Human Cardiomyopathy and HSP60

描述（由申请人提供）：热休克蛋白是一种普遍存在的保护蛋白家族。在人类心肌病中，我们发现心脏HSP60升高并异常分布，在质膜部分发现了一些HSP60。流式细胞术研究表明，三分之一的心肌细胞表面存在HSP60；HSP60在细胞表面的存在与caspase 3、7和8的激活相关。心衰的进展以线粒体HSP60升高和胞浆HSP60降低为特征。这种异常引发了异常处理的问题- pre-HSP60 (P1)，具有线粒体运输信号（MTS），在线粒体中积累，而不是返回到细胞质中。这种蛋白质的积累可能足以损害线粒体功能，或者更有可能反映线粒体中变性蛋白质的存在。血浆中存在热休克蛋白60。我们发现细胞外HSP60引起细胞凋亡。根据我们的研究结果和文献，我们的总体假设是心力衰竭中HSP60的异常通过细胞外HSP60介导的细胞死亡和HSP60向细胞结构的异常运输导致心力衰竭进展。在这一竞争性的更新中，我们建议扩展我们的研究并调查HSP60在心肌病中的转运，以及HSP60转运异常的下游影响。具体目标1 -研究线粒体异常HSP60运输与心力衰竭过渡之间的关系-在初步工作中，我们发现随着心力衰竭的发展，HSP60在线粒体中积累。HSP60作为前蛋白与MTS合成，然后在线粒体中裂解，一些HSP60返回细胞质，其余的留在线粒体中。因此，HSP60的积累表明该蛋白的加工异常。特异性目的2 -研究HSP60含外泌体的功能和命运。我们发现心肌细胞外泌体释放HSP60。外泌体中HSP60的释放随着应激的增加而增加。我们将研究心力衰竭是否会增加外泌体或改变它们的蛋白质组成，以及外泌体是否来自体内的心脏。特异性目的3 -明确细胞外热休克蛋白60 （exHSP60）在心肌病中的作用-我们还发现exHSP60可引起心肌细胞凋亡。心力衰竭患者血浆中存在热休克蛋白60。因此，我们将利用抗hsp60的F（ab）片段降低exHSP60，减少hsp60介导的细胞凋亡。确定心力衰竭患者HSP60异常与线粒体裂变/融合关键蛋白异常之间的关系。在初步实验中，我们观察到HSP60和线粒体融合的关键蛋白OPA1共同作用。在人和大鼠衰竭心脏中，OPA1均降低。我们将研究OPA1及其与HSP60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。在这一竞争性的更新中，我们建议扩展我们的研究，以调查HSP60在心肌病中的转运，以及HSP60转运异常的下游影响。我们的目标是了解异常定位的HSP60对细胞器功能和心力衰竭进展的影响。该资助的具体目的集中在HSP60和线粒体功能，外泌体和HSP60的细胞外运输，细胞外HSP60的减少以减少心肌细胞凋亡，以及HSP60在OPA1变化中的作用，OPA1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必要过程。公共卫生相关性。在这一竞争性的更新中，我们建议扩展我们的研究，以调查HSP60在心肌病中的转运，以及HSP60转运异常的下游影响。我们的目标是了解异常定位的HSP60对细胞器功能和心力衰竭进展的影响。该资助的具体目的集中在HSP60和线粒体功能，外泌体和HSP60的细胞外运输，细胞外HSP60的减少以减少心肌细胞凋亡，以及HSP60在OPA1变化中的作用，OPA1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必要过程。

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.