胰岛素及cAMP对培养人动脉平滑肌细胞HDL受体功能的影响

对胰岛素ｃＡＭＰ对培养人动脉平滑肌细胞（ＳＭＣ）ＨＤＬ受体功能的影响进行了研究,结果发现：胰岛素使ＳＭＣＨＤＬ受体的结合容量Ｂｍａｘ即受体数目显著下降,而对ＳＭＣＨＤＬ受体的Ｋｄ值亲和力无影响;ｃＡＭｐ则ＳＭＣＨＤＬ受体亲和力增加,而对受体数目无影响。     

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.     

Effects of troponin on thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8°C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48°C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70°C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100°C. However, addition of TnI, and especially of TnT, to the F-actin–Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.     

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)     

Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using α-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.     

Primary cultures of endothelial cells of the rat liver

Summary In the soleus muscle of the normal rat the number of cells containing fast troponin I decreased and those containing slow troponin I increased after birth until less than 10% stained for the fast form in the adult muscle. On denervation of soleus muscle this pattern of change was reversed with the result that the majority of cells stained for fast troponin I. The change was more rapid when denervation was carried out at 12 weeks rather than at 52 weeks of age. Denervation of extensor digitorum longus and tibialis anterior muscles produced little change in the distribution of fast and slow troponin I over a period of 12 weeks. After long periods (>24 weeks) of denervation of these fast muscles, fast troponin I was observed in cells in which originally only slow troponin I could be detected. Similar results to those obtained with troponin I in both fast and slow muscles were obtained using antibodies to the fast and slow forms of troponin C and troponin T.     

Troponin: regulatory function and disorders

Study of the molecular biology of the calcium regulation of muscle contraction was initiated by Professor Ebashi’s discovery of a protein factor that sensitized actomyosin to calcium ions. This protein factor was separated into two proteins: tropomyosin and a novel protein named troponin. Troponin is a Ca²⁺-receptive protein for the Ca²⁺-regulation of muscle contraction and, in association with tropomyosin, sensitizes actomyosin to Ca²⁺. Troponin forms an ordered regulatory complex with tropomyosin in the thin filament. Several regulatory properties of troponin, which is composed of three different components, troponins C, I, and T, are discussed in this article. Genetic studies have revealed that many mutations of genes for troponin components, especially troponins T and I, are involved in the three types of inherited cardiomyopathy. Results of functional analyses indicate that changes in the Ca²⁺-sensitivity caused by troponin mutations are the critical functional consequences leading to these disorders. Recent results of this pathophysiological aspect of troponin are also discussed.     

Background to the discovery of troponin and Setsuro Ebashi's contribution to our knowledge of the mechanism of relaxation in striated muscle

The discovery of the actomyosin system provided for the first time a model system that enabled the study of the role of the muscle protein components in the contraction and relaxation cycle to be undertaken. It soon became apparent that ATP was essential for both processes but progress really began when it became clear that components both in the myofibrillar and sarcoplasmic fractions were involved in relaxation. After it was apparent that a trace of calcium was required for the activation of the MgATPase of the myofibrils it was shown that an active calcium pump was located in the sarcoplasmic reticulum. The report by Ebashi in 1963 that a new myofibrillar protein, troponin, was the target for calcium opened up the investigation of the calcium control of the MgATPase. Troponin was shown to be a complex of troponin C, I and T, each protein being under individual genetic control and existing in isoforms specific for the muscle type. The unique forms of troponin I and T in cardiac muscle make them the biomarkers of choice for cardiac injury.     

Application of the fourth universal definition of myocardial infarction in clinical practice

Abstract

Purpose: The Fourth Universal Definition of Myocardial Infarction (MI) has highlighted the different pathophysiological mechanisms that may lead to ischaemic and non-ischaemic myocardial injury and has emphasised that the diagnosis of myocardial infarction requires the presence of acute myocardial ischaemia in the setting of acute myocardial injury. This case based review intends to illustrate basic principles on how to apply this new, revised definition in clinical practice.

Methods and Results: The distinction between different types of MIs (type 1 or type 2) and the delineation of MI from acute non-ischaemic myocardial injury may be challenging in individual patients, which is illustrated by presenting and discussing real-life routine cases.

Conclusions: Type 1?MI is a consequence of coronary plaque rupture or erosion with intracoronary thrombus formation that is usually apparent on coronary angiography. Plausible triggering mechanisms causing myocardial oxygen supply/demand mismatch must be identified for the diagnosis of type 2?MI and its treatment should focus initially on management of the underlying disease attributable to acute myocardial ischaemia.     

微丝相关新蛋白hHBRK1相互作用蛋白质的鉴定

为了鉴定hHBRK1的相互作用蛋白,通过DNA重组构建重组表达质粒pGEXhHBRK1,并以谷胱甘肽Sepharose4B亲合层析法,获得纯化的重组融合蛋白GSThHBRK1.以小鼠心肌组织为研究对象,采用GSTpulldown技术结合Western印迹法,证实hHBRK1与小鼠心肌肌钙蛋白TEa亚型(EacTnT)相互作用.结果提示,hHBRK1与EacTnT结合,可能参与心肌微丝的聚合,为小鼠cTnT众多的剪接体,提供了一种可能的功能定位.     

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca²⁺ solution (pCa> 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca2²⁺ (pCa 4.5 prior to incubation with vanadate. Transfer back to a high Ca²⁺ solution elicited no additional force (83.2% ± 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.     

肌钙蛋白I2辅助活化多种核受体的反式作用

为深入研究乳腺癌中孤儿受体ERRα1参与基因表达调控的详细机理,特别是核受体辅激活蛋白在其中的作用,以ERRα1的LBD为诱饵,用酵母双杂交系统筛选人乳腺组织cDNA文库得到了与其有明显相互作用的快速骨骼肌型肌钙蛋白I(TNNI2).应用酵母双杂交技术研究表明,TNNI2与多种核受体存在相互作用,且这种作用依赖于功能性的核受体AF2结构域.在哺乳细胞瞬时共转染实验中,TNNI2显示了对多种核受体反式激活功能的辅助活化作用.研究证明,TNNI2与许多辅激活蛋白类似,以配体依赖(对类固醇激素受体而言)或非依赖(对孤儿受体而言)的方式与核受体功能性AF2结构域相互作用,并增强多种核受体介导的反式作用.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Molecular Cloning and Comparative Characterization of the Porcine Troponin I Family

Troponin I (TnI) is a family of three muscle-specific myofibrillar proteins involved in calcium-sensitive regulation of contraction in cardiac and skeletal muscle. In this study, the full-length cDNA and genomic sequence of three genes of porcine TnI family were cloned and sequenced. The full-length cDNA of TNNI1, TNNI2, and TNNI3 genes were 989 bp, 734 bp, and 831 bp in length, which contained an open reading frame of 564, 549, and 636 nucleotides, respectively. Three Troponin I shared 54.4 ～ 58.3% similarity with each other in their predicted amino acid sequences. The TNNI1, TNNI2, and TNNI3 displayed the same genomic structure as other vertebrates and spanned over 9785 bp, 2373 bp, and 3648 bp genomic regions, respectively. The regulatory elements in the proximal promoter of TNNI2 and TNNI3 were conserved among human, mouse, and pig, but regulatory element differences existed in the TNNI1 promoter among them. Expression profiling showed that TnI genes were widely expressed in the tissues studied, with the highest expression level of TNNI1 and TNNI2 in skeletal muscle, and TNNI3 in cardiac muscle.     

Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I

Aims

The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes.

Main methods

Mechanical properties and intracellular Ca^2 + properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis.

Key findings

AMPK activator, A-769662 induced maximal velocity of shortening (+ dL/dt) and relengthening (− dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca^2 + levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser¹⁴⁹ site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662.

Significance

This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.     

急诊阵发性心房颤动患者血清肌钙蛋白水平升高的相关影响因素分析

目的:通过比较急诊就诊时不同肌钙蛋白I(TnI)水平阵发性心房颤动(48小时内)患者临床特征的差异,探讨血清肌钙蛋白水平升高的相关影响因素,为临床识别高危房颤患者提供参考依据。方法:选取2016-2017年于宣武医院急诊诊断为阵发性房颤的患者110例,记录既往病史、临床症状体征、实验室检验结果及测定血清TnI水平,并根据TnI水平将患者分为TnI正常组(0.023μg/L)和TnI升高组(0.023μg/L),结合患者既往病史及用药史给予普罗帕酮或可达龙药物转复治疗。结果:急诊阵发房颤患者TnI升高发生率为11.8%,TnI峰值的平均值0.210μg/L,中位数0.067μg/L。TnI正常组和TnI升高组性别构成、血压、心室率、胸闷、头晕、乏力、高血压、糖尿病、慢性阻塞性肺部疾病、射频消融术、转复成功率及NT-proBNP比较差异均无统计学意义(P0.05);年龄、心悸、胸痛、腹背部不适感、冠心病、房颤病史、心电图ST段压低比较差异均有统计学意义(P0.05)。二元logistic回归分析显示既往冠心病史及入室心电图ST段压低为血清TnI升高的影响因素。结论:既往冠心病史及心电图ST段压低为阵发房颤患者肌钙蛋白升高的影响因素,临床上需关注高龄以心绞痛样症状就诊的首次房颤患者。     

In vivo cell tracking of mouse embryonic myoblasts and fast fibers during development

Fast and slow TnI are co‐expressed in E11.5 embryos, and fast TnI is present from the very beginning of myogenesis. A novel green fluorescent protein (GFP) reporter mouse lines (FastTnI/GFP lines) that carry the primary and secondary enhancer elements of the mouse fast troponin I (fast TnI), in which reporter expression correlates precisely with distribution of the endogenous fTnI protein was generated. Using the FastTnI/GFP mouse model, we characterized the early myogenic events in mice, analyzing the migration of GFP+ myoblasts, and the formation of primary and secondary myotubes in transgenic embryos. Interestingly, we found that the two contractile fast and slow isoforms of TnI are expressed during the migration of myoblasts from the somites to the limbs and body wall, suggesting that both participate in these events. Since no sarcomeres are present in myoblasts, we speculate that the function of fast TnI in early myogenesis is, like Myosin and Tropomyosin, to participate in cell movement during the initial myogenic stages. genesis 52:793–808, 2014. © 2014 Wiley Periodicals, Inc.     

High-sensitivity troponin T in asymptomatic severe aortic stenosis

Background: In asymptomatic severe aortic stenosis (ASAS), treatment decisions are made on an individual basis, and case management presents a clinical conundrum.

Methods: We prospectively phenotyped consecutive patients with ASAS using echocardiography, exercise echocardiography, cardiac MRI and biomarkers (NT-proBNP, high-sensitivity troponin T (hs-TnT) and ST2) (n?=?58). The primary endpoint was a composite of cardiovascular death, new-onset symptoms, cardiac hospitalization, guideline-driven indication for valve replacement and cardiovascular death at 12?months.

Results: During the first year, 46.6% patients met primary endpoint. In multivariable analysis, aortic regurgitation ≥2 (p?=?0.01) and hs-TnT (p?=?0.007) were the only independent predictors of the primary endpoint. The best cutoff value was identified as hs-TnT >10ng/L, which was associated with a ～10-fold greater risk of the primary endpoint (HR, 9.62; 95% CI, 2.27–40.8; p?=?0.002). A baseline predictive model including age, sex and variables showing p?<?0.10 in univariable analyses showed an area under the curve (AUC) of 0.79(0.66–0.91). Incorporation of hs-TnT into this model increased the AUC to 0.90(0.81–0.98) (p?=?0.03). Patient reclassification with the model including hs-TnT yielded an NRI of 1.28(0.46–1.78), corresponding to 43% adequately reclassified patients.

Conclusions: In patients with ASAS, hs-TnT >10ng/L was associated with high risk of events within 12?months. Including hs-TnT in routine ASAS management markedly improved prediction metrics.