The Protective Effect And Mechanism Of H₂S On Cold Preserved Isolated Rat Heart

AIM Homologous heart transplantation is one of the most effective options for end stage heart disease. The shortage of donor heart is one of the main obstacles of heart transplantation. An optimal protection and preservation method may alleviate the problem of donor heart shortage. At present, the safe cold preservation duration of donor heart is only 4-6h, longer time ischemia may dramatically decrease the outcome of heart transplantation. Eric et al. found that rats could turn into a kind of suspended animation like statu, when they were put in the environment with some concentration of H2S, which is somewhat like the hibernation appears in the physiological condition of some animals. We believe that this finding may improve the preservation effect of donor organs if H2S is applied in a hypothermia condition. The aim of this study is to observe the preservation effect and its mechanism of H2S on isolated, cold preserved rat hearts.METHODS Twenty four Sprague-Dawley rats were randomized into 4 groups. The rats were anesthetized with intraperitoneal pentobarbital (60 mg/kg) and heparinized intravenously (0.2 mL). The hearts were rapidly harvested and mounted on a nonrecirculating Langendorff perfusion system (Radnoti Working Heart Recirculating System 130101)to determine the baseline performace. Retrograde perfusion was established at a pressure of 100 cm H2O with normothermic (37℃) KH solution, which was saturated with 95% oxygen and 5% carbon dioxide. Before the start of each experimental protocol, the isolated hearts were allowed to stablise at 37℃for 30min. LV performance was assessed by measurement of LV systolic pressure (LVSP, mmHg), LV end-diastolic pressure (LVEDP, mmHg), LV developed pressure (LVDP, mmHg, LVDP=LVSP-LVEDP).Positive and negative first derivatives of LVSP (+dP/dt and -dP/dt, mm Hg/s) were calculated and recorded by a BIOPAC system. Hearts were then arrested with 30 mL of different preservation solutions (see below), which were delivered at 4℃at a pressure of 100 cm H2O. Hearts were then removed from the Langendorf system and stored for 6 hours at 4℃by immersing in the same solution. At the end of the storage, heart performances were redetermined according to the method descripted above. The heart tissues were fixed for pathological examination. The arrest and preservation solution of each group were: group 1(as control), KH solution without saturation with oxygen and carbon dioxide;group 2, KH solution with NaHS (1μmol/L);group 3, KH solution with NaHS (1μmol/L) and glibenclamide (1μmol/L);group 4, St. ThomasⅡsolution. The heart tissues were examined with transparent electron microscope (TEM) and TUNEL staining, myocardial water content and ATP content were also examined.RESULTS There was no significant difference among the 4 groups of rat heart for their baseline heart function performances. Effective heart beating was recovered spontaneously in all the hearts after the arrest, storage and reperfusion. The +dp/dtmax (expressed in percentage of baseline) of the group 1, 2, 3 and 4 after reperfusion were 36.95±10.05%, 57.32±10.10%, 27.97±10.48% and 56.38±13.90%, respectively. The -dp/dtmax(expressed in percentage of baseline) of the group 1, 2, 3 and 4 after reperfusion were 33.88±6.60%, 56.12±8.18%, 28.89±16.83% and 56.59±6.31%, respectively. The LVDP (expressed in percentage of baseline) of the group 1, 2, 3 and 4 after reperfusion were 37.89±6.69%, 62.96±10.24%, 29.67±13.61% and 61.25±12.20%, respectively. The +dp/dtmax, -dp/dtmax and LVDP of the group 2 and 4 were significantly higher than those of group 1 and 3 (P<0.001). The spontaneous rhythm restoration time of the group 1, 2, 3 and 4 were 1080.5±427.8 s, 431±119 s, 173±56 s and 7±5 s, respectively, with significant differences among all the 4 groups (P<0.001). The hearts in group 4 restored their spontaneous rhythm immediately after the reperfusion, and the hearts of other 3 groups experienced a period of ventricular fibrillation before their rhythm recovery. AT the end of the experiment, the water content of the heart tissues in group 1, 2, 3 and 4 were 84.7±0.9 %,84.2±1.9%,85.2±1.2% and 84.4±0.8% respectively, with no significant differences among the 4 groups. And the ATP content (inμg/g) in of the heart tissues in group 1, 2, 3 and 4 were 155±45, 388±40, 145±26 and 187±28, respectively. It was significantly higher in group 2 compared with other 3 groups (P<0.001), and there were no significant differences among group 1, 3 and 4. The transparent electronic microscope examination revealed that the cadiocytes of the group 2 were preserved almost intact, those in group 4 were mildly impaired, and those in groups 1 and 3 were severely injured. The apoptosis index (expressed in percent of apoptotic cells compared with total cell nμMbers under microscope) of the heart tissues were investigated with TUNEL staining. The apoptosis indices of the heart tissues in group 1, 2, 3 and 4 were 34.2±6.3%, 11.2±3.3%, 38.8±5.7% and 25.2±6.4%. It was significantly lower in group 2 compared with other 3 groups (P<0.001), and it was significantly higher in group 1 and group 3 compared with group 4 (P<0.05).CONCLUSIONS KH solution containing 1μmol/L H2S has protective effect on cold preserved isolated rat heart. Its protective effect is comparable to that of St.Thomas II solution and is superior to St.Thomas II in the aspects of high energy chemical metabolism, ultrastructure presversation and apopotosis indices. The protective effect of H2S can be blocked by glibenclamide, which implys that the mechanism of its effect might be conducted by KATP openning.