A MODEL OF ANOXIC PRECONDITIONING IN THE ISOLATED RAT CARDIAC MYOCYTE- IMPORTANCE OF ADENOSINE AND INSULIN

Objective: Ischemic or hypoxic preconditioning has been shown, in mult icellular preparations, to reduce post-ischemic injury In the present study, we attempted to develop a model of preconditioning in isolated rat myocytes in order to facilitate investigation into the mechanism o f preconditioning. Methods: The protective effect of a short period (1 0 min) of anoxia and reoxygenation against a subsequent longer period of anoxia was studied in single, electrically stimulated (0.2 Hz, 37 d egrees C) adult rat cardiac myocytes. The control group received only the long period of anoxia. Three protocols were tested: Protocol 1 in which octanoate was the only substrate; Protocol 2 in which only gluco se was present during all normoxic phases and during the preconditioni ng anoxia and octanoate alone during the prolonged period of anoxia an d; Protocol 3 in which protocol 2 was repeated with the addition of ad enosine (100 mu M) and insulin (15 mu U/ml) during the prolonged anoxi c period. The end-point of assessment was loss of cell morphology i. e ., hypercontracture (death) or relengthening (survival) on reoxygenati on following the prolonged anode period. Membrane integrity was also e xamined at the end of each protocol by observing if the cells excluded trypan blue. Results: No protective effect of preconditioning on cell survival was observed in protocols 1 or 2. In contrast, in protocol 3 , a significant protection was observed in the preconditioned versus c ontrol group (55% vs 27% survival respectively; p<0.001). However: in the absence of preconditioning, adenosine and insulin provided no addi tional protection in the control group. No significant differences in trypan blue exclusion were observed between the groups in any protocol . Conclusions: These results suggest that preconditioning cannot prote ct against a subsequent period of anoxia where the accumulation of met abolic products, e.g., adenosine is prevented. However, that protectio n can be re-instated by the presence of adenosine and insulin during t he period of prolonged anoxia. Furthermore 1 this study suggests that the preconditioning by anoxia may induce a change in the A(1)-receptor or its second messenger system such that adenosine is able to provide protection.