Interaction Between Nitroso-Redox Balance and Temperature Influences SR Ca2+Leak In Cardiomyocytes: Role of Neuronal NOS Signaling

Sarcomeric reticulum (SR) Ca 2+ leak contributes to impaired Ca 2+ cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modification of RyR2 associated with the nitroso-redox state. Ca 2+ cycling is affected by both nitric oxide (NO) signaling and temperature. We studied the effect of temperature and nitroso-redox state on Ca 2+ leak. We hypothesized that nitroso-redox balance modulates leak in a temperature–dependent manner. Tetracaine was used to estimate the leak in cardiomyocytes (CMs) in mouse models of nitroso-redox imbalance (NOS1 -/- ), hyper S-nitrosylation (GSNOR -/- ) and in WT mice. Reactive oxygen species (ROS) were measured. Experiments were carried out at 23°C – 37°C. In WT CMs, Ca 2+ leak was inversely proportional to temperature whereas, in NOS1 -/- CMs, the leak suddenly increased when the temperature surpassed 30°C. Supplementing the media with NO (1 microM SNAP), reduced leak in NOS1 -/- CMs at physiologic temperature while at low temperatures, there was a trend toward an increase, a pattern similar to that of GSNOR -/- rather than WT CMs. The xanthine oxidase inhibitor, oxypurinol (100 microM), diminished the leak in NOS1 -/- CMs. SR Ca 2+ content in NOS1 -/- CMs was reduced as the temperature decreased, whereas it was not affected in WT cells. Cooling from 37°C to 23 °C increased ROS generation in WT CMs; however there was an opposite effect observed in NOS1 -/- CMs, which was further reduced by oxypurinol. Expression of the NOS isoforms (by RT-PCR) was unaffected by temperature in WT. Therefore, Ca 2+ leak is temperature dependent, is affected by the absence of NOS1 activity and increases with the rise of ROS production. Defective denitrosylation activity keeps leak low, suggesting a protective effect of S-nitrosylation against the cooling-induced increase in SR Ca 2+ leak. These results suggest that Ca 2+ leak from the SR is regulated by a temperature-dependent balance between NO and ROS.