Conditional HSF1 Expression for Ischemic Cardioprotection

描述(由申请人提供)： 热休克蛋白多基因家族的不同成员参与了抑制缺血诱导的蛋白质损伤、消除蛋白质错误折叠/聚集和防止线粒体功能障碍的研究。然而，精确的细胞机制仍有部分了解，协调它们招募的有效策略也没有明确的定义。HSP合成的强健上调需要在应激条件下通过不同刺激触发的高度保守的过程来抑制紧凑的基础抑制(对照)和完全激活热休克因子1(HSF1)。在初步研究中，我们开发了一种携带结构性活性HSF1(CaHSF1)反式激活剂的转基因小鼠模型，该模型可以方便地绕过这些应激诱导要求，并为我们提供强大的多功能性，以上调完整心脏中整体HSP的表达。这项研究的主要目的是明确地阐明caHSF1上调HSPs减少缺血后左心功能障碍和改善心肌挽救的细胞机制。具体地说，我们希望确定HSF1依赖的胞浆/未折叠蛋白反应(CyUPR)调节是否可以防止线粒体功能障碍并改善缺血后的心脏代谢。有待检验的具体假设是，组织特异性结构性活性热休克因子1(CaHSF1)的表达代表了一种新的伴侣依赖的途径，以保持线粒体通透性转变和细胞保护。以下三个特定目的旨在验证这一假说：目的1：确定在体外中到重度缺血挑战后，减少蛋白质聚集和实现缺血保护所需的组织特异性HSP表达的准确水平。目的：研究组织特异性热休克蛋白(HSP)在体外基础和缺血条件下心脏代谢和心肌耗氧量的变化。这一目标将解决热休克蛋白的伴侣依赖效应是否直接影响线粒体呼吸和能量产生。目的：研究条件性过表达热休克蛋白阻止线粒体通透性转变开放的细胞机制。条件策略非常适合于测试干预措施对上述临床结果(例如，心肌梗死、心律失常、收缩功能障碍)的有效性。在这项提案中，我们的临床前研究将解决几个及时和新颖的问题，这些问题与在功能、生化、分子和细胞水平上建立治疗干预的生物学合理性所需的时间有关。因此，我们建议，有条件的caHSF1动物模型可以在干预和临床结果之间的时间尺度上更严格地评估缺血保护的机制。鉴于与缺血相关的心力衰竭的迅速流行，越来越迫切地需要建立新的原则性证明策略，其在急性心肌梗死时的应用可能会减少缺血后的左心功能障碍，并有利于改善长期结果，如心力衰竭和心脏性猝死。 公共卫生相关性： 7.对退伍军人健康的潜在影响心脏病发作和中风分别是美国死亡率和发病率的第一和第三大原因。遗传性或后天性心力衰竭每年夺走50万人的生命，仅在美国就造成约300亿美元的损失。缺血性心脏病是男性和女性最常见的获得性心脏病，由于肥胖率和吸烟率高于普通人群，男女退伍军人的患病率都更高。这项研究计划试图了解细胞保护的机制，最终目标是在高危退伍军人中预防疾病。如果成功，这种有益的结果可能会改善在心脏移植、冠状动脉旁路手术和其他获得性心脏疾病期间患有急性缺血综合征的退伍军人的健康。

DESCRIPTION (provided by applicant): Different members of the HSP multigene family have been implicated the inhibition of ischemia- induced protein damage, abrogation of protein misfolding/aggregation, and prevention of mitochondrial dysfunction. However, the precise cellular mechanisms remain partially understood and effective strategies for their coordinated recruitment are poorly defined. Robust upregulation of HSP synthesis requires repression of tight basal repression (control) and full activation of the Heat Shock Factor 1 (HSF1) by a highly conserved process triggered by diverse stimuli under stressful conditions. In Preliminary Studies, we have developed a transgenic mouse model carrying a constitutively active HSF1 (caHSF1) transactivator that conveniently circumvents these stress-inducible requirements and provides us the robust versatility to up-regulate global HSP expression in the intact heart. The main objectives of this research application are to definitively address the cellular mechanisms that up-regulation of HSPs by caHSF1 decreases post-ischemic LV dysfunction and improves myocardial salvage. Specifically, we wish to characterize if HSF1-dependent modulation of the cytosolic/unfolded protein response (cyUPR) prevents mitochondrial dysfunction and improves post-ischemic cardiac metabolism. The specific hypothesis, to be tested, is that tissue-specific constitutively active heat shock factor 1 (caHSF1) expression represents a novel chaperone-dependent pathway for preserving mitochondrial permeability transition and cellular protection. The following three specific aims are designed to test this hypothesis: Aim 1: Determine the precise levels of tissue-specific HSP expression required to decrease protein aggregation and to achieve ischemic protection after moderate to severe ischemic challenges ex vivo. Aim 2: Characterize the tissue-specific HSP overexpression for changes in cardiac metabolism and myocardial oxygen consumption under basal and ischemic conditions ex vivo. This aim will address if the chaperone-dependent effects of HSPs directly affects mitochondrial respiration and energy production. Aim 3: Characterize the cellular mechanisms by which conditional HSP overexpression prevents mitochondrial permeability transition (MPT) opening. Conditional strategies are ideally suited for testing the efficacy of interventions to aforementioned clinical outcomes (e.g., myocardial infarction, cardiac arrhythmias, contractile dysfunction). In this proposal, our preclinical studies will address several timely and novel questions related to the elapsed times needed to establish the biological plausibility for therapeutic intervention at the functional, biochemical, molecular and cellular levels. Accordingly, we propose that a conditional caHSF1 animal model permits more rigorous assessment of the mechanisms of ischemic protection on a time scale between intervention and clinical outcomes. Given the burgeoning epidemic of ischemia-related heart failure, there is increased urgency to establish proof-of-principle novel strategies whose administration at the time of acute myocardial infarction might reduce post-ischemic left ventricular dysfunction and favorably improve long-term outcomes, such as heart failure and cardiac sudden death. PUBLIC HEALTH RELEVANCE: 7. Potential Impact on Veterans' Health Heart attacks and strokes account for the first and third leading causes of mortality and morbidity in the United States, respectively. Heart failure from either inherited or acquired conditions claims 500,000 lives annually and costs ~$30 billion dollars in the US alone. Ischemic heart disease-the most common acquired heart disease in both men and women- has a higher prevalence in veterans of both sexes due to higher rates of obesity and cigarette use than the general population. This research proposal seeks to understand the mechanisms of cellular protection with the ultimate goal for disease prevention in the high-risk veteran population. If successful, such beneficial outcomes may improve the health of veterans with acute ischemic syndromes during cardiac transplantation, coronary bypass surgery, and other acquired cardiac conditions.