There is increasing evidence that hydrogen sulfide (H2S), like nitric oxide (NO) and carbon monoxide (CO), is an endogenous gaseous mediator capable of exerting pronounced physiological effects, particularly in the central nervous system and the cardiovascular system.1,2
H2S is enzymically synthesized in the metabolic pathway that regulates tissue turnover of the sulfur-containing amino acids L-methionine, L-homocysteine and L-cysteine.3 The two pyridoxal-5’-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), act as both transulfuration and desulfhydration catalysts to catabolise L-cysteine, resulting in generation of endogenous H2S, hydrosulfide (HS–) and sulfide (S2–).3-6 The differential expression of these enzymes is tissue type specific.1,7 CSE is responsible for H2S production in myocardial, arterial and venous tissues, and its action can be inhibited by DL-propargylglycine (PPG).8-11
Recent studies have indicated that endogenous or exogenous H2S exerts physiological effects in the vertebrate cardiovascular system, possibly through the modulation of KATP channel opening.12 H2S has been well-characterized as a physiologic vasodilator and regulator of blood pressure.7,13 In vascular smooth muscle cells (VSMCs), the opening of ATP-sensitive potassium channels (KATP channels) and the entrance of extracellular calcium were reported to be involved in H2S actions.8,14 Furthermore, it has been reported that H2S could be spontaneously generated in myocardium from CSE at concentrations in the micromolar range, and that both endogenous and exogenous H2S elicit marked cardiac actions. In rat heart, exogenous H2S has been shown to produce concentration- and time-dependent decreases in left ventricular dP/dtmax. Moreover, this negative inotropic effect was abolished by glibenclamide pretreatment.9
We have previously demonstrated that H2S facilitates carotid sinus baroreflex and baroreceptor activity by opening KATP channels and further closing calcium channels.15,16 We have also demonstrated that H2S decreases the duration of the action potential (APD) in guinea pig papillary muscles in a concentration- dependent manner17 and exerts a negative chronotropic action on pacemaker cells in sinoatrial nodes of rabbits.18 The aim of our present study was to observe the electrophysiological effects of H2S on human atrial fibers and elucidate the mechanism(s) involved.
Small biopsies (<1 cm2) of atrial myocardium were obtained as part of the cannulation technique for cardiopulmonary bypass from the anterior free wall of the right atrium of 24 patients undergoing corrective open heart surgery. Prior to surgery, informed consent had been obtained. All patients were under 16 years old with congenital heart diseases, including ventricular septal defects (n=18), atrial septal defects (n=4), and tetralogy of Fallot (n=2). To ensure that the preparations of atrial tissue were physiologically normal, the following criteria were used for exclusion: congestive heart failure and/or receipt of cardiotonic, antiarrhythmic, or diuretic medications. Preoperative electrocardiograms in all patients revealed normal values for P-R interval, P-wave amplitude and P-wave duration. No patient had a history or electrocardiographic evidence of cardiac arrhythmia.
Immediately after excision, the tissue was immersed in cold Tyrode’s solution (NaCl 137 mmol/L, NaHCO3
12 mmol/L, NaH2
1.8 mmol/L, MgCl2
0.5 mmol/L, CaCl2
2.7 mmol/L, KCl 4 mmol/L, and dextrose 5.5 mmol/L) that had been saturated by a mixture of 95% O2
and 5% CO2
and was of pH 7.3–
7.4. In the laboratory, the sample was fixed with fine pins to silica gel on the base of a perfusion chamber and allowed to equilibrate for 1 hour. The preparation was then superfused (4 ml/min) with Tyrode’s solution at (36.0±0.5)°C.
Transmembrane action potentials were recorded from the atrial fibers with a glass microelectrode filled with 3 mol/L KCl (tip resistance of 10–20 MW), coupled to a high input impedance amplifier (MEZ 8201; Nihon Kohden, Tokyo, Japan). The amplified signals were fed to the A/D convertor and processed by a microcomputer. Maximal diastolic potential (MDP), amplitude of action potential (APA), maximal rate of depolarization (Vmax), velocity of diastolic (phase 4) depolarization (VDD), rate of pacemaker firing (RPF), and duration of 90% repolarization of action potential (APD90) were analyzed. Parameters were stored in the microcomputer for later analysis.
After 60 minutes of equilibration, the preparations were explored with glass microelectrodes to find cells with spontaneous electric activity. Cells were identified as atrial specialized fibers if their intracellular potentials showed the characteristics of “pacemaker” cells: specifically, a transition from the slow depolarization of phase 4 to the more rapid depolarization of phase 0.
Action potentials (APs) were recorded after an equilibration time of 60 minutes. Samples were randomly divided among four experimental groups to determine the following:
Effects of H2S on the electrophysiology of human atrial fibers
After recording three control APs, NaHS concentrations of 50, 100, and 200 μmol/L were separately applied. APs were then re-recorded at 1, 5, 10, 20, 30 and 40 minutes after NaHS exposure.
Effects of KATP channel blocker glibenclamide (Gli) on H2S-induced changes on APs of pacemaker cells
After superfusion with Gli (20 μmol/L) for 15 minutes, NaHS (100 μmol/L) was added to the superfusate and APs were recorded.
Effects of L-type calcium channel agonist Bay K8644 on H2S-induced changes in APs of pacemaker cells
After pretreatment with Bay K8644 (0.5 mmol/L) for 15 minutes, NaHS (100 mmol/L) was added and APs were recorded.
Electrophysiological effects of CSE inhibitor PPG on human atrial fibers
After recording three control APs, PPG (200 μmol/L) was applied. APs were then recorded at 5 minutes intervals for a total of 120 minutes.
In each experiment, the preparation was washed with Tyrode’s solution after application of drugs in order to observe the characteristics of AP recovery.
NaHS, Bay K8644 and PPG were purchased from Sigma, USA. Gli was purchased from the Tianjin Institute of Medical and Pharmaceutical Industry (China).
NaHS was used as a donor of H2S. NaHS was employed in our experiments because its use allows for a better definition of the concentration of H2S in solution than bubbling H2S gas. NaHS dissociates to Na+ and HS- in solution. Then, HS- associates with H+ and H2S is produced. About one-third of the H2S in solution exist in the undissociated form (H2S), while the remaining two-thirds exist as HS- which is at equilibrium with H2S.10
Gli (a KATP channel blocker) was initially dissolved in dimethylsulfoxide (DMSO; 100 μmol/L). The final concentration of DMSO in the K-H solution was 0.04% (v/v). Bay K8644, an L-type calcium channel agonist, was dissolved in 99% ethyl alcohol. PPG, an inhibitor of CSE, and NaHS were dissolved in distilled water.
All data are presented as mean ± standard deviation (SD). The differences in the parameters detected before and after drug application were analyzed by paired Student’s t-test. Differences between groups were assessed by one-way analysis of variance (ANOVA) and unpaired t-test. Statistical significance was set at P <0.05.
Effects of H2S on the electrophysiological characteristics of human atrial fibers
As compared to the control group, NaHS (50–200 μmol/L) treatment was found to decrease APA, Vmax, VDD, RPF and APD90 in a concentration-dependent manner (Table 1 and Figure). The changes in RPF induced by NaHS paralleled those of VDD. The effects occurred after 5–15 minutes of NaHS superfusion and peaked within 25–40 minutes.
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Figure. Effects of H2S on transmembrane action potentials of human atrial fibers. A: Control; B: NaHS 50 μmol/L; C: NaHS 100 μmol/L; D: NaHS 200 μmol/L; E: Wash out. The recordings were obtained from the same cell, and are representative of all other cells tested.
Effects of Gli on H2S-induced changes of pacemaker cells APs
NaHS (100 μmol/L) significantly decreased APA, Vmax, VDD, RPF and APD90, as compared to the control groups. Gli (20 μmol/L) alone had no significant effect on AP. After pretreatment with Gli, the electrophysiological effects of NaHS (100 μmol/L) were partially inhibited. APA, Vmax, VDD, RPF and APD90 were significantly different from those observed in the control group and in the NaHS treated group (Table 2).
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Table 1. Effects of H2S on transmembrane action potentials of human atrial fibers
Table 2. Effects of Gli (20 μmol/L) on the NaHS (100 μmol/L)-induced changes on action potentials of pacemaker cells
Effects of Bay K8644 on H2S-induced changes of APs
The L-type calcium channel agonist Bay K8644 (0.5 μmol/L) markedly increased APA, Vmax, VDD, RPF and APD90. Pretreatment with Bay K8644 partially inhibited the electrophysiological effects of NaHS (100 μmol/L). Furthermore, the APA, Vmax, VDD, RPF and APD90 were significantly different from those observed in the control and NaHS groups (Table 3).
Electrophysiological effects of CSE inhibition on human atrial fibers
PPG (200 μmol/L) treatment significantly increased APA, Vmax, VDD, RPF and APD90. The effects were observed after 25–30 minutes of PPG superfusion and peaked within 80–100 minutes (Table 4).
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Table 3. Effects of Bay K8644 (0.5 μmol/L) on the NaHS (100 μmol/L)-induced changes on action potentials of pacemaker cells
Table 4. Effects of PPG (200 μmol/L) on transmembrane action potentials of human atrial fibers
This study demonstrated that H2S is able to exert inhibitory actions on the cardiac automaticity and accelerate the repolarization of human atrial special fibers. H2S was found to decrease APA, Vmax, VDD, RPF and APD90 in a concentration-dependent manner. The changes in RPF induced by H2S paralleled those of VDD. Since the calcium current (ICa) is known to play an important role in pacemaker depolarization,19,20 and the action potential upstroke of human atrial fibers is generated mainly by ICa,21 the decreases of APA and Vmax induced by H2S might be attributable to the reduction of ICa. Furthermore, it is widely accepted that the decaying potassium currents (Ik) and the increasing slow inward ICa are involved in diastolic depolarization of the pacemaker and the duration of the action potential is mainly influenced by potassium efflux and calcium influx. Therefore, the decreases of VDD, RPF and APD90 induced by H2S may result from the enhanced potassium efflux and/or inhibition of calcium influx.
It has been reported that H2S is the first identified gaseous opener of KATP channels in VSMCs8 and that KATP channels are widely distributed in heart cells.22-24 Geng et al9 reported that H2S could be endogenously produced by heart tissues, where it may regulate cardiac function through actions involving the KATP channel pathway. Thus, in this study, we observed the effects of the ATP-sensitive potassium channel blocker Gli on H2S-induced changes in AP. Gli partially inhibited the electrophysiological effects of H2S. The results indicated that the effects of H2S on AP are at least in part due to the enhancement of potassium efflux through the opening of KATP channels.
In order to examine the effect of H2S on calcium influx, we employed the L-type Ca2+ channel agonist Bay K8644. We observed that Bay K8644 also partially inhibited the electrophysiological effects of H2S. These results indicated that the reduction in calcium influx may also contribute to the functional effects of H2S.
To this point, our findings had only revealed the effects of exogenous H2S. We had previously demonstrated in another study that endogenous H2S generated by papillary muscles might play an important role in regulating the action potential of guinea pig papillary muscles.17 In order to determine whether endogenous H2S is generated in human atrial fibers and to characterize its function, we evaluated the effects of PPG (an inhibitor of CSE) since CSE, but not CBS, is known to play a major role in generating H2S in cardiovascular tissues under physiological conditions.1,10,25,26 Zhao et al11 reported that PPG might be a membrane-permeable drug and that, as such, it may be useful to study the physiological functions of endogenously produced H2S. In the present study, we found that pretreatment of human atrial fibers with PPG (200 μmol/L), led to significantly increased APA, Vmax, VDD, RPF and APD90, as compared to non-treated controls. These results indicated that endogenous H2S could be generated by CSE in human atrial fibers, where it may play an important role in control of action potentials.
Recently, some reports have suggested that H2S may exert a cardiovascular protective function. Preconditioning with NaHS significantly decreased the duration and severity of ischemia/reperfusion-induced arrhythmias in isolated heart tissues and increased cell viability.27 In addition, H2S was shown to exert a marked infarct-limiting action during myocardial ischemia/reperfusion.12 It has been suggested that this cardioprotective function against ischemia/reperfusion injury may be due to an increased proportion of open cardiac myocytes KATP channels mediated by H2S.28-30 In our research, H2S was found to decrease calcium influx; as a result, intracellular Ca2+ concentration declined and myocardial contractility weakened. H2S also reduced the cardiac automaticity and accelerated the repolarization of human atrial fibers; that is to say, H2S shortened the work duration of atrial fibers and thus protected the heart. The negative inotropic and chronotropic roles of H2S in human atrial fibers may be one of mechanisms that H2S uses to exert its cardioprotective functions in response to ischemia and reperfusion injury.
However, up to now the level of H2S and CSE mRNA and protein expressions have not been quantitatively measured in human atrial fibers. It is significant to quantificationally detect the myocardial level of H2S and CSE mRNA and protein expressions in the future.
In summary, our findings indicated that H2S is able to exert a negative chronotropic action and accelerate the repolarization of human atrial specialized fibers. Furthermore, these events may be due to an increase in potassium efflux through the opening of KATP channels and a concomitant decrease in calcium influx. We also found that endogenous H2S can be generated by CSE and this event may act as an important regulator of electrophysiological properties of human atrial fibers.
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