Mitochondrial Fusion Factors and Cardiomyopathy The heart is the most mitochondrial rich of all mammalian organs; mitochondrial ATP fuels cardiac excitation- contraction coupling. Paradoxically, mitochondria are also key pathological orchestrators of apoptotic and programmed necrotic cell death in myocardial infarction or heart failure, and are sources of cardiotoxic reactive oxygen species (ROS) when mitochondrial respiration is uncoupled from ATP production. Although mitochondrial respiratory dysfunction increases with organelle and cardiac senescence, it can be moderated by functional complementation achieved through exchange of mitochondrial contents i.e. through cyclic organelle fusion, fission, and selective removal of depolarized daughter organelles. In accordance with this paradigm we found that genetic interruption of mitochondrial fusion provokes not only organelle fragmentation (by unopposed fission), but striking accumulation of toxic ROS-producing organelles. Further, by DNA sequencing a large hypertrophic cardiomyopathy (HCM) cohort my laboratory discovered the first genetic evidence linking a mitochondrial fusion/fission factor and heart disease, a rare R400Q mutation in the mitochondrial fusion factor mitofusin 2 (Mfn2). Other Mfn2 mutations cause Charcot-Marie-Tooth Syndrome, but not heart disease. By assessing the consequences of recombinantly expressed Mfn2 Q400 in mammalian cells and Drosophila heart tubes we found that it is a dominant inhibitor of normal Mfn1 and Mfn2, inducing mitochondrial fragmentation and provoking cardiomyopathy and heart failure. We postulate that loss of Mfn2 function is cardiomyopathic not because it induces mitochondrial fragmentation, but because mitochondrial fusion in general, and specifically Mfn2, plays a central role in mitochondrial quality control. Here, we will investigate the mechanisms for heart disease caused by loss of Mfn2 function. In Aim #1 we explore the organelle, cellular, and organ-level consequences of mitochondrial fragmentation caused by loss of organelle fusion (conditional Mfn1/Mfn2 cardiac knockout mice) vs. increased organelle fission (conditional Dlp1 transgenic mice). We also determine if programmed cardiomyocyte death after I-R injury can be modulated by forced mitochondrial fusion (Mfn1 or Mfn2 transgenic mice) or preventing mitochondrial fission (cardiac Dlp1 knockout mice). In Aim #2 we perform an in vitro and in vivo molecular dissection of the role of Mfn2 in PINK1-Parkin mediated mitochondrial culling, examining the hypothesis that Mfn2 is both a PINK1 substrate and the mitochondrial receptor for Parkin on depolarized mitochondrial. In Aim #3 we apply this knowledge to determine the mechanism by which the human HCM-linked Mfn2 400Q mutation induces hypertrophic cardiomyopathy using human-in-mouse models. Together, results of this work will greatly expand our understanding of mitochondrial fusion and fission in normal heart function, will define the biochemical and cellular mechanisms by which impaired mitochondrial fusion causes clinical and experimental heart disease, and will establish a foundation for a rational therapeutic approach to an entirely new class of rare heritable HCM caused by Mfn2 mutations.