Background—As shown previously, exposure to NO donors initiates protective mechanisms in cardiomyocytes that persist after removal of the donor, a form 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 were 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 (propidium iodide exclusion assay, P<0.001). Short (10-second) exposures to SNAP reversibly suppressed mitochondrial respiration without a detectable change in mitochondrial potential. In contrast, treatment with SNAP for 90 minutes caused a modest but sustained mitochondrial depolarization, as judged by JC-1 fluorescence. SNAP pretreatment limited cellular Ca²⁺ overload during 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 reoxygenation. SNAP pretreatment also significantly reduced the ability of mitochondria to accumulate Ca²⁺ in the face of a similar cytosolic Ca²⁺ load (peak rhod-2 fluorescence 133±4% versus 166±7% of baseline at similar fluo-3 levels, P=0.0004, n=52 and 25, respectively).

Conclusions—Pretreatment with an NO donor induces a modest, sustained mitochondrial depolarization and protects cardiomyocytes from sI/R injury. The demonstrated reduction in mitochondrial Ca²⁺ uptake possibly reduces cytosolic Ca²⁺ overload, providing a likely mechanism for NO-induced protection.