Abstract 72: Inhibition of Dynamin Related Protein 1 Mediated Mitochondrial Fission During Cardiac Arrest Is Cardioprotective and Associated with Therapeutic Hypothermia

Abstract

Introduction: Mitochondrial fission increases cellular reactive oxygen species (ROS) and apoptosis but its role in cardiac arrest (CA) is unknown. Dynamin related protein 1 (DRP1) enhances fission by translocating to the mitochondria and is regulated by its phosphorylation at Serine 616 (activating) and Serine 637 (inhibiting). We hypothesized that inhibiting fission and DRP1 during CA would be cardioprotective. We also tested whether therapeutic hypothermia (TH) inhibited fission and DRP1. Methods: Cardiomyocytes isolated from 1-2 day old C57BL6/J mice were infected with a modified baculovirus containing mitochondrial targeted green fluorescent protein and then exposed to simulated ischemia/reperfusion (IR) at 37C or 30C (TH) to assess mitochondrial morphology. Hearts from Sprague Dawley rats were exposed to 30 min of global IR at 37C ± 5 uM mitochondrial inhibitor of division 1 (Mdivi-1) or 30C (TH) in a Langendorff perfusion system. ROS was measured using MitoSOX tissue staining. Left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), DRP1 gene/protein expression, and mitochondrial morphology (electron microscopy) were assessed. Heart samples were also collected from C57BL/6 mice after an 8-min cardiac arrest induced by KCl. Results: IR increased cell death, mitochondrial fragmentation, ROS, and LVEDP (62±1 mm Hg vs 9±1 mmHg, n=5, p<0.05) while decreasing LVDP (26±11 vs 77±8 mm Hg, p<0.05) compared to perfusion only controls. In IR, DRP1 Serine 637 decreased two-fold (n=4, p<0.05) while total DRP1 in mitochondrial protein fractions increased two fold (n=4, p<.0001). TH and Mdivi-1 maintained mitochondrial morphology, decreased ROS, inhibited DRP1 Serine 637 dephosphorylation and mitochondrial translocation. TH and Mdivi-1 also maintained LVEDP (12±1 and 14±2 mm Hg, n=5) and LVDP (84±6 and 84±2 mm Hg, n=5) at levels similar to perfusion-only controls. Conclusions: IR is associated with increased mitochondrial fission, DRP1 activation, ROS generation, and contractile dysfunction in cellular and organ models of IR and in vivo CA. Inhibition of fission and DRP1 with Mdivi-1 and TH are cardioprotective. DRP1 may be a therapeutic target for patients suffering CA or undergoing cardiopulmonary bypass.