P668 * Extracellular mitochondrial DNA induces cell death in cardiomyocytes

Purpose: Acute myocardial infarction is associated with aseptic inflammation. Mitochondrial components can activate the innate immune system. Mitochondrial DNA is released into the circulation during acute myocardial ischemia in humans and mice. We hypothesized that mtDNA may induce an immune response through Toll like receptor 9 (TLR-9), injure cardiomyocytes in vitro, and activate nuclear factor kappa B (NFkB) in vivo. Methods: Isolated adult mice cardiomyocytes were exposed to mitochondrial DNA, total DNA, and the synthetic TLR9 agonists CPG-B and CPG-C for 30 minutes, 4, 24 and 48 hours. Cells were fixed and stained with Hoechst, mitotracker and propidium iodide. Images were taken and analysed with a high through put Olympus ScanR microscope. To investigate mitochondrial integrity after stimulation, we stained adult cardiomyocytes with the cationic dye tetramethylrhodamine methyl ester (TMRM) and exposed single lines of mitochondria to a laser beam at 2 Hz to induce ROS formation. NFkB luciferase reporter mice were used to investigate if injection of mitochondrial DNA would cause a whole-body inflammation. Results: Stimulating isolated cardiomyocytes with mitochondrial DNA for 24 hours increased the amount of necrotic cells by 67 % compared to control (p<0.05, n=6). 24 hours of total DNA exposure decreased the number of necrotic cells by 38 % (p<0.05, n=6). The TLR-9 agonist CPG-B increased cell death only after 48 hours while, no effect was found after CPG-C stimulation at any time point. Isolated adult mice cardiomyocytes incubated with mtDNA or CPG-C for 24 hours had decreased time to mitochondrial depolarization (p<0.05, n=4-6). No effect was seen with incubation of total DNA. Intraperitoneal injection of mitochondrial DNA tended to induce luciferase activity in reporter mice in vivo while CPG-C induced increased luciferase activity (p<0.05, n=6). Conclusion: Mitochondrial DNA exposure leads to cardiomyocyte death, possibly through loss of mitochondrial membrane potential. Mitochondrial DNA tended to activate nuclear factor kappa B in vivo. Thus, we provide evidence that mitochondrial DNA can act as a danger signal affecting cardiomyocyte viability.