then recruits DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation and suppressing its expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and ß-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells.

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7-14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

[Figure: see text].

Dnmt3b Deficiency in Myf5+-Brown Fat Precursor Cells Promotes Obesity in Female Mice.

Increasing energy expenditure through activation of brown fat thermogenesis is a promising therapeutic strategy for the treatment of obesity. Epigenetic regulation has emerged as a key player in regulating brown fat development and thermogenic program. Here, we aimed to study the role of DNA methyltransferase 3b (Dnmt3b), a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat function and energy homeostasis. We generated a genetic model with Dnmt3b deletion in brown fat-skeletal lineage precursor cells (3bKO mice) by crossing Dnmt3b-floxed (fl/fl) mice with Myf5-Cre mice. Female 3bKO mice are prone to diet-induced obesity, which is associated with decreased energy expenditure. Dnmt3b deficiency also impairs cold-induced thermogenic program in brown fat. Surprisingly, further RNA-seq analysis reveals a profound up-regulation of myogenic markers in brown fat of 3bKO mice, suggesting a myocyte-like remodeling in brown fat. Further motif enrichment and pyrosequencing analysis suggests myocyte enhancer factor 2C (Mef2c) as a mediator for the myogenic alteration in Dnmt3b-deficient brown fat, as indicated by decreased methylation at its promoter. Our data demonstrate that brown fat Dnmt3b is a key regulator of brown fat development, energy metabolism and obesity in female mice.

Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols.

Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.

Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle.

Mechanical stimulation has benefits for muscle mass and function. Passive stretching is widely performed in clinical rehabilitation medicine. However, the hypertrophic effects of passive repetitive stretching on senescent skeletal muscles against muscle atrophy remain unknown. We used senescence-accelerated model SAM-P8 mice. The gastrocnemius muscle was passively repetitive stretched by manual ankle dorsiflexion for 15 min, 5 days a week for 2 weeks under deep anesthesia. We examined the effects of passive stretching on muscle mass, myofiber cross-sectional area, muscle fiber type composition, satellite cell and myonuclei content, signaling pathways involved in muscle protein synthesis, and myogenic regulatory factors. The gastrocnemius muscle weight and fiber cross-sectional area of the stretched side was found greater compared with that of the unstretched side. Passive repetitive stretching increased the mRNA expression level of Akt, p70S6K, 4E-BP1, Myf5, myogenin, MuRF1.The phosphorylation level of p70S6K significantly increased in the stretched muscles, whereas of Akt and 4E-BP1 remained unchanged, compared to the unstretched side. The Pax7+ cells and myonuclei content did not differ between the stretched and unstretched muscles. These findings suggest that the hypertrophic or suppressed atrophic observation in the stretched muscles are mainly attributable to the protein turnover provoked by stretching. These findings are applicable to clinical muscle strengthening and sarcopenia prevention.

Catalytically active phospholipase A2 myotoxin from Crotalus durissus terrificus induces proliferation and differentiation of myoblasts dependent on prostaglandins produced by both COX-1 and COX-2 pathways.

Although crotoxin B (CB) is a well-established catalytically active secretory phospholipase A2 group IIA (sPLA2-IIA) myotoxin, we investigated its potential stimulatory effect on myogenesis with the involvement of prostaglandins (PGs) produced by cyclooxygenase (COX)-1 and -2 pathways. Myoblast C2C12 were cultured in proliferation or commitment protocols and incubated with CB followed by lumiracoxib (selective COX-2 inhibitor) or valeryl salicylate (selective COX-1 inhibitor) and subjected to analysis of PG release, cell proliferation and activation of myogenic regulatory factors (MRFs). Our data showed that CB in non-cytotoxic concentrations induces an increase of COX-2 protein expression and stimulates the activity of both COX isoforms to produce PGE2, PGD2 and 15d-PGJ2. CB induced an increase in the proliferation of C2C12 myoblast cells dependent on PGs from both COX-1 and COX-2 pathways. In addition, CB stimulated the activity of Pax7, MyoD, Myf5 and myogenin in proliferated cells. Otherwise, CB increased myogenin activity but not MyoD in committed cells. Our findings evidence the role of COX-1- and COX-2-derived PGs in modulating CB-induced activation of MRFs. This study contributes to the knowledge that CB promote early myogenic events via regulatory mechanisms on PG-dependent COX pathways, showing new concepts about the effect of sPLA2-IIA in skeletal muscle repair.