MAP KINASES AND H202 INDUCED MYOCARDIAL DYSFUNCTION

MAP 激酶和 H202 诱发的心肌功能障碍

• 批准号: 2898925
• 负责人: Pamela A Lucchesi
• 金额: $ 21.59万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2898925
• 关键词: calcium flux; cardiac myocytes; free radical oxygen; guanine nucleotide binding protein; heart contraction; hydrogen peroxide; immunocytochemistry; immunoprecipitation; laboratory rat; membrane transport proteins; mitogen activated protein kinase; myocardial ischemia /hypoxia; oxidative stress; protein kinase C; protooncogene; reperfusion; tissue /cell culture; video microscopy; western blottings

Reperfusion of the myocardium following an ischemic episode is associated with profound contractile and metabolic dysfunction, referred to as myocardial stunning. Reperfusion also increases the activity of the Na+/H+ exchanger (NHE), which restores intracellular pH (pHi) towards normal following ischemia-induced acidosis. However, activation of NHE also produces undesirable secondary effects leading to the exacerbation of tissue injury, a phenomenon termed the "pH paradox". Increased generation of oxygen free radicals (OFR) plays an important role in reperfusion-induced myocardial stunning and NHE activation. An in vitro model for studying the effects of OFRs on cultured neonatal rat ventricular myocytes (NRVM) has been defined, in which low concentrations of H2O2 (similar to those generated during reperfusion) cause contractile dysfunction, Ca2+ overload, and NHE activation. There is considerable interest in identifying signaling events that link H2O2 to myocardial dysfunction. H2O2 and hypoxia activate members of the mitogen activated protein kinase (MAPK) family, including p38, c-jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK1/2). Low doses of H2O2 decrease myocyte contractility and stimulate NHE activity in an ERK1/2-dependent manner. Preliminary data indicate that exposure of cardiac myocytes to H2O2 induces myofilament disassembly, Ca2+ overload, and the activation of the nonreceptor tyrosine kinase src. The hypothesis of this proposal is that the MAPK family modulates NHE activity, Ca2+ overload and contractile dysfunction induced by H2O2. In Aim 1, experiments with synthetic inhibitors and antisense oligonucleotides will determine whether MAP kinase inhibition blocks H2O2-induced phosphorylation of NHE, since phosphorylation of the exchanger protein is associated with its activation. NHE activation will be measured by fluorimetric imaging of intracellular pH and by examining the phosphorylation state of the NHE protein in vitro and in vivo. In Aim 2, the link between H2O2-induced contractile dysfunction, Ca2+ overload and MAPK activation will be investigated using pharmacological inhibitors and antisense oligonucleotides against p38, JNK, and ERK MAPKs. Contractile dysfunction will be defined as a decrease in myocyte contractility (using video edge detection), and by immunocytochemistry to measure myofibrillar assembly. In Aim 3, a studies using immunecomplex kinase assays, immunoprecipitation and Western blot analysis will identify regulatory components upstream of MAPKs that are activated by H2O2. This aim will focus on src, protein kinase C, and the Ras superfamily of monomeric GTP-binding proteins. The proposed investigations are fundamentally important to the development of therapeutic strategies targeted to signaling pathways involved in oxidant-induced injury and may have important clinical implications in the treatment myocardial ischemia.

缺血性发作后心肌的再灌注与深刻的收缩和代谢功能障碍有关，被称为心肌惊人。 再灌注还增加了Na+/H+交换剂（NHE）的活性，后者将细胞内pH（PHI）恢复到局部缺血诱导的酸中毒后正常的活性。 然而，NHE的激活还会产生不良的次要作用，导致组织损伤加剧，这种现象称为“ pH悖论”。 增加的氧自由基（OFR）的产生在再灌注引起的心肌惊人和NHE激活中起着重要作用。 已经定义了一种用于研究OFR对培养的新生大鼠心室肌细胞（NRVM）影响的体外模型，其中低浓度的H2O2（类似于再灌注过程中的H2O2）导致收缩功能障碍，CA2+过载和NHE激活。 识别将H2O2与心肌功能障碍联系起来的信号事件引起了很大的兴趣。 H2O2和缺氧激活有丝分裂原激活蛋白激酶（MAPK）家族的成员，包括p38，C-Jun NH2-末端激酶（JNK）和细胞外信号调节激酶（ERK1/2）。 低剂量的H2O2会以ERK1/2依赖性方式降低肌细胞的收缩性并刺激NHE活性。 初步数据表明，心肌细胞暴露于H2O2会诱导肌丝拆卸，Ca2+过载和非受体酪氨酸激酶SRC的激活。 该提案的假设是MAPK家族调节H2O2引起的NHE活动，Ca2+过载和收缩功能障碍。 在AIM 1中，使用合成抑制剂和反义寡核苷酸的实验将确定MAP激酶抑制阻断H2O2诱导的NHE磷酸化是否是否与换料蛋白的磷酸化有关，这与其活化有关。 NHE激活将通过细胞内pH的荧光成像以及通过在体外和体内检查NHE蛋白的磷酸化状态来测量。 在AIM 2中，将使用药理学抑制剂和针对p38，JNK和ERK MAPKS的反义寡核苷酸来研究H2O2诱导的收缩功能障碍，CA2+过载和MAPK激活之间的联系。 收缩功能障碍将定义为肌细胞收缩性（使用视频边缘检测）和免疫细胞化学来测量肌原纤维组件的降低。 在AIM 3中，使用免疫复合激酶测定，免疫沉淀和蛋白质印迹分析的研究将确定由H2O2激活的MAPK上游的调节组件。 该目标将集中于SRC，蛋白激酶C和单体GTP结合蛋白的RAS超家族。 拟议的研究对于针对参与氧化剂诱导损伤的信号传导途径的治疗策略的发展至关重要，并且在治疗心肌缺血中可能具有重要的临床意义。