A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

MDFI promotes the proliferation and tolerance to chemotherapy of colorectal cancer cells by binding ITGB4/LAMB3 to activate the AKT signaling pathway.

Colorectal cancer (CRC) is one of the most lethal cancers. Single-cell RNA sequencing (scRNA-seq) and protein-protein interactions (PPIs) have enabled the systematic study of CRC. In our research, the activation of the AKT pathway in CRC was analyzed by KEGG using single-cell sequencing data from the GSE144735 dataset. The correlation and PPIs of MDFI and ITGB4/LAMB3 were examined. The results were verified in the TCGA and CCLE and further tested by coimmunoprecipitation experiments. The effect of MDFI on the AKT pathway via ITGB4/LAMB3 was validated by knockdown and lentiviral overexpression experiments. The effect of MDFI on oxaliplatin/fluorouracil sensitivity was probed by colony formation assay and CCK8 assay. We discovered that MDFI was positively associated with ITGB4/LAMB3. In addition, MDFI was negatively associated with oxaliplatin/fluorouracil sensitivity. MDFI upregulated the AKT pathway by directly interacting with LAMB3 and ITGB4 in CRC cells, and enhanced the proliferation of CRC cells via the AKT pathway. Finally, MDFI reduced the sensitivity of CRC cells to oxaliplatin and fluorouracil. In conclusion, MDFI promotes the proliferation and tolerance to chemotherapy of colorectal cancer cells, partially through the activation of the AKT signaling pathway by the binding to ITGB4/LAMB3. Our findings provide a possible molecular target for CRC therapy.

Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) irradiation has been shown to induce various responses in different cells. It has been shown that LIPUS activates extracellular signal-regulated kinase 1/2 (ERK1/2) through integrin. PURPOSE: To study the effects of LIPUS on myogenic regulatory factors and other related myogenesis elements in a volumetric skeletal muscle loss injury model. STUDY DESIGN: Controlled laboratory study. METHODS: C57BL/6J mice were subjected to full-thickness muscle defect injury of the quadriceps and treated with direct application of LIPUS 20 min/d or non-LIPUS treatment (control) for 3, 7, and 14 days. LIPUS was also applied to C2C12 cells in culture in the presence of low and high doses of lipopolysaccharides. The expression levels of myogenic regulatory factors and the expression levels of myokine-related and angiogenic-related proteins of the control and LIPUS groups were analyzed. RESULTS: Muscle volume in the injury site was restored at day 14 with LIPUS treatment. Paired-box protein 7, myogenic factor 5, myogenin, and desmin expressions were significantly different between control and LIPUS groups at days 7 and 14. Myokine and angiogenic cytokine-related factors were significantly increased in the LIPUS group at day 3 and decreased with no significant difference between the groups by day 14. LIPUS induced different responses of myogenic regulatory factors in C2C12 cells with low and high doses of lipopolysaccharides. LIPUS promoted myogenesis through short-lived increase in interleukin-6 and heme oxygenase 1, together with activation of ERK1/2. CONCLUSION: LIPUS had a constant effect on the variables of tissue damage, from macrotrauma to microtrauma, leading to efficient muscle regeneration. CLINICAL RELEVANCE: The focus of therapeutic strategies with LIPUS has been not only for microvascular regeneration but also for skeletal muscle and related local tissue recovery from acute or chronic damage.

IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

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

Effects of thermal stress and mechanistic target of rapamycin and wingless-type mouse mammary tumor virus integration site family pathways on the proliferation and differentiation of satellite cells derived from the breast muscle of different chicken lines.

Satellite cells (SCs) are muscle stem cells responsible for muscle hypertrophic growth and the regeneration of damaged muscle. Proliferation and differentiation of the pectoralis major (p. major) muscle SCs are responsive to thermal stress in turkeys, which are, in part, regulated by mechanistic target of rapamycin (mTOR) and Frizzled7 (Fzd7)-mediated wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) pathways in a growth dependent-manner. It is not known if chicken p. major SCs respond to thermal stress in a manner similar to that of turkey p. major SCs. The objective of the current study was to investigate the effects of thermal stress and mTOR and Wnt/PCP pathways on the proliferation, differentiation, and expression of myogenic transcriptional regulatory factors in SCs isolated from the p. major muscle of a current modern commercial (MC) broiler line as compared to that of a Cornish Rock (BPM8) and Randombred (RBch) chicken line in the 1990s. The MC line SCs had lower proliferation and differentiation rates and decreased expression of myoblast determination factor 1 (MyoD) and myogenin (MyoG) compared to the BPM8 and RBch lines. Heat stress (43°C) increased proliferation and MyoD expression in all the cell lines, while cold stress (33°C) showed a suppressive effect compared to the control temperature (38°C). Satellite cell differentiation was altered with heat and cold stress in a cell line-specific manner. In general, the differentiation of the MC SCs was less responsive to both heat and cold stress compared to the BPM8 and RBch lines. Knockdown of the expression of either mTOR or Fzd7 decreased the proliferation, differentiation, and the expression of MyoD and MyoG in all the cell lines. The MC line during proliferation was more dependent on the expression of mTOR and Fzd7 than during differentiation. Thus, modern commercial meat-type chickens have decreased myogenic activity and temperature sensitivity of SCs in an mTOR- and Fzd7-dependent manner. The decrease in muscle regeneration will make modern commercial broilers more susceptible to the negative effects of myopathies with muscle fiber necrosis requiring satellite cell-mediated repair.

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.

Effect of eccentric and concentric contraction mode on myogenic regulatory factors expression in human vastus lateralis muscle.

Skeletal muscle contractions are caused to release myokines by muscle fiber. This study investigated the myogenic regulatory factors, as MHC I, IIA, IIX, Myo-D, MRF4, Murf, Atrogin-1, Decorin, Myonection, and IL-15 mRNA expression in the response of eccentric vs concentric contraction. Eighteen healthy men were randomly divided into two eccentric and concentric groups, each of 9 persons. Isokinetic contraction protocols included maximal single-leg eccentric or concentric knee extension tasks at 60°/s with the dominant leg. Contractions consisted of a maximum of 12 sets of 10 reps, and the rest time between each set was 30 s. The baseline biopsy was performed 4 weeks before the study, and post-test biopsies were taken immediately after exercise protocols from the vastus lateralis muscle. The gene expression levels were evaluated using Real-Time PCR methods. The eccentric group showed a significantly lower RPE score than the concentric group (P ≤ 0.05). A significant difference in MyoD, MRF4, Myonection, and Decorin mRNA, were observed following eccentric or concentric contractions (P ≤ 0.05). The MHC I, MHC IIA, IL-15 mRNA has been changed significantly compared to the pre-exercise in the concentric group (P ≤ 0.05). While only MHC IIX and Atrogin-1 mRNA changed significantly in the eccentric group (P ≤ 0.05). Additionally, the results showed a significant difference in MyoD, MRF4, IL-15, and Decorin at the follow-up values between eccentric or concentric groups (P ≤ 0.05). Our findings highlight the growing importance of elucidating the different responses of muscle growth factors associated with a myogenic activity such as MHC IIA, Decorin, IL-15, Myonectin, Decorin, MuRF1, and MHC IIX mRNA in following various types of exercise.

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.

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors.

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

Differential expression patterns of myogenic regulatory factors in the postnatal longissimus dorsi muscle of Jeju Native Pig and Berkshire breeds along with their co-expression with Pax7

BACKGROUND: Myogenic regulatory factors (MRFs) such as MyoD, Myf6 and Myf5 play a vital role in the growth and development of muscles. Jeju Native Pig (JNP) is the top ranker in Korea amongst the indigenous livestock reared for meat purpose. Few studies covering transcript abundance of the MRFs and related to their co-expression with Pax7 in JNP have been conducted. Despite having better quality pork, JNP does not have a comparative growth rate with respect to western breeds. Therefore, the present study was designed with the objective to study the relative transcript levels of MRFs in the postnatal myogenesis of longissimus dorsi muscles in JNP and Berkshire breeds. RESULTS: Relative transcript levels were analyzed by qRT-PCR and blot expression analysis through Western blotting. Immunocytochemistry was performed to analyze their expressions at cellular levels. ToppCluster aided in the analysis of gene ontology of biological processes. The quantitative transcript levels of MyoD and Pax7 were significantly (P < 0.05) higher in Berkshire than in JNP. Myotube formation was observed under the co-expression of MyoD and Pax7. ToppCluster helped in the understanding of the linking of biological processes of the MRFs with the different signaling pathways. MyBPH had significantly (P < 0.05) high transcript levels during the chosen age groups in JNP than Berkshire. CONCLUSIONS: The current study can be helpful in understanding the genetic basis for myogenesis in postnatal stage. Moreover, it can act as stepping stone for the identification of marker genes related to body growth and meat quality in JNP.

Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos.

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.

Gene co-expression network analysis reveals immune cell infiltration as a favorable prognostic marker in non-uterine leiomyosarcoma.

The present study aimed to improve the understanding of non-uterine leiomyosarcoma (NULMS) prognostic genes through system biology approaches. This cancer is heterogeneous and rare. Moreover, gene interaction networks have not been reported in NULMS yet. The datasets were obtained from the public gene expression databases. Seven co-expression modules were identified from 5000 most connected genes; using weighted gene co-expression network analysis. Using Cox regression, the modules showed favorable (HR = 0.6, 95% CI = 0.4-0.89, P = 0.0125), (HR = 0.65, 95% CI = 0.44-0.98, P = 0.04) and poor (HR = 1.55, 95% CI = 1.06-2.27, P = 0.025) prognosis to the overall survival (OS) (time = 3740 days). The first one was significant in multivariate HR estimates (HR = 0.4, 95% CI = 0.28-0.69, P = 0.0004). Enriched genes through the Database for Annotation, Visualization, and Integrated Discovery (DAVID) revealed significant immune-related pathways; suggesting immune cell infiltration as a favorable prognostic factor. The most significant protective genes were ICAM3, NCR3, KLRB1, and IL18RAP, which were in one of the significant modules. Moreover, genes related to angiogenesis, cell-cell adhesion, protein glycosylation, and protein transport such as PYCR1, SRM, and MDFI negatively affected the OS and were found in the other related module. In conclusion, our analysis suggests that NULMS might be a good candidate for immunotherapy. Moreover, the genes found in this study might be potential candidates for targeted therapy.

Development and patterning of rib primordia are dependent on associated musculature.

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.

A novel lncRNA, lnc403, involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6.

Skeletal muscle, the most abundant and plasticity tissue in mammals, is essential for various functions such as movement, breathing, maintaining posture and metabolism. Myogenesis is a complex and precise process, which is regulated by the sequential expression of multiple transcription factors, and accumulating evidence have confirmed that multiple lncRNAs are involved in muscle development as the important transcriptional regulator. In this study, a novel lncRNA, named lnc403 was obtained, with a full-length 2689 bp, which had poor coding ability and was mainly expressed in the nucleus of myoblasts and myotubes. The expression of lnc403 was significantly different in the proliferation and differentiation stages of muscle cells. Then we successfully constructed lnc403 loss/gain-function cell models by transfecting silnc403 and pCDNA3.1-EGFP-lnc403 into satellite cells, respectively; and found that lnc403 inhibited skeletal muscle satellite cell differentiation but had no significant effect on cell proliferation, either in the case of lnc403 knockdown or overexpression. In order to further screen the target factors regulated by lncRNA in the process of myogenic differentiation, the RNA-pull down, mass spectrometry and bioinformatics analysis were performed. The results showed that lnc403 negatively regulated the expression of the adjacent gene Myf6 and positively regulated interaction proteins KRAS expression. The above results indicate that lnc403 affects skeletal muscle cell differentiation by affecting the expression of nearby genes and interacting proteins, implying lnc403 might participate in the bovine myoblasts differentiation through multi-pathway network regulation mode. This study provides a new perspective for further understanding of the regulation mechanism of lncRNAs on bovine myogenic process.

Opposite Roles of the JMJD1A Interaction Partners MDFI and MDFIC in Colorectal Cancer.

MyoD family inhibitor (MDFI) and MDFI domain-containing (MDFIC) are homologous proteins known to regulate myogenic transcription factors. Hitherto, their role in cancer is unknown. We discovered that MDFI is up- and MDFIC downregulated in colorectal tumors. Mirroring these different expression patterns, MDFI stimulated and MDFIC inhibited growth of HCT116 colorectal cancer cells. Further, MDFI and MDFIC interacted with Jumonji C domain-containing (JMJD) 1 A, a histone demethylase and epigenetic regulator involved in colorectal cancer. JMJD1A influenced transcription of several genes that were also regulated by MDFI or MDFIC. Notably, the HIC1 tumor suppressor gene was stimulated by JMJD1A and MDFIC, but not by MDFI, and HIC1 overexpression phenocopied the growth suppressive effects of MDFIC in HCT116 cells. Similar to colorectal cancer, MDFI was up- and MDFIC downregulated in breast, ovarian and prostate cancer, but both were overexpressed in brain, gastric and pancreatic tumors that implies MDFIC to also promote tumorigenesis in certain tissues. Altogether, our data suggest a tumor modulating function for MDFI and MDFIC in colorectal and other cancers that may involve their interaction with JMJD1A and a MDFIC→HIC1 axis.

Satellite cells and their regulation in livestock.

Satellite cells are the myogenic stem and progenitor population found in skeletal muscle. These cells typically reside in a quiescent state until called upon to support repair, regeneration, or muscle growth. The activities of satellite cells are orchestrated by systemic hormones, autocrine and paracrine growth factors, and the composition of the basal lamina of the muscle fiber. Several key intracellular signaling events are initiated in response to changes in the local environment causing exit from quiescence, proliferation, and differentiation. Signals emanating from Notch, wingless-type mouse mammary tumor virus integration site family members, and transforming growth factor-ß proteins mediate the reversible exit from growth 0 phase while those initiated by members of the fibroblast growth factor and insulin-like growth factor families direct proliferation and differentiation. Many of these pathways impinge upon the myogenic regulatory factors (MRF), myogenic factor 5, myogenic differentiation factor D, myogenin and MRF4, and the lineage determinate, Paired box 7, to alter transcription and subsequent satellite cell decisions. In the recent past, insight into mouse transgenic models has led to a firm understanding of regulatory events that control satellite cell metabolism and myogenesis. Many of these niche-regulated functions offer subtle differences from their counterparts in livestock pointing to the existence of species-specific controls. The purpose of this review is to examine the mechanisms that mediate large animal satellite cell activity and their relationship to those present in rodents.

Expression of the muscle-associated gene MYF6 in hairy cell leukemia.

Hairy cell leukemia (HCL) is a purine analog-responsive B-cell malignancy containing the BRAF V600E mutation, expressing CD22, CD11c, CD103, tartrate resistant acid phosphatase (TRAP) CD25, CD123, and annexin 1A. BRAF V600E and the latter 4 markers are usually absent in the more aggressive and chemoresistant variant HCLv. To evaluate differences between HCL and HCLv, expression microarrays comparing HCL with HCLv were performed for 24694 genes using 47323 probes. Microarray data from 35 HCL and 27 HCLv purified samples showed the greatest HCL-HCLv difference in the muscle-associated gene MYF6, expressed by its 2 probes 18.5- and 10.8-fold higher in HCL than HCLv (p<0.0001). By real-time quantitative PCR (RQ-PCR), 100% of 152 classic HCL samples were MYF6-positive, vs 5 (6%) of 90 blood donors. MYF6-expression was also detected in 18 (35%) of 51 with HCLv, 11 (92%) of 12 with HCL expressing unmutated IGHV4-34, 35 (73%) of 48 with chronic lymphocytic leukemia (CLL), and 1 (8%) of 12 with mantle cell lymphoma. Hypomethylation status of MYF6 supported expression in HCL more than HCLv. Posttreatment blood samples becoming negative by flow cytometry remained MYF6+ by RQ-PCR in 42 (48%) of 87 HCL patients, and MYF6 RQ-PCR could detect 1 HCL in 105 normal cells. MYF6, universally expressed in HCL and in most CLL samples, may be a useful biomarker for these leukemias. Further studies are underway to determine the role of MYF6 in HCL.

Toxoplasma gondii Impairs Myogenesis in vitro, With Changes in Myogenic Regulatory Factors, Altered Host Cell Proliferation and Secretory Profile.

Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-ß1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-ß failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/ß-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to ß-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients.

The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis.

Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p was related to the growth of skeletal muscle. Here, we further report that miR-146b-3p is essential for the proliferation, differentiation, and apoptosis of chicken myoblast. Elevated expression of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. Besides, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can inhibit the PI3K/AKT pathway. Our study also showed that both AKT1 and MDFIC can promote the proliferation and differentiation while inhibit the apoptosis of myoblast in chicken. Overall, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts in the proliferation, differentiation, and apoptosis of myoblast in chicken.

MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis.

Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.