PROJECT SUMMARY The loss of cells through necrosis is a major cause of aging-related pathologies in many different organ systems, including the cardiovascular system. Nevertheless, our understanding of the cellular pathways that underlie necrosis is still very far from complete. Thus, our long-term goal is to identify and characterize the molecular mechanisms that drive/regulate necrotic death and how they contribute to aging-associated disease. Historically, necrosis was thought of as an “accidental”, unregulated process. However, recent work has revealed that necrosis can be tightly regulated. One form of regulated necrosis, necroptosis, is canonically activated by tumor necrosis factor-α (TNFα). TNFα receptor binding activates the kinase receptor-interacting protein 1 (RIPK1). RIPK1 then binds to, phosphorylates, and activates the kinase RIPK3, which in turn binds and phosphorylates the pseudokinase mixed lineage kinase domain-like (MLKL). MLKL then oligomerizes and translocates to the plasma membrane, where it directly perforates the membrane. Intriguingly, we have found that oxidative stress-induced necrosis is also partially dependent on activation of this pathway. However, while the “core” components of the pathway (RIPK1, RIPK3, MLKL) have been identified and characterized, next to nothing is known of the key proteins that modulate these core proteins. In particular, proteins that regulate MLKL activation, the final execution step in necroptotic signaling, have yet to be established. We have discovered the Ca2+-binding protein EF hand domain protein 2 (EFhd2) as a potential negative regulator of MLKL. We have found that EFhd2 can bind to MLKL, and its overexpression attenuates necroptotic cell death, while knockdown has the opposite effect. Thus, our central hypothesis is that EFhd2 inhibits necroptotic signaling through a Ca2+-regulated interaction with MLKL, and that loss of EFhd2 accelerates cardiac pathology during aging. The objective of the present application is to define the mechanisms by which EFhd2 inhibits necroptosis and the consequences this has for the development of cardiac dysfunction in aged mice. In Aim 1 we will determine the mechanisms by which EFhd2 inhibits MLKL activation, and the role Ca2+ plays in EFhd2's ability to inhibit necroptosis. In Aim 2 we will examine indices of necroptosis and cardiac function in aged wildtype and Efhd2-/- mice. The rationale for the proposed research is that once the key proteins that modulate necroptosis are identified, they can be targeted to improve the clinical outcome of many aging-related diseases such as cardiomyopathy.