Preconditioning of isolated rabbit cardiomyocytes: no evident separation of induction, memory and protection

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 microM 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 microM 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 nm) 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 microM) or staurosporine (100 nM) 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.