ISOLATED CARDIAC MYOCYTES ARE SENSITIZED BY HYPOXIA-REOXYGENATION TO NEUTROPHIL-RELEASED MEDIATORS

We exposed isolated rat cardiac myocytes to 20 min of hypoxia followed by 20 min of reoxygenation and observed the effect of supernatants of stimulated neutrophils [polymorphonuclear leukocytes (PMNs)] given at the beginning of reoxygenation. PMN supernatants induced cardiac myocyte injury, which was characterized by a significant (P < 0.01) reduction in cell viability to 53+/-3%, vs. 84+/-3% in rat myocytes subjected to hypoxia-reoxygenation (H/R) alone. The PMN supernatants also resulted in elevated creatine kinase (CK) activities in the myocyte medium. To examine specific PMN-released mediators that may contribute to this cell death, we studied the effects of hydrogen peroxide (H2O2), elastase, and platlet-activating factor on H/R cardiac myocytes. Incubation of myocytes after hypoxia with 10, 50, and 100 mu M H2O2 decreased viability in a concentration-dependent manner (from 83+/-2 to 37+/-2%; P < 0.01). CK release of H/R myocytes was also significantly increased by 100 mu M H2O2 (to 28+/-5 from 12+/-1% for H/R alone; P < 0.01). Similarly, elastase (5 mu g/ml) given after hypoxia significantly reduced cardiac myocyte viability during reoxygenation (viability 58+/-1 vs. 85+/-1% H/R alone; P < 0.05) and increased CK release (to 29+/-3 from 11+/-1% for H/R alone; P < 0.01), an effect that was abolished by L-680,833, an elastase inhibitor. Unlike H2O2 and elastase, platelet-activating factor had no significant effect on myocyte viability or CK release after H/R. These results indicate that neutrophil-released humoral substances enhance myocardial cell injury and death after hypoxia and reoxygenation without requiring direct contact between PMNs and cardiac myocytes.