Glycosylation and Cross-linkage of Cardiac Myosin in Diabetic Subjects: A Post-mortem Study

We have investigated the possibility that post-translational modification of myosin by protein glycosylation and cross-linking occurs in cardiac myosin. Left ventricular muscle was obtained at post-mortem from 6 diabetic and 7 non-diabetic subjects. Myosin was extracted from muscle and purified using Sephadex chromatography followed by protein concentration. Glycosylation was estimated using boronate affinity chromatography with the myosin dissolved in a pyrophosphate buffer, the glycosylated myosin being displaced with sorbitol. Cross-linkage was assessed by fluorescence at 440 nm upon excitation at 370 nm. Diabetic subjects had significantly higher levels (p<0.02) of glycosylated myosin (median 6.0% (range 3.8–6.6%)) than non-diabetic subjects (median 2.4% (range 0.3–4.2%)) but there was no difference in the degree of cross-linkage as assessed by fluorescence (diabetic median 9.8 (range 6.5–17.0) arbitrary units; non-diabetic median 9.7 (range 6.0–11.4) arbitrary units). Glycosylation of left ventricular myosin may be of relevance to the excess risk of congestive cardiac failure in diabetic patients.     

Effect of Ionic Strength on Length-dependent CaActivation in Skinned Cardiac Muscle

S. H. Smith and F. Fuchs. Effect of Ionic Strength on Length-dependent Ca²⁺Activation in Skinned Cardiac Muscle. Journal of Molecular and Cellular Cardiology (1999) 31, 2115–2125. The length-dependence of myofilament Ca²⁺sensitivity is considered to be an important component of the steep force–length relationship in cardiac muscle (Frank–Starling relation). Recent studies suggest that Ca²⁺sensitivity is a function of the number of strong-binding cross-bridge interactions formed at a given sarcomere length. However, the length-dependent step in the thin filament activation process is still unknown. This study was designed to test the hypothesis that sarcomere length influences the transition of the thin filament from the unattached (blocked) state to the weakly bound (closed) state. This hypothesis was tested by determining the length-dependence of Ca²⁺sensitivity as a function of ionic strength in skinned bovine ventricular muscle. Previous studies have shown that reduction in ionic strength below a critical level, in the absence of Ca²⁺, shifts the thin filament to the closed state. In this study normal Ca²⁺regulation was maintained at low ionic strength but the length-dependence of Ca²⁺sensitivity and the length-dependence of Ca²⁺binding were eliminated. These results are consistent with the hypothesis that the transition from the blocked to the closed state is a function of filament geometry as well as Ca²⁺and ionic strength.     

Myosin heavy chain and actin fractional rates of synthesis in normal and overload rat heart ventricles

The rates of synthesis (in per cent per day) of the total protein (ks) and myosin heavy chains (ke HC) and actin (ke A), isolated by preparative electrophoresis from myofibrils prepared in a relaxing medium, have been measured in rat heart ventricles after a continuous infusion of 50 microCi of (14C) Tyrosine (0.5 ml/h during 6 h). Normal values, in the sham-operated group, were for ks 19%, ke HC 23% and ke A 11%. Two to 4 days after an abdominal aortic stenosis there was an increase in the specific radioactivity of free plasma and intra-cellular tyrosine and of protein-bound tyrosine. The rate of synthesis also increased up to 37% for ks, 41% for ke HC and 21% for ke A but the ratio ke HC/ke A remained unchanged. We conclude that the normal heterogeneity of the rate of synthesis of the two main contractile proteins, at least when measured on myofibrils free from easily releasable myofilaments, was unmodified by cardiac overload, although both of these rates of synthesis were stimulated.     

Soleus and EDL muscle contractility across the lifespan of female C57BL/6 mice

All previous aging research on the contractility of rodent skeletal muscle has been conducted on male rodents. Because males and females age differently, we undertook this study to determine if and when age-related decrements in skeletal muscle contractility occur in female mice. Soleus and extensor digitorum longus (EDL) muscles from female C57BL/6 mice aged 4, 8, 16, 24 and 28 mo were assessed in vitro for contractility and subsequently contractile protein content. EDL muscle was resistant to age-related changes in force generation but displayed characteristics of becoming more slow-twitch like. Maximal isometric tetanic force (P_o) generated by soleus muscle declined with age. Soleus muscle size and contractile protein contents were not affected by age and thus could not explain the age-related force decrements. Soleus muscle specific P_o declined with age being 26% lower in muscles of 16–28 mo-old mice indicating that a deterioration in soleus muscle quality of female mice occurred beginning around the age of ovarian failure. Thus this study provides essential, comprehensive baseline data for future studies on age-related muscle dysfunction in the female mouse.     

血清心脏肌球蛋白结合蛋白C检测在急性心肌梗死患者诊断中的作用

目的探讨血清心脏肌球蛋白结合蛋白C(c My BP-C)在急性心肌梗死(AMI)患者诊断中的价值。方法选择2014年3月至2015年11月心血管内科收治的AMI患者62例作为病例组,选取正常体检者60例作为对照组。采用双抗体夹心酶联免疫吸附试验(ELISA)法检测血清c My BP-C浓度。比较AMI组与对照组间c My BP-C、肌钙蛋白Ⅰ(c TnⅠ)、肌酸激酶同工酶(CK-MB)和肌红蛋白(Myo)浓度的差异,分析AMI组c My BP-C与c TnⅠ、CKMB和Myo的相关性,同时分析发病时间小于4 h的AMI患者入院时血清c My BP-C和c TnⅠ浓度与对照组的差异,比较急诊经皮冠状动脉介入(PCI)术后12 h与入院时血清c My BP-C和c TnⅠ的差异。结果 AMI组患者血清c My BP-C、c TnⅠ、CK-MB和Myo浓度较对照组均明显升高(P0.05)。相关性分析显示,AMI组患者c My BP-C浓度与c TnⅠ、CK-MB和Myo浓度均存在正相关(分别为r=0.876、P0.05;r=0.632、P0.05和r=0.903、P0.05)。发病时间小于4 h的AMI患者入院时血清c My BP-C浓度较对照组明显升高(P0.05),而血清c TnⅠ浓度与对照组比较差异无统计学意义(P0.05)。行急诊PCI术后12 h血清c My BP-C浓度较入院时明显下降,而c TnⅠ浓度较入院时明显升高(P0.05)。结论 AMI患者入院时血清c My BP-C、c TnⅠ、CK-MB和Myo浓度较对照组均显著升高且c My BP-C浓度与c TnⅠ、CK-MB和Myo浓度均存在正相关;c My BP-C在发病4 h内即开始升高,提示c My BP-C可以作为诊断AMI的早期生化标志物。AMI患者行急诊PCI术后12 h血清c My BP-C浓度较入院时明显下降,表明c My BP-C可以作为评估PCI术效果的早期指标。     

Myosin phosphorylation decreases the ATPase activity of cardiac myofibrils

Our previous work showed that myosin phosphorylation decreased the ATPase activity of skeletal muscle myofibrils that were lightly fixed with glutaraldehyde. The fixation process prevented sarcomere shortening and destruction of the ordered filament array upon the addition of ATP. We have now extended these results to myofibrils prepared from hearts of rabbits, dogs and rats. Myofibrils were phosphorylated by incubation with myosin light chain kinase, calmodulin and either ATP-gamma s or ATP, for 15 minutes at 25 degrees C. The extent of myosin light chain phosphorylation was 50% to 80%. The ATPase activity of unphosphorylated myofibrils was not altered by reaction with 0.01% glutaraldehyde for 5 minutes at 0 degrees C, and the ATPase activity of unfixed myofibrils was not changed by phosphorylation. However, phosphorylation decreased the ATPase activity of fixed myofibrils by 50%. The effect on myocardial myofibrillar ATPase activity of phosphorylation was similar in the three animal species. These results suggest that in both skeletal and cardiac muscle, myosin phosphorylation decreases the rate of cross-bridge cycling resulting in decreased energy expenditure. It also appears that the effect of myosin light chain phosphorylation on ATPase activity requires an ordered myofilament structure.     

起搏/除颤螺旋电极植入心肌的急性力学研究

目的对不同构型的螺旋电极旋入及拔出犬心肌的急性力学行为进行测试，为心肌／电极界面的数字建模提供实验依据。方法将实验用犬麻醉后，开胸直视下暴露心腔，将不同头端构型的螺旋电极垂直旋入心肌，通过特殊设计的力学传感器记录电极头端的轴向及扭转受力情况，对比包括心房、心室共11个位点的差异情况；随即将旋入电极以外力强制拔除，记录受力-时间曲线进行分析。结果排除间断施加力量的影响，电极旋入过程的扭矩曲线接近指数曲线，平均扭矩分布范围为0．0003至0．0015，不同构型的电极有明显不同；拔除位点受力以左心室外膜、右心室游离壁、HIS束区域最大，同时可见到明显的不均质性。结论螺旋电极旋入及拔出心肌的受力情况随植入位点、电极头端构型等而不同；心室的受力明显高于心房，其中最牢固位点分别为左心室外膜、右心室游离壁及希氏束。     

云南不明原因心源性猝死心肌酶学研究

目的 探索发病村村民与周围自然村村民的心肌酶在本病流行期和非流行期是否存在差异,为深入开展具有针对性的病因研究提供线索.方法 选取发病村村民24人和3个对照村每村30人作为研究对象,在流行期和非流行期分别采集研究人群的血清作心肌酶、肝、肾功等检测. 结果 发病村和对照村人群的4个心肌酶指标在各次检测中异常率均较高,依次是CK-MB、CK、HBDH 、LDH.发病村异常率高于对照村;CK-MB/CK均大于5%;CK-MB最高值为上限值5倍,CK为4倍,HBDH为2.36倍,LDH为1.5倍;发病村人群非流行期LDH,HBDH较流行期明显下降. 结论 发病村村民和同在一山系的对照村村民的4种心肌酶指标异常率在流行期和非流行期均较高,提示该地区(山系)自然环境中可能存在某些致心肌酶增高的危险因素.     

血管紧张素II对心肌细胞蛋白质合成速率和肌球蛋白重链基因表达的作用

目的：研究血管紧张素ＩＩ（ＡｎｇＩＩ）对心肌细胞蛋白质合成速率和肌球蛋白重链（ｍｙｏｓｉｎｈｅａｖｙｃｈａｉｎ，ＭＨＣ）基因表达的影响。方法：采用放射性同位素氚（３Ｈ）－亮氨酸参入量方法观察培养心肌细胞蛋白质合成速率。应用斑点杂交方法分析ＡｎｇＩ对心肌细胞α－ＭＨＣ和β－ＭＨＣ基因表达的作用。结果：在培养的心肌细胞中加入ＡｎｇＩ可明显增加心肌细胞３Ｈ－亮氨酸的参入量，并可诱导心肌细胞α－ＭＨＣ和β－ＭＨＣ基因表达迅速增加，在一定范围内，两者均呈量效关系。ＡｎｇＩ阻断剂沙拉新（ｓａｒ－ａｌａｓｉｎ）可阻断ＡｎｇＩＩ的上述作用。结论：ＡｎｇＩＩ可增加心肌细胞蛋白质合成速率，促进心肌细胞α－ＭＨＣ和β－ＭＨＣ基因表达。     

载脂蛋白E在骨骼肌系统的表达研究

本研究利用骨骼肌合成和分泌非肌性蛋白质,进而分泌入血并在细胞外发挥作用的原理,构建了一个由人巨细胞病毒早期增强子和促进子驱动的人ａｐｏＥｃＤＮＡ 表达质粒（ｐＣＭＶａｐｏＥ） ,通过阳离子脂质体介导转染原代培养的小鼠骨骼肌肌母细胞,ＥＬＩＳＡ和免疫组织化学检测均证实ａｐｏＥ 在肌源性细胞成功表达并分泌至培养液中。直接肌肉注射,ｐＣＭＶａｐｏＥ 表达载体也在小鼠骨骼肌中成功表达,并且预先经氯化钡诱导损伤可使其表达量明显增加。为进一步研究以骨骼肌为靶组织的动脉粥样硬化基因治疗提供了实验依据     

Dystrophin–glycoprotein Complex Purified from Hamster Cardiac Muscle. Comparison of the Complexes from Cardiac and Skeletal Muscles of Hamster and Rabbit

The dystrophin–glycoprotein complex was isolated from hamster ventricular muscle by a method involving homogenization of muscle directly in the presence of 1% digitonin, followed by chromatography on succinylated wheat germ agglutinin agarose, Diethyl aminoethyl (DEAE) cellulose, and/or immunoaffinity agarose. Protein yield of the DEAE cellulose-purified dystrophin–glycoprotein complex was 120±30 (n=3)μg per 5 g hamster ventricular muscle. The cardiac dystrophin–glycoprotein complex, unlike the skeletal muscle counterpart, could not be solubilized from a microsomal fraction with digitonin or some other detergents. By sodium dodecyl sulfate gel electrophoresis, protein composition of the dystrophin–glycoprotein complexes from hamster cardiac muscle was found to be significantly different from that of rabbit skeletal muscle which has been extensively studied. This difference mainly arises from the species difference, because in hamster the cardiac and skeletal muscle complexes exhibited essentially the same protein composition. In rabbit, on the other hand, there are differences between the cardiac and skeletal complexes in the relative abundance of 60 and 64 kDa proteins and in the apparent M_rofα-dystroglycan. We found that the content of the dystrophin–glycoprotein complex, estimated by quantitative immunoblot assay, is at least 5 times more abundant in cardiac than in skeletal muscle in hamster and rabbit.     

亚慢性砷中毒兔心肌组织的光镜和电镜观察

本文对亚慢性砷中毒兔心肌结构的变化进行了光镜和电镜观察。结果表明:砷中毒可引起心肌纤维肿大,颗粒变性和水泡变性,线粒体肿胀。细胞膜性结构破坏,心肌蛋白变性,肌丝溶解。肌间毛细血管扩张,内皮细胞肿胀。间质水肿、出血和炎细胞浸润。上述病理改变有随As_2O_3剂量的加大而加重的趋势。     

The cellular basis of the length-tension relation in cardiac muscle

The relation between muscle length or sarcomere length and developed tension for lengths up to the optimal for contraction (Lmax) is much steeper in cardiac muscle than in skeletal muscle. The steepness of the cardiac length--tension relation arises because the degree of activation of the cardiac myofibrils by calcium increases as muscle length is increased. Two processes contribute to this length-dependence of activation: (i) the calcium sensitivity of the myofibrils increases with muscle length and (ii) the amount of calcium supplied to the myofibrils during systole increases with muscle length. Of these two, the change in calcium sensitivity is the most clearly defined and is responsible for a large part of the rapid change in developed tension when muscle length is altered. It is likely that this change in calcium sensitivity is due to a change in the affinity of troponin for calcium but the underlying mechanism has not been identified. There is good evidence that changes in the calcium supply to the myofibrils can account for the slow changes in tension that follow an alteration in length; there may also be rapid changes in calcium supply but this is less clearly established at present.     

A de novo Mutation of the Beta Cardiac Myosin Heavy Chain Gene in an Infantile Restrictive Cardiomyopathy

Here we report the first pediatric case of restrictive cardiomyopathy secondary to a de novo mutation in the cardiac myosin heavy chain gene MYH7. The clinical course is characterized by an early onset of disease, mild hypertrophy of the left ventricle and a very short evolution to death. Because of the location of the mutation in the hinge region between the rod part and the globular head of the myosin molecule, it is possible that restrictive cardiomyopathy resulted from an impairment of flexion/extension of myosin heads during the contraction/relaxation cycle.     

Biochemical adaptations in cardiac muscle: effects of physical training on sulfhydryl groups of myosin.

Myosin ATPase activity is increased in the hearts of rats exercised by swimming. There is no difference in the total amount of sulfhydryl groups in the myosin from the control and exercised groups. The difference seems to reside in the reactivity of the sulfhydryl groups at or near the active site as evidenced by (a) decreased stimulation of the ATPase activity by iodoacetamide and (b) decreased incorporation of ¹⁴C-IAA in the myosin from the hearts of the exercised animals as compared to the controls.     

巯甲丙脯酸防治心肌肥厚对血液动力学和心肌肌球蛋白重链基因表达的影响

目的 :研究巯甲丙脯酸 (captopril)在防治心肌肥厚过程中 ,对大鼠血液动力学和心肌肌球蛋白重链 (MHC)基因表达的影响。　　方法 :将 6 0只大鼠随机分 3组即 :对照组 (n=17)、心肌肥厚组 (n=19)、心肌肥厚 巯甲丙脯酸组 (n=2 4) ,观察巯甲丙脯酸对主动脉缩窄大鼠血浆和心肌血管紧张素 (Ang )含量、血液动力学以及左心室心肌α- MHC和β- MHC基因表达的作用。　　结果 :1心肌肥厚组大鼠全心重 /体重值及心肌 Ang 含量较对照组明显增加 ;收缩压、平均动脉压、左心室收缩压、左心室等容收缩期压力变化速率最大值 ( dp/ dtmax)显著升高 ;α- MHC基因表达减弱 ,β- MHC基因表达增强。 2巯甲丙脯酸组大鼠的全心重 /体重值及心肌 Ang 的含量显著低于心肌肥厚组 ;使舒张压和平均动脉压显著降低 ;使心肌α- MHC基因表达增强 ,β- MHC基因表达减弱。　　结论 :巯甲丙脯酸通过抑制 Ang 的生成 ,逆转压力超负荷所致的心肌 MHC表型的转化 ,有效地防治了心肌肥厚的发生 ,改善了心脏功能。     

Skeletal muscle myofibrillar protein oxidation in heart failure and the protective effect of Carvedilol

Heart failure is characterized by limited exercise tolerance and by a skeletal muscle myopathy with atrophy and shift toward fast fibres. An inflammatory status with elevated pro-inflammatory cytokines and exaggerated free radicals production, can worsen muscle damage. In a well established model of heart failure, the monocrotaline treated rat, we show that CHF is accompanied by oxidation of the skeletal muscle actin, tropomyosin and myosin, which further depresses muscle function and exercise capacity. We have also tested the efficacy of Carvedilol, a non-selective beta(1)-beta(2)-blocker, which has been widely used in clinical trials to improve exercise tolerance and reduce mortality in moderate and severe CHF, in preventing contractile protein oxidation in CHF rats. As comparison we used Bisoprolol a beta(1) selective agent, without known anti-oxidative properties. Carvedilol at the dose of 2 mg/kg per day was able to prevent the myofibrillar protein oxidation, while Bisoprolol (0.1 mg/kg) did it only partially, as demonstrated by the oxyblot analysis. While Carvedilol improved force production on isolated muscles, Bisoprolol did not. After the COMET trial, the anti-oxidative capacity of Carvedilol has been invoked as one of the mechanism that makes this drug superior to other selective beta-blockers in the treatment of CHF. One of the reason of Carvedilol superiority could be the effect on skeletal muscle with reduction of contractile protein peroxidation, amelioration of muscle function and improvement of exercise tolerance. Inhibition of reactive oxygen species (ROS) production, and of pro-inflammatory cytokines may also lead to a decreased muscle wastage, another factor contributing to worsening of exercise tolerance.     

T445S:肥厚型心肌病的一个新的基因突变位点

目的调查中国人群肥厚型心肌病(HCM)患者心肌肌球蛋白结合蛋白C(MyBPC3)基因突变的发生情况并对基因型与表现型之间的关系进行分析。方法对92例肥厚型心肌病患者及100名正常对照的聚合酶链反应(PCR)扩增产物进行单链构象多态性分析(SSCP)分析,于MyBPC3基因第14-15、17、25、27、33号外显子范围内寻找突变位点,并了解基因型明确的HCM患者的临床特点。结果在一例HCM患者的MyBPC3基因的第14-15号外显子上发现了一个新的突变位点T445S。由于在100名正常对照中未见该错义突变,故我们认为此突变位点为该患者的致病基因位点。结论心肌肌球蛋白结合蛋白C的C3结构域对于其连接及调节功能起着至关重要的作用,该区域的基因突变可导致肥厚型心肌病的发生。     

Cardiac calmodulin and its role in the regulation of metabolism and contraction

Calmodulin, the ubiquitous and multifunctional Ca²⁺-dependent regulatory protein, is capable of activating a number of enzymes through the formation of active ternary complexes Ca²⁺-calmodulin-enzyme, upon increase of the cytosolic Ca²⁺ level from pCa 8 to pCa 5. In cardiac muscle, calmodulin is involved in the Ca²⁺-dependent activation of cyclic nucleotide phosphodiesterase and, perhaps, inhibition of adenylate cyclase. Cardiac and vascular myosin light chain kinases are also Ca²⁺-calmodulin-dependent. Whereas light chain phosphorylation is required for vascular smooth muscle contraction, the effect of cardiac myosin phosphorylation is as yet unknown. Calmodulin is involved in the control of the phosphorylation of the sarcoplasmic reticulum Ca²⁺ pump activator, phospholamban, thereby modulating the rate of Ca²⁺ uptake. Calmodulin is likely to be a subunit of cardiac glycogen phosphorylase b kinase, that triggers glycogen breakdown and presumably inhibits glycogen synthesis. Finally calmodulin binds to other calmodulin-binding proteins of hitherto unknown function.