| Background: Donor heart shortage is the main obstacle to limit the development of heart transplantation. Heart arrest with cardioplegia infusion followed ice cold preservation solution immersing is the most populated method of donor heart preservation. HTK solution is commonly used as both cardioplegia and preservation solution in this process. But its safe preservation time is only 4~6 h, which further aggravate the situation of donor heart shortage. How to prolong the safe preservation time becomes the focus of clinical and experimental investigation. Our previous study showed that urethane could enhace the preservation effect of HTK solution on isolated rat heart, but the optimal concentration of urethane was uncertain. In this study, we further investigated the preservation effects of HTK solution with different concentrations of urethane on isolated rat hearts to determine the optimal urethane concention. And the mechamism of increased heart preservation effect of urethane enhanced HTK solution was also explored.Part one: Optimization of Urethane Concentration in Urethane Enhanced HTK Solution for Isolated Rat Heart PreservationMethods: Isolated Sprague–Dawley rat hearts were randomized into 6 groups according to the preservation solution used with 6 hearts per group: HTK group, HTK+Urethane 10 mmol/L group, HTK+Urethane 20 mmol/L group, HTK+Urethane 40 mmol/L group, HTK+Urethane 80 mmol/L group, HTK+Urethane 160 mmol/L group. All the hearts were perfused with Krebs Henseleit solution (KH) according to Langendorff procedure. After a 30 min stabilization, baseline hemodynamic parameters were recorded; hearts were arrested with preservation solutions perfusion and stored in the same solution (100 mL) at 4°C for 8 h. The hearts were reperfused with KH solution for 60 min and hemodynamic parameters were re examined. The hemodynamic parameters recovery rates were expressed as the percentage of baseline value. Cardiac troponin I (cTnl) concentrations in preservation solutions were examined with ELISA method.Results: The recovery rate of left ventricular developing pressure (LVDP) of HTK + Urethane 20 mmol/L group (90.1±1.1%) was significantly higher than other groups (57.2±3.0% ~ 67.8±3.0%, p<0.01). The recovery rate of maximal rise rate of left ventricular pressure (+dp/dt) in HTK + Urethane 20 mmol/L group (88.7±4.3%) was significantly higher than those of other groups (63.2±3.3%~70.9±3.2%, p<0.05). The recovery rate of maximal fall rate of left ventricular pressure ( dp/dt) in HTK + Urethane 20 mmol/L group (79.2±4.8%) was significantly higher than those of other groups (63.5±4.8%～65.9±3.0%, p<0.05). The recovery rate of left ventricular end systolic pressure (LVESP) in HTK + Urethane 20 mmol/L group (91.8±1.2%) was significantly higher than other groups (67.1±2.6% ~ 86.4±2.9%, p<0.05 or P<0.01). The recovery rate of heart rate (HR) in HTK + Urethane 20 mmol/L group (87.2±1.4%) was significantly higher than those in other groups (72.4±1.1% ~ 83.4±2.0%, p< 0.05). The recovery rate of Left ventricular end diastolic pressure (LVEDP) in HTK + Urethane 20 mmol/L group (190.6±18.4%) was significantly lower than other groups (259.2±18.4%~366.7±29.8%, p<0.05). The recovery rate of coronary flow (CF) in HTK + Urethane 20 mmol/L group (83.8±2.2%) was higher than those of other groups (64.7±3.0% ~ 73.1±2.9%, p<0.05 or P<0.01). The cTnI concentrations in preservation solutions of HTK + Urethane groups (31.1±1.2~32.1±1.4 ng/L) were significantly lower than it in HTK group (40.7±0.4ng/L, p<0.01).Conclusions: Tht optimal urethane concentration in urethane enhanced HTK solution for isolated rat heart preservation is 20 mmol/L.Part two: The Protective Mechanism of Urethane Enhanced HTK Solution for Isolated Rat Heart PreservationMethods: Methods: Isolated Sprague–Dawley rat hearts were randomized into 6 groups according to the preservation solution used with 6 hearts per group: HTK group, Urethane group (HTK+Urethane 20 mmol/L), 5 HD group (HTK+Urethane 20 mmol/L + 5 HD, a specific mitochondrial KATP channel blocker), glibenclamide group (HTK+Urethane 20 mmol/L + glibenclamide, a non specific KATP channel blocker) and chelethrine group (HTK+Urethane 20 mmol/L + chelethrine, a specific protein kinase C (PKC) inhibitor). All the hearts were perfused with Krebs Henseleit solution (KH) according to Langendorff procedure. After a 30 min stabilization, baseline hemodynamic parameters were recorded; hearts were arrested with preservation solutions perfusion and stored in the same solution (100 mL) at 4°C for 8 h. The hearts were reperfused with KH solution for 60 min and hemodynamic parameters were re examined. The hemodynamic parameters recovery rates were expressed as the percentage of baseline value. Cardiac troponin I (cTnl) concentrations in preservation solutions were examined with ELISA method. The cTnI degradation, Bcl 2 expression and caspase 3 activation in the myocardial tissue were examined by western blot method.Results: At the end of 60 min reperfusion, the LVDP recovery rates of 5 HD group (65.5±1.8%), glibenclamide (58.9±1.8%) and chelerythrine group (66.7±1.2%) were significantly decreased compared to urethane group (85.7±0.6%, p<0.01). The recovery rates of +dp/dt of 5 HD group (71.6±2.0%), glibenclamide group (64.5±2.2%) and chelerythrine group (74.5±2.1%) were significantly decreased compared with urethane group (83.8±0.4%, p<0.01). The recovery rates of dp/dt of 5 HD group (69.4±2.4%), glibenclamide group (61.5±3.3%) and chelerythrine group (71.4±1.1%) were significantly decreased compared with urethane group (82.0±1.0%, p<0.01). The recovery rates of LVESP of 5 HD group (81.8±1.4%), glibenclamide group (69.9±1.4%) and chelerythrine group (82.1±1.1%) were significantly decreased compared with urethane group (91.8±1.2%, p<0.01). The recovery rates of HR of 5 HD group (76.7±2.2%), glibenclamide group (72.6±2.8%) and chelerythrine group (77.4±2.0%) were significantly decreased compared with urethane group (87.2±1.4%, p<0.01). The recovery rates of LVEDP of 5 HD group (308.1±16.8%), glibenclamide group (331.5±12.1%) and chelerythrine group (318.3±10.3%) were significantly higher that it of urethane group (190.6±18.4%, p<0.01). The recovery rates of CF of 5 HD group (69.5±2.1%), glibenclamide group (62.9±2.1%) and chelerythrine group (64.6±3.0%) were significantly decreased compared with urethane group (83.8±2.2%, p<0.01). The cTnI release in preservation solution of 5 HD group (40.1±0.3 ng/L), glibenclamide group (40.8±0.5 ng/L) and chelerythrine group (36.5±0.4 ng/L) increased significantly compared with urethane group (30.7±0.2 ng/L, p<0.01). The myocardial tissue cTnI degradation (20KD/28KD ratio) of 5 HD group (1.19±0.05), glibenclamide group (1.83±0.1) and chelerythrine group (1.1±0.04) were significantly increased compared with urethane group (0.5±0.06, p<0.05 or p<0.01). The myocardial tissue Caspase 3 activation (17KD/32KD ratio) of 5 HD group (1.23±0.2), glibenclamide group (1.69±0.2) and chelerythrine group (1.06±0.08) were also increased significantly compared with urethane group (0.46±0.07, p<0.01). Linear regression analysis indicated that there was positive correlation between cTnI degradation and caspase 3 activation.Conclusion: The superior myocardial protective effects of urethane enhanced HTK solution are related to PKC activation, KATP channels opening, decreased caspase 3 activation and decreasd cTnI degradation. |
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