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 multicellular 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 of preconditioning. Methods: The protective effect of a short period (10 min) of anoxia and reoxygenation against a subsequent longer period of anoxia was studied in single, electrically stimulated (0.2 Hz, 37 degrees 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 glucose was present during all normoxic phases and during the preconditioning anoxia and octanoate alone during the prolonged period of anoxia and; Protocol 3 in which protocol 2 was repeated with the addition of adenosine (100 mu M) and insulin (15 mu U/ml) during the prolonged anoxic period. The end-point of assessment was loss of cell morphology i. e., hypercontracture (death) or relengthening (survival) on reoxygenation following the prolonged anode period. Membrane integrity was also examined 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 control group (55% vs 27% survival respectively; p<0.001). However: in the absence of preconditioning, adenosine and insulin provided no additional 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 protect against a subsequent period of anoxia where the accumulation of metabolic products, e.g., adenosine is prevented. However, that protection can be re-instated by the presence of adenosine and insulin during the 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.