骨架蛋白ENH在心血管系统中作用的研究进展

ENH是一个含有PDZ和LIM结构域的骨架蛋白,存在多种mRNA剪接体,不同的剪接体具有不同的组织表达方式和功能。ENH在心血管系统中发挥骨架蛋白的结构功能,在心脏发育和维持心脏Z线结构稳定中有重要作用。ENH还能与不同的蛋白激酶结合,作为信号分子的锚定蛋白,调控信号转导,调节心肌细胞的生长、血管平滑肌细胞的增殖与迁移。     

心肌细胞肥大过程中组蛋白脱乙酰基酶2的表达

目的 研究血管紧张素Ⅱ致心肌细胞肥大过程中组蛋白脱乙酰基酶2的表达.方法 培养原代心肌细胞,给予不同浓度的血管紧张素Ⅱ刺激心肌细胞造成肥大,取心肌细胞进行逆转录聚合酶链反应观察组蛋白脱乙酰基酶2和β-肌球蛋白重链mRNA表达,免疫组织化学法检测组蛋白脱乙酰基酶2和c-fos蛋白表达,相差显微镜观察细胞面积变化.结果 经血管紧张素Ⅱ刺激后在相差显微镜下可见心肌细胞面积变大,而且随着血管紧张素Ⅱ浓度的增加而增大.组蛋白脱乙酰基酶2和β-肌球蛋白重链mRNA水平及组蛋白脱乙酰基酶2和c-fos蛋白表达随着血管紧张素Ⅱ浓度的增加而增加.结论 血管紧张素Ⅱ致心肌细胞肥大过程中伴有组蛋白脱乙酰基酶2表达增加,后者有可能参与心肌细胞的肥大机制.     

三磷酸肌醇受体/钙调神经磷酸酶信号通路激活致心肌肥厚的研究

目的 研究激活心肌细胞三磷酸肌醇受体（IP3R）是否触发钙调神经磷酸酶（CaN)介导的心肌肥厚信号通路。方法 培养Wistar乳鼠心肌细胞检测心肌细胞蛋白和核酸合成，细胞内钙变化，CaN，活化T细胞核因子3（NFAT3）及锌指转录因子（GATA4），胚胎基因（α-actin,β-MHC）及即刻早期基因（c-fos,c-myc)表达。结果 给予IP3能时间和剂量依赖性也增加心肌细胞蛋白和核酸合成，能明显致心肌细胞内钙增加；IP3刺激心肌细胞IP3受体，能明显激活心肌细胞CaN/NFAT3/GATA4信号通路，促使心肌细胞的早期即刻基因和胚胎基因表达。结论 激活IP3R介导的CaN/NFAT3/GATA4信号通路能显地促使心肌细胞肥大，这条信号通路不同于已知的G蛋白偶联受体介导的心肌肥厚信号转导途径。     

Ⅰ型组蛋白去乙酰化酶在心肌肥厚中的靶点作用

组蛋白去乙酰化酶是一种转录后修饰的酶类,可以使组蛋白去乙酰化。Ⅰ型组蛋白去乙酰化酶可促进心肌肥厚发生。在Ⅰ型组蛋白去乙酰化酶中,组蛋白去乙酰化酶2（HDAC2）由肥大刺激和热休克蛋白70诱导活化。活化的HDAC2通过抑制级联信号KriJppel样因子4（KLF4）或肌醇多磷酸磷酸酶-5-f（Inpp5f）触发肥大。因此,调节HDAC2的酶类,例如选择性组蛋白去乙酰化酶抑制剂,被认为是治疗心脏疾病尤其是阻止心肌肥厚的一个重要靶标。该文讨论I型组蛋白去乙酰化酶在调节心肌肥厚中所起的关键作用。     

细胞因子信号转导负调控蛋白—SOCS家族研究进展

目前已知的SOCS家族共有8个成员、细胞因子信号通路能调节该家族蛋白折表达，而SOCS蛋白通过直接结合活化的JAK激酶或磷酸化的受体抑制AK／STAT信号转导通路的活性，现利用该家族保守域又得到一系列蛋白，但功能尚不详。     

三磷酸肌醇受体参与心肌钙调神经磷酸酶信号通路的激活

目的:研究激活心肌细胞三磷酸肌醇受体（IP3R）是否触发钙调神经磷酸酶（CaN）介导的心肌肥厚信号通路。方法:培养的乳鼠心肌细胞检测心肌细胞蛋白合成,细胞内钙变化,CaN、活化T细胞核因子3（NFAT3）及锌指转录因子（GATA4）,胚胎基因（αskeletalactin,βMHC）及即刻早期基因（cfos,cmyc）蛋白表达。结果:三磷酸肌醇（IP3）能显著致原代培养的心肌细胞时间和剂量依赖性地增加心肌细胞蛋白合成,能显著致心肌细胞的早期即刻基因和胚胎基因表达,能显著增加心肌细胞内游离钙。IP3剌激IP3受体,能显著激活心肌细胞CaN/NFAT3/GATA4信号通路,使心肌细胞早期即刻基因（cfos、cmyc）及胚胎基因（αskeletalactin,βMHC）表达增加。结论:激活IP3R介导的CaN/NFAT3/GATA4信号通路能显著地促使心肌细胞肥大,这条信号通路不同于已知的G蛋白偶联受体介导的心肌肥厚信号转导途径。     

益气活血方动员骨髓干细胞对心肌缺血再灌注损伤大鼠JAK2-PI3K信号转导调节的动态影响

目的探讨益气活血方动员骨髓干细胞对心肌缺血再灌注损伤(IRI)大鼠JAK2-PI3K信号转导调节的动态影响。方法大鼠144只随机为A:假手术组;B:IRI组;C:rh EPO动员组;D:益气活血方组;E:益气活血方联合动员组,F:LY294002组(益气活血方联合动员+PI3K的特异性阻断剂组),每组8只,于造模后3个不同时间段观察心肌细胞JAK2-PI3K信号途径蛋白表达及凋亡情况,结果 IRI组心肌细胞JAK2、PI3K蛋白表达均降低而凋亡指数升高,而其余各组凋亡指数降低,JAK2、PI3K蛋白表达增加,且随着时间推移逐渐明显;当使用PI3K阻断剂后,保护作用消失。结论益气活血方动员骨髓干细胞治疗心肌IRI,降低大鼠心肌细胞凋亡指数,其机制可能与调节JAK2-PI3K信号途径相关。     

蛋白乙酰化与胰岛素分泌

蛋白乙酰化直接或间接参与胰岛素分泌的调节.多项研究证实组蛋白乙酰化通过对多种组蛋白乙酰基转移酶和组蛋白去乙酰化酶的动态调节,参与调节葡萄糖刺激的胰岛β细胞分泌功能,从而直接或间接地在糖尿病的发生、发展中起重要作用.胰岛β细胞骨架蛋白乙酰化可调节细胞骨架系统运送胰岛素颗粒的活力,进而参与调节葡萄糖刺激的胰岛素分泌.对蛋白乙酰化的深入研究将为糖尿病的防治提供新的思路.     

β-Arrestins在胰岛素/胰岛素样生长因子-1信号转导中的作用

β-Arrestins是G蛋白耦联受体信号转导通路的负调节因子,越来越多的证据表明,β-arrestins也能作用于细胞内的多种信号分子,调节胰岛素/胰岛素样生长因子-1(IGF-1)信号转导通路.在胰岛素的刺激下,β-arrestin 2能够募集蛋白激酶B(Akt)和酪氨酸激酶Src到胰岛素受体,从而调节胰岛素介导的糖代谢效应;而β-arrestin 1则与胰岛素受体底物-1(IRS-1)竞争性结合泛素连接酶Mdm2,从而减少IRS-1的泛素化和降解,促进磷脂酰肌醇3激酶(PI3K)通路的信号转导.在IGF-1介导的信号转导通路中,β-arrestin 1结合并介导了IGF-1受体(IGF-1R)的内吞,促进胞外信号调节激酶活化,正性调节丝裂原活化蛋白激酶通路.此外,β-arrestin 1与IGF-1R相耦联后,能越过信号分子IRS-1而激活PI3K,进而活化Akt,表现出对P13K途径的正性调控作用.     

β肾上腺素能受体激动剂与心肌细胞凋亡

β肾上腺素能受体是调节心脏功能的主要受体。激动剂与受体结合后调节心脏功能,以适应各种应激和活动。但长期高水平的激动剂刺激受体可引起心脏的病理损害,导致心肌细胞凋亡。现就β肾上腺素能受体激动剂介导心肌细胞凋亡的蛋白激酶A(PKA)、钙/钙调磷酸酶Ⅱ(Ca2 /CaMKⅡ)、丝裂原活化蛋白激酶(MAPKs)等信号转导通路进行综述。     

Dystrophin and the Cardiomyocyte Membrane Cytoskeleton in the Healthy and Failing Heart

The cardiomyocyte membrane cytoskeleton consists of the costameric proteins that mediate force transduction from the cell to the extracellular matrix, and a sub-membrane network composed of dystrophin and associated proteins. Studies of the precise cellular distribution of dystrophin and of the consequences of genetic mutations leading to abnormal expression of the dystrophin molecule, as occurs in Duchenne and Becker's muscular dystrophies, highlight potential functional roles of this sub-membrane protein complex in cardiomyocytes. Detailed investigation of dystrophin distribution using the complementary cell imaging techniques of immunoconfocal microscopy and freeze-fracture cytochemistry at the electron-microscopical level show that, in contrast to rat cardiomyocytes, the dystrophin network in human cardiomyocytes is locally enriched at costameres. Thus located, the dystrophin network appears to have a mechanical role, involving stabilization of the peripheral plasma membrane during the repetitive distortion associated with cardiac contraction and, in the human myocyte, contributing to lateral force-transduction. Evidence from animal models of muscular dystrophy and from investigation of the interactions of the sub-membrane cytoskeleton with other membrane-associated proteins including ion channels, receptors and enzymes, further suggests a role for dystrophin in organization and regulation of membrane domains. The relative preservation of the membrane cytoskeleton in non-dystrophic dilated cardiomyopathy and in ischemic cardiomyopathy, conditions in which the myocyte contractile apparatus and internal desmin-based cytoskeleton are commonly disrupted, emphasizes the vital role of the membrane cytoskeleton in cell survival. Continued cardiomyocyte survival despite loss of contractile protein organization has implications in the potential for reversibility of left ventricular remodeling that can be achieved in the clinical setting.     

Cardiomyocyte Cytoskeleton and Myofibrillogenesis in Healthy and Diseased Heart

The unique cytoarchitecture of cardiomyocytes arises by complex interactions of different filamentous structures of the cytoskeleton. Intermediate filaments of the non-sarcomeric cytoskeleton are not essential for development but important for maintenance of myofibrils. Myofibrils consist of contractile proteins involved in force generation and the muscle cytoskeleton framework. The latter is essential for proper assembly and maintenance as well as for interaction with other cardiomyocytes or the extracellular matrix, thus being involved in force transmission. The information for sarcomere assembly is encoded in the proteins and some domains essential for faithful incorporation have been identified by epitope tagging experiments. Many KO mutations result in embryonic lethal phenotypes and new techniques e.g. using cardiomyocytes derived from ES cell-lines will have to be developed that allow to study such mutations in cardiomyocytes rather than whole organisms. Alterations in the expression levels of several proteins of the muscle cytoskeleton or impairment of their function by point mutations can result in increased mechanical stress in the cardiomyocytes which finally leads to cellular responses such as the development of dilated cardiomyopathy (DCM). MLP (muscle-LIM-protein) deficient mice develop DCM and changes in the mechanical coupling of cardiomyocytes result in alterations at the intercalated disks and enhanced accumulation of adherens junction proteins. Therefore, controlled interactions between proteins of the muscle cytoskeleton and contractile proteins are essential to ensure proper cardiac function and a more detailed insight in these processes might provide new tools to improve the contractile efficiency of the cardiomyocytes and thus working output in cardiomyopathies.     

Integrin-linked kinase at the heart of cardiac contractility, repair, and disease

Recent advances in cardiac physiology identify the integrin-linked kinase (ILK) as an essential molecule regulating cardiac growth, contractility, and repair. A key transducer of biochemical signals initiated at the plasma membrane by cell-matrix interactions, ILK now emerges as a crucial player in mechanotransduction by integrins. Animal models have been particularly instructive in dissecting the cardiac functions of ILK and its associated proteins, such as parvins and PINCH, and have clearly established ILK as a major contributor to cardiac health. ILK gene knockouts in mice, flies, and worms result in early embryonic lethality because of cell adhesion defects and cytoskeletal disorganization. Although widely distributed in mammalian tissues, ILK expression is highest in the heart, and cardiac-specific ablation of ILK causes cardiomyopathy and sudden death in mice. ILK protein complexes are found in the sarcomere, which is the basic contractile unit of myocytes. A natural inactivating mutation in the kinase domain of ILK disrupts ILK protein interactions in the sarcomere, causing a contractile defect in the zebrafish heart. The relatively subtle phenotype of mutant ILK hearts, compared with ILK-ablated hearts, suggests multiple cardiac ILK functions. Cardiac-specific expression of ILK in transgenic mice induces a hypertrophic program, pointing to ILK as a proximal regulator of multiple hypertrophic signal transduction pathways. ILK protein interactions may also be important in mediating postinfarct cell migration and myocardial repair.     

Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes

OBJECTIVE: We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS: The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS: Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS: The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.     

Characterization of cardiac myocyte and tissue beta-adrenergic signal transduction in rats with heart failure

OBJECTIVE: The cellular basis of alterations in beta-adrenergic signal transduction in rats with chronic heart failure (CHF) remains unclear. The aim of the present study was to examine this signal transduction system in isolated ventricular cardiomyocytes of rats with CHF. We focused on changes in the levels of stimulatory (Gs) and inhibitory G-proteins (Gi). METHODS: CHF was induced in male Wistar rats by coronary artery ligation (CAL). Hemodynamic and biochemical parameters were measured 8 weeks after CAL. Alterations in contractile function and Ca(2+) transients via beta-adrenergic receptor signaling of cardiomyocytes isolated from rats with CHF were characterized by simultaneous measurements of cell shortening and fura-2 fluorescence intensity. RESULTS: Coronary artery-ligated rats showed symptoms of CHF, such as decreased contractile function, increased left ventricular volume, decreased chamber stiffness, and about 40% infarct formation of the left ventricle, by 8 weeks after surgery. The contractile function and Ca(2+) dynamics of cardiomyocytes from the rats with CHF remained normal under basal conditions. Only cardiac cell length was increased. The responses of peak shortening, fura-2 fluorescence ratio amplitude, and cAMP content to beta-adrenoceptor stimulation were reduced in cardiomyocytes of the rats with CHF, whereas direct stimulation of adenylate cyclase did not affect the response of these variables. Cardiomyocyte Gsalpha protein was decreased, whereas no changes in Gialpha proteins were seen in these cells. Increases in tissue Gsalpha and Gialpha proteins in the scar zone were detected. The results on tissue levels of collagen and G-proteins in the viable left ventricle appeared to depend on the presence of nonmyocytes. CONCLUSIONS: The results suggest that impaired contractile function of cardiomyocytes is unlikely to account for global LV contractile dysfunction, and that down-regulation of beta-adrenoceptors occurs in cardiomyocytes per se. The difference in changes of G-protein between the cardiomyocyte and myocardial tissue suggests an appreciable contribution of nonmyocytes to myocardial G-protein levels.     

Expression of contractile and cytoskeletal proteins in myocardium of patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by enlargement and dilation of all heart compartments associated with serious decrease of its contractile function. DCM hallmark is the combination of dystrophic and hypertrophic alterations of cardiomyocytes. Since the power output of cardiac cells is directly related to remodeling of their contractile machinery we investigated expression of selected contractile and cytoskeletal proteins in the left ventricle of DCM patients using immunoblotting. The content of the recognized protein markers of cardiomyocyte hypertrophy such as tubulin, desmin and slow skeletal myosin heavy chain isoform, MHCbeta, was significantly elevated in DCM compared to normal myocardium. In addition, marked increase in the content of several smooth muscle proteins (smooth muscle alpha-actin, Myosin Light Chain Kinase, Kinase Related protein SM22) that are normally expressed in embryonic myocardium, was observed in DCM hearts. Thus, cardiomyocyte hypertrophy in DCM is associated with activation of embryonic protein expression program and smooth muscle proteins could serve as markers of this process. Understanding their involvement in sarcomere assembly and pathways of their expression activation during cardiac hypertrophy may bring new insights in treatment of various forms of cardiomyopathy.     

Alcoholic Cardiomyopathy: Disrupted Protein Balance and Impaired Cardiomyocyte Contractility

Alcoholic cardiomyopathy (ACM) can develop after consumption of relatively large amounts of alcohol over time or from acute binge drinking. Of the many factors implicated in the etiology of ACM, chronic perturbation in protein balance has been strongly implicated. This review focused on recent contributions (since 2010) in the area of protein metabolism and cardiac function related to ACM. Data reviewed include that from in vitro and preclinical in vivo animal studies where alcohol or an oxidative metabolite was studied and outcome measures in either cardiomyocytes or whole heart pertaining to protein synthesis or degradation were reported. Additionally, studies on the contractile properties of cardiomyocytes were also included to link signal transduction with function. Methodological differences including the potential impact of sex, dosing, and duration/timing of alcohol administration are addressed. Acute and chronic alcohol consumption decreases cardiac protein synthesis and/or activation of proteins within the regulatory mammalian/mechanistic target of rapamycin complex pathway. Albeit limited, evidence suggests that myocardial protein degradation via the ubiquitin pathway is not altered, while autophagy may be enhanced in ACM. Alcohol impairs ex vivo cardiomyocyte contractility in relation to its metabolism and expression of proteins within the growth factor pathway. Dysregulation of protein metabolism, including the rate of protein synthesis and autophagy, may contribute to contractile deficits and is a hallmark feature of ACM meriting additional sex‐inclusive, methodologically consistent studies.     

Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes

Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the α-actinin-DsRed fusion protein (SINrep5/α-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/α-actinin-DsRed exhibited a correct incorporation of the newly synthesized α-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocytes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies. Received: 15 February 2001, Returned for revision: 12 March 2001, Revision received: 16 April 2001, Accepted: 17 April 2001     

At the crossroads of myocardial signaling: the role of Z-discs in intracellular signaling and cardiac function

Understanding the molecular interactions among components of cardiac Z-discs and their role in signaling has become pivotal in explaining long- and short-term regulation of cardiac function. In striated muscle, the ends of the thin filaments from opposing sarcomeres overlap and are cross-linked by an elaborate array of proteins to form a highly ordered, yet dynamic network that is the Z-disc. We review here a current picture of the function and structure of the Z-disc of mammalian cardiac myocytes. We emphasize provocative findings that advance new theories about the place of cardiac Z-discs in myocardial intra- and intercellular signaling in myocardial physiology and pathology. Relatively new approaches, especially yeast two-hybrid screens, immunoprecipitation, and pull down assays, as well as immunohistochemical analysis have significantly altered previous views of the protein content of the Z-disc. These studies have generally defined domain structure and binding partners for Z-disc proteins, but the functional significance of the binding network and of the domains in cardiac cell biology remains an unfolding story. Yet, even at the present level of understanding, perceptions of potential functions of the Z-disc proteins are expanding greatly and leading to new and exciting experimental approaches toward mechanistic understanding. The theme of the following discussion of these Z-disc proteins centers on their potential to function not only as a physical anchor for myofilament and cytoskeletal proteins, but also as a pivot for reception, transduction, and transmission of mechanical and biochemical signals.     

TNFα在心肌缺血／再灌注重塑期的保护作用

大量研究表明,肿瘤坏死因子d（TNFa）在心肌缺血／再灌注（I／R）损伤中,既能促进心肌损伤,又具有保护心肌的作用,这种看似相互矛盾的作用,系因在心肌I／R的不同时期由不同的信号转导通路所介导。在心肌I／R的急性期,缺血区冠脉血管床的内皮细胞受损,黏附血中巨噬细胞与淋巴细胞可释放大量可溶型TNFa（sTNFα）。高浓度的sTNFα可激活TNF的1型受体（TNFR1）及其下游信号转导通路,引起心肌细胞凋亡。在心肌I／R的重构期或修复期,虽然血中sTNFα的水平高于生理浓度,但血中可溶型TNFR的水平增加可中和sTNFa,阻止其对正常心肌组织的损害。心肌局部缺血可刺激心肌细胞与成纤维细胞合成TNFα,在细胞膜上装配为跨膜型TNFa,激活相邻细胞的2型TNF受体（TNFR2）及其下游信号转导通路,增强心肌细胞钙转运而增强其收缩功能,同时拯救缺血边界区可存活的心肌细胞。心肌局部跨膜型TNFα还能募集间充质干细胞,分泌多种细胞生长因子,改善心功能。TNFa可通过组蛋白去乙酰化酶1（HDAC1）调节核转录因子KB（NF-KB）的转录活性,保护心肌细胞在持续的TNFa作用下得以存活。跨膜型TNFα还可激活心肌中固有的干细胞向心肌细胞分化,修复受损的心肌组织。