Conditional HSF1 Expression for Ischemic Cardioprotection

描述（由申请人提供）： HSP多基因家族的不同成员已经涉及抑制缺血诱导的蛋白质损伤、消除蛋白质错误折叠/聚集和预防线粒体功能障碍。然而，精确的细胞机制仍然是部分理解和有效的策略，其协调招聘定义不清。HSP合成的稳健上调需要通过在应激条件下由不同刺激触发的高度保守过程来抑制紧密基础抑制（对照）和完全激活热休克因子1（HSF 1）。在初步研究中，我们已经开发了一种转基因小鼠模型，携带组成型活性HSF 1（caHSF 1）反式激活因子，方便地规避这些应激诱导的要求，并为我们提供了强大的通用性上调全球HSP表达在完整的心脏。本研究应用的主要目的是明确地解决细胞机制，即通过caHSF 1上调HSP降低缺血后LV功能障碍并改善心肌挽救。具体来说，我们希望表征HSF 1依赖性调节胞质/未折叠蛋白反应（cyUPR）是否能预防线粒体功能障碍并改善缺血后心脏代谢。有待检验的具体假设是，组织特异性组成型活性热休克因子1（caHSF 1）表达代表了一种新的分子伴侣依赖性途径，用于保护线粒体通透性转换和细胞保护。以下三个具体的目的是为了测试这一假设：目的1：确定精确的组织特异性HSP表达水平所需的减少蛋白质聚集和实现缺血保护后，中度至重度缺血的挑战离体。目标二：在离体基础和缺血条件下，表征组织特异性HSP过表达对心脏代谢和心肌耗氧量变化的影响。这一目标将解决热休克蛋白的伴侣依赖性效应是否直接影响线粒体呼吸和能量产生。目的3：探讨条件性HSP过表达阻止线粒体通透性转换（MPT）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的有效性（例如，心肌梗塞、心律失常、收缩功能障碍）。在本提案中，我们的临床前研究将解决几个及时和新的问题，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相容性所需的时间有关。因此，我们建议，一个条件性的caHSF 1动物模型允许更严格的评估缺血保护机制的干预和临床结果之间的时间尺度。鉴于缺血相关心力衰竭的流行日益迅速，迫切需要建立原理性新策略，其在急性心肌梗死时给药可能减少缺血后左心室功能障碍并有利地改善长期结局，如心力衰竭和心源性猝死。 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占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去50万人的生命，仅在美国就花费了约300亿美元。缺血性心脏病是男性和女性中最常见的后天性心脏病，由于肥胖和吸烟的比例高于普通人群，因此在男女退伍军人中的患病率都较高。这项研究计划旨在了解细胞保护机制，最终目标是预防高风险退伍军人群体的疾病。如果成功，这些有益的结果可能会改善心脏移植，冠状动脉搭桥手术和其他获得性心脏病期间患有急性缺血综合征的退伍军人的健康状况。

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.