Protection of Cardiac Myocytes from Hypothermic Injury by Cardiac Fibroblasts Isolated from Neonatal Rat Ventricle

Abstract

In this study, we evaluated the functional and bio-chemical effects of cardiac fibroblasts (CFs) on cardiac myocytes (CMs) incubated under hypothermic conditions. CMs and CFs were isolated from neonatal rat ventricles and cultures of myocytes only or myocytes in coculture with CFs were established. A CM anti CF concentration of 2.5 ×105 cells/ml was chosen and the optimum proportion of CF was determined to be 33% (ml CM:ml CF) as follows: 6:0 (group C-a), 4:0 (group C-b), 4:2 (group M-F). On the fourth day of culture, cells were incubated at 4?C for 6, 12, 18, 24, 36 and 48 hr. After each incubation, creatine phosphokinase ((PK) and lactate dehydrogenase (LDH) were measured in the medium, and then the cells were cultured for an additional 24 hr at 37°C to evaluate the recovery of the CM contracting rate. In groups C-a and C-b (n = 6 each), the recovery ratio of CM contracting rate decreased at 18 hr (C-a, 59.0; C-b, 55.6% of control; i.e., contracting rate prior to hypothermic incubation), reaching null levels at 48 hr. However, group M-F (n = 6) showed a significantly increased recovery, at 18 hr (95.7%, P < 0.05; at 48 hrs, 27.6%). Release of CPK and LDH in groups C-a and C-b increased gradually by 24 hr and showed a marked increase at48 hr (C-a, 114.3 × 10-2, 194.2 × 10-2; C-b, 81.6 × 10-2, 118.6 × 10-2 IU flask, respectively). In addition, the enzyme values of each control group at each incubation time correlated with the myocyte cell number per flask. In contrast, the levels of CPK and LDH in group M-F were significantly lower at 18 hr, and the greatest difference was observed at 48 hr (CPK, 21.6 × 102; LDH, 47.3 × 10-2; P < 0.001 vs group C-b). In conclusion, the presence of CFs protects CMs from hypothermic injury, which suggests that fibroblasts may play an important role in preserving cardiac myocyte function in the neonatal period.