Genetic Mechanisms of Resistance against Cardiac Preconditioning

描述(由申请人提供)： 作为退伍军人管理局的麻醉师和生理学家，我研究预防心肌梗死，这是退伍军人面临的一个重大医学问题。例如，在缺血预适应(IPC)中，持续缺血/再灌注(IR)前的几个较短的缺血期可以减轻脑梗塞。已经描述了IPC的各种触发机制，包括一氧化氮(NO)和超氧化物的形成。然而，当试图将其转移到临床实践时，遗传易感性可能是一个重要的混杂因素；一些患者可能受益，而另一些患者则不然。缺血耐受性的深刻差异不仅存在于某些物种之间，而且存在于某些物种内部。例如，Dahl盐敏感(SS)大鼠比Brown Norways(BN)大鼠更容易受到IR损伤，这使它们成为研究与非裔美国人患者相似的疾病表型的理想模型。在威斯康星医学院构建的一个独特的染色体替换(共体体)模型中，将BN 6染色体导入SS使所产生的SS6BN共体体更耐IR，同时将遗传差异缩小到单个染色体。这为研究IPC等心脏保护策略的遗传学基础提供了一个很好的起点。虽然NO参与了BN与SS相比无IPC的SS的缩小梗塞范围，但尚不清楚SS是否对IPC有反应，以及对IPC的抵抗是否与内皮一氧化氮合酶(ENOS)活性的差异有关，可能受DNA结合蛋白抑制剂Id2和过氧化体增殖物激活受体3(PPAR3)的调节。ENOS可产生NO或超氧化物。两者都可以调节线粒体的功能，而线粒体又可以作为IPC的触发器和效应器。因此，我的总体假设是，IPC心脏保护的成功或失败是由调控NO产生和线粒体功能的基因介导的。因此，我建议使用这个协整模型来研究两个特定的目标：i)确定不同的遗传背景是否对IPC的细胞和线粒体的差异保护负责；ii)eNOS和/或其上游调节因子Id2和PPAR3是否是负责这种差异保护的候选基因。验证了四个假说：1)BN染色体6上的基因是IPC所必需的，BN和SS相比，BN和SS6BN的心功能更好，心肌梗死更少。2)BN染色体6上的基因是IPC所必需的，BN和SS6BN的线粒体功能比SS更有效。3)IPC对完整心脏细胞和线粒体功能的保护依赖于大鼠6号染色体上基因调控的信号通路Id2-&gt；PPAR3-&gt；eNOS-&gt；4)对分离的心肌细胞细胞和线粒体功能的差异性保护依赖于NO的供应。方法1和2：在完整的不停跳动的心脏中测量各种心脏和线粒体功能、一氧化氮的产生和梗塞面积，以评估IPC基因决定的保护的数量和质量。方法3和4：在存在NOS抑制剂、NO供体或PPAR3激动剂或拮抗剂的情况下进行额外的心脏跳动实验，并确定Id2、PPAR3和eNOS的表达以及NO水平和定位的差异。此外，我们还在分离的心肌细胞中检测了在没有或存在NO的情况下线粒体的不同功能，以确定在这种遗传模型中，NO在介导IPC对细胞和线粒体的差异性保护中的作用和来源。遗传工具对于研究遗传易感性的作用至关重要。将功能结果、梗塞面积和线粒体功能与IPC、NO水平、蛋白表达和基因型别相关联，将使我们能够确定NO和线粒体功能在心脏保护中的信号通路和作用，并描述与心脏保护基因变异相关的亚细胞和线粒体表型。这项CDA为进一步研究遗传学在心脏保护中的作用奠定了不可或缺的基础，并将成为退伍军人管理局心血管研究实现研究独立性的关键里程碑。 公共卫生相关性： 作为退伍军人管理局的麻醉师和心脏生理学家，我研究预防心肌梗死的保护策略，这是退伍军人面临的一个重大医学问题。其中一种策略是“预适应”。然而，当试图将其应用于临床实践时，遗传易感性可能是一个混杂因素。在某些物种之间，甚至在某些物种内部，对缺血的耐受性存在着深刻的差异。利用一种独特的大鼠遗传模型，可以证明，将一条染色体从更耐药的品系转移到更敏感的品系中，可以使所产生的共生体对脑梗塞的抵抗力更强，同时将遗传差异缩小到与敏感的亲本品系相比的单一染色体。这为研究从完整的心脏到分离的线粒体的心脏保护的遗传基础和机制提供了一个很好的起点。这一建议是我进一步研究遗传学在心脏保护中的作用所不可或缺的基础，并代表着在VA系统的心血管研究中实现独立的一个关键里程碑。

DESCRIPTION (provided by applicant): As a VA anesthesiologist and physiologist I study protection against myocardial infarction, a significant medical problem in aging Veterans. In Ischemic Preconditioning (IPC), for example, several shorter ischemic periods before sustained ischemia/reperfusion (IR) attenuate infarction. Various triggering mechanisms have been described for IPC, including nitric oxide (NO) and superoxide formation. Genetic predisposition, however, may be an important confounding factor when trying to transfer it into clinical practice; while some patients may benefit others do not. Profound differences in ischemic tolerance exist not just among but also within certain species. For example, Dahl Salt Sensitive (SS) rats are more susceptible to IR injury than Brown Norways (BN), making them an ideal model to study the genotype of diseases phenotypically similar to African-American patients. In a unique chromosomal substitution (consomic) model constructed at the Medical College of Wisconsin introgression of BN chromosome 6 into SS renders the resulting SS6BN consomic more IR resistant while narrowing the genetic difference to a single chromosome. This offers an excellent starting point to study the genetic basis of cardioprotective strategies like IPC. Although NO has been implicated in decreasing infarct size in BN vs SS without IPC, it is yet unknown if SS can respond to IPC and if resistance to IPC is related to differences in endothelial NO synthase (eNOS) activity possibly modulated by the DNA-binding protein inhibitor Id2 and the peroxisome proliferator-activated receptor 3 (PPAR3). eNOS can produce NO or superoxide. Both can modulate mitochondrial function, which in turn can function as a trigger and effector of IPC. Thus, my overall hypothesis is that failure/success of cardioprotection by IPC is mediated by genes regulating NO production and mitochondrial function. I therefore propose to use this consomic model to study two specific aims: I) Determine if a different genetic background is responsible for differential cellular and mitochondrial protection by IPC, and II) if eNOS and/or its upstream modulators Id2 and PPAR3 are candidate genes responsible for this differential protection. Four hypotheses are tested: 1) Genes on BN chromosome 6 are necessary for IPC as evidenced by better cardiac function and less infarction in BN & SS6BN vs SS. 2) Genes on BN chromosome 6 are necessary for IPC as evidenced by more efficient mitochondrial function in BN & SS6BN vs SS. 3) Protection of cellular and mitochondrial function by IPC in intact hearts depends on the signaling pathway Id2->PPAR3->eNOS->NO modulated by genes on rat chromosome 6. 4) Differential protection of cellular and mitochondrial function in isolated cardiomyocytes depends on NO availability. Approaches for 1 & 2: Various cardiac and mitochondrial functions, NO production and infarct size are measured in intact, beating hearts to assess quantity and quality of genetically determined protection by IPC. Approaches for 3 & 4: Additional beating heart experiments are conducted in the presence of NOS inhibitors, an NO-donor, or a PPAR3 agonist or antagonist, and differences in Id2, PPAR3, and eNOS expression and NO levels and localization are determined. In addition, different mitochondrial functions in the absence or presence of NO are measured in isolated myocytes to determine the role and origin of NO in mediating differential cellular and mitochondrial protection by IPC in this genetic model. Genetic tools are essential to study the role of genetic predisposition. Correlating functional outcomes, infarct size, and mitochondrial functions with IPC, NO levels, protein expressions and genotype will allow us to define the signaling pathway and role of NO and mitochondrial function in cardioprotection and to delineate the subcellular and mitochondrial phenotypes associated with the cardioprotective genotype variation. This CDA marks the indispensable basis for further investigations on the role of genetics in cardioprotection and will be a critical milestone to achieve investigative independence in cardiovascular research at the VA. PUBLIC HEALTH RELEVANCE: As a VA anesthesiologist and cardiac physiologist I study protective strategies against myocardial infarction, a significant medical problem in Veterans. One such strategy is "Preconditioning". Genetic predisposition, however, may be a confounding factor when trying to transfer its use into clinical practice. Profound differences in tolerance to ischemia exist among and even within certain species. Utilizing a unique genetic rat model it could be shown that transfer of one chromosome from a more resistant strain into a more sensitive strain renders the resulting "consomic" more resistant against infarction while narrowing the genetic difference to a single chromosome compared to the sensitive parental strain. This offers an excellent starting point to study the genetic basis and mechanisms of cardioprotection, from intact hearts to isolated mitochondria. This proposal is the indispensable basis for me to further investigate the role of genetics in cardioprotection and represents a critical milestone to achieve independence in cardiovascular research in the VA system.