Kana Fukui

Activation of chloride current by P2‐purinoceptors in rat ventricular myocytes

1 Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration. 2 ATP initially induced an inward current followed by an outward current at − 50 mV. With a Cs‐rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATPs) suggesting that a K+conductance underlies the late response. 3 When the extracellular Cl−concentration was changed, the reversal potential of IATPs corresponded well to the shift of the Cl−equilibrium potential. IATPs was reversibly blocked by the chloride channel blocker, 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS). 4 The concentration‐response curve of IATPs had a Hill coefficient of 0.98 and an EC50 value of 5.2 × 10−6m. 5 ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2‐purinoceptor activation induced IATPs. 6 The activation of IATPs was depressed by depleting the extracellular Mg2+and increased by adding Mg2+. 7 Our results strongly suggest that P2‐purinoceptor activation by ATP induces both a Cl−‐conductance (IATPs) and a K+‐conductance in rat ventricular myocytes.

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

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.

In vitro evaluation of phosphate, bicarbonate, and hepes buffered storage solutions on hypothermic injury to immature myocytes

SummaryIn this study we evaluated cardiac myocyte viability and function under hypothermic conditions using three types of buffer solutions: phosphate buffer solution (PBS), Krebs-Henseleit bicarbonate buffer solution (KHB), and Hepes buffered minimum salt solution (MSS). As a control, normal saline solution (NSS) was used. Cardiac myocytes were isolated from neonatal rat ventricles. Myocytes (12.5 × 105 myocytes/culture flask) were then incubated at 4°C for 6, 12, 18, and 24 hours in various buffer solutions. After each incubation time, CPK and LDH were measured. The myocytes were then incubated for an additional 24 hours at 37°C to evaluate the recovery of the myocyte beating rate. Group MSS had a significantly better beating rate recovery than group NSS (control) after 18 hours (MSS, 32.7%, NSS, 0.0% of control; i.e., beating rate prior to hypothermic incubation). In contrast, group KHB showed a significantly lower recovery ratio than group NSS at 12 hours (41.0%, 78.8%, respectively), and the lowest recovery was observed in group PBS beginning at 6 hours of hypothermic incubation (27.6%). Group MSS significantly suppressed the release of CPK and LDH compared to group NSS at 24 hours (MSS, 246.7 and 440.2 mIU/flask; NSS, 369.7 and 821.3 mIU/flask, respectively). In contrast, groups PBS and KHB showed significantly increased CPK and LDH levels compared to group NSS after 12 hours (PBS, 388.6 and 721.4 mIU/flask; KHB, 340.5 and 540.5 mIU/flask; NSS, 91.5 and 222.7 mIU/flask, respectively). In conclusion, Hepes buffer has cytoprotective characteristics that may be suitable for long-term hypothermic preservation of immature myocardium compared to phosphate or bicarbonate buffer.

In vitro evaluation of diltiazem on hypothermic injury to immature myocytes

SummaryThe purpose of the present study was to evaluate the functional and biochemical effects of diltiazem (DTZ) on cardiac myocytes incubated under hypothermic conditions. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for 4 days with MCDB 107 medium. Then, myocytes (12.5×105 myocytes/flask) were incubated at 4°C for 24 hours in media with or without DTZ at concentrations of 0 M (group C), 10−7 M (Group D1), 10−6 M (group D2), 10−5 M (group D3), or 10−4 M (group D4). After 24 hours at 4°C, CPK and LDH were measured. The myocytes were then cultured for 24 hours at 37°C to evaluate the recovery of the myocyte beating rate. In group C (n=7), the recovery ratio of the myocyte beating rate was 29.9% of control (beating rate prior to hypothermic incubation). Groups D1 and D2 (n=7 each) had approximately the same recovery ratios as group C (24.0% and 24.7%, respectively); however, groups D3 and D4 (n=7 each) showed no beating rate recovery. Release of CPK and LDH in group C was 112.3 mIU/flask and 457.4 mIU/flask, respectively. Groups D1 and D2 showed no significant differences in both enzymes compared to group C. However, the levels of CPK were significantly higher in group D4 (203.3, p<0.05), and LDH levels were significantly higher in groups D3 and D4 (669.3, p<0.05; 883.4, p<0.02). In conclusion, DTZ showed no protective effects on hypothermic injury to immature cardiac myocytes; moreover, it accelerated cellular injury at the concentrations of 10−5 and 10−4 M both functionally and biochemically. Therefore, diltiazem may not be suitable for cardiac preservation during the neonatal period.

Possible Deleterious Effects of Glucose on Immature Myocytes Under Hypothermic Conditions

The purpose of the present study was to evaluate the functional and biochemical effects of glucose-based solutions in combination with potassium or insulin (or both) on immature myocytes under hypothermic conditions. Myocytes were isolated from neonatal rat ventricles and cultured for 4 days with MCDB 107 (University of Colorado solution). Initially, myocytes (12.5 x 10(5) myocytes/flask) were incubated at 4 degrees C for 6 hours in 5% glucose solution containing various potassium concentrations ranging from 0 to 80 mEq/L to evaluate the protective effects. Next, myocytes were incubated at 4 degrees C for 3, 6, 12, 18, and 24 hours in three types of solutions: normal saline solution (control), glucose-potassium solution, and glucose-insulin-potassium solution (glucose: 50 g/L; NaHCO3, 20 mEq; potassium, 20 mEq; insulin, 20 IU/L). After each incubation, creatine kinase and lactate dehydrogenase levels were measured in the incubation solutions. The myocytes then were cultured for an additional 24 hours at 37 degrees C to evaluate the recovery of myocyte beating rate. The 20-mEq potassium treatment showed significantly better beating rate recovery and lower enzymal release than the glucose-only control. The saline solution showed the best protection of all three solutions, both functionally and biochemically, by 12 hours. The greatest damage was observed with glucose-potassium solution, beginning at 3 hours of hypothermic incubation. Although potassium and insulin have additional protective effects on hypothermic preservation, the high concentration of glucose has noxious characteristics for immature myocytes that may not be suitable for cardiac preservation in the neonatal period.

In vitro protective effects of nicorandil on hypothermic injury to immature cardiac myocytes: Comparison with nitroglycerin

SummaryThe purpose of the present study was to evaluate the functional and biochemical effects of nicorandil and nitroglycerin on cardiac myocytes incubated under hypothermic conditions. Nicorandil is a coronary vasodilator with mixed nitrate-potassium channel agonist activity. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for 4 days with MCDB 197 medium. Myocytes (12.5 × 105 myocytes/flask) were then incubated at 4°C for 24 hours in media containing various concentrations of nicorandil (NRD) or nitroglycerin (NTG). After hypothermic incubation, CPK and LDH were measured. The myocytes were cultured for an additional 24 hours at 37°C to evaluate the recovery of the myocyte beating rate. In the nicorandil group, 10−4 M NRD showed a significant beating rate recovery compared to control (44.2% vs. 24.6%, respectively, as a percent of control; i.e., beating rate prior to hypothermic incubation). Nitroglycerin treatment had no effect on either beating rate recovery or release of CPK and LDH from myocytes. However, the release of CPK and LDH was significantly suppressed by 10−4 M nicorandil compared to the control (10−4 M NRD: 24.1, 257.2; control: 125.4 mIU/flask, 459.5 mIU/flask, respectively). Thus nicorandil showed an approximate two-fold recovery of myocyte functional activity after hypothermic incubation with only minor biochemical effects, and therefore may be suitable for cardiac preservation.