Preconditioning of Isolated Rabbit Cardiomyocytes: No Evident Separation of Induction, Memory and Protection☆

Abstract Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 μ m adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60–180 min ischemia. Repetitive sub-maximal stimulations with 1 μ m adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 n m ) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 μ m ) or staurosporine (100 n m ) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.