Myocardial Afterload Is a Key Biomechanical Regulator of Atrioventricular Myocyte Differentiation in Zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development.

MicroRNA-202 Induces Myoblast to Myocyte Differentiation through Targeting Rock-1

The expression patterns of microRNAs (small non-coding RNAs) are altered in many biological processes such as myogenesis. In this study, we aimed to investigate the impact of predicted miR-202, its target genes Akt2 and Rock-1 as a potential regulator of myoblast in the myocyte differentiation process using the C2C12 cell line. After confirmation of the differentiation process induced by 3% horse serum, the expression level of miRNA and its targets were evaluated. In the following, a luciferase assay was conducted to approve the effect of miRNA on its target. Our results indicated that miR-202 and Akt2 were significantly up-regulated during differentiation, while Rock-1 was downregulated. Co-transfection of miRNA with psiCHECK2-Rock-1 significantly presented that Rock-1 was directly targeted by miR-202. On the contrary, miR-202 has failed to enforce its inhibitory effect on Akt2 expression. In particular, miR-202 seems to be a regulator of muscle differentiation pathway thought targeting Rock-1.

The miR-133a, TPM4 and TAp63γ Role in Myocyte Differentiation Microfilament Remodelling and Colon Cancer Progression

MicroRNAs (miRNAs) play an essential role in the regulation of a number of physiological functions. miR-133a and other muscular miRs (myomiRs) play a key role in muscle cell growth and in some type of cancers. Here, we show that miR133a is upregulated in individuals that undertake physical exercise. We used a skeletal muscle differentiation model to dissect miR-133a’s role and to identify new targets, identifying Tropomyosin-4 (TPM4). This protein is expressed during muscle differentiation, but importantly it is an essential component of microfilament cytoskeleton and stress fibres formation. The microfilament scaffold remodelling is an essential step in cell transformation and tumour progression. Using the muscle system, we obtained valuable information about the microfilament proteins, and the knowledge on these molecular players can be transferred to the cytoskeleton rearrangement observed in cancer cells. Further investigations showed a role of TPM4 in cancer physiology, specifically, we found that miR-133a downregulation leads to TPM4 upregulation in colon carcinoma (CRC), and this correlates with a lower patient survival. At molecular level, we demonstrated in myocyte differentiation that TPM4 is positively regulated by the TA isoform of the p63 transcription factor. In muscles, miR-133a generates a myogenic stimulus, reducing the differentiation by downregulating TPM4. In this system, miR-133a counteracts the differentiative TAp63 activity. Interestingly, in CRC cell lines and in patient biopsies, miR-133a is able to regulate TPM4 activity, while TAp63 is not active. The downregulation of the miR leads to TPM4 overexpression, this modifies the architecture of the cell cytoskeleton contributing to increase the invasiveness of the tumour and associating with a poor prognosis. These results add data to the interesting question about the link between physical activity, muscle physiology and protection against colorectal cancer. The two phenomena have in common the cytoskeleton remodelling, due to the TPM4 activity, that is involved in stress fibres formation.

EMBR-15. PRC2 COMPLEX ENFORCES NEURONAL LINEAGE AND SUPPRESSES TUMOR GROWTH IN SHH MEDULLOBLASTOMA

Hyperactivation of Sonic Hedgehog (SHH) signaling pathway drives tumor progression in the largest medulloblastoma subgroup. During cerebellar development, promoters of SHH target genes show inhibitory trimethylation of histone H3 at lysine 27 (H3K27me3), mediated by the Polycomb Repressive Complex 2 (PRC2). Here, we explored the regulation of cerebellar growth and medulloblastoma tumorigenesis by PRC2 complex components EED and EZH2. For developmental studies, we conditionally deleted Eed or Ezh2 in the Atoh1 lineage that gives rise to the cerebellar granule neuron progenitors (CGNP) that are cells of origin for SHH medulloblastomas. For tumor studies, we bred the conditional Eed- or Ezh2-deleted mouse lines with mice genetically engineered to develop SHH medulloblastoma. Our developmental studies showed that Eed was absolutely required for cerebellar growth. Eed-deleted CGNPs underwent aberrant, myocyte-like differentiation and spontaneous apoptosis, resulting in cerebellar hypoplasia. In contrast, Ezh2 deletion produced no developmental phenotype, despite blocking all H3K27me3 in CGNPs. Our tumor studies showed that Eed-deleted medulloblastomas similarly showed aberrant, myocyte differentiation, but unlike CGNPs, did not undergo widespread apoptosis. Eed-deleted medulloblastomas progressed more rapidly than control tumors, indicating that the inappropriate, muscle-like differentiation did not slow tumor growth. Ezh2-deleted medulloblastomas similarly progressed more rapidly than controls. Our data show that the PRC2 complex acts to enforce neuronal lineage commitment in both development and tumorigenesis and to restrain tumor growth in SHH medulloblastoma. Myocyte differentiation in Eed-deleted tumors suggests that PRC2 loss of function may contribute to the medullomyoblastomas that have been observed in patients. The differences in developmental phenotype show that EZH2 and EED functions are non-identical and can be dissociated, while similar increase in tumor progression show tumor suppressive functions for both EED and EZH2.

miR-322/miR-503 clusters regulate defective myoblast differentiation in myotonic dystrophy RNA-toxic by targeting Celf1

Myotonic dystrophy (DM) is a genetic disorder featured by muscular dystrophy. It is caused by CUG expansion in the myotonic dystrophy protein kinase gene that leads to aberrant signaling and impaired myocyte differentiation. Many studies have shown that microRNAs are involved in the differentiation process of myoblasts. The purpose of this study was to investigate how the miR-322/miR-503 cluster regulates intracellular signaling to affect cell differentiation. The cell model of DM1 was employed by expressing GFP-CUG200 or CUGBP Elav-like family member 1 (Celf1) in myoblasts. Immunostaining of MF-20 was performed to examine myocyte differentiation. qRT-PCR and western blot were used to determine the levels of Celf1, MyoD, MyoG, Mef2c, miR-322/miR-503, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling. Dual luciferase assay was performed to validate the interaction between miR-322/miR-503 and Celf1. CUG expansion in myoblasts impaired the cell differentiation, increased the Celf1 level, but it decreased the miR-322/miR-503 levels. miR-322/miR-503 mimics restored the impaired differentiation caused by CUG expansion, while miR-322/miR-503 inhibitors further suppressed. miR-322/miR-503 directly targeted Celf1 and negatively regulated its expression. Knockdown of Celf1 promoted myocyte differentiation. Further, miR-322/miR-503 mimics rescued the impaired differentiation of myocytes caused by CUG expansion or Celf1 overexpression through suppressing of MEK/ERK signaling. miR-322/miR-503 cluster recover the defective myocyte differentiation caused by RNA-toxic via targeting Celf1. Restoring miR-322/miR-503 levels could be an avenue for DM1 therapy.

Interleukin-4 Promotes Myogenesis and Boosts Myocyte Insulin Efficacy

Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy metabolism in muscle, the largest insulin-targeting organ, remain obscure. The study aimed at addressing the roles of IL-4 in myocyte differentiation (myogenesis) and energy metabolism of muscle cells. Effects of IL-4 on myogenesis, and interaction between IL-4 and insulin on glucose metabolism of C2C12 myoblasts and the terminal differentiated myocytes were analyzed. IL-4 improved GLUT4 translocation and tended to elevate glucose uptake by boosting insulin signaling. In diabetic mice, transient and long-term IL-4 showed differential effects on insulin signaling and efficacy. The study provides evidence to address the roles of IL-4 in mediating whole-body muscle reservoir and glucose metabolism, as well as the interaction between immune responses and energy homeostasis. IL-4 has dual potential to act as an adjuvant therapeutic target for sarcopenia to preserve muscle mass and insulin resistance to improve insulin sensitivity, which implicates the regulation of immune system to the muscle differentiation and exercise performance.

Analysis on correlation between polymorphism of MyoG gene exon I and body size traits of sheep

Myogenin (MyoG) is responsible for centering control over myocyte differentiation process and can influence the meat production of animals directly. Large tailed Han sheep, small tailed Han sheep, Yuxi fatty tailed sheep, Lanzhou large tailed sheep, Mongolia sheep and Tong Sheep were used as the experimental materials in this study. Polymorphism of MyoG gene exon I was tested by native polyacrylamide gel electrophoresis. The correlation between polymorphism of MyoG gene exon I and body size traits was analyzed. Influences of MyoG gene exon I on growth traits of sheep were discussed. Results demonstrated that 2 alleles (A, B) and 3 genotypes (AA, BB and AB) were detected in MyoG gene exon I of all 6 sheep varieties. The A allele frequencies in MyoG gene exon I of small tailed Han sheep, large tailed Han sheep, Yuxi fatty tailed sheep, Lanzhou large tailed sheep, Mongolia sheep and Tong Sheep were 0.5167, 0.2500, 0.4376, 0.6500, 0.5750 and 0.7125, while the B allele frequencies were 0.4833, 0.7500, 0.5625, 0.3500, 0.4250 and 0.2875, respectively. Chest width and neck length of the AA genotype of MyoG gene exon I were significantly higher than those of the AB genotype (P less than 0.01) and BB genotype (P less than 0.05). Body length and rump length of the AA genotype were significantly higher than those of the AB genotype (P less than 0.05). The chest depth and hip width of the AA genotype was far lower than that of the AB genotype (P less than 0.01). The hip height of the AA genotype was far lower than that of the BB genotype (P less than 0.05).

A Missense Mutation in KCNJ12 Was Strongly Associated with Beef Cattle Stature

Potassium inwardly-rectifying channel, subfamily J, member 12 (KCNJ12) gene is one promising candidate for economic traits because of its crucial roles in myoblast development. Here, a missense mutation (Cys>Arg), was firstly detected to locate in exon 3 of KCNJ12 from three Chinese cattle breeds by DNA-pool sequencing. Then, we performed the association analysis of this SNP with stature in three Chinese cattle populations (n = 820). Significantly positive correlation was revealed by reduced animal general linear model and the genotype of CC is the most excellent genotype in three breeds. Further, we measured the expression profiling of the KCNJ12 gene in various cattle tissues and primary bovine skeletal muscle cells. Ubiquitous expression with high abundance in muscle was observed. Further, in primary bovine skeletal muscle cells, the KCNJ12 mRNA expression was gradually up-regulated in differentiation medium (DM) compared with that in growth medium (GM), suggesting that KCNJ12 gene is involved in bovine myocyte differentiation. Conclusively, KCNJ12 gene is a functional candidate gene which can be used as molecular marker for beef cattle breeding.