Hsp90, NOS3 and Cardioprotection

我们对介导心肌缺血抵抗的机制的理解仍不清楚。确定机制， 为了增加对缺血的抵抗力，我们使来自Brown Norway（BN/Mcw）和Dahl S（SS/Mcw）大鼠的心脏经受全脑缺血。 BN/Mcw大鼠心脏比SS/Mcw大鼠心脏具有更强的抗缺血性。检查细胞机制 揭示了尽管两种品系的心脏表现出相同水平的内皮型一氧化氮合酶（NOS 3）和热休克， 蛋白90（hsp 90）表达，BN/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（O2-）。 SS/Mcw大鼠心脏。基础蛋白质组学研究表明，在细胞中NO和C>2~-产生的平衡向NO的转变， BN/Mcw心脏至少部分是由于改变了热休克蛋白90（hsp 90）与NOS 3的联系，并可能与GTP的联系 环化水解酶I（GTPCH-1）。我们观察到BN/Mcw心脏中hsp 90与NOS 3的结合增加了近2倍 与SS/Mcw心脏中的相关性相比;总生物蝶呤，四氢生物蝶呤（BH 4）的分析指数， BN/Mcw心脏80%。BN/Mcw大鼠心脏中的总生物蝶呤浓度与GTPCH-I增加70%直接相关 蛋白水平，有趣的是，与SS/Mcw心脏中的水平相比，GTPCH-1与hsp 90的结合增加了2.2倍。 这些数据表明，热休克蛋白90依赖的伴侣活性可能在控制NOS 3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）hsp 90依赖性信号调节NOS 3产生NO（偶联 活性）和OV”（非偶联活性）; 2）调节BH 4水平，这反过来又调节偶联和非偶联的NOS 3活性。 本提案的总体目标是确定BN/Mcw心脏的细胞、分子和酶机制， 大鼠比来自SS/Mcw大鼠的心脏更耐缺血。通过阐明这些途径，这项提案的发现将 提供了新的，基本的洞察hsp 90伴侣活性如何增加NOS 3-NO的产生，以增加对 缺血这些研究的观察结果实际上可能统一了两个假设，许多人认为这两个假设是相互竞争的。 心血管生理学，也可能导致治疗缺血性心脏病的新策略的发展。

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 resistance 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 ¿NOand C>2~ 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 associationwith 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 OV" (uncoupled activity); and, 2) modulation of BH4levels, 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.