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，称为心肌顿抑。 再灌注还增加了Na +/H+交换器（NHE）的活性，其在缺血诱导的酸中毒后将细胞内pH（pHi）恢复至正常。 然而，NHE的活化也产生不期望的二次效应，导致组织损伤的恶化，这种现象被称为"pH悖论"。 氧自由基（OFR）的产生增加在再灌注诱导的心肌顿抑和NHE激活中起重要作用。 一种用于研究OFR对培养的新生大鼠心室肌细胞（NRVM）的影响的体外模型已经被定义，其中低浓度的H2O2（类似于在再灌注期间产生的那些）引起收缩功能障碍、Ca 2+过载和NHE激活。 有相当大的兴趣，在确定信号事件，连接H2O2心肌功能障碍。 H2O2和缺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（ERK 1/2）。 低剂量的H2O2降低心肌细胞的收缩力，并以ERK 1/2依赖的方式刺激NHE活性。 初步数据表明，暴露于H2O2的心肌细胞诱导肌丝解体，Ca2+超载，和非受体酪氨酸激酶src的激活。 这一建议的假设是，MAPK家族调节NHE活性，Ca2+超载和收缩功能障碍诱导的H2O2。 在目的1中，合成抑制剂和反义寡核苷酸的实验将确定MAP激酶抑制是否阻断H2O2诱导的NHE磷酸化，因为交换蛋白的磷酸化与其活化相关。 NHE活化将通过细胞内pH的荧光成像和通过检查体外和体内NHE蛋白的磷酸化状态来测量。 在目标2中，H2O2诱导的收缩功能障碍，Ca2+超载和MAPK激活之间的联系将使用药理学抑制剂和反义寡核苷酸对p38，JNK和ERK MAPK进行研究。 收缩功能障碍将被定义为肌细胞收缩性降低（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。 在目标3中，使用免疫复合物激酶测定、免疫沉淀和Western印迹分析的研究将鉴定被H2O2激活的MAPK上游的调节组分。 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。 所提出的调查是根本上重要的针对参与氧化剂诱导的损伤的信号通路的治疗策略的发展，并可能在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.