New insights on myocardial pyridine nucleotides and thiol redox state in ischemia and reperfusion damage.

OBJECTIVE: to investigate the changes of pyridine nucleotides and thiol redox state in cardiac tissue following ischemia and reperfusion. NADH/NAD and NADPH/NADP redox couples were specifically studied and the influence of NADPH availability on cellular thiol redox was also investigated. METHODS: isolated rabbit hearts were Langendorff perfused and subjected to a protocol of ischemia and reperfusion. An improved technique for extraction and selective quantitation of pyridine nucleotides was applied. RESULTS: ischemia and reperfusion induced an increase in diastolic pressure, limited recovery in developed pressure and loss of creatine phosphokinase. Creatine phosphate and ATP were decreased by ischemia and only partially recovered during reperfusion. NADH was increased (from 0. 36+/-0.04 to 1.96+/-0.15 micromol/g dry wt. in ischemia, P<0.001), whereas NADPH decreased during ischemia (from 0.78+/-0.04 to 0. 50+/-0.06 micromol/g dry wt., P<0.01) and reperfusion (0.45+/-0.03 micromol/g dry wt.). Furthermore, we observed: (a) release of reduced (GSH) and oxidised glutathione (GSSG) during reperfusion; (b) decreased content of reduced sulfhydryl groups during ischemia and reperfusion (GSH: from 10.02+/-0.76 to 7.11+/-0.81 nmol/mg protein, P<0.05, and to 5.48+/-0.57 nmol/mg protein; protein-SH: from 280.42+/-12.16 to 135.11+/-17.00 nmol/mg protein, P<0.001, and to 190.21+/-11.98 nmol/mg protein); (c) increased content in GSSG during reperfusion (from 0.17+/-0.02 to 0.36+/-0.02 nmol/mg protein, P<0.001); (d) increased content in mixed disulphides during ischemia (from 6.14+/-0.13 to 8.31+/-0.44 nmol/mg protein, P<0.01) and reperfusion (to 9.87+/-0.82 nmol/mg protein, P<0.01). CONCLUSIONS: under severe low-flow ischemia, myocardial NADPH levels can decrease despite the accumulation of NADH. The reduced myocardial capacity to maintain NADPH/NADP redox potential can result in thiol redox state changes. These abnormalities may have important consequences on cellular function and viability.