Project Summary Mitochondrial dysfunction in cardiac myocytes occurs early in the pathogenesis of heart failure. In the heart, the primary function of mitochondria is to meet the high energy demand of the beating myocytes by providing ATP through oxidative phosphorylation. However, mitochondria can quickly change into death–promoting organelles. In response to changes in the intracellular environment, they can become excessive producers of reactive oxygen species and release pro-death proteins. Not surprisingly, cells have developed defense mechanisms against aberrant mitochondria that can cause harm to it. The ability of a cell to repair itself and prevent unnecessary death is particularly important in a post-mitotic cell such as a myocyte that cannot be easily replaced. Studies have found that dysfunctional mitochondria can be sequestered by autophagosomes and subsequently delivered to lysosomes for degradation. However, the mechanism and regulation of mitochondrial removal are not well characterized and whether additional mechanisms of mitochondrial clearance exist is currently unclear. We have previously found that the E3 ubiquitin ligase Parkin plays an important role in clearing dysfunctional mitochondria in the heart in response to stress and lack of Parkin leads to accumulation of dysfunctional mitochondria after a myocardial infarction. Parkin is known to induce autophagy of mitochondria but our preliminary studies have uncovered evidence that Parkin can also promote clearance of mitochondria via an autophagy-independent mechanism. In this proposal, we will explore the hypothesis that the small GTPase Rab5 and the endosomal degradation pathway play an important role in clearing dysfunctional mitochondria in myocytes. This hypotheses will be tested with two aims. Aim 1 will define the functional importance of Rab5 and endosomal-mediated mitochondrial clearance in myocytes in vitro and in vivo. We will also examine the relationships between the endosomal pathway and traditional/alternative autophagy pathways in the heart. In Aim 2, we will delineate the role of Beclin1 in initiating the endosomal pathway in response to mitochondrial damage. We will examine whether Beclin1 regulates activation of the endosomal degradation pathway in response to cellular stress by forming a specific pro-endosomal complex with Rab5 and Vps34. Loss-of-function studies in vitro and in vivo using unique cardiac specific inducible Beclin1 deficient mice will be utilized to confirm the functional importance of Beclin1 initiating formation of early endosomes in response to mitochondrial damage and stress. These studies will provide important novel insight into how dysfunctional and potentially dangerous mitochondria are cleared in the heart. These studies will also provide insights into new potential therapeutic targets in this pathway.