Novel ubiquitin-like modification regulating mitochondrial dynamics in myocardium

Disruption of mitochondrial dynamics (fission and fusion) underlies the development of cardiomyopathies and heart failure but the upstream mechanisms governing this process remains poorly understood. This grant addresses a novel link between neddylation and mitochondrial dynamics in cardiomyocytes (CMs). Neddylation is a protein modification that covalently attaches a ubiquitin-like protein, NEDD8, to target proteins. Neddylation requires NEDD8-specific E1-E2-E3 enzymes and impacts diverse physiological processes and disease states, yet very little is known about its significance in fully differentiated, post-mitotic organs. Preliminary data from this application have identified a key role of neddylation in maintaining mitochondrial dynamics. Pharmacological inhibition of neddylation increased mitochondrial size, disrupted mitochondrial membrane potential, impaired mitochondrial respiration, and diminished fatty acid utilization in CMs. Deletion of NAE1, a regulatory subunit of the NEDD8 E1 enzyme, also led to mitochondrial elongation and enhanced sensitivity to mitochondrial depolarization and cell death, which was accompanied by dysregulation of multiple key mitochondrial dynamic proteins. Unbiased proteomics analysis further identified Drp1, a GTPase protein directing mitochondrial fission, as a putative NEDD8 target. Moreover, conditional knockout (KO) of NAE1 in adult mice resulted in heart failure and early mortality. These compelling findings formulate our central hypothesis that neddylation is required for normal heart function through the regulation of mitochondrial dynamics and function. Using the inducible NAE1KO mice and a pharmacological inhibitor, Aim 1 will examine the impact of neddylation inhibition on mitochondrial and cardiac homeostasis, and test whether restoration of mitochondrial dynamics rescues NAE1KO adult hearts. Aim 2 will define the role of neddylation in mitochondrial morphology, delineate the underlying molecular basis and determine the effects of Drp1 neddylation on mitochondrial dynamics. This project will be the first to study this novel protein modification in mitochondrial fitness and in postmitotic CMs. We will employ a combination of cardiac gene manipulation, cardiac phenotyping, sequencing, proteomics, and live CM research to address the questions. The findings are expected to have a positive impact on the field by yielding new paradigms and novel insights into adult CMs regulatory mechanisms. (AHA Program: Transformational Project Award)