Gasotransmitters are endogenous gases of small molecular weight, which exert important physiological functions. Their production and metabolism are enzymatically regulated, and their effects are not dependent on specific membrane receptors. In recent years, another naturally occurring gas, hydrogen sulfide (H2S), has been found to be of importance (1). The endogenous metabolism and physiological functions of H2S may well place this gas in the group of already well-established gasotransmitters.
Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) are the key enzymes responsible for the endogenous production of H2S in mammalian cells and tissues. Both enzymes are pyridoxal-5-phosphate-dependent and use l-cysteine as the main substrate. The expression of CBS and CSE is tissue-specific. Although other enzymes can catalyze the production of H2S, CBS seems to be the main H2S-forming enzyme in the central nervous system, whereas CSE is the main H2S-forming enzyme in the cardiovascular system (2).
Although we know relatively little about the importance of H2S in the regulation of blood pressure and/or organ perfusion, there is evidence that alterations in the synthesis of H2S play a role in the physiology and pathophysiology of the cardiovascular system. For instance, recent studies demonstrate that H2S dilates blood vessels in vitro and in vivo by a mechanism that involves the opening of vascular smooth muscle ATP-sensitive K+ (KATP) channels (3, 4). Moreover, Mok and colleagues have recently reported that the inhibitor of hydrogen sulfide synthesis, dl-propargylglycine (PAG), attenuated the excessive vasodilatation in rats subjected to prolonged periods of hemorrhagic shock. In addition, treatment with the KATP channel blocker glibenclamide also partially reversed the hypotension associated with hemorrhagic shock, leading the authors to suggest that an excessive formation of H2S by CSE may open KATP channels within the vasculature and contribute to the excessive vasodilatation associated with hemorrhagic shock (5). We have recently shown that the enhanced formation of H2S contributes to the pathophysiology of the organ injury in endotoxemia (6).
It should also be noted that a reduction in the expression and the activity of CSE along with a reduction in the plasma concentration of H2S have been reported in a rat model of hypoxic pulmonary vasoconstriction (7). Interestingly, the parenteral administration of H2S in these animals attenuated the rise in pulmonary arterial pressure, which suggests that reduced formation of H2S may play a role in the pathophysiology of hypoxic pulmonary vasoconstriction (7). Observations made by Geng and colleagues provide evidence that the H2S-producing enzyme, CSE, is expressed in the rat heart and that endogenous production of H2S occurs in the rat myocardium (8). These studies have also demonstrated that administration of the H2S donor, sodium hydrosulfide (NaHS), causes a reduction in cardiac function in vitro and in vivo and that the effect was partially inhibited by the KATP channel blocker glibenclamide (8). Interestingly, in a clinical setting, moderate use of H2S balneotherapy raised the tolerance to exercise and reduced the daily need for short-acting nitrates in patients with coronary heart disease (9). However, the exact role of H2S in the pathophysiology of ischemia-reperfusion injury of the heart (and/or other organs) is still uncertain. A recent study by Bian and colleagues demonstrate that H2S protects (1) isolated cardiac myocytes by increasing cell viability and improving cell function and (2) isolated perfused hearts against ischemia and reperfusion injury (10).
Hence, the aim of this study was to investigate whether endogenous H2S protects the heart against injury caused by ischemia-reperfusion. Having demonstrated that PAG, a specific and irreversible inhibitor of CSE, enhances the tissue injury caused by myocardial ischemia and reperfusion in the rat, we have subsequently investigated the role of endogenous H2S production in the cardioprotection afforded by (1) ischemic preconditioning (acute cardioprotection caused by short cycles of ischemia and reperfusion) and (2) delayed cardioprotection after treatment with endotoxin (second window of protection caused by LPS).
The opening of KATP channels in the heart has long been known to play a pivotal role in the cardioprotective mechanism(s) of ischemic preconditioning (11-14). In recent years, the mitochondrial KATP channel has been believed to be the main KATP channel involved in cardiac protection (12,15-18). However, there is still controversy as to whether opening mitochondrial KATP channels in the heart is a trigger, mediator, or an end-effector of the protection afforded by ischemic preconditioning (16-18).
In light of the previously published observations linking the production of H2S within the vasculature to the opening of KATP channels, we have used the selective mitochondrial KATP channel blocker 5-hydroxydecanoate (5-HD)(12,14,15,19) to investigate whether the opening of KATP channels contributes to the cardioprotective effects of endogenous H2S in the heart.
METHODS AND MATERIALS
The experiments described in this article were performed in adherence to National Institutes of Health guidelines on the use of experimental animals. All experiments were performed in adherence with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 published by Her Majesty's Stationery Office, London.
This study was carried out on 87 male Wistar rats (Charles River UK Limited, Margate, UK) weighing 220 to 300 g and receiving a standard diet and water ad libitum as previously described (20). All animals were anesthetized with thiopentone sodium (Intraval, 120 mg/kg i.p.). Anesthesia was maintained by supplementary injections of thiopentone sodium as required. The trachea was cannulated, and the animals were ventilated with a Harvard ventilator (inspiratory oxygen concentration, 30%; 70 strokes per minute; tidal volume, 8-10 mL/kg). Body temperature was maintained at 37 ± 1°C with the aid of a rectal probe thermometer attached to a homeothermic blanket unit (Harvard Apparatus Ltd, Edenbridge, Kent, UK). The right carotid artery was cannulated and connected to a pressure transducer (Senso-Nor 840, Senso-Nor, Horten, Norway) to monitor mean arterial pressure (MAP) and heart rate (HR), which were displayed on a data acquisition system (MacLab 8e, ADI Instruments, Hastings, UK) installed on a Dell Dimension 4100 (Dell Inc, Round Rock, Tex). The right jugular vein was then cannulated to facilitate the administration of drugs. A parasternal thoracotomy was then performed, using an electrosurgery device to cauterize the intercostal arteries before cutting through 3 ribs. The chest was retracted and the pericardium was dissected from the heart. The left anterior descending (LAD) coronary artery was isolated and a snare occluder was placed around the LAD. The retractor was then removed and the animal was allowed to stabilize for 15 min.
Ischemia and reperfusion
The occluder was tightened at time 0. After 15 or 25 min of LAD occlusion, the occluder was released to allow reperfusion of the previously ischemic myocardium (2 h). Hemodynamic parameters were continuously monitored. Baseline readings were taken before treatment and myocardial ischemia. The pressure rate index (PRI), a relative indicator of myocardial oxygen consumption (21), was calculated as a product of the MAP and HR and expressed in mmHg beats per minute 10−3. Saline was administered immediately after reperfusion and throughout reperfusion at a rate of 2 mL/kg/h.
Quantification of myocardial tissue injury
At the end of the 2-h reperfusion period, the LAD was re-occluded and 1 mL of Evans blue dye (2% wt/vol) was injected into the animal via the jugular vein. The Evans blue dye stains the tissue through which it is able to circulate, so that the nonperfused vascular (occluded) tissue remains uncolored. Each animal was killed with an overdose of anesthetic, the heart excised, and excess dye washed off. The heart was then sectioned into slices of 3 to 4 mm, the right ventricle wall was removed, and the area at risk (AAR; the nonperfused and, hence, nonstained myocardium) was separated from the nonischemic (blue) tissue. The ischemic and nonischemic tissue was weighed, and the AAR was expressed as a percentage of the left ventricle. The tissue from the AAR was cut into small pieces and incubated with p-nitroblue tetrazolium (0.5 mg/mL) for 30 min at 37°C. p-Nitroblue tetrazolium is a reducing agent that reacts with dehydrogenases present in viable (noninfarcted) tissue to produce a dark blue formazan (22). Infarcted tissue (nonviable) will not possess dehydrogenase activity and will therefore fail to stain. The stained tissue was separated from the infarcted tissue and weighed, and the infarct size was expressed as a percentage of the AAR.
The protocols of the 4 experimental groups have been displayed in Table 1.
PAG, NaHS, and Escherichia coli LPS (serotype 0127:B8) were obtained from Sigma-Aldrich Company Ltd, Poole, Dorset, UK. 5-HD was purchased from Affiniti Research Products Ltd, Exeter, UK. Thiopentone sodium (Intraval Sodium) was purchased from Rhône Mérieux Ltd, Harlow, Essex, UK. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd, Thetford, Norfolk, UK).
All data are expressed as a mean ± SEM of n observations, where n represents the number of animals in the group. Hemodynamic parameters were analyzed via a 2-way ANOVA followed by a Bonferroni post-test. AAR and infarct size were analyzed via a 1-factorial ANOVA followed by a Bonferroni post-test for multiple comparisons. A P value of less than 0.05 denotes a statistically significant difference when compared with the MI Vehicle or MI LPS where applicable.
Baseline values of MAP, HR, and PRI were not significantly different between groups. There was no significant difference in body weight (data not shown) or AAR between groups (Table 2). When compared with sham-operated animals, 15 or 25 min of LAD occlusion followed by 2 h of reperfusion did not cause significant alterations in MAP, HR, or PRI (Tables 3-6). When compared with their respective controls, neither preconditioning nor administration of NaHS, PAG, 5-HD, or LPS (alone or in combination) had any significant effect on MAP, HR, or PRI(Tables 3-6).
The cardioprotective effects of NaHS are abolished by a mitochondrial KATP channel blocker (Study 1)
The mean values for the AAR were similar in all animal groups studied and ranged from 49 ± 2% to 53 ± 3% (P > 0.05, Table 2). When compared with sham-operated animals, 25 min of LAD occlusion followed by 2 h of reperfusion caused a significant increase in infarct size (Fig. 1). When compared with vehicle (saline), administration of the H2S-donor NaHS (3 mg/kg i.v.) 15 min before the onset of myocardial ischemia caused a significant reduction in myocardial infarct size by approximately 26% (Fig. 1). To gain further insight into the possible mechanism for the cardioprotection afforded by NaHS, the aforementioned experiment was repeated after pretreatment with the mitochondrial KATP channel blocker, 5-HD. The dose of 5-HD used during this investigation abolished the cardioprotective effect of ischemic preconditioning and/or endothelin in the anesthetized rabbit (13, 14). Administration of 5-HD (5 mg/kg i.v.) 30 min before the onset of myocardial ischemia attenuated the cardioprotective effect of NaHS (Fig. 1).
PAG and 5-HD augment the myocardial infarct size caused by regional myocardial ischemia and reperfusion (Study 2)
The dose of PAG used throughout this study has been shown to inhibit the formation of H2S in the liver homogenates of rodents (23). To investigate whether the formation of endogenous H2S is able to protect the heart against the injury caused by ischemia-reperfusion, we investigated the effects of PAG, a well-documented, irreversible inhibitor of CSE, on myocardial infarct size. The mean values for the AAR were similar in all animal groups studied and ranged from 46 ± 2% to 53 ± 3% (P > 0.05, Table 2). When compared with sham-operated animals, 15 min of LAD occlusion followed by 2 h of reperfusion caused a significant increase in infarct size. Administration of the CSE-inhibitor PAG (50 mg/kg i.v.) at 15 min before the onset of ischemia significantly augmented the infarct size caused by ischemia and reperfusion (Fig. 2). To investigate whether ischemia-reperfusion of the heart, in principle, leads to the release of a mediator (presumably H2S) that opens KATP channels, we investigated the effects of the KATP channel blocker 5-HD on myocardial infarct size. Administration of 5-HD (5 mg/kg) 30 min before ischemia augmented cardiac injury (Fig. 2). Taken together, these results suggest endogenous cardioprotection afforded by H2S may indeed be mediated by mitochondrial KATP channels.
The cardioprotective effects of ischemic preconditioning are not affected by the specific inhibitor of CSE (Study 3)
The mean values for the AAR were similar in all animal groups studied and ranged from 49 ± 3% to 50 ± 3% (P > 0.05, Table 2). When compared with sham-operated animals, 25 min of LAD occlusion followed by 2 h of reperfusion caused a significant increase in infarct size. When compared with vehicle-treated animals (saline), 2 cycles of preconditioning with ischemia resulted in a significant reduction in infarct size by approximately 48% (Fig. 3). Administration of PAG (50 mg/kg) had no effect on the reduction in infarct size afforded by ischemic preconditioning in the rat heart (Fig. 3).
The cardioprotective effects of endotoxin (second window of protection) are abolished by a CSE inhibitor and a mitochondrial channel blocker (Study 4)
The mean values for the AAR were similar in all animal groups studied and ranged from 48 ± 2% to 53 ± 3% (P > 0.05, Table 2). When compared with sham-operated animals, 25 min of LAD occlusion followed by 2 h of reperfusion caused a significant increase in infarct size. When compared with vehicle-treated animals (saline), pretreatment of rats with LPS (1 mg/kg i.p.) at 16 h before the onset of LAD occlusion resulted in a significant reduction in myocardial infarct size by approximately 41%. Interestingly, the specific CSE inhibitor PAG (50 mg/kg i.v.) attenuated the cardioprotective effect afforded by LPS (Fig. 4), indicating that the synthesis of endogenous H2S by CSE contributes to the second window of protection afforded by LPS. In addition, the mitochondrial KATP channel blocker 5-HD also attenuated the cardioprotection afforded by LPS (Fig. 4). These findings support the view that the delayed cardioprotection afforded by LPS is mediated by endogenous H2S, with subsequent opening of opening mitochondrial KATP channels being one component underlying this effect.
Previous findings by Geng and colleagues have shown that exogenous administration of H2S caused a significant reduction in the lipid peroxidation and the rise in plasma levels of creatine phosphokinase in a rat model of isoproterenol-induced cardiac injury (2). Although these and other previous findings suggest that H2S can be synthesized endogenously within the myocardium as a physiological regulator of cardiac function, it is not clear whether H2S is being produced during myocardial ischemia and reperfusion and, if so, whether the amounts produced are sufficient to protect the heart against the tissue injury associated with myocardial ischemia and reperfusion. This study demonstrates that intravenous administration of the H2S donor, NaHS, reduced the myocardial infarct size caused by regional ischemia (25 min) and reperfusion (2 h) in the anesthetized rat. Interestingly, these data complement recent findings whereby the plasma levels of H2S in coronary heart disease patients were significantly lower than that observed in angiographically normal control subjects (24). Having confirmed that H2S is cardioprotective, we then carried out further investigations to gain a better insight into the mechanism(s) of action.
Using the KATP channel blocker glibenclamide as a pharmacological tool, several authors have demonstrated that the biological effects of H2S such as vasorelaxation (1, 3, 4) and (more recently) inhibition of nociception in a rodent model of colitis (25) are mediated via the opening of KATP channels. Although several subtypes of KATP channels have been identified in cardiomyocytes, since the discovery of the mitochondrial KATP channel (26, 27), numerous studies have established the link between the opening of mitochondrial KATP channels and protection against ischemic injury in isolated myocytes and intact hearts (19, 28, 29). Liu and colleagues have shown that the protection of cardiac myocytes by diazoxide, a selective mitochondrial KATP channel opener having no effect on sarcolemmal KATP currents, was completely blocked by 5-HD (29). Hence, we have used the selective mitochondrial KATP channel blocker 5-HD (12, 14, 15) to investigate whether the opening of KATP channels contributes to the cardioprotective effects of endogenous H2S in the heart. In this study, we have demonstrated that 5-HD attenuated the cardioprotective effects of NaHS, indicating that the cardioprotective effects of H2S are (at least in part) due to the opening of mitochondrial KATP channels.
This study also demonstrates that the specific and irreversible inhibitor of CSE, PAG, caused a small but significant increase in the infarct size caused by shorter periods (15 min) of myocardial ischemia and reperfusion in the anesthetized rat. Interestingly, pretreatment with the mitochondrial KATP channel blocker 5-HD also enhanced the tissue injury caused by shorter periods of myocardial injury to a similar degree. Taken together, these data suggest that endogenous H2S protects the heart during episodes of ischemic injury and that this mediator may exert its cardioprotective effects by the opening of mitochondrial KATP channels.
Having discovered that endogenous H2S protects the heart against ischemia-reperfusion injury, we then investigated whether endogenous H2S may contribute to the potent cardioprotective effects caused by preconditioning of the heart with short cycles of regional ischemia (2 cycles of 5 min). Although we were able to demonstrate a very substantial reduction in infarct size caused by ischemic preconditioning, pretreatment of rats with the specific CSE inhibitor PAG did not affect the cardioprotective effects of ischemic preconditioning. Thus, endogenous H2S is unlikely to contribute to the cardioprotective effects of (acute) ischemic preconditioning.
There is also good evidence that pretreatment of animals with cell-wall fragments of Gram-negative (endotoxin, LPS) bacteria reduces the degree of myocardial tissue injury (and infarct size) caused by LAD occlusion and reperfusion at 8 to 24 h after injection of small amounts (1 mg/kg) of LPS (30, 31). We confirm here that LPS (1 mg/kg) causes a significant reduction in the infarct size after LAD occlusion (25 min) and reperfusion (2 h) at 16 h after the administration of LPS. Before this investigation, the contribution of endogenous H2S to the second window of cardioprotection afforded by LPS was unknown. We report here for the first time that the specific CSE inhibitor PAG largely attenuated the cardioprotective effects of LPS in the rat. Moreover, pretreatment of rats with 5-HD also attenuated the delayed cardioprotection afforded by LPS. Taken together, these results indicate that the delayed cardioprotective effects of LPS are-at least in part-mediated by endogenous production of H2S, and this possible cardioprotective effect of H2S may be mediated via the opening of mitochondrial KATP channels. We have shown that the reversal of the second window of cardioprotection by PAG was slightly more pronounced than that observed with 5-HD. Therefore, we cannot rule out that endogenously produced H2S after pretreatment with LPS may also exert cardioprotective effects via a mechanism that is independent of the opening of mitochondrial KATP. It should be noted that the estimated blood levels of the H2S donor used here in vivo are similar to the effective concentration shown to scavenge superoxide anions and hydrogen peroxide in vitro (2).
In summary, this article reports for the first time that (1) endogenous hydrogen sulfide is produced by myocardial ischemia in sufficient amounts to limit myocardial injury and (2) the synthesis or formation of H2S hydrogen sulfide by cystathionine-γ-lyase may contribute to the second window of protection caused by endotoxin. The protection mediated by endogenous hydrogen sulfide in part is mediated by the opening of mitochondrial KATP channels.
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