Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: Role of nitroso-redox balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca²⁺) leak. Methods and results: We measured SR Ca²⁺ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1^-/-]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR) mice. In WT cardiomyocytes, SR Ca²⁺ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1^-/- cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1^-/- cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1^-/- cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1^-/- cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca²⁺ content in NOS1^-/- cells but had no effect in WT or GSNOR. Conclusions: Ca²⁺ leak and temperature are normally inversely proportional, whereas NOS1^-/- deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca²⁺.