Abstract 18498: Regulator of Calcineurin 1 (RCAN1): A New Factor in the Control of Mitochondrial Dynamics, Metabolism, and Cardiomyocyte Survival

Abstract

Dysregulation of calcium handling and mitochondrial function are hallmarks of heart disease. Furthermore, mitochondrial dynamics and metabolism influence the susceptibility of the heart to ischemia reperfusion (I/R). Regulator of Calcineurin 1 (RCAN1) is an endogenous feedback inhibitor of calcineurin, a calcium-activated phosphatase involved in pathological cardiac remodeling. The hearts of mice with a targeted disruption of Rcan1 are more susceptible to damage from I/R in vivo. Here we have used an in vitro model of simulated I/R (sim-I/R) and advanced mitochondrial imaging techniques to dissect RCAN1’s cardiomyocyte-autonomous role in mitochondrial dynamics and cell survival. SiRNA was used to deplete the RCAN1.4 and RCAN1.1 isoforms from neonatal rat ventricular myocytes (NRVMs). Cultures were subjected to 6 hours of sim-I/R followed by 12 hours of reperfusion. Death, measured as LDH release, was significantly greater in the siRCAN1-depeleted cultures than in si-controls. Si-depleted NRVMs were dyed with Mitotracker-green and three-dimensional images obtained by confocal microscopy. Z-stacks of thresholded images were volume-reconstituted. Our results show that siRCAN1-depleted NRVMs have a decrease in the mean volume of mitochondrial particles and an increase in the total number of these organelles. Consistent with increased mitochondrial fission, the rate of ATP production and the mitochondrial membrane potential were lower in siRCAN1-depleted NRVMs compared to control siRNA treated cells. There was also a profound redistribution of the mitochondrial network, decreasing the functional coupling between mitochondria and endoplasmic reticulum, assayed as a decrease in mitochondrial calcium uptake capacity. In vivo and in vitro ultraestructural EM studies showed increased mitochondrial fragmentation and a loss of the cristae structure. Finally, there was evidence of sarcomeric hypertrophy in NRVMs depleted of RCAN1, correlating with a switch to a more glycolytic metabolism. These studies suggest that RCAN1 helps preserve a functional mitochondrial network, providing new insights into the role of mitochondrial dynamics and calcineurin/RCAN1 regulation in adaptive and pathological cardiac remodeling.