Human Cardiomyopathy and HSP60

描述（由申请人提供）：热休克蛋白是普遍存在的保护性蛋白家族。在人类心肌病中，我们发现心脏HSP 60增加且分布异常，在质膜部分中发现一些HSP 60。流式细胞术研究表明，热休克蛋白60存在于三分之一的细胞的心肌细胞的表面上，细胞表面上的热休克蛋白60的存在与半胱天冬酶3，7和8的活化相关。心力衰竭进展的特征在于线粒体HSP 60的增加和细胞溶质HSP 60的减少。这种异常引起了异常处理的问题-具有线粒体转运信号（MTS）的前HSP 60（P1）在线粒体中积累，而不是一些返回到胞质溶胶。这种蛋白质的积累可能足以损害线粒体功能，或者更可能反映了线粒体中变性蛋白质的存在。HSP 60在血浆中存在。我们发现细胞外HSP 60引起细胞凋亡。根据我们的研究结果和文献，我们的总体假设是，在心力衰竭HSP 60的异常有助于通过细胞外HSP 60介导的细胞死亡和通过HSP 60的异常贩运到细胞结构的心力衰竭进展。在这个竞争性更新中，我们建议扩展我们的研究，并调查心肌病中HSP 60的运输，以及HSP 60运输异常的下游效应。4具体目的将解决我们的假设：具体目的1 -调查异常线粒体HSP 60运输和向心力衰竭转变之间的关系-在初步工作中，我们发现随着心力衰竭的发展，HSP 60在线粒体中积累。HSP 60作为前蛋白与MTS合成，然后在线粒体中裂解，一些HSP 60返回到胞质溶胶，其余的留在线粒体中。因此，HSP 60的积累表明蛋白质的异常加工。具体目标2 -研究含HSP 60的外泌体的功能和命运。我们已经发现心肌细胞在外泌体中释放HSP 60。HSP 60的外泌体释放随应激增加而增加。我们将研究心力衰竭是否会增加外泌体或改变其蛋白质组成，以及外泌体是否来自体内心脏。具体目标3 -定义细胞外HSP 60（exHSP 60）在心肌病中的作用-我们还发现exHSP 60会导致心肌细胞凋亡。HSP 60存在于心力衰竭的血浆中。因此，将进行使用抗HSP 60的F（ab）片段减少exHSP 60和减少HSP 60介导的凋亡的研究。具体目标4 -确定心力衰竭中HSP 60异常与线粒体分裂/融合关键蛋白异常之间的关系。在初步的实验中，我们观察到HSP 60和OPA 1，一个线粒体融合的关键蛋白，co-IP。在人类和大鼠衰竭的心脏中，OPA 1都降低。我们将研究OPA 1及其与HSP 60的相互作用在心力衰竭进展中的作用。计划中的工作将进一步加深我们对导致心力衰竭进展的潜在机制的理解。在这一竞争更新中，我们建议扩展我们的研究，以调查心肌病中HSP 60的运输，以及HSP 60运输异常的下游影响。我们的目标是了解异常定位的HSP 60对细胞器功能和心力衰竭进展的影响。该补助金的具体目标集中在HSP 60和线粒体功能，HSP 60的外泌体和细胞外运输，减少细胞外HSP 60以减少心肌细胞凋亡，以及HSP 60在OPA 1变化中的作用，OPA 1在心肌病中减少，对线粒体融合至关重要，这是维持线粒体功能的重要过程。公共卫生相关性。在这一竞争性更新中，我们建议将我们的研究扩展到研究心肌病中HSP 60的运输，以及HSP 60运输异常的下游影响。我们的目标是了解异常定位的HSP 60对细胞器功能和心力衰竭进展的影响。该补助金的具体目标集中在HSP 60和线粒体功能，HSP 60的外泌体和细胞外运输，减少细胞外HSP 60以减少心肌细胞凋亡，以及HSP 60在OPA 1变化中的作用，OPA 1在心肌病中减少，对线粒体融合至关重要，这是维持线粒体功能的重要过程。

DESCRIPTION (provided by applicant): Heat shock proteins are a ubiquitous family of protective proteins. In human cardiomyopathy, we have found that cardiac HSP60 is increased and abnormally distributed, with some HSP60 found in the plasma membrane fraction. Flow cytometry studies demonstrated that HSP60 was present on the surface of the cardiac myocyte in a third of cells; the presence of HSP60 on the cell surface correlated with activation of caspase 3, 7 and 8. Progression of heart failure was characterized by an increase in mitochondrial HSP60 and a decrease in cytosolic HSP60. This abnormality raises the issue of abnormal processing - that the pre-HSP60 (P1), which has a mitochondrial transport signal (MTS), accumulates in the mitochondria, rather than some returning to the cytosol. Accumulation of this protein could be sufficient to damage mitochondrial function, or more likely, reflects the presence of denatured proteins in the mitochondria. HSP60 was present in the plasma. We found that extracellular HSP60 causes apoptosis. Based on our findings and the literature, our overall hypothesis is that abnormalities in HSP60 in heart failure contribute to heart failure progression through cell death mediated by extracellular HSP60 and through abnormal trafficking of HSP60 to cellular structures. In this competing renewal, we propose to extend our studies and investigate the trafficking of HSP60 in cardiomyopathy, andthe downstream effectsof abnormalitiesin HSP60 trafficking. 4 SpecificAims will address our hypothesis: Specific Aim 1 - Investigate the relation between abnormal mitochondrial HSP60 trafficking and the transition to heart failure - In preliminary work we found that HSP60 accumulated in the mitochondria as heart failure developed. HSP60 is synthesized as a pre-protein with an MTS, and then cleaved in the mitochondria with some HSP60 returning to the cytosol and the rest remaining in the mitochondria. Therefore, accumulation of HSP60 suggests abnormal processing of the protein.Specific Aim 2 - Investigate the function and fate of HSP60 containing exosomes. We have found that cardiac myocytes release HSP60 in exosomes. The exosomal release of HSP60 increases with stress. We will investigate whether heart failure increases exosomes or alters their protein composition and whether exosomes arise from the heart in vivo.Specific Aim 3 - Define role of extracellular HSP60 (exHSP60) in Cardiomyopathy - We have also found that exHSP60 causes apoptosis in cardiac myocytes. HSP60 is present in the plasma in heart failure. Therefore, studies will be undertaken using the F(ab) fragment of anti-HSP60 to reduce exHSP60 and reduce HSP60-mediated apoptosis. Specific Aim 4 - Determine relation between HSP60 abnormalities in heart failure and abnormalities in key proteins for mitochondrial fission/fusion. In preliminary experiments, we observed that HSP60 and OPA1, a key protein for mitochondrial fusion, co-IP. In both human and rat failing hearts OPA1 was decreased. We will investigate the role of OPA1 and its interaction with HSP60 in the progression of heart failure. The planned work will further our understanding of the underlying mechanisms contributing to the progression of heart failure.In this competing renewal, we propose to extend our studies to investigate the trafficking of HSP60 in cardiomyopathy, and the downstream effects of abnormalities in HSP60 trafficking. Our goal is to understand the effects of abnormallylocalized HSP60 on organelle function and the progression of heart failure. The specific aims of the grant focus on HSP60 and mitochondrial function, exosomes and extracellular trafficking of HSP60, reduction in extracellular HSP60 to reduce cardiac myocyte apoptosis, and the role of HSP60 in changes in OPA1,which is reduced in cardiomyopathy, and vital for mitochondrial fusion, an essential process for maintaining mitochondrial function. PUBLIC HEALTH RELEVANCE. In this competing renewal, we propose to extend our studies to investigate the trafficking of HSP60 in cardiomyopathy, and the downstream effects of abnormalities in HSP60 trafficking. Our goal is to understand the effects of abnormally localized HSP60 on organelle function and the progression of heart failure. The specific aims of the grant focus on HSP60 and mitochondrial function, exosomes and extracellular trafficking of HSP60, reduction in extracellular HSP60 to reduce cardiac myocyte apoptosis, and the role of HSP60 in changes in OPA1,which is reduced in cardiomyopathy, and vital for mitochondrial fusion, an essential process for maintaining mitochondrial function.