Intracellular pH (pHi) is an important endogenous modulator of cardiac function. Inhibition of Na+/H+ exchanger-1 (NHE-1) protects the heart by preventing Ca2+ overload during ischemia/reperfusion. Hydrogen sulfide (H2S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H2S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 μM, produced sustained decreases in pHi in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl4 prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) significantly attenuated NaHS-suppressed NHE-1 activity and/or NaHS-induced cardioprotection. Although KT5823 failed to affect NaHS-induced Akt phosphorylation, Akt inhibitor did attenuate NaHS-stimulated PKG activity. In conclusion, this work demonstrated for the first time that H2S produced cardioprotection via the suppression of NHE-1 activity involving a PI3K/Akt/PKG-dependent mechanism.
Hydrogen sulfide (H2S) has been positioned as the third candidate of the newly emerging “gasotransmitters” family along with nitric oxide (NO) and carbon monoxide (Wang, 2002). It is produced endogenously from cysteine and homocysteine in reactions catalyzed by cystathionine-β-synthase and cystathionine-γ-lyase. The expression of these two enzymes is highly tissue-specific. Whereas cystathionine-γ-lyase is largely expressed in the cardiovascular system, cystathionine-β-synthase predominates in the central nervous system (Chen et al., 1999; Geng et al., 2004b). H2S plays an important role in the regulation of heart function. Together with other groups, we have found that both endogenous and exogenous H2S protect the heart from isoproterenol-induced myocardial injury by directly scavenging oxygen free radicals (Geng et al., 2004a) and inhibiting the adenylyl cyclase/cAMP pathway or L-type calcium channel (Yong et al., 2008b). Moreover, researchers have found that preconditioning and postconditioning with H2S produced cardioprotective effects against ischemic injury via the regulation of protein kinase C, KATP channels, cyclooxygenase-2, NO, p42/44-mitogen-activated protein kinase, phosphoinositol-3-kinase (PI3K)/Akt, and GSK3β pathways (Bian et al., 2006; Hu et al., 2008; Yong et al., 2008a; Yao et al., 2010). More importantly, endogenous H2S was found to contribute to the cardioprotection induced by ischemic preconditioning and postconditioning (Bian et al., 2006; Pan et al., 2006; Yong et al., 2008a). In addition, H2S may produce a proangiogenic effect (Cai et al., 2007) that can contribute to its cardioprotective action. These results suggest that H2S not only ameliorates the pathological process of ischemic heart disease but may also act as a cardioprotective regulator.
Intracellular pH (pHi) is an important modulator of cardiac function, influencing processes as varied as contraction, excitation, and electrical rhythm. Regulation of pHi is required for the maintenance of an environment appropriate for cellular activities. Hence, pHi has to be tightly controlled within a narrow range, largely through the activity of transporters such as Na+/H+ exchanger-1 (NHE-1) and Cl−/HCO3− exchanger (CBE). Protons are produced metabolically within the heart. These ions are highly reactive with cellular proteins and must be removed if cardiac function is to be maintained. During ischemia, lactic acid accumulation causes significant intracellular acidosis, which stimulates NHE-1. This minimizes intracellular acidosis and causes an increase in intracellular sodium. The protons leaving the cell produce extracellular acidosis. During reperfusion, the extracellular protons are flushed away, and the activity of NHE-1 then leads to a rapid recovery of pHi and a rise in intracellular sodium. The latter could eventually result in Ca2+ entry through the activity of Na+/Ca2+ exchangers. Therefore, it is well accepted that the inhibition of NHE-1 protects against some of the damaging effects of ischemia. We have reported that H2S regulates pHi in vascular smooth muscle cells (Lee et al., 2007) and glial cells (Lu et al., 2010a). Whether or not H2S can also regulate pHi in hearts by affecting NHE-1 activity is still unknown. The present study was therefore designed to determine the effect of H2S on pHi and NHE-1 activity in isolated cardiac myocytes and investigate whether H2S could protect the heart against ischemia/reperfusion via this mechanism.
Materials and Methods
The study protocol was approved by the Institutional Animal Care and Use Committee of National University of Singapore.
Isolation of Rat Ventricular Cardiac Myocytes.
Sprague-Dawley male rats (∼190–210 g) were anesthetized with 200 mg/kg pentobarbitone intraperitoneally. Heparin (1000 IU) was administered intraperitoneally to prevent coagulation during heart removal. The rat hearts were quickly excised and mounted on a Langendorff apparatus. Hearts were then perfused in a retrograde fashion via the aorta with calcium-free Tyrode's solution (137 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 37°C. After 5 min, the perfusion solution was changed to Tyrode's solution containing 1 mg/ml collagenase type I and 0.28 mg/ml protease (type XIV) and perfused for another 25 to 30 min. The perfusion solution was then changed to Ca2+-Tyrode's solution containing 0.2 mM CaCl2 without enzymes for an additional 5 min. The ventricles were then cut into small pieces in a Petri dish containing prewarmed Ca2+-containing Tyrode's solution and shaken gently to ensure adequate dispersion of the dissociated cardiac myocytes. A 2.5 × 10−4 μM mesh screen was used to separate the isolated myocytes from cardiac tissue. The cells were then washed three times in Ca2+-Tyrode's solution and collected by centrifugation (700 rpm, 1 min). Ca2+ concentration was increased gradually to 1.25 mM in 20 min. More than 80% of the cells were rod-shaped and impermeable to Trypan blue. The cells were allowed to stabilize for 30 min before further experimentation.
pHi Measurements in Rat Ventricular Cardiac Myocytes.
The method of pHi measurement has been described previously (Lee et al., 2007). In brief, cardiac myocytes were incubated with 1 μM 2,7-biscarboxyethyl-5,6-carboxyfluorescein/acetoxymethyl ester (BCECF/AM) for 30 min in the dark at room temperature. The unincorporated dye was then removed by washing the cardiac myocytes twice in fresh incubation solution. The membrane-permeable ester was trapped inside the myocytes because of hydrolyzation by esterases and fluoresced pH-dependently. The loaded rat cardiac myocytes were kept in the dark at room temperature for another 30 min before pHi measurement to allow the BCECF/AM in the cytosol to de-esterify.
The BCECF/AM-loaded rat cardiac myocytes were transferred to the stage of an inverted microscope (Nikon, Tokyo, Japan) in a perfusion chamber at room temperature. The inverted microscope was coupled with a dual-wavelength excitation spectrofluorometer (Intracellular Imaging Inc., Cincinnati, OH). Cells were perfused with Krebs' solution (117 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.25 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose, pH 7.4). Drugs were then added directly into the bath solution during pHi measurements, and the change in fluorescent intensity was monitored. The pH-dependent fluorescent signal of BCECF/AM was obtained by illuminating at excitation wavelengths of 490 nm (F490) and 440 nm (F440). The ratio of signals obtained at F490 and F440 was used to represent pHi. The calibration of BCECF/AM signals was performed by setting pHi to extracellular pH with 10 μM nigericin in Krebs' solution. The extracellular pH was changed by perfusion with Krebs' solution at pH 6.8, 7.4, 8, or 10. From these corresponding pH and fluorescence measurements, a graph was constructed and used for the translation of fluorescence values into pHi values.
Determination of NHE-1 Activity.
NHE-1 activity in cardiac myocytes was assessed by measuring the recovery rate of cells from intracellular acidification. Cells were perfused with HEPES-buffered solution containing 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose, adjusted to pH 7.4 with NaOH. Myocytes were maintained at 25°C throughout and subjected to intracellular acidosis by transient exposure to 20 mM NH4Cl (6 min at 25°C) with subsequent washout for 8 min. Because NH4+ enters the cell at a slow, but significant, rate on transporter pathways, this feature is used to acid-load the cytoplasm (Fröhlich and Wallert, 1995). Washout of NH4Cl therefore imposes an acid load by trapping protons into the myocytes. Subsequently, myocytes recovered from this acid load via the activity of NHE-1. The slope of pHi recovery determines the sarcolemmal NHE-1 activity (Fröhlich and Wallert, 1995). To assess NHE-1 activity of myocytes in the presence of H2S, 0.1 mM sodium hydrosulfide (NaHS) was added to the cells 10 min before intracellular alkalinization, followed by perfusion with NaHS-containing HEPES-buffered solution.
Determination of CBE Activity.
CBE activity was assessed by measuring the recovery rate of cells from intracellular alkalinization. Intracellular alkalinization was introduced by a rapid addition of 20 mM NH4Cl to bathing solution. Exposure of cells to NH4Cl results in diffusion of NH3 across cell membranes, leading to rapid intracellular alkalinization (Furtado, 1987). pHi gradually decreases from the peak of alkalinization because of the efflux of HCO3− via activity of CBE, that is, a recovery from alkali load (Xu and Spitzer, 1994). The slope of the pHi decrease determines the rate of recovery from the peak of alkalinization and is measured in ΔpH/ms. To assess the CBE activity of cells in the presence of H2S, 0.1 mM NaHS was added 10 min before intracellular alkalinization, and myocytes were perfused with NaHS-containing HEPES-buffered solution.
Ischemia/Reperfusion in Isolated Rat Ventricular Myocytes.
For the cardiac myocyte ischemia/reperfusion experiments, simulated ischemia solution [i.e., glucose-free Krebs buffer containing 10 mM 2-deoxy-d-glucose, an inhibitor of glycolysis, and 10 mM sodium dithionite (Na2S2O4), an oxygen scavenger, pH 6.6] was applied. The use of simulated ischemia solution in this way produces a mixture of effects including metabolic inhibition, anoxia, and acidosis. This method was adapted from a previous publication (Ho et al., 2002). In brief, after dissociation, the cardiac ventricular myocytes were allowed to stabilize for 30 min before the experiment commenced. Ventricular myocytes were subjected to ischemia solution for 30 min followed by reperfusion with Dulbecco's modified Eagle's medium solution for up to 1 h. NaHS or cariporide was applied 10 min before and during ischemia, respectively. The viability of cardiac myocytes was determined at the end of reperfusion for a certain period as specified in the individual results.
Cell Viability Assay for Rat Ventricular Myocytes.
Trypan blue exclusion was used as an index of myocyte viability. At the end of reperfusion, cardiac myocytes were incubated with 0.4% (w/v) Trypan blue dye (Sigma-Aldrich, St. Louis, MO) for 3 min. Those that were unstained were designated as nonblue cells. The ratio of nonblue cells/total cells was determined in a hemocytometer chamber under light microscopy.
PKG Activity Assay.
cGMP-dependent protein kinase assay (Cyclex; MBL International, Woburn, MA) was used to measure PKG activity. The isolated cardiac myocytes were divided into different treatment groups: vehicle + ischemia (myocytes treated with vehicle and subjected to simulated ischemia solution for 30 min), NaHS + ischemia (myocytes were pretreated with NaHS at 0.1 mM for 10 min before being subjected to ischemia), Akt VIII + NaHS + ischemia (myocytes were treated with Akt VIII at 1 μM for 10 min and then NaHS at 0.1 mM for 10 min followed by ischemia for 30 min), and KR + NaHS + ischemia [myocytes were treated with [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568) at 1 μM for 10 min and then NaHS at 0.1 mM for 10 min followed by ischemia for 30 min]. Protein extraction was performed in accordance with the manufacturer's instructions (MBL International, Woburn, MA). Experiments of test sample cGMP-minus and ATP-minus groups were conducted as quality controls of our assay.
Western Blotting Analysis.
The procedure of detecting Akt phosphorylation by Western blotting was adopted from our previous publication (Hu et al., 2008). To examine the effect of NaHS on nonischemic myocytes, the isolated cardiac myocytes were subjected to NaHS or vehicle treatment for 30 min. To test the action of NaHS on cardiac myocytes subjected to ischemic insults, NaHS (0.1 mM) was added to the myocytes for 10 min before and during ischemia for 30 min. To examine the regulatory effect of PKG on Akt phosphorylation, (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) (0.5 μM), a specific inhibitor of PKG, was added 10 min before NaHS treatment. At the end of treatment or ischemia, myocytes were gently washed twice with ice-cold PBS, homogenized in chilled lysis buffer containing 125 mM NaCl, 25 mM Tris, pH 7.5, 5 mM EDTA, 1% nonidet P-40, 1 mM NaF, 2 mM Na3VO4, and protease inhibitor (Roche Diagnostics, Indianapolis, IN) and then shaken on ice for 1 h. After that, the lysates were centrifuged at 13,000g for 10 min at 4°C. The supernatants were then collected and denatured by SDS sample buffer, and the epitopes were exposed by boiling the protein samples for 5 min in a dry heat block. Equal amounts of proteins were loaded and separated on 12% SDS-polyacrylamide gel electrophoresis gel, and then transferred onto nitrocellulose membrane. The membrane was then probed with antibodies against phosphorylated and total Akt (1:1000; Cell Signaling Technology, Danvers, MA) and a second antibody. Immunoreactivity was detected by using an ECL Western blot detection kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
Langendorff Heart Preparation and Hemodynamic Assessment.
Hearts were quickly excised, mounted on a Langendorff apparatus, and perfused in a retrograde fashion via the aorta with Kreb's buffer (137 mM NaCl, 2.5 mM CaCl2, 5 mM KCl, 1.2 mM MgSO4, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 37°C. The hearts were perfused at a constant pressure of 80 mm Hg and subsequently submitted to 40-min stabilization, 30-min global no-flow ischemia, and 1-h reperfusion. NaHS or cariporide in Kreb's solution was perfused into the heart for 10 min before global no-flow ischemia was commenced. During the no-flow ischemic period, no solution was perfused. Hearts continued to be exposed to the solution containing NaHS or cariporide during the no-flow ischemic period.
Left ventricular pressure was monitored with a latex balloon connected to a pressure transducer (Powerlab; ADInstruments Pty Ltd., Castle Hill, Australia). The balloon volume was adjusted to obtain a left ventricular end-diastolic pressure (LVEDP) of 5 to 8 mm Hg. All data were digitally converted and stored on a computer for analysis (Powerlab).
Chemicals and Reagents.
Nigericin, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), NH4Cl, NaHS, KR-32568, collagenase I, protease XIV, and Trypan blue dye (0.4%) were purchased from Sigma-Aldrich. BCECF/AM was obtained from Molecular Probes (Carlsbad, CA). KT5823, Akt VIII, and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were obtained from Merck Sharp and Dohme (Hoddesdon, UK). Nigericin was dissolved in ethanol. All other chemicals were dissolved in water except KR-32568, KT5823, LY294002, BCECF/AM, and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, which were dissolved in dimethyl sulfoxide.
Cariporide ([4-isopropyl-3-(methylsulfonyl)benzoyl]guanidine methanesulfonate; HOE-642) was a gift from sanofi-aventis (Frankfurt, Germany). In our preliminary study, we used cariporide at doses of 1, 5, and 7 μM to determine the dose-dependent effect on cell viability and NHE activity in the isolated rat cardiac myocytes. We found that cariporide produced significant effects only at a concentration of 7 μM. This concentration was used in all further experiments.
NaHS was used as a source of H2S. The use of NaHS enabled us to define the concentrations of H2S in solution more accurately than bubbling H2S gas. NaHS at concentrations used in our work did not change the pH of the medium (Geng et al., 2004b).
Values presented are mean ± S.E.M. Paired Student's t test was used to determine the difference in fluorescent signal before and after treatment in the same cell. One-way analysis of variance was used with a post hoc (Bonferroni) test to determine the differences among groups. The significance level was set at P < 0.05.
Effect of NaHS on pHi in the Rat Ventricular Myocytes.
As shown in Fig. 1A, application of NaHS (an H2S donor, 100 μM), but not Krebs' solution, induced an obvious decrease in pHi in the isolated ventricular myocytes. The effect of H2S occurred rapidly after NaHS administration, suggesting that H2S readily diffused through the cell membrane and influenced pHi. The decrease in pHi by NaHS mimicked the effect of cariporide (7 μM), a selective NHE-1 inhibitor.
The concentration-dependent response is shown in Fig. 1B. NaHS at a concentration range from 10 to 1000 μM (yielding approximately 3.3–330 μM H2S) produced concentration-dependent decreases in pHi in the rat cardiac myocytes. The strongest effect was obtained with NaHS at 100 μM. For this reason, 100 μM NaHS was applied in the subsequent experiments.
Effect of U50,488H on pHi in the Presence and Absence of NaHS or Cariporide.
We had reported previously that U50,488H stimulates NHE-1 activity in cardiac myocytes (Bian et al., 1998). As shown in Fig. 2A, U50,488H at 30 μM significantly increased the pHi in the isolated ventricular myocytes. Both cariporide (7 μM) and NaHS (100 μM) abolished this effect, suggesting that, like cariporide, H2S may also inhibit NHE-1 activity.
Effect of NaHS on NHE-1 Activity in Rat Ventricular Myocytes.
To verify the involvement of NHE-1 in the action of NaHS, we determined the activity of NHE-1 in cardiac myocytes by measuring the recovery rate of cells from intracellular acidification (Fröhlich and Wallert, 1995). Figure 3A shows the representative tracings of pHi in three individual ventricular myocytes subjected to intracellular alkalinization by transient exposure to NH4Cl. Transient application of NH4Cl loaded the cell with alkali, thus inducing a significant elevation in pHi. Subsequent washout with NH4Cl induced a strong acid load by trapping protons in the myocytes, thus inducing intracellular acidification. As shown in Fig. 3B, both NaHS and cariporide significantly attenuated the recovery slope of pHi after washout with NH4Cl, suggesting that H2S decreased NHE-1 activity.
Effect of NaHS on CBE Activity in the Isolated Ventricular Myocytes.
Given that CBE is the main acid loader in cardiac myocytes, we also determined its activity by perfusing NH4Cl in the presence and absence of NaHS. NH4Cl perfusion is a widely adopted method to measure the capacity of CBE to maintain pHi (Xu and Spitzer, 1994). NH4Cl loaded the cells with alkali, resulting in a significant elevation in pHi. As an acid loader, CBE may consequently activate and extrude HCO3− in exchange for Cl−, which eventually restores the pHi to normal. Therefore, the slope of the pHi recovery phase provides a measure of CBE activity. As shown in Fig. 4, H2S inhibited the activity of CBE. Because inhibition of CBE alone induces intracellular alkalization, instead of acidification, these data indicate that the intracellular acidification induced by H2S was mediated by NHE-1 instead of CBE.
Effects of NaHS on Cell Viability in Rat Cardiac Myocytes Subjected to Ischemia/Reperfusion Insults.
Because NHE-1 inhibition may protect hearts against ischemia/reperfusion-induced injury (Karmazyn, 2001), we further tested whether NaHS could also protect the cardiac myocytes against ischemia/reperfusion injury. The experimental protocol is shown in Fig. 5A and described under Materials and Methods. As shown in Fig. 5B, both NaHS and cariporide significantly increased viability of ventricular myocytes subjected to ischemia (30 min)/reperfusion (10 min), suggesting that, like the NHE-1 inhibitor, NaHS may also protect cardiac myocytes against ischemia/reperfusion-induced injury. We further examined the time course for the cardioprotection offered by NaHS. As shown in Fig. 5C, NaHS (100 μM) protected cardiac myocytes subjected to ischemia (30 min)/reperfusion for up to 60 min. These data suggest that the cardioprotective effects of NaHS may last at least 1 h after reperfusion.
To examine whether the effect of NaHS was additive to that caused by NHE-1 inhibition, we compared the cardioprotective effects conferred by treatment with NaHS and KR-32568 (1 μM; another potent NHE-1 inhibitor) alone or in combination (NaHS + KR-32568). As shown in Fig. 5D, NaHS produced comparable protective effects to those caused by KR-32568 alone and in combination treatment. These data suggest that H2S and the NHE-1 inhibitor were not synergistic.
The Effect of NaHS on NHE-1 Activity Is Mediated by PI3K/Akt and Protein Kinase G Pathways.
Because activation of PI3K/Akt (Snabaitis et al., 2008) or PKG (Pérez et al., 2007) inhibits NHE-1 activity, we examined whether the effect of H2S involves these pathways. As shown in Fig. 6A, blockade of PI3K with LY294002 (1 μM) or PKG with KT5823 (0.5 μM) (neither of which had a significant effect on NHE-1 activity) produced marked attenuation of the inhibitory effect of NaHS on NHE-1 activity. These data suggest that the activation of PI3K/Akt and PKG may somehow be involved in the inhibitory effect of NaHS on NHE-1 activity.
We then investigated the effect of NaHS on PKG by measuring its activity with a semiquantitative immunoassay kit. As shown in Fig. 6B, NaHS significantly increased the PKG activity in the myocytes subjected to ischemia. Pretreatment with Akt VIII, the Akt inhibitor, but not KR-32568 (1 μM), an NHE-1 inhibitor, abolished this effect. This implies that activation of Akt by H2S is upstream to that of PKG.
Moreover, pretreatment with KT5823 for 10 min failed to attenuate NaHS-stimulated Akt phosphorylation (Fig. 6C). The similar effect was also observed under ischemic conditions. Ischemia for 30 min significantly down-regulated Akt phosphorylation. This effect was reversed by NaHS (100 μM) pretreatment for 10 min. Blockade of PKG with KT5823 also failed to attenuate the effect of H2S on Akt phosphorylation during ischemia (Fig. 6D). Taken together, our data clearly suggest that the activation of PKG is secondary to the NaHS-induced Akt activation but is upstream to NHE-1 inhibition.
The Effects of NaHS on Postischemic Recovery of Left Ventricular Function during Ischemia/Reperfusion Are Mediated by the PI3K/Akt/PKG/NHE-1 Pathway.
We also examined the effect of NaHS on the contractile function of isolated heart during ischemic reperfusion. As shown in Fig. 7, during the preischemic period, all measured parameters such as left ventricular developed pressure (LVDP), LVEDP, and maximum and minimum rate of developed pressure (±dP/dt) were comparable among the groups. Representative tracings of control, NaHS (100 μM), and cariporide (7 μM), are shown in Fig. 7A. Time-dependent changes in LVDP (Fig. 7B), LVEDP (Fig. 7C), and ±dP/dt (Fig. 7, D and E) in the control, NaHS, and cariporide treatment groups are shown. Treatment with NaHS and cariporide at 5, 10, and 15 min of reperfusion improved the postischemic contractile function significantly, compared with the untreated hearts (control).
We further confirmed that the cardioprotection offered by NaHS is mediated by the PI3K/Akt/PKG pathway. As shown in Fig. 8, the effects of NaHS on LVDP and ±dp/dt in the isolated hearts subjected to ischemia/reperfusion were also alleviated by LY294002 (1 μM; a PI3K inhibitor), Akt VIII (1 μM; a specific Akt inhibitor), and KT5823 (0.5 μM; a PKG inhibitor). These data suggest that NaHS-induced cardioprotection is, at least partly, mediated by the PI3K/Akt and PKG pathways. It is interesting to note that the blockade of PI3K/Akt or PKG had no significant effect on NaHS-induced protection on LVEDP. These data imply that the protective effects of NaHS may not be solely mediated by these signaling pathways.
pHi is a fundamental modulator of protein function in cells (Zaniboni et al., 2003). In the heart, pHi influences contractility (Vaughan-Jones et al., 1987; Kohmoto et al., 1990) and affects the generation of arrhythmias (Orchard and Cingolani, 1994; Ch'en et al., 1998). Previous studies from ours and other groups have found that H2S plays a critical role in protecting the heart against ischemic injury (Geng et al., 2004a,b). In the present study, we investigated the effect of H2S on pHi in the cardiac myocytes and the underlying mechanisms. We found that NaHS over a concentration range of 10 to 1000 μM decreased pHi in a concentration-dependent manner.
In the present study, U50488H was used as a tool compound to stimulate NHE. U50488H is a κ-opioid receptor agonist. At lower concentrations (<10 μM), it produces cardioprotective effects via activation of PI3K/Akt and suppression of cAMP/PKA pathways (Yu et al., 1999; Wang et al., 2001; Kim et al., 2011). However, at higher concentrations (∼30 μM or higher), it induces intracellular alkalization and cardiac arrhythmias via stimulation of protein kinase C/NHE pathways (Yu et al., 1999; Bian et al., 2000). We found that NaHS abolished the U50,488H-stimulated intracellular alkalization. The NHE activity assay further confirms that H2S-induced intracellular acidosis is mediated by inhibition of NHE-1 activity.
NHE-1 is an important acid extruder in cells. It is well recognized that NHE-1 inhibition may produce cardioprotection. We also found in the present study that both NaHS and NHE-1 inhibitor protected hearts against ischemic injury. However, the combination of two drugs did not produce any additive or synergistic cardioprotective effect. Our study therefore suggests that NaHS produced cardioprotection via the inhibition of NHE-1 activity.
The signaling mechanism for the action of H2S on NHE-1 was also investigated. It has been reported previously that the activation of Akt phosphorylates NHE-1 and thus inhibits its activity (Snabaitis et al., 2008). We therefore tested whether the inhibition of NHE-1 activity caused by H2S is mediated by the PI3K/Akt pathway. We found that NaHS treatment stimulated Akt phosphorylation in both normal and ischemic cardiac myocytes. Our finding is in agreement with a report that H2S protects cardiac myocytes from hypoxia/reoxygenation-induced apoptosis via stimulation of Akt phosphorylation (Yao et al., 2010). Moreover, blockade of PI3K with LY294002 or Akt with Akt VIII significantly attenuated the inhibitory effect by NaHS on NHE-1 activity and its subsequent cardioprotection. To confirm the contribution of the PI3K/Akt pathway, we also observed the protective action of NaHS on heart contractile function in the presence or absence of Akt VIII, a specific inhibitor of Akt. Like LY294002, Akt VIII also attenuated the effect of H2S on cardiac contractile function. These data suggest that the cardioprotection of H2S involves the PI3K/Akt/NHE-1 pathway. It is noteworthy that recent studies showed that the inhibition of NHE-1 may also stimulate Akt (Jung et al., 2010). These data suggest that the activation of Akt and inhibition of NHE-1 may be codependent. In addition to Akt activation, Yao et al. (2010) found H2S inhibited GSK3β and subsequently inhibited the opening of mitochondrial permeability transition pore (mPTP). NHE-1 inhibition was also noted to produce cardioprotection via the phosphorylation and inhibition of GSK3β (Jung et al., 2010). These findings suggest that H2S may inhibit GSK3β through the inhibition of NHE-1 activity.
NHE-1 activity can also be regulated by PKG. A previous study showed that blockade of PKG restored the suppressed NHE-1 activity to normal (Pérez et al., 2007). We therefore examined the involvement of PKG on the actions of NaHS. We found that blockade of PKG with KT5823 attenuated the effect of NaHS on NHE-1 activity and its protective effects on postischemia-induced contractile function injury. This implies that PKG may also be involved in the regulation of NHE-1 activity by NaHS. This was verified by the PKG activity assay. In fact, NaHS treatment enhanced PKG activity in ischemic cardiac myocytes. Moreover, the increase of PKG activity could be blocked by the Akt inhibitor, indicating that Akt is upstream of PKG activation. The failure of the PKG inhibitor, KT5823, to attenuate NaHS-stimulated Akt activation again supports the evidence that the activation of PKG is secondary to PI3K/Akt activation.
CBE is the major acid loader in the cardiovascular myocyte. Stimulation of this transporter would be expected to “dampen” the ability of the cell to maintain a normal pHi range and therefore decrease pHi. For this reason, we also observed the effect of H2S on CBE activity. We found that H2S inhibits, but not stimulates, CBE. These data exclude the possibility that CBE contributes to the action of H2S in regulating pHi in the cardiac myocytes.
The mechanisms for cardioprotection resulting from NHE-1 inhibition have been well studied previously. Ischemia-induced protons leaving the myocytes via NHE-1 during reperfusion may lead to Na+ loading, which may subsequently induce Ca2+ overloading as Na+ leaves the cell via the Na+/Ca2+ exchanger. The resultant rise in intracellular calcium concentration is believed to trigger Ca2+-activated proteases and phospholipases that cause cellular damage (Tani, 1990; Pierce and Czubryt, 1995). In addition, ischemia/reperfusion may open mPTP to disrupt the permeability barrier of the inner membrane. This will result in mitochondrial swelling and cause the outer membrane of mitochondria to break. The rupture of the outer membrane will lead to the release of proteins in the intermembrane space such as cytochrome c, apoptosis-inducing factor, and other factors that play a critical role in apoptotic cell death. Once released, these factors activate caspase-9, which in turn activates caspase-3. This protease mediates the proteolytic cleavage of a range of proteins responsible for the rearrangement of the cytoskeleton, plasma membrane, and nucleus that are characteristic of apoptosis. A potent inhibitor of mPTP opening is low pH (Halestrap et al., 2004). Therefore, in this way, NHE-1inhibition produces cardioprotection.
However, despite the great promise of cardioprotection offered by NHE inhibitors from extensive preclinical work (Avkiran et al., 2008), clinical studies have not been as convincing. It was found that direct and global NHE inhibition may trigger noncardiac adverse effects. Compared with NHE inhibitors, H2S may have better clinical application potential. This is because, apart from inhibition of NHE-1, H2S also protects the heart via suppression of β-adrenoceptor function and stimulation of various protein kinases and cardioprotective mediators such as prostaglandin E2 and NO (Bian et al., 2006; Hu et al., 2008; Pan et al., 2008, 2009; Yong et al., 2008b). In addition, H2S has been found to be useful in treating renin-dependent hypertension (Lu et al., 2010b), chronic heart failure (Calvert et al., 2010), and hypertrophy (Shi et al., 2007). These data suggest that H2S treatment may provide multiple cardioprotective effects. However, bearing in mind that the NHE inhibitors were not very successful in clinical development for cardioprotection despite the great promise shown in preclinical studies, the outcome of this preclinical study does not rule out the possibility of failed clinical application of H2S in ischemic heart conditions. Clinical investigations are thus warranted to confirm the usefulness of H2S.
In summary, we demonstrate in the present study that H2S decreases pHi in cardiac myocytes by inhibiting NHE-1 activity via a PI3K/Akt/PKG-dependent mechanism. This further offers cardioprotective effects against ischemia/reperfusion-induced injury. The unique action of H2S suggests a potential therapeutic application for its use in ischemic conditions of the heart.
Participated in research design: Hu, Li, Neo, and Bian.
Conducted experiments: Hu, Li, and Neo.
Contributed new reagents or analytic tools: Yong.
Performed data analysis: Hu, Li, Lee, and Bian.
Wrote or contributed to the writing of the manuscript: Hu, Neo, Tan, and Bian.
We thank Ester Khin Sanda Win and Soong Mei Leng for technical assistance.
This work was supported by the Singapore Biomedical Research Council Research Fund [Grant 07/1/21/19/509].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- hydrogen sulfide
- nitric oxide
- glycogen synthase kinase 3β
- Cl−/HCO3− exchanger
- sodium hydrosulfide
- left ventricular developed pressure
- left ventricular end-diastolic pressure
- change in pressure/change in time
- Na+/H+ exchanger
- phosphoinositide 3-kinase
- protein kinase G
- intracellular pH
- 2,7-biscarboxyethyl-5,6-carboxyfluorescein/acetoxymethyl ester
- mitochondrial permeability transition pore
- trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate
- (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester.
- Received June 3, 2011.
- Accepted August 18, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics