Human Cardiomyopathy and HSP60

热休克蛋白是一个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。流式细胞术研究表明，HSP 60是 HSP 60在心肌细胞表面的存在，在三分之一的细胞; HSP 60的存在， 细胞表面与半胱天冬酶3、7和8的活化相关。心力衰竭的进展是 其特征在于线粒体HSP 60的增加和细胞溶质HSP 60的减少。这 异常引起了异常处理的问题-即前HSP 60（P1），其具有 线粒体转运信号（MTS）在线粒体中积累，而不是一些返回 到细胞质中。这种蛋白质的积累可能足以损害线粒体功能， 或者更可能反映了线粒体中变性蛋白的存在。HSP 60是 存在于血浆中。我们发现细胞外HSP 60引起细胞凋亡。基于我们 结合研究结果和文献，我们的总体假设是心脏中HSP 60的异常 心力衰竭通过细胞外介导的细胞死亡导致心力衰竭进展 HSP 60和通过HSP 60向细胞结构的异常运输。在这场竞争中， 更新，我们建议扩大我们的研究，并调查贩运的HSP 60在 心肌病和HSP 60运输异常的下游效应。4具体目标 将解决我们的假设：具体目标1 -调查异常之间的关系 线粒体HSP 60运输和心力衰竭的转变-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是 作为前蛋白与MTS合成，然后在线粒体中与一些HSP 60裂解 返回到胞质溶胶，其余的留在线粒体中。因此，积累 HSP 60提示蛋白质的异常加工。具体目标2 -研究其功能 和含有HSP 60的外泌体的命运。我们发现心肌细胞释放HSP 60， 在外泌体中。HSP 60的外泌体释放随应激增加而增加。我们将调查是否 心力衰竭会增加外泌体或改变其蛋白质组成， 特异性目的3 -确定细胞外HSP 60（exHSP 60）在心肌细胞中的作用 心肌病-我们还发现exHSP 60引起心肌细胞凋亡。 HSP 60存在于心力衰竭的血浆中。因此，将利用 抗HSP 60的F（ab）片段以减少exHSP 60和减少HSP 60介导的凋亡。 2 具体目标4 -确定心力衰竭中HSP 60异常与 线粒体分裂/融合的关键蛋白质异常。在初步实验中， 我们观察到HSP 60和线粒体融合的关键蛋白OPA 1共IP。在人 降低心衰大鼠OPA 1。我们将研究OPA 1的作用及其在 HSP 60在心力衰竭进展中的相互作用计划中的工作将进一步促进我们的 了解导致心力衰竭进展的潜在机制。在这一竞争性更新中，我们建议将我们的研究扩展到调查HSP 60的贩运 以及HSP 60运输异常的下游效应。我们的目标是 了解HSP 60异常定位对细胞器功能的影响， 心脏衰竭的症状赠款的具体目标集中在HSP 60和线粒体功能， 外泌体和HSP 60的细胞外运输，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在心肌细胞凋亡中减少， 心肌病，并对线粒体融合至关重要，这是维持 线粒体功能

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 effects of abnormalities in 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. 2 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.