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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Clinical and Integrative Cardiovascular Sciences Study Section (CICS)
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Keleti, Julianna
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University of California Davis
Internal Medicine/Medicine
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United States
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