Myosin Heavy Chain IIA Research Articles

The curious incident of a cavum velum interpositum cyst in twins of a mother carrying May-Hegglin anomaly: a case report and short literature review

BackgroundMay-Hegglin anomaly is an autosomal dominant inherited condition, characterized by thrombocytopenia, giant platelets and Dohle-like bodies. Incidence is unknown and affected individuals can show from mild to moderate-severe haemorrhagic symptoms. The cyst of cavum veli interpositi (a virtual space filled with fluid within the third ventricle) is rarely reported in the foetal period. Furthermore, it is unclear whether isolated cavum veli interpositi cysts are a normal variant or developmental malformations. The simultaneous presence of these two anomalies was never described.Case presentationWe describe a very rare case of a twin monochorionic pregnancy in a woman with the May-Hegglin anomaly, whose foetuses carried cavum veli interpositi cysts. Since childhood, our patient had shown macro-thrombocytopenia, deafness and bleeding (epistaxis and menorrhagia), but she was misdiagnosed until the age of 30 years when our Centre identified a de novo allelic variant in the gene MYH9 coding for the non-muscle myosin heavy chain IIa. Our patient bled neither during the pregnancy, nor in the peripartum period. Children are now eight-months-old and have never bled, although both inherited the MYH9 variant and have thrombocytopenia with giant platelets. Furthermore, none of them developed psychomotor disorders.ConclusionsTo the best of our knowledge, this is the sixth case of twin pregnancy in a woman carrying May-Hegglin anomaly and the first one with cavum veli interpositi cysts in the neonates. We speculate that MYH9 could have, at least in part, played a role in the development of both conditions, as this gene has a pleiotropic effect.

Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth.

Feeding ractopamine (RAC), a β-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P < 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P < 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P < 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, β 1-adrenergic receptor (AR), and β 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P < 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P < 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P < 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. β 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the β 2-AR gene was decreased (P < 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and β 2-AR gene expression in porcine skeletal muscles independent of growth.

Myosin heavy chain expression relationships to power-load and velocity-load curves

Velocity- and power-based training are popular methods of determining training session loads and volumes. One factor that may influence load-velocity and load-power properties of an individual is the myosin heavy chain (MHC) composition of the muscle. The aim of this study was to examine the relationship between MHC composition and both load-velocity and load-power properties of muscle performance. Forty-two men with a variety of training backgrounds took part in this study (mean±SD; age=22.4±3.5 yrs, hgt=1.78±0.07 m, BW=78.7±13.3 kg). After testing leg extension one repetition maximum (1 RM), subjects performed maximal effort leg extensions at loads from 30% to 90% 1 RM. Muscle biopsies from the vastus lateralis were analyzed via SDS-PAGE electrophoresis technique for MHC content (IIx=13.8±12.9%, IIa=49.4±10.3%, I=36.8±11.3%). Leg extension rotational velocity and power were plotted against relative loads for all subjects. Significant correlations (P<0.05) were observed for MHC IIa with all performance variables (i.e. slopes, intercepts, peaks and relative loads). Relationships indicated that greater %MHC IIa was associated with greater velocity intercepts, more negative load-velocity slopes, greater maximal power, and with maximal power occurring at a lower relative intensity (% 1 RM). These data indicate that muscle velocity and power characteristics appear to be partially influenced by MHC content in a manner consistent with single muscle fiber contractile properties.

Super-relaxed state of myosin in human skeletal muscle is fiber-type dependent.

The myosin super-relaxed state (SRX) in skeletal muscle is hypothesized to play an important role in regulating muscle contractility and thermogenesis in humans but has only been examined in model organisms. Here we report the first human skeletal muscle SRX measurements, using quantitative epifluorescence microscopy of fluorescent 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) single-nucleotide turnover. Myosin heavy chain (MHC) isoform expression was determined using gel electrophoresis for each permeabilized vastus lateralis fiber, to allow for novel comparisons of SRX between fiber types. We find that the fraction of myosin in SRX is less in MHC IIA fibers than in MHC I and IIAX fibers (P = 0.008). ATP turnover of SRX is faster in MHC IIAX fibers compared with MHC I and IIA fibers (P = 0.001). We conclude that SRX biochemistry is measurable in human skeletal muscle, and our data indicate that SRX depends on fiber type as classified by MHC isoform. Extension from this preliminary work would provide further understanding regarding the role of SRX in human muscle physiology.

Fermented Feed Modulates Meat Quality and Promotes the Growth of Longissimus Thoracis of Late-Finishing Pigs.

Simple SummaryLittle is known about the effect of fermented feed on lean mass production of finishing pigs and the underlying mechanism. This study aimed to investigate the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. Our results found that fermented diet significantly increased loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. Fermented diet also increased the abundance of insulin, insulin receptor, myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of protein kinase B (AKT), mammalian target of rapamycin (mTORC1), initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1) in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Our results indicated that fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy. This study investigated the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. A total of 48 finishing pigs [Duroc × (Landrace × Large White), male, 126 ± 5-d-old] weighing 98.76 ± 1.27 kg were randomly assigned to two treatments (eight pens per treatment and three pigs per pen) for a 28-d feeding trial, including control diet and fermented diet. Fermented diet significantly increased the loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing the shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. A fermented diet also significantly increased the abundance of insulin, insulin receptor (IR), myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of AKT, mTORC1, 4EBP1 and S6K1 in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Moreover, proteomic analysis revealed that differentially expressed proteins predominantly involved in protein synthesis and muscle development were modulated by fermented diet. Our results indicated that a fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating the Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy.

MYH9 suppresses melanoma tumorigenesis, metastasis and regulates tumor microenvironment.

Non-muscle myosin IIA heavy chain (MYH9) has been implicated in many physiological and pathological functions including cell adhesion, polarity, motility to cancer. However, its role in melanoma remains unexplored. The aim of our study was to evaluate the role of MYH9 in melanoma tumor development and metastasis and further to find out the potential underlying mechanisms. In this study, we evaluated the in vitro migratory and invasive properties and in vivo tumor development and metastasis in C57BL/6 mice by silencing MYH9 in B16F10 melanoma cells. Knocking down MYH9 enhanced migration and invasiveness of B16F10 cells in vitro. Furthermore, MYH9 silencing accelerated tumor growth and metastasis in melanoma subcutaneous and intravenous mouse models. Next, oncogenes analysis revealed epithelial-mesenchymal transition and Erk signaling pathway are being regulated with MYH9 expression. Finally, MYH9 silencing in B16F10 cells modulates the tumor microenvironment by manipulating the leukocytes and macrophages infiltration in tumors. These findings established the opposing role of MYH9 as a tumor suppressor in melanoma suggesting specific MYH9 based approaches in therapeutics.

Myosin Heavy Chain Composition, Creatine Analogues, and the Relationship of Muscle Creatine Content and Fast-Twitch Proportion to Wilks Coefficient in Powerlifters.

Machek, SB, Hwang, PS, Cardaci, TD, Wilburn, DT, Bagley, JR, Blake, DT, Galpin, AJ, and Willoughby, DS. Myosin heavy chain composition, creatine analogues, and the relationship of muscle creatine content and fast-twitch proportion to Wilks coefficient in powerlifters. J Strength Cond Res 34(11): 3022-3030, 2020-Little data exist on powerlifting-specific skeletal muscle adaptations, and none elucidate sex differences in powerlifters. Powerlifters tend to display higher fast-twitch fiber content and phosphagen system dependence. Nevertheless, it is unknown whether fast-twitch fiber or muscle creatine content are predictive of competitive powerlifting performance (via Wilks coefficient). Twelve actively competing powerlifters (PL; n = 6M/6F; age = 21.3 ± 1.0; 3.0 ± 1.8 year competing; 7.3 ± 6.6 meets attended) and 10 sedentary controls (CON; n = 5M/5F; age = 19.4 ± 2.0 year) underwent vastus lateralis muscle biopsies and venipuncture to compare the myosin heavy chain (MHC) fiber type and creatine analogue profiles between groups of both sexes, and determine whether MHC IIa and muscle total creatine (MTC) composition predict powerlifting performance. Samples were analyzed for specific MHC isoform (I, IIa, and IIx) content via mixed homogenate SDS-PAGE, and creatine analogues (MTC, muscle creatine transporter [SLC6A8], serum total creatine [STC], and serum creatinine [CRT]). Furthermore, MHC IIa and MTC content were compared with Wilks coefficient using Pearson correlation coefficients. Male PL MHC content was 50 ± 6% I, 45 ± 6% IIa, and 5 ± 11% IIx, versus 46 ± 6% I, 53 ± 6 IIa, and 0% IIx in female PL. Conversely, male CON MHC content was 33 ± 5% I, 38 ± 7% IIa, and 30 ± 8% IIx, vs. 35 ± 9% I, 44 ± 8% IIa, and 21 ± 17% IIx in female CON. Muscle total creatine, SLC6A8, STC, and CRT did not significantly differ between groups nor sexes. Finally, neither MHC IIa content (r = -0.288; p = 0.364) nor MTC (r = 0.488; p = 0.108) significantly predicted Wilks coefficient, suggesting these characteristics alone do not determine powerlifting skill variation.

Mechanism of thyroid hormone signaling in skeletal muscle of aging mice.

Skeletal muscle (SM) has been shown as a target of thyroid hormones (THs). However, the status of TH signaling in aged SM remains unclear. This study aimed to explore the mechanism of TH signaling in SM of aging mice. Thirty C57BL/6J male mice were divided into 6-, 15- and 22-month (6, 15 and 22M) groups according to different age. Physical parameters were evaluated by analytical balance, grip strength test and histological analysis. Thyroid function was detected by enzyme-linked immunosorbent assay. TH signaling was compared among the three groups by real-time PCR and western blotting analysis. p16, p21, and p53 mRNA levels in SM increased in age-dependent manner. The muscle weight and strength decreased in 22M group compared to 6 and 15M groups. Concentrations of thyroid hormones, including free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in 22 M mice were not shown significant difference compared to 6M or 15M mice, although FT3 showed slightly decrease and TSH appeared a mild increase accompanying with age. mRNA levels of TH transporters, including MCT8 and MCT10, as well as iodothyronine deiodinase type 2 (DIO2) and type 3 (DIO3), were higher in 22M, while TH receptor α (TRα) mRNA and protein expression was lower in 22M, compared to the other groups. Type-I myosin heavy chain (MyHC I), MyHC IIx, and MyHC IIa were upregulated and Type-IIb MyHC (MyHC IIb) was downregulated in SM with advancing age. TH signaling in SM changes with aging.

GSK3 inhibition with low dose lithium supplementation augments murine muscle fatigue resistance and specific force production.

Calcineurin is a Ca2+‐dependent serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT), allowing for NFAT entry into the nucleus. In skeletal muscle, calcineurin signaling and NFAT activation increases the expression of proliferator‐activated receptor‐gamma coactivator 1‐alpha (PGC‐1α) and slow myosin heavy chain (MHC) I ultimately promoting fatigue resistance. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that antagonizes calcineurin by re‐phosphorylating NFAT preventing its entry into the nucleus. Here, we tested whether GSK3 inhibition in vivo with low dose lithium chloride (LiCl) supplementation (10 mg kg−1 day−1 for 6 weeks) in male C57BL/6J mice would enhance muscle fatigue resistance in soleus and extensor digitorum longus (EDL) muscles by activating NFAT and augmenting PGC‐1α and MHC I expression. LiCl treatment inhibited GSK3 by elevating Ser9 phosphorylation in soleus (+1.8‐fold, p = .007) and EDL (+1.3‐fold p = .04) muscles. This was associated with a significant reduction in NFAT phosphorylation (−50%, p = .04) and a significant increase in PGC‐1α (+1.5‐fold, p = .05) in the soleus but not the EDL. MHC isoform analyses in the soleus also revealed a 1.2‐fold increase in MHC I (p = .04) with no change in MHC IIa. In turn, a significant enhancement in soleus muscle fatigue (p = .04), but not EDL (p = .26) was found with LiCl supplementation. Lastly, LiCl enhanced specific force production in both soleus (p < .0001) and EDL (p = .002) muscles. Altogether, our findings show the skleletal muscle contractile benefits of LiCl‐mediated GSK3 inhibition in mice.

Influenza Infection has Fiber Type-Specific Effects on Cellular and Molecular Skeletal Muscle Function in Aged Mice.

Skeletal muscle myopathies represent a common non-pulmonary manifestation of influenza infection, leading to reduced physical function and hospitalization in older adults. However, underlying mechanisms remain poorly understood. Our study examined the effects of influenza virus A pulmonary infection on contractile function at the cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) levels in soleus and extensor digitorum longus muscles of aged (20 months) C57BL/6 male mice that were healthy or flu-infected for 7 (7-days post-infection; 7-DPI) or 12 days (12-DPI). Cross-sectional area (CSA) of myosin heavy chain (MHC) IIA and IIB fibers was reduced at 12-DPI relative to 7-DPI and healthy. Maximal isometric force in MHC IIA fibers was also reduced at 12-DPI relative to 7-DPI and healthy, resulting in no change in specific force (maximal isometric force divided by CSA). In contrast, MHC IIB fibers produced greater isometric force and specific force at 7-DPI compared to 12-DPI or healthy. The increased specific force in MHC IIB fibers was likely due to greater myofilament lattice stiffness and/or an increased number or stiffness of strongly bound myosin-actin cross-bridges. At the molecular level, cross-bridge kinetics were slower in MHC IIA fibers with infection, while changes in MHC IIB fibers were largely absent. In both fiber types, greater myofilament lattice stiffness was positively related to specific force. This study provides novel evidence that cellular and molecular contractile function is impacted by influenza infection in a fiber type-specific manner, suggesting potential molecular mechanisms to help explain the impact of flu-induced myopathies.

P0086FOCAL SEGMENTAL GLOMERULOSCLEROSIS AND TROMBOCYTOPENIA WITHOUT BLEEDING DIATHESIS: WHEN GENETICS MATTER

Abstract Background and Aims A 45-year old patient presented to our renal unit because of increased serum creatinine (2 mg/dL), proteinuria (2g/24h collection) and haematuria; platelets were 12000/mcL. His family history was negative. He had already undergone cataract surgery and was wearing a hearing device. Alport syndrome was suspected and kidney biopsy performed. Light microscopy showed focal segmental glomerulosclerosis (FSGS), while immunofluorescence was negative. Electron microscopy was not available. No specific treatment was undertaken and he progressed to end-stage renal disease (ESRD) in 10 years. The patient chose peritoneal dialysis. He was considered at high risk of bleeding but the Tenchkoff catheter was inserted without problems. After 3 months he received kidney transplant and again no pathological bleeding occurred. Method A genetic test was requested using next generation sequencing (NGS). Results A mutation of myosin heavy chain 9 (MYH9) gene which encodes the non-muscle myosin heavy chain-IIA (NMMH-IIA) was found. This mutation is associated with thrombocytopenia and proteinuria; platelets are giant and display normal aggregation, even in those patients who have a very low count. Proteinuria can be mild or in the nephrotic range, with or without microhaematuria. Kidney involvement is highly heterogeneous and leads to ESRD in half of the cases. Light microscopy shows FSGS as the prevalent pattern whereas electron microscopy is characterized by podocyte foot process effacement. In fact, NMMH-IIA is expressed in podocytes and controls cytoskeleton and cell migration. The differential diagnosis with Alport disease is challenging because additional features of MYH9 mutation are sensorineural deafness and pre-senile cataract. For all these reasons, accurate kidney biopsy study, family history and genetics are decisive. Conclusion Genetic testing has a pivotal role in understanding kidney diseases and should be more and more used.

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease

AbstractMegakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9–related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/−GFP+/−, NMIIAD1424N+/−, and NMIIAE1841K+/−) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms.Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Effect of feeding regimen on meat quality, MyHC isoforms, AMPK, and PGC-1α genes expression in the biceps femoris muscle of Mongolia sheep.

The effects of two feeding regimens on meat quality, myosin heavy chain (MyHC) types, and key factors regulating muscle fiber type (AMP‐activated protein kinase [AMPK] and peroxisome proliferator‐activated receptor‐coactivator‐1α [PGC‐1α]) in the biceps femoris muscle of Mongolia sheep were investigated. A total of 20 Mongolia sheep were weaning for 90 days and divided into two groups (pasture group (P) and confinement group (C)) at 10.36 ± 0.35 kg of weaning weight. After weaning, sheep were pasture fed or confinement fed for 9 months. The results showed that live weights, carcass weight, intramuscular fat (IMF), and Warner–Bratzler shear force (WBSF) in P group were significantly lower (p < .05) than that in C group. Compared with P group, color evaluations with respect to L* and b* values were significantly higher (p < .05) in C group. Expression of the MyHC I gene in the P group was significantly higher, while MyHC IIa and MyHC IIb genes expression was significantly lower (p < .05) than that in C group. Also, AMPK activity and expression of AMPKα2 and PGC‐1α genes were significantly higher (p < .05) in P group compared with C group. The present study indicated that muscle fiber composition was one of the key differences leading to the differences of meat quality in different feeding regimens. AMPK, particularly AMPKα2, and PGC‐1α were considered to be two key factors regulating muscle fiber types in Mongolia sheep. The results support that AMPK activity and the expression of AMPKα2 and PGC‐1α genes may affect the composition of muscle fibers; thus, AMPK activity and the expression of AMPKα2 and PGC‐1α genes had an effect on meat quality by changed composition of muscle fibers.

The long noncoding RNA MyHC IIA/X-AS contributes to skeletal muscle myogenesis and maintains the fast fiber phenotype

Mammalian skeletal muscles comprise different types of muscle fibers, and this muscle fiber heterogeneity is generally characterized by the expression of myosin heavy chain (MyHC) isoforms. A switch in MyHC expression leads to muscle fiber-type transition under various physiological and pathological conditions, but the underlying regulator coordinating the switch of MyHC expression remains largely unknown. Experiments reported in this study revealed the presence of a skeletal muscle-specific antisense transcript generated from the intergenic region between porcine MyHC IIa and IIx and is referred to here as MyHC IIA/X-AS. We found that MyHC IIA/X-AS is identified as a long noncoding RNA (lncRNA) that is strictly expressed in skeletal muscles and is predominantly distributed in the cytoplasm. Genetic analysis disclosed that MyHC IIA/X-AS stimulates cell cycle exit of skeletal satellite cells and their fusion into myotubes. Moreover, we observed that MyHC IIA/X-AS is more enriched in fast-twitch muscle and represses slow-type gene expression and thereby maintains the fast phenotype. Furthermore, we found that MyHC IIA/X-AS acts as a competing endogenous RNA that sponges microRNA-130b (miR-130b) and thereby maintains MyHC IIx expression and the fast fiber type. We also noted that miR-130b was proved to down-regulate MyHC IIx by directly targeting its 3'-UTR. Together, the results of our study uncovered a novel pathway, which revealed that lncRNA derived from the skeletal MyHC cluster could modulate local MyHC expression in trans, highlighting the role of lncRNAs in muscle fiber-type switching.

Manipulating Levels of Stress‐Response Proteins in a Drosophila Model of Myosin‐Based Inclusion Body Myopathy 3 Worsens Muscle Dysfunction

Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with &gt;1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.Support or Funding InformationThis work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.

Myosin heavy chain isoforms expression, calpain system and quality characteristics of different muscles in goats

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7–10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.

Regulation of myosin heavy chain antisense long noncoding RNA in human vastus lateralis in response to exercise training

Alterations to muscle activity or loading state can induce changes in expression of myosin heavy chain (MHC). For example, sedentary individuals that initiate exercise training can induce a pronounced shift from IIx to IIa MHC. We sought to examine the regulatory response of MHC RNA in human subjects in response to exercise training. In particular, we examined how natural antisense RNA transcripts (NATs) are regulated throughout the MHC gene locus that includes MYH2 (IIa), MYH1 (IIx), MYH4 (IIb), and MYH8 (Neonatal) in vastus lateralis before and after a 5-wk training regime that consisted of a combination of aerobic and resistance types of exercise. The exercise program induced a IIx to IIa MHC shift that was associated with a corresponding increase in transcription on the antisense strand of the IIx MHC gene and a decrease in antisense transcription of the IIa MHC gene, suggesting an inhibitory mechanism mediated by NATs. We also report that the absence of expression of IIb MHC in human limb muscle is associated with the abundant expression of antisense transcript overlapping the IIb MHC coding gene, which is the opposite expression pattern as compared with that previously observed in rats. The NAT provides a possible regulatory mechanism for the suppressed expression of IIb MHC in humans. These data indicate that NATs may play a regulatory role with regard to the coordinated shifts in MHC gene expression that occur in human muscle in response to exercise training.

Procyanidin B2 Promotes Skeletal Slow-Twitch Myofiber Gene Expression through the AMPK Signaling Pathway in C2C12 Myotubes.

Dimer procyanidin B2 [epicatechin-(4β-8)-epicatechin] (PB2) has attracted a lot of interest in nutrition and medicine because of its significant health-promoting abilities. However, the function of PB2 on different types of skeletal myofiber is still unclear. Here, we have found that PB2 significantly increased protein expression of the slow myosin heavy chain (MyHC) and decreased fast MyHC protein in C2C12 myotubes, accompanied by upregulation of mRNA expression of MyHC I, MyHC IIa, and Tnni1 and downregulation of MyHC IIx and MyHC IIb. We have also found that PB2 enhanced the activities of malate dehydrogenase and succinic dehydrogenase and reduced lactate dehydrogenase activity. PB2 promoted phosphorylation of AMPK and significantly increased mRNA expression of AMPKα1. The upstream factors of AMPK, such as phospho-LKB1, NRF1, and CaMKKβ, and the downstream factors of AMPK, including Sirt1 and PGC-1α, were also increased by PB2. Specific suppression of AMPK signaling by AMPKα1 siRNA or by AMPK inhibitor compound C significantly attenuated the PB2-induced upregulation of phospho-AMPK, PGC-1α, and slow MyHC and downregulation of fast MyHC. Our findings suggested that PB2 promotes skeletal slow-twitch myofiber gene expression through the AMPK signaling pathway in C2C12 myotubes.

Sex and fiber type independently influence AMPK, TBC1D1, and TBC1D4 at rest and during recovery from high-intensity exercise in humans.

Women and men present different metabolic responses to exercise, yet whether this phenomenon results from differences in fiber type (FT) composition or other sex-specific factors remains unclear. Therefore, our aim was to examine the effects of sex and FT independently on AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), Tre-2/BUB2/CDC1 domain family (TBC1D)1, and TBC1D4 in response to acute exercise. Segregated pools of myosin heavy chain (MHC) I and MHC IIa fibers were prepared from vastus lateralis biopsies of young trained men and women at rest and during recovery (0 min, 45 min, 90 min, or 180 min) from high-intensity interval exercise (6 × 1.5 min at 95% maximum oxygen uptake). In resting MHC I vs. IIa fibers, AMPKα2, AMPKγ3, and TBC1D1 were higher and TBC1D4 expression was lower in both sexes, along with higher phospho (p)-TBC1D1Ser660 and lower p-TBC1D4Thr642. Women expressed higher ACC than men in MHC IIa fibers and higher AMPKβ1, AMPKβ2, TBC1D1, and TBC1D4 in both FTs. Immediately after exercise, p-AMPKαThr172 increased only in MHC IIa fibers, whereas p-ACCSer221 increased in both FTs, with no change in p-TBC1D1Ser660 or p-TBC1D4Thr642. During recovery, delayed responses were observed for p-AMPKαThr172 in MHC I (45 min), p-TBC1D4Thr642 in both FTs (45 min), and p-TBC1D1Ser660 (180 min). FT-specific phosphorylation responses to exercise were similar between men and women. Data indicate that sex and FT independently influence expression of AMPK and its substrates. Thus failing to account for sex or FT may reduce accuracy and precision of metabolic protein measurements and conceal key findings.NEW & NOTEWORTHY This investigation is the first to compare muscle fiber type (FT)-specific analysis of proteins between the sexes, providing comprehensive data on AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), Tre-2/BUB2/CDC1 domain family (TBC1D)1, and TBC1D4 before and in the hours following high-intensity interval exercise (HIIT). Expression and phosphorylation of specific AMPK isoforms, ACC, TBC1D1, and TBC1D4 were shown to be FT dependent, sex dependent, or both, and TBC1D1 showed an unexpected delay in FT-dependent phosphorylation in the time period following HIIT.