- Email: [email protected]
Possible Role of Hydrogen Sulfide on the Preservation of Donor Rat Hearts
Possible Role of Hydrogen Sulfide on the Preservation of Donor Rat Hearts X. Hu, T. Li, S. Bi, Z. Jin, G. Zhou, C. Bai, L. Li, Q. Cui, and W. Liu ABSTRACT Objective. The aim of this study was to observe the preservative effect of hydrogen sulfide (H2S) on donor rat hearts. Materials and Methods. The hearts of 24 Sprague-Dawley rats were perfused on a Langendorff perfusion column for 30 minutes. We calculated and recorded the left ventricular-developed pressure (LVDP), and positive and negative derivatives of left ventricular systolic pressure (LVSP; ⫹dP/dt and ⫺dP/dt). Hearts were then arrested and stored for 6 hours at 4°C: group 1, Krebs-Henseleit (KH) solution; group 2, KH solution with 1 mol/L NaHS; group 3, KH solution with 1 mol/L NaHS and 10 mol/L glibenclamide; group 4, St. Thomas II solution. Hearts were transferred back to the Langendorff column. After stabilizing for 30 minutes, LV performance was assessed as before. The donor hearts were kept for pathological study including myocardial water ratio, ATP content, and myocyte apoptosis index. Results. The recovery rates of ⫹dp/dtmax, ⫺dp/dtmax, and LVDP of groups 2 and 4 were much better than those of groups 1 and 3. The hearts contracted immediately after reperfusion in group 4. Ventricular fibrillation was seen before contraction in the other 3 groups, with the longest duration in group. No significant difference in myocardial water ratio was found. The ATP content was the highest in group 2. Apoptosis was observed in the 4 groups with the lowest apoptosis index in group 2. Conclusions. H2S has a protective effect on rat donor hearts at the concentration of 1 mol/L. The protective effect is better than that of St. Thomas II solution. The protective effect of H2S can be blocked by glibenclamide.
T
HE MOST IMPORTANT obstacle for heart transplantation is the shortage of donor hearts. At present, heart transplantations are performed much less frequently than liver and kidney transplantations. Part of the reason is that hearts cannot endure ischemia as long as the liver or kidney. Thus the protection and preservation of donor hearts are of interest in transplant research. It is well known that hydrogen sulfide (H2S) is a toxic gas. Recent evidence indicates that following nitric oxide and carbon monoxide, H2S is the third endogenous gaseous mediator with pronounced physiological effects, particularly in the cardiovascular and central nervous systems.1,2 Endogenous or exogenous H2S causes relaxation of various smooth muscle types, including those of the vascular, intestinal, and reproductive tract systems.3 A number of studies have confirmed that H2S is a potent vasodilator in vivo,4 as well as in several isolated vascular preparations
including rat aorta,4,5 rat mesenteric bed,6 trout efferent branchial arteries,7 and pulmonary arteries among a range of vertebrate species from hagfish (phylogenetically the most primitive vertebrate) to rats.8 At present the mechanisms of H2S-induced vasorelaxation are not completely established. However, as glibenclamide is a selective KATP blocker, there is evidence that the action is at least partially
From the Cardiovascular Surgery Department, Xijing Hospital, Fourth Military Medical University, XI’an, People’s Republic of China (X.H., S.B., Z.J., G.Z., C.B., L.L., Q.C., W.L.), and the Cardiovascular Surgery Department, Third Center Hospital, Tianjin, People’s Republic of China (X.H., T.L.). Address reprint requests to Weiyong Liu, MD, Cardiovascular Surgery Department, Xijing Hospital, Fourth Military Medical University, West Changle Road 15, XI’an, China PRC, 710032. E-mail: [email protected]
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.086
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3024
Transplantation Proceedings, 39, 3024 –3029 (2007)
H2S IN PRESERVATION OF DONOR RAT HEARTS
3025
glibenclamide-sensitive, suggesting involvement in the opening of ATP-dependent K⫹ channels (KATP) in vascular smooth muscle.4,9 Moreover, one study indicated that H2S induced a suspended animation-like state in mice,10 leading to the hypothesis that H2S is a specific, potent, and reversible inhibitor of complex IV (cytochrome c oxidase), the terminal enzyme complex in the electron transport chain. The physiological roles of H2S in the myocardium and coronary vasculature have received little attention. Recent reports have indicated that in rat hearts, exogenous H2S produces concentration- and time-dependent decreases in left ventricular dP/dtmax.11 The negative inotropic effect was abolished by glibenclamide pretreatment. In a subsequent study, Geng et al12 reported down-regulation of cystathionine-␥-lyase gene expression, enzyme activity, and H2S production in an in vivo rat model of myocardial injury induced by subacute isoprenaline treatment. In contrast, in vitro exogenous H2S reduced markers of lipid peroxidation in myocardial homogenates exposed to reactive oxygen species. A number of documents have suggested that the KATP opener could benefit isolated heart preservation.13–15 Consequently, we have reason to believe that H2S improves the preservation of donor rat hearts via KATP opening. The aim of this study was to examine the effects of exogenous H2S on the preservation of donor rat hearts, and to examine the modifying effects of glibenclamide, a blocker of KATP opening.
minutes. LV performance was assessed by measurement of LV systolic pressure (LVSP; mm Hg) and LV end-diastolic pressure (LVEP; mm Hg; LVSP ⫺ LVEP ⫽ LV-developed pressure [LVDP]). Calculated positive and negative first derivatives of LVSP (⫹dP/dt and ⫺dP/dt, mm Hg/s) were recorded by a BIOPAC system. Hearts were then arrested with 30 mL of 1 of the 4 heart preservation solutions (see below), delivered at 4°C under a pressure of 75 mm Hg. Hearts were removed from the perfusion apparatus and stored for 6 hours at 4°C in glass containers filled with the same solution. On completion of the storage, the hearts were transferred back to the Langendorff column. After stabilizing for 30 minutes with normothermic Krebs-Henseleit (KH) bicarbonate buffer perfusion, LV performance was assessed with the same methods. LVDP, ⫹dP/dt, and ⫺dP/dt were calculated and recorded as previously. The isolated hearts taken off the Langendorff column were kept for later pathologic study.
Experimental Group Twenty-four Sprague-Dawley rats were randomly divided into 4 groups. Group 1 was the control group of rat hearts arrested and stored in KH solution without NaHS. Group 2 were rat hearts arrested and stored in KH solution with NaHS (1 mol/L). Group 3 were rat hearts expressed to NaHS (1 mol/L) in the presence of glibenclamide (10 mol/L). Group 4 were rat hearts exposed to St. Thomas II solution.
Myocardial Tissue Water Ratio Measurement A piece of myocardial tissue was weighed and put into a 100°C drying cabinet for 24 hours after which we calculated the water ratio.
MATERIALS AND METHODS Langendorff Perfusion
Myocardial ATP Content Measurement Myocardial tissue (50 mg) was extracted from liquid nitrogen and crushed with 20 times to volume of 0.4 mol/L HClO4 solution at 0°C. After centrifugation, the pH of 2 mL of supernate was adjusted to 7.80 using 5 mmol/L K2CO3. After centrifugation for KClO4 elimination, the supernate was diluted 2-fold with 0.02 mol/L Tris acetic buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 2.1 mmol/L EDTA, pH 7.87) before ATP measurement. The above manipulation was performed in an iced bath. ATP content was measured by high-performance liquid chromatography (HPLC) using a mobile phase of NH4H2PO4 buffer.
Male Sprague-Dawley rats (220 –270 g) were intravenously heparinized (0.2 mL) and anesthetized with intraperitoneal pentobarbital (60 mg/kg). The rapidly harvested hearts were mounted on a nonrecirculating Langendorff perfusion column (Radnoti Working Heart Recirculating System 130101). Retrograde perfusion was established at a pressure of 75 mm Hg with an oxygenated (95% oxygen, 5% carbon dioxide), normothermic (37°C) KrebsHenseleit bicarbonate buffer ultrafiltered through 5-mm pores. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). A fluid-filled latex balloon inserted via the left atrium into the left ventricle (LV) was inflated to set a left ventricular end-diastolic pressure (LEDP) between 5 and 10 mm Hg. Before each experimental protocol was initiated, the isolated hearts were allowed to stabilize at 37°C for 30
TUNEL Staining Ventricular tissues fixed in formalin for 24 hours were embedded in paraffin and sectioned. The apoptotic cells were identified by TUNEL using an apoptosis detection kit according to the manu-
Table 1. Hemodynamics Group 1
Group 2
Group 3
Group 4
Variables
Initial
30-Minute RP
Initial
30-Minute RP
Initial
30-Minute RP
Initial
30-Minute RP
⫹dp/dt (mm Hg/s) ⫺dp/dt (mm Hg/s) LVDP (mm Hg)
2348 ⫾ 382† 2177 ⫾ 15.5† 103 ⫾ 3†
1042 ⫾ 276* 739 ⫾ 154* 39 ⫾ 7*
2494 ⫾ 326 2172 ⫾ 349 108 ⫾ 8
1439 ⫾ 365* 1220 ⫾ 263* 68 ⫾ 15*
2844 ⫾ 407 2110 ⫾ 459 113 ⫾ 16
782 ⫾ 301* 575 ⫾ 276* 33 ⫾ 16*
2514 ⫾ 361 1943 ⫾ 232 97 ⫾ 3
1434 ⫾ 435* 1093 ⫾ 126* 60 ⫾ 14*
RP, reperfusion; LVDP, left ventricular-developed pressure. Values are means ⫾ SEM; n ⫽ 6 for each group. *p ⬍ .001 vs initial. † p ⬍ .05 vs groups 2– 4.
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HU, LI, BI ET AL
facturer’s protocol (Roche). From 10 photographs (magnification, ⫻400) of each tissue section, the number of cardiomyocytes with brown nuclear staining, indicating apoptosis, was expressed as the number of TUNEL-positive cells. Apoptosis in cardiomyocytes was quantified by the number of apoptotic nuclei in the total nuclei in 10 continuous microscopic fields under ⫻400 magnifications using the following formula: apoptosis index ⫽ (apoptotic nuclei/total nuclei) ⫻ 100.
Drugs and Solutions
Statistical Analysis The results were expressed as the mean values ⫾ SEM. Paired Student t test was used for the hemodynamic index comparison before and after reperfusion. Recontract time (RCT) values were expressed as log-normal distribution with F tests of the logarithms of each value, one paired values compared by LSD-t test. Other values were analyzed by one-way ANOVA (F test); paired values were compared using the LSD-t test.
RESULTS Left Ventricular Function
Effective contraction was regained by all 24 isolated hearts. No significant difference was observed among the 4 groups before arrest and reperfusion (Table 1). LV function decreased significantly after arrest and reperfusion in each group. In groups 2 and 4, ⫹dp/dtmax, ⫺dp/dtmax, and LVDP were better than those of the other 2 groups after effective contraction was regained after reperfusion. The recovery rates of ⫹dp/dtmax, ⫺dp/dtmax, and LVDP of groups 2 and 4 were better than those of groups 1 and 3 (Fig 1). There was a significant difference among the time duration before effective contraction was regained after reperfusion. The isolated heart contracted immediately after reperfusion in group 4; ventricular fibrillation was
Fig 2. Time duration before contraction after reperfusion. RPT, reperfusion time. †P ⬍ .001 vs groups 2– 4.
seen before contraction in the other 3 groups, and the duration was longest in group 1 (Fig 2). Myocardial Water Ratio
Myocardial water ratios were 84.7% ⫾ 0.9%, 84.2% ⫾ 1.9%, 85.2% ⫾ 1.2%, and 84.4% ⫾ 0.8% in each group, respectively. There was no significant difference (P ⬎ .05). ATP Content
After preservation and reperfusion the myocardial ATP values were 155 ⫾ 45, 388 ⫾ 40, 145 ⫾ 26, and 187 ⫾ 28 in each group, respectively. It was the highest in group 2 (P ⬍ .001). No significant difference was observed among the other 3 groups (P ⬎ .05; Fig 3). TUNEL Staining
Apoptosis could be found in the 4 groups (Fig 4). The apoptosis index was the lowest in group 2 (P ⬍ .001
‡
400 ATP(ug/g)
H2S was generated by the spontaneous dissociation of the H2S donor, sodium hydrosulfide (NaHS). Glibenclamide (Gli, glyburide) was obtained from Sigma. Glibenclamide was dissolved in DMSO, such that the final concentration of DMSO in the heart perfusate was 0.01%. All other chemicals were of analytical or ultrapure quality. All drugs were dissolved in ultrapure distilled water. All solutions were prepared on the day of the study. The Krebs-Henseleit buffer contained (in mmol/L): NaCl 118; KCl 4.7; MgSO4 1.2; NaHCO3 25; KH2PO4 1.2; CaCl2 2.5; glucose 11. St. Thomas II solution contained (in mmol/L): NaCl 120; KCl 16; MgCl2 16.6; CaCl2 1.2; NaHCO3 10.
300 †
200 100 0 1
2
3
4
Groups Fig 1. Left ventricular function recovery rate after reperfusion. †P ⬍ .05 vs groups ⫽ 2 and 4. ‡P ⬍ .05 vs groups.
Fig 3. ATP content after reperfusion. ‡P ⬍ .001 vs groups 1, 3, and 4. †P ⬍ .05 vs groups 3 and 4. Group 2 refers to hearts arrested and stored in KH solution with NaHS (1 mol/L).
H2S IN PRESERVATION OF DONOR RAT HEARTS
A
3027
B
C compared with the other 3 groups). Apoptosis was obvious in groups 1 and 3 and the difference between group 4 and groups 1 and 3 was significant (P ⬍ .05; Fig 5). DISCUSSION
We found KH solution with addition of 1 mol/L NaHS to improve the preservative effect of isolated rat hearts. Its preservative effect equaled St. Thomas II solution (STH) in hemodynamic aspects and was better than STH in myocar-
Fig 4. Apoptosis comparison. (A) TUNEL staining cardiomyocytes without after staining (⫻400). (B) TUNEL staining cardiomyocytes with hematoxylin after staining (⫻400). (C) Hematoxylin-eosin staining cardiomyocytes (⫻400). Arrows point to TUNEL-positive myocyte nuclei.
dial energetic metabolism and prevention of apoptosis. The study showed that H2S protected the myocardium by opening potassium channels, for this function was blocked with glibenclamide, which blocks KATP. KH solution is only a buffer which shows no protective effect on the myocardium. Its protection of the myocardium in this study depended on hypothermia, which decreases energetic metabolism rate and tissue energy demand. But hypothermia destroys the normal physiological status for it
3028
Fig 5. Apoptosis index (AI) by TUNEL staining. †P ⬍ .05 vs groups 2 and 4. ‡P ⬍ .001 vs groups 3 and 4. §P ⬍ .001 vs group 4.
decreases the metabolism rate and inhibits energy production at the same time. After preservation and reperfusion with KH solution, the hemodynamic index recovered poorly resulting in severe apoptosis. The myocardial ATP content decreased dramatically. All 6 hearts fibrillated before contraction after reperfusion. Possibly due to a small sample size, the water ratio was similar to the other 3 groups. St. Thomas II solution is a commonly used cardioplegic solution, which is representative of a high potassium intracellular solution. The high concentration of potassium decreases the transmembrane potential, reducing inward sodium current. When the potential reaches ⫺50 mV the sodium ionic channel is inactivated. At this time, an action potential cannot be generated or propagated and the heart is arrested. By heart arrest and hypothermia, STH decreases myocardial energetic metabolism and demand, thus protecting the myocardium. As a high potassium cardioplegic solution, it has its own shortcomings. Hypothermia affects enzymatic functions, stability of cellular membranes, utilization of glucose, production and utilization of ATP, and the balance between pH and intracellular osmotic levels; reperfusion injury may result.16 Another problem is that STH is a crystaline solution that cannot contain oxygen. It takes the myocardium into an anaerobic metabolic status after cross clamping the aorta, and may cause severe intracellular metabolic acidosis. High concentrations of potassium increase membrane permeability and intracellular calcium overload may result, thereby increasing ventricular wall tension and oxygen free radical production, producing irreversible ischemia reperfusion injury. High potassium concentrations may cause coronary endothelial cell injury and thereby affect cardiac functional recovery after reperfusion. In this study, hearts preserved with STH regained contraction immediately after reperfusion without ventricular fibrillation. The hemodynamic recovery was satisfactory; apoptosis was somewhat severe and ATP content was not optimal.
HU, LI, BI ET AL
It has been shown that KATP is the end effect substance in ischemia preconditioning. KATP is a highly selective potassium channel manipulated by intracellular ATP concentration. The KATP opener improves cardiac function, reducing infarction areas after ischemia. The KATP opener may be the main element of ischemie preconditioning and endogenous myocardium protection. The mechanism of KATP opener protective effect may relate to the following factors: membrane hyperpolarization and reduction of action potential duration, reduction of inward calcium, relaxation of vascular smooth muscle, prevention of intracellular calcium overload, production of oxygen free radicals, and protection of coronary endothelium cells. KATP has the following effects: improved systolic and diastolic function after reperfusion,17 antireperfusion arrhythmia functions18 decreased energetic consumption, increased energetic reserve,19,20 prevention of calcium overload,21 attenuated chondrosomal edema,22 relaxed vascular smooth muscle and thus increased coronary blood flow for better perfusion,23 suppressed neutrophil function and oxygen free radical production, participation in fatty acid metabolism and prostacylin generation, and improved coronary endothelial cell functions. H2S acts like a KATP opener attenuating ischemia reperfusion injury.4,9,24 This study suggested that KH solution supplemented with 1 mol/L NaHS as a preservation solution (group 2) protected the isolated heart equal to STH solution and better than STH in some aspects. In group 2, the hearts fibrillated after reperfusion, but the fibrillation time from reperfusion and regain of effective contraction was shorter than that of hearts preserved in KH solution without NaHS. In group 2, the hemodynamics recovered as well as that of the STH solution group after reperfusion. It has been widely accepted that myocardial protection closely relates to intracellular ATP preservation, since ATP content is one of the best indices to evaluate the myocyte recovery.25 ATP content, myocardial contractility, and supermicrostructure of the myocardium closely relate to each other. This study proved that the solution used in group 2 protected the myocardium based upon ATP content and pathological study by TUNEL staining. Glibenclamide (10 mol/L in this study) blocks the KATP current selectively in a concentration-dependent fashion.26 The myocardial protective effect of KH solution with NaHS was completely blocked by adding glibenclamid (10 mol/L) in group 3. Surprisingly, although the protective effect was blocked, the duration before contraction after reperfusion was shorter than that of group 2. We do not know the mechanism. A clinical test comparing glibenclamide and bigunaides in diabetic patients suggested that the probability of ventricular precontraction, paroxysmal ventricular tachycardia, and ventricular fibrillation was smaller in the glibenclamide group under conditions of acute myocardial ischemia or infarction.27 Pasnani and Ferrier28 observed that small doses of glibenclamide (3 or 30 mmol/L) shortened the duration of the action potential and the effective refractory period significantly in the early
H2S IN PRESERVATION OF DONOR RAT HEARTS
period of ischemia and reperfusion. This may explain the phenomenon we encountered in group 3. In the present experiment H2S showed myocardial protective effectiveness after reperfusion. It attenuated myocardial ischemia injury and increased hemodynamic recovery rate at least after reperfusion. But the main limit of this research was that we studies the protectiveness after reperfusion, and no observation was made during the preservation period. Thus it is hard to tell whether H2S protects the myocardium during preservation or reperfusion or both. Whether H2S opens chondriosome or membrane KATP is still not clear. Further observation should be made on cardiac preservation solution containing H2S.
REFERENCES 1. Moore PK, Bhatia M, Moochhala S: Hydrogen sulfide: from the smell of the past to the mediator of the future? Trends Pharmacol Sci 24:609, 2003 2. Wang R: The gasotransmitter role of hydrogen sulfide. Antioxid Redox Signal 5:493, 2003 3. Teague B, Asiedu S, Moore PK: The smooth muscle relaxant effect of hydrogen sulphide in vitro: evidence for a physiological role to control intestinal contractility. Br J Pharmacol 137:139, 2002 4. Zhao W, Zhang J, Lu Y, et al: The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J 20:6008, 2001 5. Zhao W, Wang R: H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol 283:H474, 2002 6. Budas GR, Jovanovic S, Crawford RM, et al: Hypoxiainduced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal KATP channels. FASEB J 18:1046, 2004 7. Dombkowski RA, Russell MJ, Olson KR: Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am J Physiol Regul Integr Comp Physiol 286:R678, 2004 8. Dombkowski RA, Russell MJ, Schulman AA, et al: Vertebrate phylogeny of hydrogen sulfide vasoactivity. Am J Physiol Regul Integr Comp Physiol 288:R243, 2005 9. Cheng Y, Ndisang JF, Tang G, et al: Hydrogen sulfideinduced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol 287:H2316, 2004 10. Blackstone E, Morrison M, Roth MB: H2S induces a suspended animation-like state in mice. Science 308:518, 2005 11. Geng B, Yang J, Qi Y, et al: H2S generated by heart in rat and its effects on cardiac function. Biochem Biophys Res Commun 313:362, 2004 12. Geng B, Chang L, Pan C, et al: Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol. Biochem Biophys Res Commun 318:75, 2004
3029 13. Gu K, Kin S, Saitoh Y, et al: The HTK solution with nicorandil can improve cardiac function after simple cold storage. Transplant Proc 28:77, 1996 14. Dorman BH, Hebbar L, Hinton RB, et al: Preservation of myocyte contractile function after hyperthermic cardioplegic arrest by activation of ATP-sensitive potassium channels. Circulation 96:2376, 1997 15. Hoenicke EM, Peterseim DS, Ducko CT, et al: Donor heart preservation with the potassium channel opener pinacidil: comparison with University of Wisconsin and St. Thomas’ solution. J Heart Lung Transplant 19:286, 2000 16. Lee J, Drinkwater DC Jr, Laks H, et al: Preservation of endothelium-dependent vasodilation with low-potassium University of Wisconsin solution. J Thorac Cardiovasc Surg 112:103, 1996 17. Lawton JS, Hsia PW, Damiano RJ Jr: The adenosinetriphosphate-sensitive potassium-channel opener pinacidil is effective in blood cardioplegia. Ann Thorac Surg 66:768, 1998 18. Tanaka H, Okazaki K, Shigenobu K: Cardioprotective effects of NIP-121, a novel ATP-sensitive potassium channel opener, during ischemia and reperfusion in coronary perfused guinea pig myocardium. J Cardiovasc Pharmacol 27:695, 1996 19. Grover GJ: Protective effects of ATP-sensitive potassiumchannel openers in experimental myocardial ischemia. J Cardiovasc Pharmacol 24(suppl 4):S18, 1994 20. Jayawant AM, Stephenson ER Jr, Matte GS, et al: Potassium-channel opener cardioplegia is superior to St. Thomas’ solution in the intact animal. Ann Thorac Surg 68:67, 1999 21. Behling RW, Malone HJ: KATP-channel openers protect against increased cytosolic calcium during ischaemia and reperfusion. J Mol Cell Cardiol 27:1809, 1995 22. Monticello TM, Sargent CA, McGill JR, et al: Amelioration of ischemia/reperfusion injury in isolated rat hearts by the ATPsensitive potassium channel opener BMS-180448. Cardiovasc Res 31:93, 1996 23. Satoh K, Yamada H, Taira N: Differential antagonism by glibenclamide of the relaxant effects of cromakalim, pinacidil and nicorandil on canine large coronary arteries. Naunyn Schmiedebergs Arch Pharmacol 343:76, 1991 24. Johansen D, Ytrehus K, Baxter GF: Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemiareperfusion injury— evidence for a role of K ATP channels. Basic Res Cardiol 101:53, 2006 25. Wicomb WN, Hill DJ, Avery JG, et al: Donor heart preservation—limitations of cardioplegia and warm ischemia. Transplantation 53:947, 1992 26. Quayle JM, Nelson MT, Standen NB: ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev 77:1165, 1997 27. Schotborgh CE, Wilde AA: Sulfonylurea derivatives in cardiovascular research and in cardiovascular patients. Cardiovasc Res 34:73, 1997 28. Pasnani JS, Ferrier GR: Differential effects of glyburide on premature beats and ventricular tachycardia in an isolated tissue model of ischemia and reperfusion. J Pharmacol Exp Ther 262: 1076, 1992
T
HE MOST IMPORTANT obstacle for heart transplantation is the shortage of donor hearts. At present, heart transplantations are performed much less frequently than liver and kidney transplantations. Part of the reason is that hearts cannot endure ischemia as long as the liver or kidney. Thus the protection and preservation of donor hearts are of interest in transplant research. It is well known that hydrogen sulfide (H2S) is a toxic gas. Recent evidence indicates that following nitric oxide and carbon monoxide, H2S is the third endogenous gaseous mediator with pronounced physiological effects, particularly in the cardiovascular and central nervous systems.1,2 Endogenous or exogenous H2S causes relaxation of various smooth muscle types, including those of the vascular, intestinal, and reproductive tract systems.3 A number of studies have confirmed that H2S is a potent vasodilator in vivo,4 as well as in several isolated vascular preparations
including rat aorta,4,5 rat mesenteric bed,6 trout efferent branchial arteries,7 and pulmonary arteries among a range of vertebrate species from hagfish (phylogenetically the most primitive vertebrate) to rats.8 At present the mechanisms of H2S-induced vasorelaxation are not completely established. However, as glibenclamide is a selective KATP blocker, there is evidence that the action is at least partially
From the Cardiovascular Surgery Department, Xijing Hospital, Fourth Military Medical University, XI’an, People’s Republic of China (X.H., S.B., Z.J., G.Z., C.B., L.L., Q.C., W.L.), and the Cardiovascular Surgery Department, Third Center Hospital, Tianjin, People’s Republic of China (X.H., T.L.). Address reprint requests to Weiyong Liu, MD, Cardiovascular Surgery Department, Xijing Hospital, Fourth Military Medical University, West Changle Road 15, XI’an, China PRC, 710032. E-mail: [email protected]
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.086
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3024
Transplantation Proceedings, 39, 3024 –3029 (2007)
H2S IN PRESERVATION OF DONOR RAT HEARTS
3025
glibenclamide-sensitive, suggesting involvement in the opening of ATP-dependent K⫹ channels (KATP) in vascular smooth muscle.4,9 Moreover, one study indicated that H2S induced a suspended animation-like state in mice,10 leading to the hypothesis that H2S is a specific, potent, and reversible inhibitor of complex IV (cytochrome c oxidase), the terminal enzyme complex in the electron transport chain. The physiological roles of H2S in the myocardium and coronary vasculature have received little attention. Recent reports have indicated that in rat hearts, exogenous H2S produces concentration- and time-dependent decreases in left ventricular dP/dtmax.11 The negative inotropic effect was abolished by glibenclamide pretreatment. In a subsequent study, Geng et al12 reported down-regulation of cystathionine-␥-lyase gene expression, enzyme activity, and H2S production in an in vivo rat model of myocardial injury induced by subacute isoprenaline treatment. In contrast, in vitro exogenous H2S reduced markers of lipid peroxidation in myocardial homogenates exposed to reactive oxygen species. A number of documents have suggested that the KATP opener could benefit isolated heart preservation.13–15 Consequently, we have reason to believe that H2S improves the preservation of donor rat hearts via KATP opening. The aim of this study was to examine the effects of exogenous H2S on the preservation of donor rat hearts, and to examine the modifying effects of glibenclamide, a blocker of KATP opening.
minutes. LV performance was assessed by measurement of LV systolic pressure (LVSP; mm Hg) and LV end-diastolic pressure (LVEP; mm Hg; LVSP ⫺ LVEP ⫽ LV-developed pressure [LVDP]). Calculated positive and negative first derivatives of LVSP (⫹dP/dt and ⫺dP/dt, mm Hg/s) were recorded by a BIOPAC system. Hearts were then arrested with 30 mL of 1 of the 4 heart preservation solutions (see below), delivered at 4°C under a pressure of 75 mm Hg. Hearts were removed from the perfusion apparatus and stored for 6 hours at 4°C in glass containers filled with the same solution. On completion of the storage, the hearts were transferred back to the Langendorff column. After stabilizing for 30 minutes with normothermic Krebs-Henseleit (KH) bicarbonate buffer perfusion, LV performance was assessed with the same methods. LVDP, ⫹dP/dt, and ⫺dP/dt were calculated and recorded as previously. The isolated hearts taken off the Langendorff column were kept for later pathologic study.
Experimental Group Twenty-four Sprague-Dawley rats were randomly divided into 4 groups. Group 1 was the control group of rat hearts arrested and stored in KH solution without NaHS. Group 2 were rat hearts arrested and stored in KH solution with NaHS (1 mol/L). Group 3 were rat hearts expressed to NaHS (1 mol/L) in the presence of glibenclamide (10 mol/L). Group 4 were rat hearts exposed to St. Thomas II solution.
Myocardial Tissue Water Ratio Measurement A piece of myocardial tissue was weighed and put into a 100°C drying cabinet for 24 hours after which we calculated the water ratio.
MATERIALS AND METHODS Langendorff Perfusion
Myocardial ATP Content Measurement Myocardial tissue (50 mg) was extracted from liquid nitrogen and crushed with 20 times to volume of 0.4 mol/L HClO4 solution at 0°C. After centrifugation, the pH of 2 mL of supernate was adjusted to 7.80 using 5 mmol/L K2CO3. After centrifugation for KClO4 elimination, the supernate was diluted 2-fold with 0.02 mol/L Tris acetic buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 2.1 mmol/L EDTA, pH 7.87) before ATP measurement. The above manipulation was performed in an iced bath. ATP content was measured by high-performance liquid chromatography (HPLC) using a mobile phase of NH4H2PO4 buffer.
Male Sprague-Dawley rats (220 –270 g) were intravenously heparinized (0.2 mL) and anesthetized with intraperitoneal pentobarbital (60 mg/kg). The rapidly harvested hearts were mounted on a nonrecirculating Langendorff perfusion column (Radnoti Working Heart Recirculating System 130101). Retrograde perfusion was established at a pressure of 75 mm Hg with an oxygenated (95% oxygen, 5% carbon dioxide), normothermic (37°C) KrebsHenseleit bicarbonate buffer ultrafiltered through 5-mm pores. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). A fluid-filled latex balloon inserted via the left atrium into the left ventricle (LV) was inflated to set a left ventricular end-diastolic pressure (LEDP) between 5 and 10 mm Hg. Before each experimental protocol was initiated, the isolated hearts were allowed to stabilize at 37°C for 30
TUNEL Staining Ventricular tissues fixed in formalin for 24 hours were embedded in paraffin and sectioned. The apoptotic cells were identified by TUNEL using an apoptosis detection kit according to the manu-
Table 1. Hemodynamics Group 1
Group 2
Group 3
Group 4
Variables
Initial
30-Minute RP
Initial
30-Minute RP
Initial
30-Minute RP
Initial
30-Minute RP
⫹dp/dt (mm Hg/s) ⫺dp/dt (mm Hg/s) LVDP (mm Hg)
2348 ⫾ 382† 2177 ⫾ 15.5† 103 ⫾ 3†
1042 ⫾ 276* 739 ⫾ 154* 39 ⫾ 7*
2494 ⫾ 326 2172 ⫾ 349 108 ⫾ 8
1439 ⫾ 365* 1220 ⫾ 263* 68 ⫾ 15*
2844 ⫾ 407 2110 ⫾ 459 113 ⫾ 16
782 ⫾ 301* 575 ⫾ 276* 33 ⫾ 16*
2514 ⫾ 361 1943 ⫾ 232 97 ⫾ 3
1434 ⫾ 435* 1093 ⫾ 126* 60 ⫾ 14*
RP, reperfusion; LVDP, left ventricular-developed pressure. Values are means ⫾ SEM; n ⫽ 6 for each group. *p ⬍ .001 vs initial. † p ⬍ .05 vs groups 2– 4.
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HU, LI, BI ET AL
facturer’s protocol (Roche). From 10 photographs (magnification, ⫻400) of each tissue section, the number of cardiomyocytes with brown nuclear staining, indicating apoptosis, was expressed as the number of TUNEL-positive cells. Apoptosis in cardiomyocytes was quantified by the number of apoptotic nuclei in the total nuclei in 10 continuous microscopic fields under ⫻400 magnifications using the following formula: apoptosis index ⫽ (apoptotic nuclei/total nuclei) ⫻ 100.
Drugs and Solutions
Statistical Analysis The results were expressed as the mean values ⫾ SEM. Paired Student t test was used for the hemodynamic index comparison before and after reperfusion. Recontract time (RCT) values were expressed as log-normal distribution with F tests of the logarithms of each value, one paired values compared by LSD-t test. Other values were analyzed by one-way ANOVA (F test); paired values were compared using the LSD-t test.
RESULTS Left Ventricular Function
Effective contraction was regained by all 24 isolated hearts. No significant difference was observed among the 4 groups before arrest and reperfusion (Table 1). LV function decreased significantly after arrest and reperfusion in each group. In groups 2 and 4, ⫹dp/dtmax, ⫺dp/dtmax, and LVDP were better than those of the other 2 groups after effective contraction was regained after reperfusion. The recovery rates of ⫹dp/dtmax, ⫺dp/dtmax, and LVDP of groups 2 and 4 were better than those of groups 1 and 3 (Fig 1). There was a significant difference among the time duration before effective contraction was regained after reperfusion. The isolated heart contracted immediately after reperfusion in group 4; ventricular fibrillation was
Fig 2. Time duration before contraction after reperfusion. RPT, reperfusion time. †P ⬍ .001 vs groups 2– 4.
seen before contraction in the other 3 groups, and the duration was longest in group 1 (Fig 2). Myocardial Water Ratio
Myocardial water ratios were 84.7% ⫾ 0.9%, 84.2% ⫾ 1.9%, 85.2% ⫾ 1.2%, and 84.4% ⫾ 0.8% in each group, respectively. There was no significant difference (P ⬎ .05). ATP Content
After preservation and reperfusion the myocardial ATP values were 155 ⫾ 45, 388 ⫾ 40, 145 ⫾ 26, and 187 ⫾ 28 in each group, respectively. It was the highest in group 2 (P ⬍ .001). No significant difference was observed among the other 3 groups (P ⬎ .05; Fig 3). TUNEL Staining
Apoptosis could be found in the 4 groups (Fig 4). The apoptosis index was the lowest in group 2 (P ⬍ .001
‡
400 ATP(ug/g)
H2S was generated by the spontaneous dissociation of the H2S donor, sodium hydrosulfide (NaHS). Glibenclamide (Gli, glyburide) was obtained from Sigma. Glibenclamide was dissolved in DMSO, such that the final concentration of DMSO in the heart perfusate was 0.01%. All other chemicals were of analytical or ultrapure quality. All drugs were dissolved in ultrapure distilled water. All solutions were prepared on the day of the study. The Krebs-Henseleit buffer contained (in mmol/L): NaCl 118; KCl 4.7; MgSO4 1.2; NaHCO3 25; KH2PO4 1.2; CaCl2 2.5; glucose 11. St. Thomas II solution contained (in mmol/L): NaCl 120; KCl 16; MgCl2 16.6; CaCl2 1.2; NaHCO3 10.
300 †
200 100 0 1
2
3
4
Groups Fig 1. Left ventricular function recovery rate after reperfusion. †P ⬍ .05 vs groups ⫽ 2 and 4. ‡P ⬍ .05 vs groups.
Fig 3. ATP content after reperfusion. ‡P ⬍ .001 vs groups 1, 3, and 4. †P ⬍ .05 vs groups 3 and 4. Group 2 refers to hearts arrested and stored in KH solution with NaHS (1 mol/L).
H2S IN PRESERVATION OF DONOR RAT HEARTS
A
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B
C compared with the other 3 groups). Apoptosis was obvious in groups 1 and 3 and the difference between group 4 and groups 1 and 3 was significant (P ⬍ .05; Fig 5). DISCUSSION
We found KH solution with addition of 1 mol/L NaHS to improve the preservative effect of isolated rat hearts. Its preservative effect equaled St. Thomas II solution (STH) in hemodynamic aspects and was better than STH in myocar-
Fig 4. Apoptosis comparison. (A) TUNEL staining cardiomyocytes without after staining (⫻400). (B) TUNEL staining cardiomyocytes with hematoxylin after staining (⫻400). (C) Hematoxylin-eosin staining cardiomyocytes (⫻400). Arrows point to TUNEL-positive myocyte nuclei.
dial energetic metabolism and prevention of apoptosis. The study showed that H2S protected the myocardium by opening potassium channels, for this function was blocked with glibenclamide, which blocks KATP. KH solution is only a buffer which shows no protective effect on the myocardium. Its protection of the myocardium in this study depended on hypothermia, which decreases energetic metabolism rate and tissue energy demand. But hypothermia destroys the normal physiological status for it
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Fig 5. Apoptosis index (AI) by TUNEL staining. †P ⬍ .05 vs groups 2 and 4. ‡P ⬍ .001 vs groups 3 and 4. §P ⬍ .001 vs group 4.
decreases the metabolism rate and inhibits energy production at the same time. After preservation and reperfusion with KH solution, the hemodynamic index recovered poorly resulting in severe apoptosis. The myocardial ATP content decreased dramatically. All 6 hearts fibrillated before contraction after reperfusion. Possibly due to a small sample size, the water ratio was similar to the other 3 groups. St. Thomas II solution is a commonly used cardioplegic solution, which is representative of a high potassium intracellular solution. The high concentration of potassium decreases the transmembrane potential, reducing inward sodium current. When the potential reaches ⫺50 mV the sodium ionic channel is inactivated. At this time, an action potential cannot be generated or propagated and the heart is arrested. By heart arrest and hypothermia, STH decreases myocardial energetic metabolism and demand, thus protecting the myocardium. As a high potassium cardioplegic solution, it has its own shortcomings. Hypothermia affects enzymatic functions, stability of cellular membranes, utilization of glucose, production and utilization of ATP, and the balance between pH and intracellular osmotic levels; reperfusion injury may result.16 Another problem is that STH is a crystaline solution that cannot contain oxygen. It takes the myocardium into an anaerobic metabolic status after cross clamping the aorta, and may cause severe intracellular metabolic acidosis. High concentrations of potassium increase membrane permeability and intracellular calcium overload may result, thereby increasing ventricular wall tension and oxygen free radical production, producing irreversible ischemia reperfusion injury. High potassium concentrations may cause coronary endothelial cell injury and thereby affect cardiac functional recovery after reperfusion. In this study, hearts preserved with STH regained contraction immediately after reperfusion without ventricular fibrillation. The hemodynamic recovery was satisfactory; apoptosis was somewhat severe and ATP content was not optimal.
HU, LI, BI ET AL
It has been shown that KATP is the end effect substance in ischemia preconditioning. KATP is a highly selective potassium channel manipulated by intracellular ATP concentration. The KATP opener improves cardiac function, reducing infarction areas after ischemia. The KATP opener may be the main element of ischemie preconditioning and endogenous myocardium protection. The mechanism of KATP opener protective effect may relate to the following factors: membrane hyperpolarization and reduction of action potential duration, reduction of inward calcium, relaxation of vascular smooth muscle, prevention of intracellular calcium overload, production of oxygen free radicals, and protection of coronary endothelium cells. KATP has the following effects: improved systolic and diastolic function after reperfusion,17 antireperfusion arrhythmia functions18 decreased energetic consumption, increased energetic reserve,19,20 prevention of calcium overload,21 attenuated chondrosomal edema,22 relaxed vascular smooth muscle and thus increased coronary blood flow for better perfusion,23 suppressed neutrophil function and oxygen free radical production, participation in fatty acid metabolism and prostacylin generation, and improved coronary endothelial cell functions. H2S acts like a KATP opener attenuating ischemia reperfusion injury.4,9,24 This study suggested that KH solution supplemented with 1 mol/L NaHS as a preservation solution (group 2) protected the isolated heart equal to STH solution and better than STH in some aspects. In group 2, the hearts fibrillated after reperfusion, but the fibrillation time from reperfusion and regain of effective contraction was shorter than that of hearts preserved in KH solution without NaHS. In group 2, the hemodynamics recovered as well as that of the STH solution group after reperfusion. It has been widely accepted that myocardial protection closely relates to intracellular ATP preservation, since ATP content is one of the best indices to evaluate the myocyte recovery.25 ATP content, myocardial contractility, and supermicrostructure of the myocardium closely relate to each other. This study proved that the solution used in group 2 protected the myocardium based upon ATP content and pathological study by TUNEL staining. Glibenclamide (10 mol/L in this study) blocks the KATP current selectively in a concentration-dependent fashion.26 The myocardial protective effect of KH solution with NaHS was completely blocked by adding glibenclamid (10 mol/L) in group 3. Surprisingly, although the protective effect was blocked, the duration before contraction after reperfusion was shorter than that of group 2. We do not know the mechanism. A clinical test comparing glibenclamide and bigunaides in diabetic patients suggested that the probability of ventricular precontraction, paroxysmal ventricular tachycardia, and ventricular fibrillation was smaller in the glibenclamide group under conditions of acute myocardial ischemia or infarction.27 Pasnani and Ferrier28 observed that small doses of glibenclamide (3 or 30 mmol/L) shortened the duration of the action potential and the effective refractory period significantly in the early
H2S IN PRESERVATION OF DONOR RAT HEARTS
period of ischemia and reperfusion. This may explain the phenomenon we encountered in group 3. In the present experiment H2S showed myocardial protective effectiveness after reperfusion. It attenuated myocardial ischemia injury and increased hemodynamic recovery rate at least after reperfusion. But the main limit of this research was that we studies the protectiveness after reperfusion, and no observation was made during the preservation period. Thus it is hard to tell whether H2S protects the myocardium during preservation or reperfusion or both. Whether H2S opens chondriosome or membrane KATP is still not clear. Further observation should be made on cardiac preservation solution containing H2S.
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