MAP KINASES AND H202 INDUCED MYOCARDIAL DYSFUNCTION

缺血发作后心肌的再灌注与严重的收缩和代谢功能障碍有关，称为心肌休克。再灌注还会增加Na+/H+交换器（NHE）的活性，使缺血引起的酸中毒后细胞内pH值（pHi）恢复正常。然而，NHE的激活也会产生不良的继发性影响，导致组织损伤加剧，这种现象被称为“pH悖论”。氧自由基（OFR）生成的增加在再灌注诱导的心肌休克和NHE激活中起重要作用。研究ofr对培养的新生大鼠心室肌细胞（NRVM）影响的体外模型已经定义，在该模型中，低浓度的H2O2（类似于再灌注过程中产生的H2O2）导致收缩功能障碍、Ca2+过载和NHE激活。人们对鉴定H2O2与心肌功能障碍之间的信号事件非常感兴趣。H2O2和缺氧可激活丝裂原活化蛋白激酶（MAPK）家族成员，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以erk1 /2依赖性的方式降低心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可诱导肌丝断裂、Ca2+超载和非受体酪氨酸激酶src的激活。本研究假设MAPK家族调控H2O2诱导的NHE活性、Ca2+超载和收缩功能障碍。在Aim 1中，合成抑制剂和反义寡核苷酸的实验将确定MAP激酶抑制是否会阻断h2o2诱导的NHE磷酸化，因为交换蛋白的磷酸化与其激活相关。NHE激活将通过细胞内pH值的荧光成像和检查体外和体内NHE蛋白的磷酸化状态来测量。在Aim 2中，我们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+超载和MAPK激活之间的联系。收缩功能障碍将被定义为肌细胞收缩能力下降（使用视频边缘检测），并通过免疫细胞化学测量肌原纤维组装。在Aim 3中，一项使用免疫复合物激酶测定、免疫沉淀和Western blot分析的研究将识别被H2O2激活的MAPKs上游的调节成分。这一目标将集中在src，蛋白激酶C和Ras超家族的单体gtp结合蛋白。所提出的研究对于针对氧化诱导损伤信号通路的治疗策略的发展至关重要，并且可能在治疗心肌缺血方面具有重要的临床意义。

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.