Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Induction of Cardiomyocyte Apoptosis by Anti-Cardiac Myosin Heavy Chain Antibodies in Patients with Acute Myocardial Infarction

Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from patients with AMI on rat cardiomyocyte apoptosis.Cardiomyocyte apoptosis was observed and measured by DNA end labeling and Annexin-Ⅴ/PI double-staining assay.The expression of apoptosis related p53 and Bcl-2 protein and the second messenger calcium were detected respectively by Western blotting, patch clamp and confocal calcium imaging.The results showed that AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner.Apoptosis-accelerating nucleoprotein p53 was up-regulated, while apoptosis-inhibiting cytoplasmic protein Bcl-2 was down-regulated.In parallel, cytoplasmic calcium concentration was elevated.There was no effect on L-type calcium currents.It is concluded that AMHCA in patients with AMI as a novel triggering factor can induce cardiomyocyte apoptosis, which contributes to ventricular remodeling.

Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

Objective To study the effect of 4-6 weeks’ treadmill training of male SD rats on the contractile function of their gastrocnemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4-6 weeks at an intensity of about 75% VO2max (18.5-24 m/min,gradient of 0°,each training session lasting 50 minutes,twice a day). The content of gastrocnemius MHC mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR),and the changes of muscle fibre and its cross-section area (CSA) were measured using immunohistochemistry. Electric stimulation tests were used to determine the maximal tension of isometric contraction of the post-training gastrocnemius. Results ① After continuous treadmill training for 4-6 weeks,we found that the content of the total MHC,MHC Ⅰ,MHC Ⅱx,MHC Ⅱa mRNAs was 105%,105%,109% and 108% of that in the resting control group,respectively,and the MHC Ⅱb mRNA content did not change significantly. The percentage of MHC Ⅰ mRNA in the total MHC mRNA increased while that of MHC Ⅱ mRNA decreased after aerobic training. ② The slow type of fibre type Ⅰ was the main part of the MHC after training and the CSA of the muscle fibres increased simultaneously. ③ The maximal tension of isometric contraction by pulse stimulation of square wave in the training group increased significantly compared with that in the control group (P<0.01). Conclusion The findings indicate that aerobic exercise may promote an increase in the contractile function of MHC.

Alterations in myosin heavy chain isoform gene expression during the transition from compensatory hypertrophy to congestive heart failure in rats

Objective To explore the molecular mechanism underlying the decreased velocity of tension rise in rat myocardium during congestive heart failure (CHF) and left ventricular hypertrophy (LVH) induced by aortic stenosis.Methods The maximum velocity of tension rise (+dT/dtmax) was measured in left ventricular papillary muscle and the mRNA level of myosin heavy chain (MHC) isoforms in the left ventricle were detected by Northern blot analysis.Results The value of +dT/dtmax in CHF and LVH group were 64.17% and 37.15% lower than sham-operated controls (Sham) (P<0.01); values in the CHF group were 42.99% lower than that of LVH (P<0.01). The level of α-MHC mRNA in LVH was not different from that of the Sham (P>0.05), but decreased significantly in CHF to 42.3% of Sham and 56.1% of LVH (P<0.01). The level of β-MHC mRNA was up-regulated by 88.3% (P<0.01) in LVH compared with Sham and the level of β-MHC in CHF was 1.5-fold and 3.7-fold higher than that in LVH and Sham respectively (P<0.01). The ratio of α-MHC/β-MHC mRNA in LVH and CHF decreased to 42.4% and 9.8% respectively of the value in Sham (P<0.01). Correlation between α-MHC/β-MHC mRNA level and +dT/dtmax was analyzed which showed that these values were positively correlated with a correlation coefficient of 0.875 (P<0.01).Conclusion The decreased ratio of α-MHC/β-MHC mRNA was the major molecular mechanism underlying the decreased +dT/dtmax in CHF and LVH myocardium. The decreased ratio of α-MHC/β-MHC mRNA in LVH was mainly due to the up regulation of β-MHC mRNA while in CHF both down regulation of α-MHC and up regulation of β-MHC were involved.

Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain composition of rat gastrocnemius muscles

Background and objective:Botulinum toxin type A(BoNT/A)is a metalloprotease that blocks synaptic transmission via the cleavage of a synaptosomal-associated protein of 25 kDa(SNAP-25).It has gained widespread use as a treatment for cerebral palsy and skeletal muscle hypertrophy.In China,Chinese botulinum toxin type A(CBTX-A),a type of BoNT/A,is in widespread clinical use.However,the changes in the morphological and biochemical properties of treated muscles and in remote muscles from the CBTX-A injection site are relatively unknown.Therefore,we investigated the changes in histomorphology and myosin heavy chain(MyHC)isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A.Methods:The weakness of the injected muscles was assessed periodically to identify their functional deficiency.Muscle slices were stained with hematoxylin-eosin(HE)and adenosine triphosphatase(ATPase).MyHC isoform composition was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)to uncover changes in morphological and biochemical properties.Results:Our findings demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles,shifts in MyHC isoform composition emerged on the third day after injection and peaked in the fourth week.The composition remained distinctly different from that of the control group after the twelfth week.More specifically,there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx,type IIa,and type I isoforms.Conclusions:Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that the MyHC composition remained far from normal six months after a single injection.However,no noticeable remote muscle weakness was induced.

Mutation of Arg723Gly in β-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy

Background Hypertrophic cardiomyopathy （HCM） is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain （β-MHC） are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. Methods The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. Results The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13 619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. Conclusion The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining（TIE） is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining（SIE）. The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens（A4.74 for My HC-IIA, BA-F8 for My HC-I） and alkaline phosphatase staining system for the third antigen（BF-F3 for My HC-IIB）, followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers（100%）. All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

Restricted nutrient intake does not alter serum-mediated measures of implant response in cell culture

Background： During nutritional stress, reduced intake may reduce the efficacy of anabolic implants. This study was conducted to evaluate basic cellular responses to a growth promotant implant at two intake levels. Methods： Sixteen crossbred steers （293 ± 19.3 kg） were used to evaluate the impact of anabolic implants in either an adequate or a restricted nutritional state. Steers were trained to individual Calan gates, and then randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Treatments consisted off presence or absence of an anabolic growth implant （Revalor-XS, 200 mg TBA and 40 mg estradiol; IMPLANT or CONTROL） and a moderate energy, pelleted, starting cattle diet fed at either 2.0 × or 1.0 × maintenance energy （NEM） requirements （HIGH or LOW）. Serum （d O, 14, and 28） was used for application to bovine muscle satellite cells. After treatment with the serum （20% of total media） from the trial cattle, the satellite cells were incubated for 72 h. Protein abundance of myosin heavy chain （MHC）, phosphorylated extracellular signal-related kinase （phospho-ERK）, and phosphorylated mammalian target of rapamycin （phospho-mTOR） were analyzed to determine the effects of implant, intake, and their interaction （applied via the serum）. Results： Intake had no effect on MHC （P = 0.85） but IMPLANT increased （P 〈 0.01） MHC abundance vs. CONTROL. Implant status, intake status, and the interaction had no effect on the abundance of phospho-ERK （P〉0.23）. Implanting increased phospho-mTOR （P 〈 0.01） but there was no effect （P 〉 0.51） of intake or intake x implant. Conclusions： The nearly complete lack of interaction between implant and nutritional status indicates that the signaling molecules measured herein respond to implants and nutritional status independently. Furthermore, results suggest that the muscle hypertrophic effects of anabolic implants may not be mediated by circulating IGF-1.

Effects and mechanism of different adrenergic receptor antagonists on left ventricular hypertrophy subsequent to coarctation of abdominal aorta in rats

Objective: To study the changes of a collagen-binding protein (Colligin) and myosin heavy chain isoform (α/β-MHC) gene and protein in left ventricular hypertrophy subsequent to coarctation of abdominal aorta in rats and the effects of three kinds of adrenergic receptor blockers: Carvedilol (CAR) , Metoprolol (MET) and Terazosin (TER) on these changes, and to elucidate the effects and new mechanism of CAR on left ventricular hypertrophy regression. Methods: A model of hypertrophy induced by coarctation of abdominal aorta(CAA)was used in this study. Thirty two male wistar rats were divided randomly into four groups 4 weeks after CAA operation: CAA, CAR, MET and TER. Hemodynamics, ventricular remodeling parameters, expressions of Colligin and α/β-MHC mRNA, protein expressions of Collagen Ⅰ / Ⅲ and Colligin were investigated in the four groups and sham operation group. Results: Left ventricle hypertrophy was observed clearly 16 weeks after operation. The ratio of α/β-MHC mRNA decreased, while expressions of Collagen Ⅰ /Ⅲ proteins and Colligin mRNA/protein increased( P < 0.05). CAR could ameliorate left ventricle hypertrophy prior to MET and TER. CAR could also change the expressions of α/β-MHC, Collagen Ⅰ /Ⅲ and Colligin in both gene and protein levels ( P < 0.05), while MET and TER have no effect on them ( P > 0.05). Conclusion: The effects of CAR on extracellular matrix proteins and MHC isoform shift regression of left ventricle may be due to antiproliferative or antioxidative mechanism, which was independent of beta-adrenergic receptor antagonist.

Ruscogenin alleviates LPS-triggered pulmonary endothelial barrier dysfunction through targeting NMMHC IIA to modulate TLR4signaling

Pulmonary endothelial barrier dysfunction is a hallmark of clinical pulmonary edema and contributes to the development of acute lung injury(ALI).Here we reported that ruscogenin(RUS),an effective steroidal sapogenin of Radix Ophiopogon japonicus,attenuated lipopolysaccharides(LPS)-induced pulmonary endothelial barrier disruption through mediating non-muscle myosin heavy chain IIA(NMMHC IIA)-Toll-like receptor 4(TLR4)interactions.By in vivo and in vitro experiments,we observed that RUS administration significantly ameliorated LPS-triggered pulmonary endothelial barrier dysfunction and ALI.Moreover,we identified that RUS directly targeted NMMHC IIA on its N-terminal and head domain by serial affinity chromatography,molecular docking,biolayer interferometry,and microscale thermophoresis analyses.Downregulation of endothelial NMMHC IIA expression in vivo and in vitro abolished the protective effect of RUS.It was also observed that NMMHC IIA was dissociated from TLR4 and then activating TLR4 downstream Src/vascular endothelial cadherin(VE-cadherin)signaling in pulmonary vascular endothelial cells after LPS treatment,which could be restored by RUS.Collectively,these findings provide pharmacological evidence showing that RUS attenuates LPS-induced pulmonary endothelial barrier dysfunction by inhibiting TLR4/Src/VE-cadherin pathway through targeting NMMHC IIA and mediating NMMHC IIA-TLR4 interactions.

Skeletal Muscle Fiber Type and Morphology in a Middle-Aged Elite Male Powerlifter Using Anabolic Steroids

Powerlifting regularly exposes athletes to extreme stimuli such as chronic heavy resistance training(HRT),and many powerlifters choose to augment their performance with anabolic-androgenic steroids(AAS).However,little is known about the myocellular adaptations that occur from long-term HRT and AAS use,especially into middle age.We were presented with the unique opportunity to study muscle cells from an elite-level powerlifter(EPL;age 40 years)with≥30 years of HRT experience and≥15 years of AAS use.The purpose of this case study was to identify myocellular characteristics[myosin heavy chain(MHC)fiber type,fiber size,and myonuclear content]in EPL,as well as compare these data to existing litera-ture.The participant underwent a resting vastus lateralis muscle biopsy and single fibers were analyzed for MHC content via SDS-PAGE.A subset of fibers underwent MHC-specific imaging analysis via confocal microscopy to identify cell size(cross-sectional area,CSA)and myonuclear domain(MND)size.MHC fiber type distribution was 9% I,12% I/IIa,79% IIa,and 0% other isoforms.This pure MHC IIa(fast-twitch)fiber content was amongst the highest reported in the literature.Imaging analysis of MHC IIa fibers revealed a mean CSA of 4218±933μm^(2) and MND of 12,548±3181μm^(3).While the fast-twitch fiber CSA was comparable to values in previous literature,mean MND was smaller than has been reported in untrained men,implying greater capacity for growth and repair.These findings showcase the unique muscle cell structure of an elite powerlifter,extending the known physiological limits of human muscle size and strength.