MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken.

BACKGROUND: Skeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development. METHODS: We provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay. RESULTS: A total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway. CONCLUSIONS: Our results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.

Myosin heavy-chain myopathy in 2 American quarter horses.

A 1.5-year-old American quarter horse gelding (case 1) and an 11-month-old American quarter horse filly (case 2) were presented for acute onset pelvic lameness and lethargy. Case 1 had nasal discharge, while case 2 developed rapid muscle atrophy. Both horses had elevated serum creatine kinase activity. The horses showed similar polyphasic histiocytic and lymphoplasmacytic myositis with necrosis, mineralization, and regeneration. Additionally, case 1 had Streptococcus equi subsp. equi-induced suppurative retropharyngeal lymphadenitis with renal purpura hemorrhagica and myoglobinuric nephropathy. A focal pulmonary abscess caused by Actinobacillus equuli was found in case 2. Genetic testing revealed case 1 as heterozygous and case 2 as homozygous for the E321G MYH1 variant, supporting the diagnosis of myosin heavy-chain myopathy, with concomitant bacterial disease as potential triggers.

MYH1F promotes the proliferation and differentiation of chicken skeletal muscle satellite cells into myotubes.

In diploid organisms, interactions between alleles determine phenotypic variation. In previous experiments, only MYH1F was found to show both ASE (spatiotemporal allele-specific expression) and TRD (allelic transmission ratio distortion) characteristics in the pectoral muscle by comparing the genome-wide allele lists of hybrid populations (F1) of meat- and egg- type chickens. In addition, MYH1F is a member of the MYH gene family, which plays an important role in skeletal muscle and non-muscle cells of animals, but the specific expression and function of this gene in chickens are still unknown. Therefore, qRT-PCR was used to detect the expression of MYH1F in different tissues of chicken. Proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs) have been detected by transfection of MYH1F-specific small interfering RNA (siRNA). The results showed that the expression of MYH1F in chicken skeletal muscle was higher than that in other tissues. Combined with CCK-8 assay, EdU assay, immunofluorescence, and Western blot Assay, it was found that MYH1F knockdown could significantly suppress the proliferation of chicken SMSCs and depress the differentiation and fusion of the cells. These results suggest that MYH1F plays a critical role in myogenesis in poultry, which is of great significance for exploring the regulatory mechanisms of muscle development and improving animal productivity.

MYH1 is a candidate gene for recurrent rhabdomyolysis in humans.

Rhabdomyolysis is a serious medical condition characterized by muscle injury, and there are recognized genetic causes especially in recurrent forms. The majority of these cases, however, remain unexplained. Here, we describe a patient with recurrent rhabdomyolysis in whom extensive clinical testing failed to identify a likely etiology. Whole-exome sequencing revealed a novel missense variant in MYH1, which encodes a major adult muscle fiber protein. Structural biology analysis revealed that the mutated residue is extremely well conserved and is located in the actin binding cleft. Furthermore, immediately adjacent mutations in that cleft in other myosins are pathogenic in humans. Our results are consistent with the finding that MYH1 is mutated in rhabdomyolysis in horses and suggest that this gene should be investigated in cases with recurrent rhabdomyolysis.

Ectopic Overexpression of Porcine Myh1 Increased in Slow Muscle Fibers and Enhanced Endurance Exercise in Transgenic Mice.

Myosin heavy chain (MyHC) isoforms consist of Myh7, Myh2, Myh1, and Myh4, which are expressed in skeletal muscle tissues during postnatal development. These genes influence the contraction⁻relaxation activity in skeletal muscles and are involved in determining muscle composition such as the proportion of fast-to-slow and/or slow-to-fast fiber types. Among them, Myh1 is associated with skeletal muscle contraction and is involved in both slow-to-fast and fast-to-slow transition. However, the muscle transition mechanism is not well understood. For this study, we first produced porcine Myh1 transgenic (TG) mice to study whether the ectopic expressed porcine Myh1 gene had any effects on muscle composition, especially on slow-type muscle components. Our results showed that the factors associated with slow muscles, such as Myh7, Myoglobin, Troponin (slow-type units), and cytochrome C, were highly expressed in the quadriceps muscles of Myh1 transgenic mice. Furthermore, the ectopic porcine MYH1 protein was located only in the slow-type muscle fibers of the quadriceps muscles in Myh1 transgenic mice. In physical endurance tests, Myh1 transgenic mice ran longer and further on a treadmill than wild-type (WT) mice. These data fully supported our hypothesis that Myh1 is associated with slow muscle composition, with overexpression of Myh1 in muscle tissues possibly being a new key in modulating muscle fiber types. Our study provides a better understanding of muscle composition metabolism, physical mobility, and genetic factors in muscle fatigue.

Layer-specific proteomic profiling of human normal heart.

BACKGROUND: The organized functioning of the anisotropic myocardial layers-including the inner longitudinal, middle circular, and outer longitudinal layers-is essential for stable systemic circulation. However, the proteomic profile of each myocardial layer has not been studied yet. Here, we aimed to elucidate the layer-specific proteomic profile of human cardiac tissue using microscopic sampling. METHODS: Normal hearts were obtained from five autopsy cases, and cardiomyocytes were microdissected separately from the three myocardial layers of the left ventricle. Histological analysis and shotgun proteomic profiling were performed, followed by immunohistochemical analysis. RESULTS: Histologically, no significant changes were observed among the three layers regarding cardiomyocyte diameter and myocardial fibrosis. Totally 1220 proteins-comprising 9404 peptides-were identified from 15 samples, of which the expression levels of 92 proteins were significantly altered among the layers. Gene ontology enrichment analysis revealed that the proteins specifically elevated in the inner and outer layers mostly belonged to the actin filament-binding protein group. In particular, MYH1 was highly expressed in cardiomyocytes in the outer layer, and CTNNA3 was highly expressed at the intercalated disc in the inner layer. CONCLUSIONS: This is the first report on layer-specific proteomic profiling of human normal hearts. Anisotropic profiles of actin filament-binding proteins in myocardial layers may contribute to the anisotropic contractile and conductive abilities of the heart. Knowledge of the layer-specific proteome profiles of a human heart in the normal state can aid in further research on cardiac pathology, such as the prognosis and treatment of focal myocardial infarction.

LncRNA lncMGR regulates skeletal muscle development and regeneration by recruiting CDK9 and sponging miRNAs.

Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1 A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.

Prevalence of the E321G MYH1 variant in Brazilian Quarter Horses.

BACKGROUND: In the Quarter Horse (QH), myosin heavy chain myopathy (MYHM), which is characterised by nonexertional rhabdomyolysis or immune-mediated myositis (IMM) with acute muscle atrophy, is strongly associated with the missense E321G MYH1 mutation. OBJECTIVES: To document the existence of MYHM in the Brazilian QH population, this study includes a case report of two related QH foals with the E321G MYH1 mutation that had clinical signs of MYHM, with histological confirmation of IMM in one of the foals. This prompted an investigation the aim of which was to determine the allele frequency of the E321G MYH1 variant across QHs using a DNA archive in Brazil. Study design Cross sectional. METHODS: To estimate the allele frequency of the E321G MYH1 variant in Brazilian QHs, 299 DNA samples from QHs used in different disciplines (reining, barrel racing, halter, cutting and racing) were analysed. DNA fragments containing the region with the mutation were amplified by PCR and used for direct genomic sequencing. RESULTS: Of the 299 genotyped QHs, 44 animals (14.7%) were heterozygous (My/N) for the E321G MYH1 variant, and 255 (85.3%) were homozygous for the wild-type allele (N/N), implying an allele frequency of 0.074. Reining horses had a significantly higher prevalence of heterozygosity than horses in other disciplines (P = .008). MAIN LIMITATIONS: The DNA samples were collected from 2010 to 2014. As only registered QHs were evaluated, the results may not reflect the actual incidence in the general population of Brazilian QHs. CONCLUSIONS: The reported cases of MYHM and the high prevalence of the MYH1 mutation found in the assessed Brazilian QH population, particularly in reining QHs, suggests that MYHM should be included in genetic screening. Reasonable control measures are important to prevent an increase in the incidence of MYHM in QHs in Brazil.

Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G.

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G-/-) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/-) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/- and MYH1E321G-/- horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

Differential Expression of IGF1, IGFBP5, MSTN, and MYH1 Across Different Age Classes in American Quarter Horses.

The objective of this study was to determine the influence of age on expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein (IGFBP5), myostatin (MSTN), and myosin (MYH1) genes which are related to growth and muscle development in the American Quarter Horse. Thus, horses (n = 10) from weanling, yearling, 2-, 3-, and 10-year-old age classes were sampled and gene expression was assessed by RT-qPCR. ΔCT was calculated using the hypoxanthine-guanine phosphoribosyltransferase gene as an internal normalizer. The generalized linear model was used to determine differentially expressed genes, by pairwise comparison between ages. Among technical replicates, the coefficient of variation ranged from 1.0 to 5.2% and was lower than the variation observed between biological replicates (2.1-12.9%). IGF1 demonstrated significantly lower expression in the 3-year-old age class than in weanlings and yearlings, but the 10-year-old age class displayed a significantly higher level than 2- and 3-year-old age classes. Expression of IGFBP5 was highest in weanlings compared with all other age classes. Expression of MSTN was significantly higher in weanlings than in other age classes, whereas 10-year-old horses had an intermediate level of expression, but significantly different from yearlings, 2- and 3-year-old fillies. Finally, expression of MYH1 was lower in 2- and 10-year-old horses than in weanlings and yearlings, whereas 3-year-old fillies demonstrated an intermediate level of expression. Differential expression patterns observed in this preliminary study provide insight into the physiological changes occurring throughout the life span of horses. These patterns could also help explain the variation in performance and endurance between individuals at different developmental stages.

Prevalence of the E321G MYH1 variant for immune-mediated myositis and nonexertional rhabdomyolysis in performance subgroups of American Quarter Horses.

BACKGROUND: Immune-mediated myositis (IMM) in American Quarter Horses (QHs) causes acute muscle atrophy and lymphocytic infiltration of myofibers. Recently, an E321G mutation in a highly conserved region of the myosin heavy chain 1 (MYH1) gene was associated with susceptibility to IMM and nonexertional rhabdomyolysis. OBJECTIVES: To estimate prevalence of the E321G MYH1 variant in the QH breed and performance subgroups. ANIMALS: Three-hundred seven elite performance QHs and 146 random registered QH controls. METHODS: Prospective genetic survey. Elite QHs from barrel racing, cutting, halter, racing, reining, Western Pleasure, and working cow disciplines and randomly selected registered QHs were genotyped for the E321G MYH1 variant and allele frequencies were calculated. RESULTS: The E321G MYH1 variant allele frequency was 0.034 ± 0.011 in the general QH population (6.8% of individuals in the breed) and the highest among the reining (0.135 ± 0.040; 24.3% of reiners), working cow (0.085 ± 0.031), and halter (0.080 ± 0.027) performance subgroups. The E321G MYH1 variant was present in cutting (0.044 ± 0.022) and Western Pleasure (0.021 ± 0.015) QHs at lower frequency and was not observed in barrel racing or racing QHs. CONCLUSIONS AND CLINICAL IMPORTANCE: Knowing that reining and working cow QHs have the highest prevalence of the E321G MYH1 variant and that the variant is more prevalent than the alleles for hereditary equine regional dermal asthenia and hyperkalemic periodic paralysis in the general QH population will guide the use of genetic testing for diagnostic and breeding purposes.

Associations of MYH3 gene copy number variations with transcriptional expression and growth traits in Chinese cattle.

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.