A cardiac myocyte culture system as an in vitro experimental model for the evaluation of hypothermic preservation

Abstract

In cardiac transplantation, the donor heart is exposed to severe hypothermic and ischemic conditions. The purpose of the present study was to evaluate the functional and biochemical effects on cardiac myocytes cultured under hypothermic conditions. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for 4 days, then incubated (1.5×10⁶ myocytes/culture flask) for 24 h in media at 4, 10, 15, 20, and 37°C. In addition, myocytes were incubated at 4°C for 6, 12, 18, 24, 36, and 48 h. After each incubation, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were measured and the myocytes then cultured for an additional 24 h at 37°C to evaluate the recovery of the myocyte beating rate. The recovery ratio of the myocyte beating rate following 24 h of varying temperature incubations was complete for the 10, 15, 20, and 37°C groups, although it was markedly decreased in the 4°C group, at 25.1% of the control; taken as the beating rate prior to hypothermic incubation. The release of CPK and LDH in the 4°C group showed a three-fold increase compared to the other four groups, with a CPK of 147.2 mIU/flask and a LDH of 487.5 mIU/flask. The recovery of the beating rate for varying time incubations at 4°C was complete for the 6- and 12-h groups, but decreased significantly in the other four groups, being 59.0% at 18 h, 28.2% at 24 h, 16.3% at 36 h, and 0% at 48 h. The CPK and LDH levels increased gradually over 24 h, then markedly at 36 and 48 h, to 301.3 and 940.5 at 36 h, and 1143.6 and 1942.9 at 48 h, respectively. Thus, 4°C hypothermia induced myocyte injury both functionally and biochemically which increased with the incubation time.