PAR1 as a Therapeutic Target in Doxorubicin-induced Cardiotoxicity

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Even with current regimens designed to reduce cardiac injury, Dox cardiotoxicity (DoxTox) still occurs. Its prevention and management remain a major problem for both cardiologists and oncologists, limiting the maximal lifetime dosage and compromising the patient’s cancer outcomes. Protease-activated receptor 1 (PAR1) is a G- protein coupled receptor (GPCR) that is expressed by cardiac myocytes (CM) and cardiac fibroblasts (CFs). It is the main thrombin receptor on human platelets and is the target of the FDA-approved drug vorapaxar. In contrast to human platelets, mouse platelets do not express PAR1 making them an excellent model to study platelet-independent effects of PAR1. PAR1 is activated by a variety of proteases. As with other GPCRs, PAR1 exhibits biased signaling. For instance, PAR1 activation by thrombin or matrix metalloproteinases (MMPs) is cytotoxic whereas activation by activated protein C (APC) is cytoprotective. Importantly, we recently demonstrated that activation of PAR1 with a thrombin agonist peptide (AP) enhanced Dox-induced cell death of CMs and CFs in vitro. In addition, PAR1 deficient mice and wild-type mice treated with vorapaxar were protected from acute DoxTox. We hypothesize that administration of Dox leads to increased generation of thrombin and other proteases, such as MMPs, that activate PAR1 on CMs and CFs contributes to oxidative stress, leading to DoxToxOur proposal has 2 aims. 1. Determine the effect of different PAR1 activating proteases on DoxTox. We hypothesize that PAR1/Gαq/Ca2+-dependent signaling activated by thrombin and MMPs enhances DoxTox. In contrast, we hypothesize that the APC/PAR1/β-arrestin2 pathway will reduce DoxTox. Specific Aim 2. Elucidate the cell-specific PAR1- and protease-dependent pathways contributing to DoxTox. We hypothesize that PAR1 expressed by CMs and CFs both contribute via common PAR1/G-protein and distinct CF-specific PAR1/β- arrestin1 mechanisms to acute and chronic DoxTox. We hypothesize that thrombin and MMPs contribute to DoxTox whereas exogenous APC will reduce DoxTox in vivo. In this proposal, we will analyze the role of PAR1 expressed on CMs versus CFs on DoxTox. In addition, we will use a pharmacologic approach to investigate the PAR1-dependent contribution of thrombin or MMPs to DoxTox. Finally, we will test whether PAR1 inhibitors or APC reduce acute and chronic DoxTox in mice. We propose that vorapaxar will reduce DoxTox by blocking all PAR1. In addition, APC treatment prior to Dox treatment will reduce DoxTox by enhancing cytoprotective PAR1/β-arrestin2 signaling. Finally, the biased PAR1 inhibitor Parmodulin 2 will reduce primarily pathologic PAR1/G-protein while enhancing protective APC-like signaling in DoxTox. The overall goals of this proposal are to (i) understand the pathologic roles of PAR1 in DoxTox and (ii) develop new therapies to prevent anthracycline- induced cardiac injury.