Project Summary/Abstract Cardiac cell death by apoptosis and/or necrosis is a hallmark of cardiac ischemic injury, pathological remodeling, and end-stage heart failure. In contrast to apoptosis, the role of necrosis in the pathogenesis of heart disease has been largely understudied. Emerging evidence has identified several forms of “programmed necrosis”, such as death receptor-mediated necrosis (termed “necroptosis”), mitochondria-mediated necrosis, and oxidative stress-induced necrosis. How programmed necrosis is regulated in the heart remains largely unknown, and preventing necrosis is still an important challenge. Moreover, currently no molecular strategies are available to simultaneously target multiple cell death processes in heart disease. Here, our preliminary studies identified an unexpected role for CYLD (cylindromatosis), a lysine 63 (K63)-specific deubiquitinase, as a key regulator of multiple cell death pathways in cardiomyocytes, including apoptosis, necroptosis, and oxidative stress-induced necrosis. Intriguingly, CYLD expression was markedly upregulated in the heart following ischemic injury. Using Cyld knockout and transgenic mouse models, our preliminary data further show that ablation of CYLD attenuated, whereas overexpression of CYLD exacerbated, cardiac ischemic injury. Importantly, ablation of CYLD inhibited apoptosis, necroptosis, and necrosis in cardiomyocytes, whereas overexpression of CYLD showed the opposite effect. Mechanistically, our data reveal a K63-linked polyubiquitination (K63-Ub) dependent cell death signaling mechanism whereby CYLD controls the ubiquitination status and activity of three cell death regulators: TRAF2, TAK1 and AKT/PKB. Therefore, we hypothesize the deubiquitinase CYLD is a key regulator of multiple cell death pathways and a promising therapeutic target for cardiac ischemic injury and remodeling. Using genetic loss- and gain-of-function strategies, we will address two specific aims: Aim 1) To investigate the novel role of CYLD as a key regulator of myocardial cell death, ischemic injury, and remodeling using Cyld knockout and transgenic models and AAV9-shCYLD vectors. Aim 2) To define a CYLD-mediated, K63-Ub dependent cell death signaling network regulating apoptosis, necroptosis, and necrosis in cardiomyocytes. This project investigates a novel CYLD-mediated cell death signaling network in the heart and its functional relevance in cardiac ischemic injury and remodeling. Moreover, the proposed studies will define a K63-Ub dependent mechanism regulating apoptosis, necroptosis, and necrosis, which constitutes a new paradigm of cell death regulation. These studies also have important translational implications by providing new anti-cell death strategies, given our preliminary results revealing CYLD as a molecular target for multiple cell death processes.