Minoru Kohi

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.

Myocardial α1A-adrenoceptor subtypes in rabbit: differentiation by a selective antagonist, HV723

The influence of a newly developed alpha 1-adrenoceptor antagonist, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxy-phenoxy)ethyl)-ami no)- propyl) benzeneacetonitrile fumarate), on the positive inotropic effect and acceleration of phosphoinositide hydrolysis induced by phenylephrine via alpha 1-adrenoceptors in the presence of bupranolol (0.3 microM) was studied in the rabbit ventricular muscle: (1) HV723 at low concentrations (1-100 pM) attenuated the maximal inotropic response by 15-20% without altering the pD2 value for and [3H]inositol monophosphate accumulation induced by phenylephrine. The inhibitory action of HV723 (1-100 pM) showed a close resemblance to that of a selective alpha 1A antagonist, (+)-niguldipine. (2) HV723 (> or = 1 nM) shifted the concentration-response curve for phenylephrine to the right and downwards in association with a partial inhibition (42.5%) of [3H]inositol monophosphate accumulation. The IC50 values of HV723 for the inhibition of the inotropic response and phosphoinositide hydrolysis were approximately equal to the Kilow value determined by displacement of [3H]prazosin-specific binding. The mode of the inhibitory action of HV723 (> or = 1 nM) resembled that of another alpha 1A antagonist, WB 4101. These results indicate that HV723 shows a differential antagonistic action on the alpha 1A-mediated responses depending on the concentration: HV723 (1-100 pM) selectively inhibits the (+)-niguldipine-sensitive subclass to lead to a decrease in the maximal inotropic response with no change in phosphoinositide hydrolysis; HV723 (> or = 1 nM) may antagonize the WB 4101-sensitive subtype coupled to acceleration of phosphoinositide hydrolysis.

Intramuscular hemangioma in the right ventricle.

Intramuscular cardiac hemangiomas are extremely rare. We describe a 74-year-old man with a tumor occupying the apex of the right ventricle that was incidentally diagnosed by echocardiography. Computed tomography and coronary angiography showed that the tumor arose from the ventricular septum and that the feeding artery was a branch of the right coronary artery. The tumor was completely excised, and the postoperative course was uneventful. Histological examination revealed that the tumor was a cardiac hemangioma (intramuscular type). To our knowledge, this is only the second case report of an intramuscular cardiac hemangioma in the world literature.

Early definitive surgery for total colon aganglionosis: Duhamel procedure with right colon patch graft using GIA stapler

A report is presented of an infant with total colon aganglionosis (TCA) with ileal involvement in whom a modified Duhamel procedure was used accompanied by a right colon patch graft 12 cm in length using a GIA-90 stapler at 3 months of age. The patient with TCA is usually treated by an initial diverting ileostomy followed by a definitive operation performed several months later. However, the management during the ileostomy period is associated with a variety of problems. In to minimize the resulting complications, we developed a procedure to allow a much earlier definitive operation.

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.