Hsp90, NOS3 and Cardioprotection

描述(由申请人提供)：我们对介导心肌缺血抵抗的机制的理解仍不清楚。为了确定增加缺血抵抗的机制，我们用棕色挪威(BN/MCW)和达尔·S(SS/MCW)大鼠的心脏进行全脑缺血。BN/MCW大鼠的心脏比SS/MCW大鼠的心脏更耐缺血。细胞机制研究表明，尽管两个品系的心脏表现出相同水平的内皮型一氧化氮合酶(NOS3)和热休克蛋白90(HSP90)的表达，但BN/MCW大鼠的心脏比SS/MCW大鼠的心脏产生更多的一氧化氮(.NO)和更少的超氧阴离子(02.-)。基本的蛋白质组学研究表明，BN/MCW心脏中NO和O2-产生的平衡向NO的转变至少部分是由于热休克蛋白90(HSP90)与NOS3的结合，并可能与GTP环水解酶I(GTPCH-I)有关。我们观察到，与SS/MCW心脏相比，BN/MCW心脏中HSP90与NOS3的关联性增加了近2倍；BN/MCW心脏中四氢生物蝶呤(BH4)的分析指数总生物蝶呤增加了80%。BN/MCW大鼠心脏中的总生物蝶呤浓度与GTPCH-I蛋白水平增加70%直接相关，有趣的是，与SS/MCW大鼠心脏中的水平相比，GTPCH-I与HSP90的相关性增加2.2倍。这些数据表明，HSP90依赖的伴侣活性可能在调控NOS3功能的机制中发挥关键作用，许多人认为这是两个独立的假设：1)依赖HSP90的信号调节NOS3产生NO(偶联活性)和O2-(非偶联活性)；2)BH4水平的调节，进而调节偶联和非偶联的NOS3活性。该提案的总体目标是确定BN/MCW大鼠的心脏比SS/MCW大鼠的心脏更耐缺血的细胞、分子和酶机制。通过阐明这些途径，这一提议的发现将为HSP90伴侣活性如何增加NOS3提供新的、基本的见解。NO的产生增加对缺血的抵抗。这些研究的观察结果可能实际上统一了两个假说，许多人认为这两个假说在心血管生理学中是相互竞争的，也可能导致开发治疗缺血性心脏病的新策略。

DESCRIPTION (provided by applicant): Our understanding of the mechanisms mediating resistance to myocardial ischemia remains unclear. To determine mechanisms for increasing resistance to ischemia, we subjected hearts from Brown Norway (BN/Mcw) and Dahl S (SS/Mcw) rats to global ischemia. Hearts from BN/Mcw rats were more resistant to ischemia than hearts from SS/Mcw rats. Examination of cellular mechanisms revealed that although hearts from both strains exhibited the same levels of endothelial nitric oxide synthase (NOS3) and heat shock protein 90 (hsp90) expression, hearts from BN/Mcw rats generated more nitric oxide (.NO) and less superoxide anion (02.-) than hearts from SS/Mcw rats. Basic proteomic studies revealed that the shift in the balance in NO and O2.- production toward NO in the BN/Mcw hearts was due, at least in part, to altered heat shock protein 90 (hsp90) association with NOS3 and possibly, with GTP cyclohydrolase I (GTPCH-I). We observed that hsp90 association with NOS3 in BN/Mcw hearts was increased nearly 2-fold compared to association in SS/Mcw hearts; and that total biopterin, an analytical index of tetrahydrobiopterin (BH4) was increased in BN/Mcw hearts by 80%. Total biopterin concentrations in hearts from BN/Mcw rats directly correlated with a 70% increase in GTPCH-I protein levels and interestingly, a 2.2-fold increase in association of GTPCH-I with hsp90 compared to the levels in SS/Mcw hearts. These data suggest that hsp90-dependent chaperone activity may play a critical role in mechanisms governing NOS3 function that many consider to be two independent hypotheses: 1) hsp90-dependent signaling modulates NOS3 generation of .NO (coupled activity) and O2.- (uncoupled activity); and, 2) modulation of BH4 levels, which in turn, regulate coupled and uncoupled NOS3 activity. The overall objective of this proposal is to determine the cellular, molecular and enzymatic mechanisms by which hearts from BN/Mcw rats are more resistant to ischemia than hearts from SS/Mcw rats. By elucidating these pathways, findings from this proposal will provide new, fundamental insight into how hsp90 chaperone activity increases NOS3 .NO generation to increase resistance to ischemia. Observations from these studies may actually unify two hypotheses that many consider competing hypotheses in cardiovascular physiology and may also lead to the development of new strategies for treating ischemic heart disease.