| Objective:1. To investigate the cardioprotective effects of St. Thomas (ST) solution, Histidine-Tryptophan-Ketoglutarate (HTK) solution and Non-depolarizing (NDP) solution after preservation in the three solutions at room temperature and4℃, separately.2. To investigate the effects of ST solution, HTK solution and NDP solution on the electrophysiological characteristics of action potential (AP), fast sodium current (INa), L-type calcium current (Ica-L and transient outward potassium current (Ito) from new-born rat cardiomyocytes.3. To investigate the effects of ST solution, HTK solution and NDP solution on the cytoplasmic and mitochondrial calcium metabolism and their membrane potential migration.4. Compare the cardioprotective and electrophysiological effects of ST solution, HTK solution and NDP solution.Methods:1. Sixty-four isolated SD rat hearts were perfused with37℃oxygen-saturated KH solution for15min and then divided into8groups,8for each group.(1) for the hearts preserved under room temperature:we used24～26℃calcium-free KH solution, ST solution, HTK solution and NDP solution for cardiac arrest and the1hour-preservation for the Con group, the ST group, the HTK group and the NDP group separately. After preservation, hearts were reperfused with37℃oxygen-saturated KH solution for lhr;(2) for the hearts preserved under icy temperature:we used4℃calcium-free KH solution, ST solution, HTK solution and NDP solution for cardiac arrest and the8hour-preservation for the Con group, the ST group, the HTK group and the NDP group separately. After preservation, hearts were rewarmed with each solution and reperfused with37℃oxygen-saturated KH solution for1hr. Heart function recovery, Cardiac troponin I (cTnI) level, myocardial infarction area, ATP and lactic acid (LA) content and morphology variations were compared between each group.2. We cultured the new-born rat cardiomyocytes. Cells after36hours’culturation were usded for whole-cell patch clamp detection. Five groups were included:(1) the Control (Con) group:normal cardiomyocytes;(2) the ischemia/reperfusion (I/R) group:cells were treated with3-hour ischemia and1-hour-reperfusion;(3) the ST group, the HTK group and the NDP group:during the ischemia-reperfusion (I-R) treatment, ST solution, HTK solution and NDP solution were added in the culture medium separately. The characteristics of the AP, INa、ICa-L and Ito were compared between each group.3. We cultured the new-born rat cardiomyocytes. Cells after5days’culturation were usded for laser confocal microscopy scanning. Cells were incubated with four different fluorochromes. Under each condition, four groups were included:the Con group were scanned for35min continuously; after5min scanning, the ST group, the HTK group and the NDP group were treated with ST solution, HTK solution and NDP solution separately, and then followed with30min continuous scanning.Results:1. Under the1-hour-room temperature condition:the heart function recovery in the HTK group and the NDP group were superior to the ST group (P<0.05); the coronary flow in the NDP group increased compared with the ST group and the HTK group (P<0.05); the infarction percentage and cTnI level in the HTK group were lower than the ST and NDP groups (P<0.05); there was no significant difference in the ATP and LA content among the three groups.2. Under the8-hour-icy temperature condition:the heart function of the ST group could not effectively recover; the heart function of the NDP group could recover to the level of90%of the preischemic value; the cTnI levels of the HTK and NDP groups were lower than the ST group. Compared with the ST group and the HTK group, the infarction area of the NDP group decreased, and the ATP content increased. There was no significant difference in the LA content among the three groups.3. The electrophysiological changes of each group were as follows.(1) AP:the action potential amplified (APA) and action potential duration (APD) of the I/R group decreased and the resting membrane potential (RMP) depolarized compared with the Con group. The RMP, APA, APD of the NDP group recovered almost to the normal level.(2) INa:the peak current density of the I/R group decreased significantly than the Con group (P<0.05), the activation curve right-shifted and the inactivation curve left-shifted. Compared with the I/R, ST and the HTK groups, the peak current density increased (P<0.05), and the inactivation curve right-shifted.(3) ICa-L:the Peak current density of the I/R group decreased significantly than the Con group (P<0.05), the activation curve right-shifted and the inactivation curve left-shifted. Compared with the I/R, ST and the HTK groups, the peak current density increased (P<0.05), and the inactivation curve right-shifted.(4) Ito:the peak current density of the I/R group decreased significantly than the Con group (P<0.05), the activation curve right-shifted and the inactivation curve left-shifted. Compared with the I/R, ST and the HTK groups, the peak current density of the NDP group increased (P<0.05), and the inactivation curve left-shifted.4. The calcium and membrane potential variations of each group were as follows. After the addition of each cardioplegia:(1) cytoplasmic calcium:the fluorescent intensity baseline in the NDP group kept staying at a lower level compared with the ST and the HTK groups.(2) cell membrane potential:the potential of the NDP group maintained at a hyperpolarization level, while of the ST and the HTK groups, the membrane potential kept at a depolarization level.(3) mitochondrial calcium:the fluorescent intensity in the ST group increased than the Con and the HTK groups, while the NDP group decreased.(4) mitochondrial membrane potential:the depolarization level of the ST group increased gradually, while the NDP and the Con groups tended to be more hyperpolarized. Conclusion:1. Under the1-hour-room temperature condition, the NDP solution could effectively protect the coronary function. The HTK solution and the NDP solution could improve the I-R heart function recovery, while there was no significant difference between the two groups. The result suggested that the application advantage of NDP solution in the regular cardiac surgery needed further studies.2. Under the8-hour-icy temperature condition, the NDP solution could improve the heart function to an almost normal level. This result suggested, compared with the HTK solution, that the NDP solution was a more ideal preservation solution during the organ transferation process in heart transplantation.3. The NDP solution could superiorly recover the electrophysiological characteristic changes caused by I-R injury.4. The NDP solution could maintain the cytoplasm and the mitochondria calcium concentration at a continuous low level. These results indicated that the NDP solution could effectively reduce the risk of the intracellular calcium overload, and was beneficial for the mitochondrial calcium absorption function, which could intensify the myocardiual anti-I-R effects.5. The NDP solution tended to induce the cell and the mitochondria membrane potential at hyperpolarization level, which could help maintain the cell calcium homeostatic and prevent the heart against oxidative damges during the I-R procedure. |
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