[Experimental procedure for evaluation of myocardial protection using cultured cardiac myocyte--functional and biochemical effects of hypothermic preservation].

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 by collagenase dispersion and cultured for 4 days with MCDB 107 medium containing 2% FCS. Thereafter, the myocytes (12.5 x 10(5) myocytes/culture flask) were incubated at 4 degrees C for 6, 12, 18, 24, 36 or 48 hours. After each incubation, CPK and LDH were measured. The myocytes were then cultured for additional 24 hours at 37 degrees C to evaluate the recovery of myocyte beating rate. The recovery ratio of myocyte beating rate was well maintained at 6 and 12 hours (94.5, 103.4 percent of control: ie, beating rate prior to hypothermic incubation, respectively). Thereafter, the recovery ratio significantly decreased at 18 hours (58.4 percent, p < 0.05), and reached null levels at 48 hours. Release of CPK and LDH increased significantly at 18 hours (CPK: 131.8 mIU/flask, p < 0.05; LDH: 356.0 mIU/flask, p < 0.05), and showed a marked increase at 48 hours (CPK: 961.3, LDH: 1729.5). In addition, the CPK and LDH levels did not significantly increased at 37 degrees C for 48 hours compared to those for 6 hours (6 hours: 7.95, 193.7; 48 hours: 13.5, 199.4, respectively). In summery, 4 degrees C hypothermia induced the myocyte injury both functionally and biochemically, which increased with an increasing incubation time. In addition, it was suggested that an optimal temperature in hypothermic preservation might be 10 degrees C. Thus, this cell-culture system may provide a useful and simple method for in vitro evaluation of hypothermic preservation.