Ethanol induced delayed cellular protection in mouse cardiac myocytes: role of inducible nitric oxide synthase

This study has examined the hypotheses that, firstly, the ethanol induces delayed cellular protection in mouse cardiac myocytes against subsequent sustained simulated ischemia (SI). Secondly, the delayed cyto-protective effect induced by the ethanol depends more on time than the dose in mouse cardiac myocytes. Finally, ethanol-induced delayed cellular protection in mouse cardiac myocytes is mediated through inducible nitric oxide synthase (iNOS). Accordingly, we planned the following groups of BALB/c mouse cultured cardiac myocytes in our study: (a) SI, (b) 5 mM ethanol (ETOH)/15 min + SI, (c) 5 mM ETOH/30 min + SI, (d) 10 mM ETOH/15 min + SI, (e) 10 mM ETOH/30 min + SI, (f) 25 mM ETOH/15 min + SI, (g) 25 mM ETOH/30 min + SI, (h) 25 mM ETOH/60 min + SI, (i) 50 mM ETOH/15 min + SI, (j) 50 mM ETOH/30 min + SI, (k) 50 mM ETOH/60 min + SI, (l) 250 mM ETOH/15 min + SI and (m) 250 mM ETOH/30 min + SI. Another set of experiments we designed with iNOS-/- and its wild-type (iNOS+/+) mice cardiac myocytes as follows: SI, 5 mM ETOH/30 min + SI, 10 mM ETOH/30 min + SI, 25 mM ETOH/30 min + SI, 50 mM ETOH/30 min + SI and 250 mM ETOH/30 min + SI. Cellular injury was measured by the release of creatinine kinase (CK, U/l) into the medium. BALB/c mouse cardiac myocytes subjected to SI demonstrated significant increase in CK release as compared to the ethanol-treated cells. Ethanol-induced delayed cellular protection resulted in a significant (p < 0.001) attenuation in the cellular injury as indicated by reduction in the release of CK (U/l) from 9.25 +/- 0.52 to 5.16 +/- 0.44 (5 mM ETOH/30 min), from 7.50 +/- 0.43 (10 mM ETOH/15 min) to 4.16 +/- 0.64 (10 mM ETOH/30 min), from 5.91 +/- 0.41 (25 mM ETOH/15 min) to 2.50 +/- 0.58 (25 mM ETOH/30 min) and to 2.25 +/- 0.37 (25 mM ETOH/60 min), from 5.41 +/- 0.28 (50 mM ETOH/15 min) to 1.66 +/- 0.56(50 mM ETOH/30 min) and to 1.25 +/- 0.30 (50 mM ETOH/60 min), and from 5.25 +/- 0.21(250 mM ETOH/15 min) to 1.66 +/- 0.51(250 mM ETOH/30 min). Reduction in CK release from ethanol-treated iNOS-/-mouse cardiac myocytes was insignificant (p > 0.05) compared to non-treated wild-type (iNOS+/+) mouse cardiac myocytes subjected to SI alone. Our data suggest that ethanol induces delayed cellular protection in mouse cardiac myocytes against sustained simulated ischemia. Further, ethanol-induced delayed cellular protection depends more on time than the dose. Furthermore, ethanol-induced delayed cellular protection is dependent on iNOS.