Expression Of MYH Research Articles

Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice

BackgroundTitinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin—titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses.ResultsThe overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles.ConclusionsWe were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

Diet and Exercise Restores Muscle Fiber Phenotype Following Long Term High Fat/High Sugar Diet Yet mRNA Expression Remains Unchanged

Obesity affects roughly 42% of the US population. High fat/high sugar diets (HFHS) often referred as “western diet” contributes to this prevalence. Diet‐induced obesity (DIO) results in impaired metabolic responses and associated disease states (i.e., Type 2 Diabetes). Metabolic impairments in DIO are a result of changes in muscle metabolism and muscle fiber phenotype. This is determined by both mRNA expression and protein isoform‐content of myosin heavy chain (MHC). Fast muscle fiber phenotype (i.e., type IIb in mice) is characterized by lower capacity for lipid utilization, implicated in the pathogenesis of Type 2 Diabetes. Regular exercise expresses mRNA in a fiber type specific manner, shifting MHC proportions under healthy circumstances. However, diet and exercise‐driven fiber type shifts in DIO are less understood. We aimed to determine the impact of exercise and diet on fiber‐type proportions in mice with DIO. We hypothesized exercise shifts mouse gastroc muscle phenotype induced by HFHS diet away from IIb fast fiber types. 49 C57BL/6 mice were split into 4 groups: 1) Lean Control (n = 9) fed standard chow and water for 24 wks, 2) HFHS (n = 10), fed HFHS diet (60% of calories from fat, high sugar/fructose: 42 g/L in drinking water) for 24 wks, 3) HFHS Control (n = 10) fed HFHS diet for 12 wks followed by standard chow and water for 12 wks (i.e., simulating traditional diet approach), and 4) HFHS + exercise group (n = 10) fed HFHS diet for 24 wks and performed aerobic exercise (30 min of treadmill running 5 d/wk) in the last 12 wks. Gastroc muscles were collected, homogenized, and analyzed for MHC mRNA expression of MYH 1 (type IIx), MYH 2 (type IIa), MYH 4 (type IIb), and protein isoforms via q‐PCR and SDS‐PAGE respectively. Intensity of bands corresponding to MHC isoforms were quantified using Image J. mRNA expression was calculated using comparative CT analysis (2‐∆∆CT). Paired sample t‐tests were conducted to test differences between MHC isoforms across groups. Proportions of MHC IIb isoform increased (91 ± 3%) in HFHS compared to Control (81 ± 6.2%, p>0.05), HFHS Control (77 ± 3%, p=0.004), and HFHS + exercise groups (79 ± 5%, p=0.057). Additionally, MHC IIa/x proportions in the HFHS (8 ± 3%) compared to Control (17 ± 5.9%, p>0.05), HFHS Control (20 ± 2%, p=0.004), and HFHS + exercise (18 ± 4%, p=0.054) groups were reduced. There were no differences in mRNA expression data. These data suggest performing aerobic exercise with HFHS diet or switching to a standard diet restores muscle fiber phenotype in mouse gastroc. Yet, this was not reflected in mRNA expression data. Changes in MHC isoforms are likely better detected at the protein level rather than mRNA expression. We conclude that exercise and dietary intervention may be a good strategy to shift MHC isoforms away from extreme fast fiber phenotype with lower capacity for lipid utilization. Future research should aim to determine single muscle fiber phenotype shifts with long‐term diet and exercise in humans with obesity to better understand regulation of muscle fiber phenotypes impact on human metabolism.

Ostarine-Induced Myogenic Differentiation in C2C12, L6, and Rat Muscles.

Ostarine (also known as enobosarm or Gtx-024) belongs to the selective androgen receptor modulators (SARMs). It is a substance with an aryl-propionamide structure, classified as a non-steroidal compound that is not subjected to the typical steroid transformations of aromatization and reduction by α5 reductase. Despite ongoing research on ostarine, knowledge about it is still limited. Earlier studies indicated that ostarine may affect the metabolism of muscle tissue, but this mechanism has not been yet described. We aimed to investigate the effect of ostarine on the differentiation and metabolism of muscle. Using C2C12 and L6 cells, as well as muscles obtained from rats administered ostarine, we showed that ostarine stimulates C2C12 and L6 proliferation and cell viability and that this effect is mediated by androgen receptor (AR) and ERK1/2 kinase activation (p < 0.01). We also found that ostarine stimulates muscle cell differentiation by increasing myogenin, MyoD, and MyH expression in both types of cells (p < 0.01). Moreover, pharmacological blocking of AR inhibits the stimulatory effect of ostarine. We further demonstrated that 30 days of ostarine administration increases myogenin, MyoD, and MyH expression, as well as muscle mass, in rats (p < 0.01). Based on our research, we conclude that ostarine stimulates muscle tissue proliferation and differentiation via the androgen receptor.

Quantitative Proteome Analysis in Response to Glucose Concentration in C2C12 Myotubes

Glucose is important for the maintenance of muscle function; however, it is still unclear how changes in glucose concentration affect muscle. Here, we analyzed the effect of glucose concentration on protein expression under different glucose concentration media in C2C12 myotubes. First, we performed proteome analysis in C2C12 myotubes cultured in Low (1.0 g/L), Medium (2.0 g/L), and High (4.5 g/L) glucose media. Proteome analysis revealed 113 proteins were significantly changed in group cultured in Low or Medium glucose media compared to group cultured in High glucose media. Furthermore, glycolysis, oxidative phosphorylation, and fatty acid metabolism were increased in the Medium and Low groups. Among these pathways, HK2, PFKP, NDUFA11, and FABP3 were especially upregulated proteins in Low and Medium groups. In this context, ATP production in C2C12 myotubes cultured in Low and Medium glucose media was increased. There was no significant change in myotubes morphology and myogenic differentiation factors in all groups. Finally, we examined the effect on glucose concentration in culture media on myosin isoforms expression by qRT-PCR. As a result, Myh2 and Myh4 were significantly increased in Low and Medium conditions. Altogether, Low and Medium glucose conditions induced Myh expression probably via enhancement glucose utilization.

A standardized herbal combination of Astragalus membranaceus and Paeonia japonica, protects against muscle atrophy in a C26 colon cancer cachexia mouse model

Ethnopharmacological relevanceAstragalus membranaceus (Fisch.) and Bunge and Paeonia japonica (Makino)Miyabe & H.Takeda have been traditionally used to improve the poor quality of life such as weakness, lack of appetite, fatigue, and malaise which is considered with cachexia condition. Aim of the studyWe investigated anti-cachectic effects of a herbal formula composed of Astragalus membranaceus and Paeonia japonica (APX) and the molecular mechanisms of APX in C26 cancer-induced cachexia mice and TNF-a-treated C2C12 myotubes. Additionally synergistic anti-cachectic effects of APX were compared to those of individual herbal extracts and megestrol acetate. Methods and materialsThe forty-two BALB/c mice were randomly divided into 6 groups: normal (nontreatment), control (C26 injection), AM (C26 injection with Astragalus membranaceus), PJ (C26 injection with Paeonia japonica), APX (C26 injection with combination of Astragalus membranaceus and Paeonia japonica and MA (C26 injection with megestrol acetate). All mice were orally administered DW (normal and control groups) or 100 mg/kg AM, PJ, APX or MA for 10 days. In the animal model, several tissues were weighed, and muscle tissue and blood were used to measure pro-inflammatory cytokines. C2C12 myotubes were exposed to 100 ng/mL TNF- α with or without 10 μg/mL of AM, PJ, APX or MA for 48 h. The cells were used to immunofluorescence staining and western blot analyses. ResultsC26 injection induced notable body and muscle weight loss while APX administration significantly attenuated these alterations and the decrease of muscle weights and strength. APX also significantly attenuated the abnormal elevations in the concentration of three muscle atrophy-inducible cytokines; serum and muscle TNF-α,muscle TWEAK and IL-6 in C26 tumor-bearing mice. In the TNF-α-treated C2C12 myotube model, TNF-α treatment notably decreased MyH but activated atrophic proteins (MuRF and Fbx32) along with p38 and NFκB while these molecular alterations were significantly ameliorated by APX treatment. These pharmacological actions of APX were supported by the results of immunofluorescence staining to MyH expression and the translocation of NFκB into the nucleus in C2C12 myotubes. ConclusionsOur data indicate the potential of an herbal formula, APX as an anti-cachexia agent; the effect of APX was superior to that of megestrol acetate overall especially for muscle atrophy. The underlying mechanisms of this herbal formula may involve the modulation of muscle atrophy-promoting molecules including p38, NFκB, TNF-α and TWEAK.

Identification of histamine receptor subtypes in skeletal myogenesis.

To date, conventional and/or novel histamine receptors (HRs) have not been investigated in mouse skeletal myogenesis. Therefore, the present study aimed to investigate the HR-subtypes in skeletal myogenesis. The myogenesis of C2C12 skeletal myoblasts was evaluated using desmin, myogenin and myosin heavy chain (Myh) as early, intermediate and late differentiation markers, respectively. Reverse transcription-quantitative polymerase chain reaction and immunostaining were performed and the messenger RNA (mRNA) expression levels of the HR-subtypes and markers were determined. H1R mRNA was found to be highly expressed in myoblasts at day 0; however, the expression levels were reduced as differentiation progressed. By contrast, H2R mRNA expression remained constant, while H3R mRNA expression increased by 28-, 103- and 198-fold at days 2, 4 and 6 compared with the baseline level (day 0), respectively. In addition, Myh expression increased by 7,718-, 94,487- and 286,288-fold on days 2, 4 and 6 compared with the baseline expression level (day 0). Weak positive staining of the cells for H3R protein was observed on day 2, whereas highly positive staining was observed on days 4 and 6. HR expression during myogenesis was, in part, regulated by the stage of differentiation. These results along with previous findings indicated possible involvement of H1R in the regulation of progenitor cell mitogenesis and of H2R in the relaxation of acetylcholine-stimulated contraction of muscle cells, following the activation of professional histamine-producing cells, including mast cells. By contrast, H3R may participate in the regulation of specialized myocyte functions, potentially by maintaining the relaxed state under the influence of constitutive H3R activity and low histamine concentrations, locally produced/released by non-professional histamine-producing cells.

Molecular changes in detrained &amp; retrained adult jaw muscle

A hypofunctional masticatory system was developed in 21-day-old male rats by feeding them a soft diet for 27 weeks. Retraining of a parallel group for 6 weeks was achieved by switching back to a hard diet after 21 weeks. A control group was fed a hard diet for 27 weeks. At the end of the experimental period, the expression levels of the myosin heavy chain isoform genes MYH 1 and 2 (fast), 3 (embryonic) and 7 (slow) in the deep masseter were compared using qRT-PCR analysis. The gene expressions of MYH 3 and MYH 7 were significantly higher in the rehabilitation group compared with the normal and hypofunctional group, but no significant differences were found in regards to the gene expression of MYH 1 and 2. Retraining made it possible for the slow (MYH 7) isoform levels to adapt to the increased mechanical load. The increased level of embryonic (MYH 3) isoform could be due to the need of creation of new MYH isoforms.

Myostatin facilitates slow and inhibits fast myosin heavy chain expression during myogenic differentiation

Skeletal muscles in the limb and body trunk are composed of heterogeneous myofibers expressing different isoforms of myosin heavy chain (Myh), including type I (slow, Myh7), IIA (intermediate, Myh2), IIX (fast, Myh1), and IIB (very fast, Myh4). While the contraction force and speed of a muscle are known to be determined by the relative abundance of myofibers expressing each Myh isoform, it is unclear how specific combinations of myofiber types are formed and regulated at the cellular and molecular level. We report here that myostatin (Mstn) positively regulates slow but negatively regulates fast Myh isoforms. Mstn was expressed at higher levels in the fast muscle myoblasts and myofibers than in the slow muscle counterparts. Interestingly, Mstn knockout led to a shift of Myh towards faster isoforms, suggesting an inhibitory role of Mstn in fast Myh expression. Consistently, when induced to differentiate, Mstn null myoblasts formed myotubes preferentially expressing fast Myh. Conversely, treatment of myoblasts with a recombinant Mstn protein upregulated Myh7 but downregulated Myh4 gene expression in newly formed myotubes. Importantly, both Mstn antibody and soluble activin type 2B receptor inhibited slow Myh7 and promoted fast Myh4 expression, indicating that myostatin acts through canonical activin receptor to regulate the expression of Myh genes. These results demonstrate a role of myostatin in the specification of myofiber types during myogenic differentiation.

Expression and clinical significance of the DNA repair enzyme MYH in esophageal squamous cell carcinoma

MYH is an important enzyme in combating DNA oxidative stress in the occurrence and development of various types of tumors. To investigate the correlation between expression of the DNA repair enzyme MYH in esophageal squamous cell carcinoma and 8-oxoguanine (8-oxoG) oxidative damage, as well as the clinical significance of altered MYH expression, tissues from 175 esophageal carcinoma cases were investigated in the present study. MYH expression and 8-oxoG oxidative damage in squamous cell carcinoma and adjacent normal tissue were assessed by immunohistochemistry and Western blotting. In 82.9% (145/175) of the cases, MYH protein expression in esophageal squamous cell carcinoma was lower than that of adjacent normal tissue (t=4.24, P<0.001). Additionally, 8-oxoG staining was higher in the tumors than in the normal tissue. Lower expression of MYH in esophageal squamous cell carcinoma was associated with depth of invasion, venous invasion, TNM stage and lymph node metastasis (P<0.05). In conclusion, a lower MYH expression level in esophageal cell carcinoma tissue was inversely associated with more severe 8-oxoG oxidative damage, suggesting that changes in MYH activity correspond to increased DNA damage in tumor cells. The use of MYH expression as a postoperative index for esophageal squamous cell carcinoma may guide the formulation of individualized chemotherapy for patients after surgery.

P226 Decreased expression of MYH is associated with oral carcinogenesis in betel-nut chewers

P226 Decreased expression of MYH is associated with oral carcinogenesis in betel-nut chewers

Immunohistochemical Expression of MYH Protein Can Be Used to Identify Patients With MYH-Associated Polyposis

MYH-associated polyposis is a recently described, autosomal-recessive disease characterized by multiple colorectal adenomas and cancer. There are only few immunohistochemical studies of the MYH protein. We investigated the expression pattern of the MYH protein to evaluate whether a immunohistochemical approach could be used in clinical practice to screen patients for germline mutations in the MYH gene. The expression of MYH, MSH2, MLH1, and MSH6 proteins was studied by immunohistochemistry in 20 samples (colorectal adenomas or cancer) from 18 patients with biallelic MYH mutation, in 11 samples from patients with germline adenomatous polyposis coli (APC) mutations, in 20 samples from patients with sporadic colorectal cancers, and in 10 samples from patients with normal colonic mucosa without malignancies. In all cases the mismatch repair proteins were expressed normally. Nuclear and cytoplasmic immunoreactivity for the MYH protein were observed in normal colorectal mucosa, in sporadic colorectal carcinomas, and in adenomas and carcinomas from patients carrying APC germline mutations. Adenomas and carcinomas from patients with MYH biallelic mutation showed a different pattern of expression: a strong granular cytoplasmic staining was observed without any nuclear expression. The same immunophenotype was observed in the surrounding normal mucosa. Patients with biallelic MYH mutations showed disappearance of staining from the nucleus, and segregation of immunoreactivity in the cytoplasm, both in neoplastic and surrounding healthy mucosa. Because this pattern of expression seems to be specific for biallelic mutations, it follows that immunohistochemistry might be used in clinical practice to screen patients at risk for MYH-associated polyposis.

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease.

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.

Replication-Associated Repair of Adenine:8-Oxoguanine Mispairs by MYH

Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG.

Mice Expressing Mutant Myosin Heavy Chains Are a Model for Familial Hypertrophic Cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myocellular disarray, arrhythmias, and sudden death. Mutations in several contractile proteins, including cardiac myosin heavy chains, have been described in families with this disease, leading to the hypothesis that HCM is a disease of the sarcomere. A mutation in the myosin heavy chain (Myh) predicted to interfere strongly with myosin's binding to actin was designed and used to create an animal model for HCM. Five independent lines of transgenic mice were produced with cardiac-specific expression of the mutant Myh. Although the mutant Myh represents a small proportion (1-12%) of the heart's myosin, the mice exhibit the cardiac histopathology seen in HCM patients. Histopathology is absent from the atria and primarily restricted to the left ventricle. The line exhibiting the highest level of mutant Myh expression demonstrates ventricular hypertrophy by 12 weeks of age, but the further course of the disease is strongly affected by the sex of the animal. Hypertrophy increases with age in female animals while the hearts of male show severe dilation by 8 months of age, in the absence of increased mass. The low levels of the transgene protein in the presence of the phenotypic features of HCM suggest that the mutant protein acts as a dominant negative. In addition, the distinct phenotypes developed by aging male or female transgenic mice suggest that extragenic factors strongly influence the development of the disease phenotype.