Mitochondria as targets for nitric oxide-induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes

Background-As shown previously, exposure to NO donors initiates protective mechanisms in cardiomyocytes that persist after removal of the donor, a for m of pharmacological preconditioning. Because NO also affects mitochondrial respiration, we studied the effect of NO on mitochondrial Ca2+ uptake. Methods and Results-Neonatal rat ventricular myocytes in primary culture we re exposed to 1 hour of simulated ischemia and 1 hour of reoxygenation (sI/ R). Pretreatment with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (1 mmol/L for 90 minutes), followed by washing and incubation for 10 to 30 minutes, reduced sI/R-induced cell death to 25.4% compared with control (pr opidium iodide exclusion assay, P<0.001). Short (10-second) exposures to SN AP reversibly suppressed mitochondrial respiration without a detectable cha nge in mitochondrial potential. In contrast, treatment with SNAP for 90 min utes caused a modest but sustained mitochondrial depolarization, as judged by JC-1 fluorescence. SNAP pretreatment limited cellular Ca2+ overload duri ng ischemia (fura-2 ratio rose to 226+/-40% versus 516+/-170% of baseline, n=5, P<0.05) and prevented loss of cell membrane integrity during reoxygena tion. SNAP pretreatment also significantly reduced the ability of mitochond ria to accumulate Ca2+ in the face of a similar cytosolic Ca2+ load (peak r hod-2 fluorescence 133+/-4% versus 166+/-7% of baseline at similar fluo-3 l evels. P=0.0004, n=52 and 25, respectively). Conclusions-Pretreatment with an NO donor induces a modest, sustained mitoc hondrial depolarization and protects cardiomyocytes from sI/R injury. The d emonstrated reduction in mitochondrial Ca2+ uptake possibly reduces cytosol ic Ca2+ overload, providing a likely mechanism for NO-induced protection.