부산대학교 도서관

Introduction: The heart is a "mitochondrion-dense" organ, and as much as 30% of the volume of the cardiomyocyte is occupied by mitochondria. Mitochondria are both the target and origin of many of the pathogenic pathways in the development of heart failure, including metabolic remodeling and increased oxidative stress. Recent evidence suggests that mitochondria are the main source of inflammasome-activating short-lived reactive oxygen species (ROS), and as such may constitute the signal-integrating organelle for NLRP3 inflammasome activation. Methods: Adult mouse cardiomyocytes were isolated from normal C57Bl6 (WT) or mice deficient in NLRP3. Cardiomyocytes were incubated with the mitochondrial uncoupler (carbonyl cyanide m -chlorophenylhydrazone; CCCP) for 24 h. Cell viability was visualized by Annexin V (apoptosis) and propidium iodide (necrosis) staining. The number of positive cells was calculated. Cardiomyocytes were also loaded with the dye tetramethylrhodamine methyl ester (TMRM) and 20 min later subjected to ROS injury evoked by laser confocal microscopy. The time to membrane depolarization was measured and compared with control cells. Mitochondria were isolated from heart tissue after ex vivo ischemia reperfusion (Langendorff technique) and oxygen consumption by high-resolution respirometry was measured. NLRP3 protein was visualized using antibody staining and confocal microscopy. Results: Adult NLRP3–/– cardiomyocytes had reduced CCCP-induced cell death as compared to WT cardiomyocytes, as evident by decreased number of Annexin V and propidium iodide positive cells, Moreover, NLRP3-/- cells had slower mitochondrial membrane depolarization evaluated in TMRM loaded cardiomyocytes during exposure to reactive ROS induced by laser confocal microscopy, indicating protection of mitochondrial function. WT mitochondria from heart had reduced oxygen consumption after ischemia reperfusion compared to NLRP3–/– mitochondria demonstrating mitochondrial respiration impairment. Perinuclear NLRP3 protein was detected by laser confocal microscopy in the cardiomyocytes. Conclusions: NLRP3 in adult cardiac myocytes may play a role in regulating mitochondrial and cellular viability. If this is an effect dependent on inflammasome activity remains to be determined, but our results are compatible with the notion that ROS generated by mitochondria having reduced membrane potential can lead to NLRP3 inflammasome activation in cardiomyocytes and further to cardiac dysfunction. [ABSTRACT FROM AUTHOR]