Pyruvate (PYR) supplementation protects myocardium from ischemia reperfusion injury. This study was designed to characterize and quantify the mechanism underlying this protection: specifically whether this ability resides in PYR's metabolic effect or in its antioxidant effect. Isolated perfused rat hearts (n = 6/group) were subjected to 15 min of equilibration (EQ), 25 min of ischemia, and 10 min of reperfusion (RP). Glucose was the sole metabolic substrate (Control) or was supplemented with PYR (5 mM) during (a) EQ only (PYREQ group), (b) RP only (PYRRP group), or (c) EQ and RP (PYREQ-RP group). Left ventricular developed pressure (DP) and ±dP/dt were recorded throughout the experiment. ATP concentrations and intracellular pH were determined by 31P NMR spectroscopy. Myocardial creatine kinase (CK) activity was assayed at end EQ and end RP. In vitro, purified CK was assayed and, after exposure to H2O2 (200 μM) and increasing concentrations of PYR (0-6 mM) for 10 min, reassayed to determine the antioxidant effect of PYR. In all cases PYR improved recovery of mechanical function at end RP (DP: Control, 11 ± 1%; PYRRP, 23 ± 6%; PYREQ, 34 ± 8%; PYREQ&RP, 53 ± 7%; P < 0.05 between all groups and Control). Ischemic contracture was delayed in hearts that received PYR during EQ (PYREQ and PYREQ&RP: 17.8 ± 0.2 vs 12.5 ± 0.3 min, P < 0.001). PYR during EQ (PYREQ and PYREQ&RP) led to higher end ischemic ATP levels (32 ± 4% vs 14 ± 3%, P < 0.001) and a more acidic end ischemic pH (5.92 ± 0.02 vs 5.98 ± 0.03 in Control and PYRRP, P < 0.05). PYREQ&RP showed the highest end reperfusion ATP levels (55 ± 7% vs 38 ± 4 %, P < 0.05 vs other groups). PYR during reperfusion (PYREQ&RP and PYRRP) showed more end reperfusion CK activity (501 ± 9 IU/gV vs 446 ± 13 IU/gV, P < 0.001). In vitro, CK exposure to H2O2 abolished 65 ± 6% of its activity, while in the presence of PYR 6 mM abolished only 9 ± 2% (P < 0.001). We conclude that PYR given during EQ stimulates anaerobic glycolysis during ischemia as seen by the lower pH and higher end ischemic ATP with a correlative delay in onset of contracture and improvement in DP at end RP. PYR during RP protects CK from hydrogen peroxide-induced inactivation and improves recovery of function. PYR given during EQ and RP has a cumulative effect leading to an even greater recovery of function. PYR must be present both prior to ischemia and during reperfusion for best utilization of its metabolic and antioxidant effects.
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