MAP KINASES AND H202 INDUCED MYOCARDIAL DYSFUNCTION

心肌缺血后的再灌流与严重的收缩和代谢功能障碍有关，称为心肌顿抑。再灌流还会增加Na+/H+交换器(NHE)的活性，使细胞内pH(Phi)在缺血诱导的酸中毒后恢复到正常水平。然而，NHE的激活也会产生不良的继发效应，导致组织损伤的加剧，这种现象被称为“pH悖论”。氧自由基(OFR)的产生增加在再灌流诱导的心肌顿抑和NHE激活中起重要作用。建立了一种研究OFRs对培养的新生大鼠心室肌细胞(NRVM)影响的体外模型，在该模型中，低浓度的H_2O_2(与再灌流时产生的H_2O_2相似)引起收缩功能障碍、钙超载和NHE激活。人们对识别将过氧化氢与心肌功能障碍联系在一起的信号事件很感兴趣。过氧化氢和低氧激活丝裂原活化蛋白激酶(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H_2O_2以ERK1/2依赖的方式降低心肌细胞的收缩能力并刺激NHE活性。初步数据表明，心肌细胞暴露在过氧化氢中会导致肌丝解体、钙超载和非受体酪氨酸激酶src的激活。这一假设是MAPK家族调节NHE活性、钙超载和H_2O_2引起的收缩功能障碍。在目标1中，使用合成的抑制剂和反义寡核苷酸的实验将确定MAP激酶抑制是否能阻止H_2O_2诱导的NHE的磷酸化，因为交换蛋白的磷酸化与其激活有关。NHE的激活将通过细胞内pH的荧光成像和在体外和体内检测NHE蛋白的磷酸化状态来测量。在目标2中，将使用药物抑制剂和针对p38、JNK和ERK MAPK的反义寡核苷酸来研究H_2O_2诱导的收缩功能障碍、钙超载和MAPK激活之间的联系。收缩功能障碍将被定义为心肌细胞收缩能力的降低(使用视频边缘检测)，并通过免疫细胞化学来测量肌原纤维组装。在目标3中，一项使用免疫复合体激酶分析、免疫沉淀和蛋白质印迹分析的研究将确定被过氧化氢激活的MAPKs上游的调节成分。这个目标将集中在src、蛋白激酶C和单体GTP结合蛋白的RAS超家族上。这些研究对于开发针对氧化剂诱导损伤的信号通路的治疗策略具有重要意义，并可能对治疗心肌缺血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.