Cellular characterization of an in-vitro cell culture model of seal-induced cardiac ischaemia

The lack of a well-characterized in-vitro cell culture model of load-induced cardiac ischaemia has hampered investigations into the mechanism of ischemic injury. We therefore developed a new in-vitro model of cardiac ischaemia that mimics distinct features of ischaemic injury. Neonatal rat heart cells were cultured in a sealed flask for 24-72 h. In this environment, the cells were exposed to stresses of hypoxia, acidosis and stagnant incubation medium. The pO2 and pH of the medium gradually decreased during the ischaemic insult and ultimately fell to a level of 14 mmHg and pH 6.8, respectively. The model triggered severe cell injury, including morphological degeneration, CPK release, beating impairment and ATP depletion. Apoptosis occurred in some cardiomyocytes as early as 24 h after onset of seal-induced ischaemia. This was evidenced by positive nuclear staining using Hoechst 33258 and by the induction of caspase-3 mRNA. By 72 h, internucleosomal DNA fragmentation was observed in 45% of the myocytes; however, a non-myocyte preparation subjected to the same ischaemic insult exhibited no evidence of DNA fragmentation. These results demonstrate that neonatal cardiomyocytes subjected to the new simulated ischaemia model exhibit several similarities to cardiac ischaemia, including the simultaneous appearance of necrosis, breakdown of cellular ATP, beating cessation and apoptosis. The new model should prove useful in unravelling the molecular alterations underlying ischaemic injury and myocardial apoptosis.