Myosin Heavy Chain IIb Expression Research Articles

Palmitic Acid Inhibits Myogenic Activity and Expression of Myosin Heavy Chain MHC IIb in Muscle Cells through Phosphorylation-Dependent MyoD Inactivation.

Sarcopenia associated with aging and obesity is characterized by the atrophy of fast-twitch muscle fibers and an increase in intramuscular fat deposits. However, the mechanism of fast-twitch fiber-specific atrophy remains unclear. In this study, we aimed to assess the effect of palmitic acid (PA), the most common fatty acid component of human fat, on muscle fiber type, focusing on the expression of fiber-type-specific myosin heavy chain (MHC). Myotubes differentiated from C2C12 myoblasts were treated with PA. The PA treatment inhibited myotube formation and hypertrophy while reducing the gene expression of MHC IIb and IIx, specific isoforms of fast-twitch fibers. Consistent with this, a significant suppression of MHC IIb protein expression in PA-treated cells was observed. A reporter assay using plasmids containing the MHC IIb gene promoter revealed that the PA-induced reduction in MHC IIb gene expression was caused by the suppression of MyoD transcriptional activity through its phosphorylation. Treatment with a specific protein kinase C (PKC) inhibitor recovered the reduction in MHC IIb gene expression levels in PA-treated cells, suggesting the involvement of the PA-induced activation of PKC. Thus, PA selectively suppresses the mRNA and protein expression of fast-twitch MHC by modulating MyoD activity. This finding provides a potential pathogenic mechanism for age-related sarcopenia.

17 Generation of myostatin gene knockout boars by somatic cell nuclear transfer

Skeletal muscle is the most economically valuable tissue part in meat-producing animals, and enhancing muscle growth in these species may increase the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and nonfunctional mutations of this MSTN gene in various animal species have led to dramatic hypermuscularity. A porcine fetal fibroblast cell line with biallelic MSTN mutations (MSTN −/−), consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, was used as a donor to generate cloned pigs via somatic cell nuclear transfer. Cloned embryos were transferred into seven surrogates and 38 live piglets were delivered. Using these MSTN null piglets, the detailed morphological, gene and protein expression analyses were performed not only in biceps femoris, semitendinosus, and diaphragm of MSTN null piglets but also in heart, liver, spleen, lung, kidney, and tongue. The results showed that MSTN −/− boars have visually clear hypermuscular characteristics, which was supported by the increased carcass dressing percentage and loin eye size and decreased backfat thickness. The fibre cross-sectional areas of the semitendinosus and semimembranosus muscles were much larger in MSTN −/− piglets than in wild-type (WT) piglets (98.0±8.2 vs. 62.7±4.7, 105.7±11.6 vs. 72.3±8.4mm2, respectively; P<0.05). MSTN −/− pigs showed a higher proportion of fast-type fibres (Type-II fibre%, 95±1.8 vs. 86±2.4; P<0.05) and lower succinate dehydrogenase activity in muscles than WT pigs. The mRNA expression of myosin heavy-chain IIB in both two muscles was higher in MSTN −/− pigs compared with WT pigs (P<0.05). In organs, the heart and liver were lighter in MSTN −/− piglets than in WT piglets (8.1±0.3 vs. 10.3±0.4, 26.9±1.8 vs. 34.2±1.2g kg−1, respectively; P<0.05), whereas the tongue was heavier in MSTN −/− piglets than in WT piglets and myofibres of the tongue were significantly larger in the former piglets than in the latter piglets (P<0.01). Messenger RNA expression of MSTN in all organs was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01) and follistatin in the heart and liver was significantly higher in MSTN −/− piglets than in WT piglets (P<0.05). Protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01). In conclusion, MSTN −/− pigs showed a phenotype of supermuscular ratio is associated with increased muscle fibres and smaller internal organs and has a complicated gene expression patterns.

Succinate induces skeletal muscle fiber remodeling via SUNCR1 signaling.

The conversion of skeletal muscle fiber from fast twitch to slow‐twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber‐type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss‐of‐function models generated by phospholipase Cβ antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius‐specific knockdown, we found that the effects of succinate on skeletal muscle fiber‐type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate‐based compounds in both athletic and sedentary populations.

Effect of L-lysine on expression of selected genes, serum concentration of amino acids, muscle growth and performance of growing pigs.

Lysine (Lys) is the first limiting amino acid (AA) in most feed formulations for pigs and most abundant, along with leucine, in muscle proteins. An experiment was conducted with 17 pigs (17.7±0.05kg initial BW) to identify a role of dietary Lys in the control of protein synthesis in pigs. Fourteen pigs were randomly assigned to one of the two wheat-based dietary treatments: Lys-deficient, 3.0g/kg (DEF) and Lys-adequate, 10.8g/kg (ADE). Samples from jejunum mucosa, liver, Longissumus and Semitendinosus muscles, and blood were collected. The other three pigs were sacrificed at the beginning of the trial to measure basal carcass composition. Weight gain, gain:feed ratio, Lys intake and loin eye area were greater in ADE than in DEF pigs (p<0.01). Muscle-related carcass characteristics were better, and myosin heavy chain IIb expression (MyHC IIb) in Semitendinosus was higher in ADE than in DEF pigs. Expression of AA transporters CAT-1 was lower (p<0.05), serum Lys was higher and serum Val was lower in pigs fed the ADE diet. The higher muscularity, MyHC IIb expression in Semitendinosus muscle and Lys serum of pigs fed the ADE diet suggest that Lys increases growth rate not only by functioning as protein construction unit but also as potential control of the protein synthesis process.

The homoeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle

Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fibre-type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2C12 mouse myoblasts were grown in standard conditions. Cells were cotransfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4days of differentiation. In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8-fold (±0.1, P<0.05). With decreased Six1, MHC IIB expression decreased 2.7-fold (±0.7, P=0.04). Proportion of muscle fibres expressing MHC IIB decreased (45.3±4.8% vs. 65.1±7.3% in control group, P=0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6±4.3% vs. 75.2±5.4% in control group, P<0.05). Decreased Six1 increased MHC IIA expression 1.9-fold (±0.3, P=0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9-fold (±0.3, P<0.01). Six1 knockdown repressed MHC IIB promoter 2.9-fold (±0.1, P<0.05) and MHC IIX 3.7-fold (±0.08, P<0.01). Six1 knockdown caused a fast-to-slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fibre phenotype.

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

Relationship between myosin heavy chain isoform expression and muscling in several diverse pig breeds1

The objective of this study was to examine the relationship of the relative abundance of transcripts of myosin heavy chain (MyHC) isoforms and muscling in several diverse pig breeds. The animals used were from 3 pure breeds (Pietrain, Duroc, and Mongcai) and 2 crosses [Duroc x Pietrain (DUPI) and Duroc x Berlin Miniature pigs (DUMI)]. Real-time PCR quantification of MyHC isoforms I, IIa, IIx, and IIb showed that the relative expression of MyHC IIb was greater in pigs with large LM areas in both DUPI (69.6 vs. 53.0%) and DUMI (60.5 vs. 47.5%). In DUPI, similar transcript levels of MyHC I were found in both large and small LM (14.7 and 15.2%), whereas in DUMI animals, these values were 18.4 and 33.5% (P < 0.05). The groups of animals with large and small LM area in the DUPI also tended to differ in MyHC IIa and IIx transcripts. The comparison among different breeds confirmed the trend of high MyHC IIb transcript abundance together with high muscularity. In Pietrain, Duroc, DUPI, and DUMI, MyHC IIb accounted for more than half of the MyHC transcripts (65.4, 59.7, 54.0, and 54.0%). Mongcai showed low MyHC IIb (11.4%) but high type I, IIa, and IIx relative RNA levels (24.1, 28.5, and 35.9%). Frequencies of fibers, determined by muscle fiber staining with ATPase, and relative abundance of MyHC isoforms, determined by quantitative reverse transcription-PCR, of corresponding pairs of type I, IIa, and IIx/ IIb were correlated (r = 0.71, 0.67, and 0.52, respectively). The study demonstrates that MyHC IIb fibers are the most prominent in pigs having large LM area and implies that MyHC IIb is the determining fiber contributing to the differentiation of large and small loin eye muscle area in the pig.

Contractile protein content reflects myosin heavy-chain isoform gene expression1

Muscle fiber types are classified based on contractile speed and type of metabolism. Fast-contracting fibers involve mainly glycolytic-based metabolism, whereas slow-contracting fibers involve a more oxidative type of energy metabolism. The relationship between expression of the genes controlling these functional characteristics and their relative protein abundance in porcine muscle is unknown. The objective of this study was to determine the expression of adult myosin heavy-chain (MyHC) genes and their corresponding protein content in various porcine muscles. Moreover, changes in expression of 2 genes involved in energy metabolism (glycogen synthase and citrate synthase) were determined on muscles varying in MyHC. Using real-time PCR, the relative transcript abundance was determined for the adult MyHC isoforms (types I, IIA, IIX, and IIB), glycogen synthase, and citrate synthase in the masseter (MAS), diaphragm, longissimus, cutaneous trunci, and red and white semitendinosus muscles of 7 pigs. Each muscle was subjected to SDS-PAGE analyses to determine the relative abundance of each MyHC. The relative transcript abundance of type IIB MyHC was greatest (P < 0.05) in the longissimus, white semitendinosus, and cutaneous trunci muscles, whereas type I MyHC expression was greatest (P < 0.05) in the MAS, diaphragm, and red semitendinosus muscles. Glycogen synthase gene expression was least in the MAS (P < 0.01) but exhibited a pattern similar to MyHC IIB expression across muscles. Citrate synthase transcript abundance, however, varied (P < 0.05) independently of MyHC gene expression. Expression of types I and IIB MyHC was correlated with their tissue protein content (R2 = 0.76 and 0.78, respectively), whereas type IIA and X MyHC expression did not correlate with the SDS-PAGE-determined protein content. These data show differences in MyHC gene expression across various porcine muscles and suggest that expression of these genes is reflective of the type of myosin contained within the muscle. Moreover, these data show that expression of energy-specific genes differs greatly across porcine muscles with different functions.

Stapedius muscle fiber characterization during postnatal development in the rat

The stapedius muscle (SM) is reported to prevent cochlear damage by noise. Functional demands are then the ability of fast contraction with long endurance. At the end of the third postnatal week, the middle ear of the rat is completely pneumatized and according to electrophysiological data, the auditory function starts to match the adult. We investigated the developmental changes in myosin composition of SM fibres using consecutive complete SM cross-sections (taken from rats on post natal day (PND) 7, 14, 16, 21, 28, 42 and 84) which were processed by enzymehistochemistry to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) and by immunohistochemistry using myosin heavy chain (MHC) antibodies (mAb). Fibres were assigned to mATPase type I, IIA, IIB, IIX or ‘Miscellaneous’ categories. Per mATPase category, the fibres were attributed to groups with specific MHC isoform compositions. Neonatal MHC expression could not be documented with the mAb used. However, embryonal (Emb) MHC was expressed at PND 7, very little at PND 14; at later PND fibres did not show Emb MHC. In general, the mATPase-based classification did not show large alterations after PND 21. Expression of MHC IIB, which was present in almost 50% of the fibres at PND 7 and 14, diminished to 3% at PND 84. A decrease in number of fibres expressing more than one MHC isoform was found. These results show that the SM is a precociously developing muscle compared to limb muscles and even to the diaphragm. Moreover, it is shown that the expression of the adult MHC isoform phenotype coincides with the onset of auditory function in the third postnatal week.

Calcineurin Mediated Slow Muscle Fiber Program In Hindlimb Suspension And Reloading Conditions

Calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes and in muscle hypertrophy. We used the hindlimb suspension (HS) and reloading model with or without administration of FK506, a specific inhibitor of CaN, to elucidate the hypothesis that CaN regulates muscle regrowth and fiber type transformation toward a slow profile during reloading conditions. PURPOSE Our aim was to investigate the functional role of CaN as a regulator of muscle growth and/or muscle fiber type under conditions of recovery from inactivity. METHODS Female ICR mice (8 weeks of age, 28–32 g) were used. To examine the effects of HS and reloading on skeletal muscle fiber size and muscle fiber type, animals were designated to 8 weeks of HS and subsequent reloading for 4 weeks. During reloading, animals were treated with FK506 (Fujisawa, Japan) by intraperitoneal administration (3–5 mg/kg/day). After each experimental period, slow-twitchdominant antigravitational soleus muscle was analyzed. Myosin heavy chain (MHC) isoform composition was analyzed with SDS-PAGE. Fiber cross sectional area was determined with immunohistochemical analysis. Expression level of CaN signaling factors were assessed by Western blotting analyse. RESULTS HS treatment resulted in obvious muscle atrophy and slow-to-fast fiber-type transformation in the mouse soleus muscle (decrease in MHC I compositon and new expression of MHC IIb in HS group). Subsequent reloading for 4 weeks following HS induced muscle regrowth and fiber-type reversion toward a slow profile. FK506 administration prevented this kind of reloading-induced transformation of muscle fiber type. Furthermore, we confirmed that FK506 administration attenuated maintenance of fiber cross-sectional area and reloading-induced fiber regrowth, specifically in slow-type muscle fibers. Unexpectedly, we observed an increase in protein contents of CaN and its downstream effectors after HS, although the muscle fiber type transformed toward a fast profile and obvious muscle atrophy was induced. CONCLUSIONS CaN signaling pathway is required for the slow-type muscle fiber program under suspension and reloading conditions. We also confirmed that CaN signaling factors were activated in the soleus muscle under suspended conditions. These results imply that the CaN signaling pathway may have undefined opposing roles in the regulation of muscle fiber type and size under these conditions. Supported by Grants #15200048 and in part by the Nishihira/Tsukuba Project of COE (Center of Excellence) from the Japan Ministry of Education, Culture, Sports, Science and Technology.

Transcriptional Control of Myosin Heavy Chain IIB Gene Expression in Normal and Overloaded Rat Plantaris

1038 The rat plantaris (P) muscle predominantly expresses fast-contracting isoforms of myosin heavy chain (MHC) proteins. During hypertrophy induced by chronic mechanical overload, the expression of the fastest type IIb MHC isoform decreases as the muscle shifts phenotype toward expression of slower-contracting MHC isoforms. PURPOSE: To determine the transcriptional control elements that reside downstream of the transcription start site (tss) and regulate MHC IIb expression in normal (Con) and functional overload (FO) rat P. METHODS: Female SD rats (N = 8/group) underwent 7 d FO of the left P. MHC IIb pre-mRNA and mRNA expression were measured by RT-PCR. The transcriptional activities of five MHC IIb promoters were tested by direct gene transfer into the P. Each promoter fragment began −1.431 kb upstream of the tss, and extended into either exon 1 (+0.013 kb), intron 2 (+1.008, +1.568, +2.054 kb), or exon 3 (+2.814 kb). Promoter activity was measured by a firefly luciferase (fLuc) reporter assay 7 d after bilateral intramuscular injection of plasmid constructs containing the fLuc gene controlled by the inserted IIb promoter fragment. To control for plasmid uptake efficiency, the fLuc reporter activity was corrected to the renilla luciferase activity of a co-injected alpha-actin promoter-reporter plasmid. RESULTS: After 7 d FO, the wet weight of the P increased ∼31% (P < 0.05). IIb pre-mRNA and mRNA each decreased by ∼60% after FO (P < 0.05). Activities of the +2.054 and +2.814 kb promoters were similar, and decreased ∼50% after FO (P < 0.05). The three promoters that extended downstream between +0.013 and +1.568 kb were 80–99% less active (P < 0.05) than the +2.814 kb promoter, and did not decrease after FO. CONCLUSION: MHC IIb expression in Con and FO P is controlled by transacting factor(s) located downstream of the tss, and specifically within the intron 2 region between +1.568 and +2.054 kb. Further studies are aimed at identifying the trans-acting factors that bind this region of DNA to control transcription of the IIb gene in the normal rat P and its down-regulation during hypertrophy. Supported by NIH grant AR30346

Effect of Dietary Creatine on Skeletal Muscle Myosin Heavy Chain Isoform Expression in an Animal Model of Uremia

Background: Chronic renal failure is accompanied with muscle dysfunction and myopathy, characterized by muscle weakness and increased fatigue. Myosin heavy chain (MHC) is the principal structural protein that controls the intrinsic contractile properties of striated muscle. Creatine is widely used as an ergogenic nutritional supplement in sportsmen. This study investigates the effect of creatine supplementation on MHC expression in the setting of uremic myopathy. Methods: Male Wistar rats were either sham operated or subtotally nephrectomized and received a control diet or creatine (2% w/w)-supplemented diet. After 4 weeks of treatment, serum creatinine, creatine, urea and creatinine clearances were determined. MHC isoforms were determined electrophoretically in the extensor digitorum longus and soleus muscles. Results: Creatinine clearances were lower in nephrectomized animals, but similar in creatine-supplemented and control diet animals. Nephrectomized animals had significantly higher MHC IIb and lower MHC IIx isoform expression in the extensor digitorum longus muscle compared to sham-operated animals. In the soleus muscle, MHC IIb expression was increased in nephrectomized animals. Creatine supplementation reversed the MHC transitions observed in uremia in the soleus muscle, but not in the extensor digitorum longus muscle. Conclusion: We observed altered expression of MHC isoforms in uremia. In uremic animals, fast MHC IIb isoforms were increased, whereas MHC I and IIx isoforms predominate in control animals. Dietary creatine supplementation reversed the altered MHC expression during uremia in slow-twitch, but not in fast-twitch muscles.