The growth and development of bovine skeletal muscle and beef yield is closely intertwined. Our previous research found that forkhead box O1 (FOXO1) plays an important role in the regulation of beef muscle formation, but its specific mechanism is still unknown. In this study, we aimed to clarify the regulatory mechanism of FOXO1 in proliferation and differentiation of bovine skeletal muscle cells (BSMCs). The results showed that interfering with FOXO1 can promote proliferation and the cell G1/S phase of BSMCs by up-regulating the expression of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2. Besides, interfering with FOXO1 inhibited the apoptosis of BSMCs by up-regulating the expression of anti-apoptosis gene BCL2, while simultaneously down-regulating the expression of the pro-apoptosis genes BAD and BAX. Inversely, interfering with FOXO1 can promote the differentiation of BSMCs by up-regulating the expression of myogenic differentiation marker genes MYOD, MYOG, MYF5, MYF6 and MYHC. Furthermore, RNA-seq combined with western bolt, immunofluorescence and chromatin immunoprecipitation analysis showed that FOXO1 could regulate BSMCs differentiation process by influencing PI3K-Akt, Relaxin and TGF-beta signaling pathways, and target MYH3 for transcriptional inhibition. In conclusion, this study provides a basis for studying the role and molecular mechanism of FOXO1 in BSMCs.

Improving meat quality has become the main goal of modern pig breeding. Intramuscular fat content (IMF) is an important trait influencing meat quality of livestock, but the molecular mechanism behind this trait is still unclear. Recently, Cho et al. reported the discovery of the first causal mutation affecting IMF and red flesh color (a*) in pigs, namely XM_013981330.2:g.-1805_-1810del, a 6-bp deletion variant in the porcine MYH3 promoter region. The objective of this study was to reassess the causality of this mutation for its potential commercial application. By Sanger sequencing, we firstly identified several new variants (including a 4-bp deletion) at or near the 6-bp deletion site, which formed four haplotypes in multiple breeds. Unexpectedly, the 6-bp deletion allele, previously determined as the MYH3 Q allele because of its significantly positive effect on IMF and a*, was found not only in Chinese indigenous breeds, but also in four western commercial breeds with relatively lower IMF levels, including Duroc, Large White, Landrace and Pietrain. More surprisingly, we found that the MYH3 Q allele and the haplotypes harboring it had no significant effects on IMF, marbling and color score in three large-scale divergent pig populations: the heterogeneous F6 and F7 pigs and commercial crossbred Duroc × (Landrace × Yorkshire) pigs. Transient transfection analysis in porcine satellite cells showed that the 6-bp deletion variants had a negligible effect on transcription of reporter gene, but could attenuate the MRF (myogenesis regulatory factors)-induced increase in luciferase activity of the MYH3 promoter vector. The MYH3 protein level in muscle did not differ significantly among the haplotype groups. Therefore, our results cannot support the causal relationship between the 6-bp deletion in MYH3 and IMF trait, suggesting that the causal mutation for the IMF QTL on SSC12 needs to be further identified.

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

Sheldon-Hall syndrome is the most common type of distal arthrogryposis syndromes, also known as distal arthrogryposis 2B (DA2B). Sheldon-Hall syndrome is caused by mutations in the TPM2, TNNI2, TNNT3 or MYH3 gene and characterized by ulnar deviation, camptodactyly, overlapping fingers and scoliosis from birth. We investigated a Chinese family with multiple members who clinically presented with distal arthrogryposis of the hands. In total, 261 subjects including one proband and ten family members from the non-consanguineous Chinese family and 250 healthy volunteers were included and had their genomic DNA extracted. A novel missense mutation in exon 13 of the MYH3 gene, c.1160A > G (p.Tyr387Cys), was identified in the proband and his father through whole-exome sequencing. The proband and six affected family members were confirmed to carry this mutation by Sanger sequencing, although the mutation was not detected in the four unaffected individuals or 250 volunteers. This is the first report of a novel MYH3 mutation being identified as the cause of DA2B in a Chinese family. Our findings confirm that MYH3 gene mutations can be a pathogenic cause of DA2B in Asian patients. This study increases the mutational spectrum in MYH3 and aids genetic counseling and prenatal diagnosis.

Copy number variations (CNVs) have been recently recognized as another important genetic variability complementary to single nucleotide polymorphisms (SNPs). Compelling evidence has indicated that CNVs are responsible for phenotypic traits by changing the copy numbers of functional genes. Myosin heavy chain 3 (MYH3) gene is a critical regulatory factor in skeletal muscle development, and has been detected in the CNVs region by comparative genomic hybridization (CGH) array. This study was conducted to validate and detect the distribution of MYH3 copy numbers (relative to Angus cattle) in four Chinese cattle breeds (NY, QC, LX, and CY), and further to investigate the associations of the copy number changes with its transcriptional expression and cattle growth traits. Substantial genetic differences of MYH3 copy numbers were identified between NY and the other three breeds (P<0.01). The copy numbers of MYH3 gene presented the positive correlations with the transcript level of MYH3 gene in both fetal and adult skeletal muscles (P<0.05). Statistical analysis revealed that CNVs of MYH3 gene were significantly associated with growth traits of NY cattle, and the individuals with copy number gain showed better phenotypes than the loss and/or median groups (P<0.05). This study firstly attempted to establish the correlations between CNVs of candidate genes and growth traits, and our results suggested that the CNVs of MYH3 gene may be utilized as the potential markers for economic traits in selection breeding programs of Chinese cattle.

Distal arthrogryposes (DAs), a clinically and genetically heterogeneous group of disorders characterized by congenital contractures with predominant involvement of the hands and feet, can be classified into at least 12 different forms. These autosomal dominant disorders are of variable expressivity and reduced penetrance. Mutations in sarcomeric protein genes, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosin 2 (TPM2), embryonic myosin heavy chain 3 (MYH3), and myosin binding protein C1 (MYBPC1), have been identified in distal arthrogryposis type 1 (DA1, MIM 108120), type 2B (DA2B, MIM 601680) and type 2A (DA2A)/Freeman-Sheldon syndrome (FSS, MIM 193700). However, mutations causing FSS have only been reported in MYH3. Herein we describe a Chinese DA family whose members meet classical strict criteria for FSS, as well as one member of the family who has isolated facial features consistent with FSS. No disease-causing mutation was found in MYH3. Segregation of microsatellite markers flanking the TNNI2 and TNNT3 genes at 11p15.5 was compatible with linkage. Subsequent sequencing of TNNI2 revealed a novel mutation, c.A493T (p.I165F), located in the C-terminal region, which is critical for proper protein function. This mutation was found to cosegregate with the FSS phenotype in this family, and assessment using SIFT and PolyPhen-2 predicted a damaging effect. To the best of our knowledge, we report the first TNNI2 mutation in classical FSS and describe an atypical adult FSS case with only facial contractures resulting from somatic mosaicism. We infer that DA1, DA2B and FSS represent a phenotypic continuum of the same disorder and provide further genetic evidence for this hypothesis.