18 - Sarco-endoplasmic reticulum ATPase participates in the regulation of mitochondrial calcium

Abstract

Background Although sarco-endoplasmic reticulum ATPase-2 (SERCA) is located in close proximity to mitochondria, its role in the regulation of mitochondrial calcium ([Ca2+]m) is not clear. Reactive oxygen species (ROS) such as H2O2 can oxidize SERCA at C674, thereby inhibiting its activity and potentially affecting [Ca2+]m in conditions associated with oxidative stress in the myocardium. Methods In adult rat ventricular myocytes (ARVM) we used the SERCA inhibitor thapsigargin (TG) or H2O2 to inhibit SERCA and measured the effects on [Ca2+]m using a mitochondrially-targeted [Ca2+] probe (CAMmito). CAMmito was excited at 446nm and emission was detected at 480nm/535nm to derive a CFP/YFP ratio. WT and C674S SERCA, and CAMmito were expressed in (ARVM) using adenoviral vectors. Results TG (1 uM, 30 min) increased the CFP/YFP ratio 11 + 5% (p=0.07, n=4). H2O2 (100 uM, 10 min) similarly increased the ratio by 11 + 1% (p=0.02, n=7). The basal CFP/YFP ratio was not different in ARVM expressing WT vs. C674S SERCA (p=0.89, n=4). In ARVM expressing WT SERCA, H2O2 increased the CFP/YFP ratio 21 + 4% (p=0.02, n=4), whereas in ARVM expressing C674S SERCA the H2O2-stimulated increase was decreased to 12 + %, (p=0.03 vs. baseline, 0.2 vs. WT SERCA, n=4). Conclusion In cardiac myocytes, SERCA inhibition with TG increased [Ca2+]m, indicating that SERCA participates in regulation of [Ca2+]m under basal conditions. ROS also increased [Ca2+]m, although to a greater magnitude (21 vs. 11%) than TG. The redox-insensitive SERCA mutant C674S decreased the ROS-stimulated increase to 12%, suggesting that oxidative inhibition of SERCA contributes to the ROS-stimulated increase in [Ca2+]m. SERCA may be an important regulator of [Ca2+]m in cardiac myocytes in health and in disease states associated with increased oxidative stress.