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 x 10(6) myocytes/culture flask) for 24 h in media at 4, 10, 15, 20, and 37 degrees C. In addition, myocytes were incubated at 4 degrees 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 degrees 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 degrees C groups, although it was markedly decreased in the 4 degrees 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 degrees 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation

“…In addition to organ models, like coronary artery occlusion, the ex vivo model of IR, cold storage, and organ transplantation, cell in vitro models are used to simulate cardiac IR injury. These models entail the use of cultured neonatal/adult cardiomyocytes [8–11], mouse atrial HL-1 cells, or rat ventricular H9c2 cells (myoblasts) [12–20]. Neonatal cardiomyocytes can be considered as a very useful model for cardiac ventricular cells, in particular to study various adaptive mechanisms.…”

Cytotoxic effects of cardioplegic solutions and cytoprotective effects of insulin on immature cardiac myocytes during hypothermic preservation

“…Cardiac myocyte cultures were prepared as previously described. 4 " 6 ' 9 Hearts were removed from 1-2 day-old neonatal Wistar male rats after decapitation. The ventricles from 20 hearts were minced into fine fragments with scissors in 0.025 M Hepes buffered minimum salt solution (MSS, Gibco, Grand Island, NY).…”

“…A myocyte concentration of 2.5 x 10 5 cells/ml was chosen and the total number of cells/flask was 12.5 x 10 5 cells. 4 -69 All animals have received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institute of Health (NIH Publication No. 86-23, revised 1985).…”

“…Cardiac myocytes isolated from neonatal rat ventricles formed confluent monolayers by the fourth day of culture, at which time they almost all beat synchronously and steadily at a constant frequency. 4 ' 5 Therefore, the myocyte contraction rate for each flask was observed and the mean contraction or beating rate of 10 myocytes/culture flask was determined microscopically. The myocyte beating rate was observed immediately prior to and 24 hours after hypothermic incubation.…”

“…Previously, we reported that hypothermia at 4 °C induced immature myocytic injury which was diminished or accelerated by various cryopreservation solutions, and demonstrated that an in vitro cell culture system would provide a model for evaluating cellular injury under various preservation conditions. 4 " 6 The purpose of the present study was to evaluate the cytotoxic effects on immature cardiac myocytes under hypothermic conditions of three types of cardioplegic solutions: glucose based potassium solution, lactated Ringer's solution and St. Thomas' Hospital solution. In addition, the effects of insulin as an additive were evaluated in each solution.…”