Analysis of decreases in systemic arterial pressure and heart rate in response to the hydrogen sulfide donor sodium sulfide

H₂S is a gasotransmitter capable of producing a decrease in mean arterial pressure and heart rate. The hypotensive and bradycardic effects of H₂S can be dissociated, as shown with cardiac pacing experiments. Responses were not blocked by diltiazem, ivabradine, methylene blue, or glybenclamide.

Abstract

The actions of hydrogen sulfide (H₂S) on the heart and vasculature have been extensively reported. However, the mechanisms underlying the effects of H₂S are unclear in the anesthetized rat. The objective of the present study was to investigate the effect of H₂S on the electrocardiogram and examine the relationship between H₂S-induced changes in heart rate (HR), mean arterial pressure (MAP), and respiratory function. Intravenous administration of the H₂S donor Na₂S in the anesthetized Sprague-Dawley rat decreased MAP and HR and produced changes in respiratory function. The administration of Na₂S significantly increased the RR interval at some doses but had no effect on PR or corrected QT(n)-B intervals. In experiments where respiration was maintained with a mechanical ventilator, we observed that Na₂S-induced decreases in MAP and HR were independent of respiration. In experiments where respiration was maintained by mechanical ventilation and HR was maintained by cardiac pacing, Na₂S-induced changes in MAP were not significantly altered, whereas changes in HR were abolished. Coadministration of glybenclamide significantly increased MAP and HR responses at some doses, but methylene blue, diltiazem, and ivabradine had no significant effect compared with control. The decreases in MAP and HR in response to Na₂S could be dissociated and were independent of changes in respiratory function, ATP-sensitive K⁺ channels, methylene blue-sensitive mechanism involving L-type voltage-sensitive Ca²⁺ channels, or hyperpolarization-activated cyclic nucleotide-gated channels. Cardiovascular responses observed in spontaneously hypertensive rats were more robust than those in Sprague-Dawley rats.

NEW & NOTEWORTHY H₂S is a gasotransmitter capable of producing a decrease in mean arterial pressure and heart rate. The hypotensive and bradycardic effects of H₂S can be dissociated, as shown with cardiac pacing experiments. Responses were not blocked by diltiazem, ivabradine, methylene blue, or glybenclamide.

hydrogen sulfide (H₂S), nitric oxide (NO), and carbon monoxide (CO) are gaseous agents that have diverse concentration-dependent actions on the cardiovascular system (14, 22, 23, 42). Exposure to all three gaseous agents in high concentration can produce death, whereas administration of these agents in lower doses causes vasodilation and has a beneficial effect in a number of disease models (20, 34, 36). In low concentrations, H₂S can act as an endothelium-derived vasodilator factor that can help maintain systemic arterial pressure at physiological levels (11, 43). In contrast to a beneficial physiological role in blood pressure regulation at low concentration, a high concentration of H₂S may act as a chemical poison, causing hypotension, cardiac failure, respiratory failure, and death (18, 19, 27, 55). Acute, potentially fatal H₂S exposures in humans occur intentionally as a method of suicide and unintentionally in the petroleum, pulp and paper, agriculture, aquaculture, and other industries (28, 65).

Sulfur pools in vivo are typically broken down into free H₂S (which exists in equilibrium with HS⁻ and S²⁻), acid-labile sulfur, and sulfane sulfur, the chemistry of which has been previously reviewed (50, 62). Endogenous enzymatic production of H₂S is mediated by cystathionine γ-lyase (CSE), cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase (48, 51, 57). H₂S production in the vascular system is largely mediated by CSE, and CSE knockout mice develop hypertension similar to that observed in endothelial NO synthase knockout mice (66, 70). Modulation of endogenous CSE levels affects disease progression in myocardial ischemia-reperfusion and pressure overload models, whereas CSE overexpression demonstrated protective effects and CSE deletion demonstrated deleterious effects (6, 12, 29, 35). In rodent models of myocardial ischemia, the CSE-selective inhibitor propargylglycine worsened outcomes, whereas treatment with the H₂S donor sodium hydrosulfide (NaHS) improved outcomes (72). H₂S donors have demonstrated therapeutic benefit in a number of cardiovascular disease models (5, 7, 17, 26, 46).

H₂S has been shown to be endogenously produced by the heart and NaHS in ex vivo Langendorff preparations reduced heart rate (HR), coronary perfusive flow, and left ventricular pressure (17, 47). Administration of NaHS reduced action potential duration and decelerated sinus rhythm in the isolated rat atria and inhibited the second half of slow diastolic depolarization in the murine sinus node (1, 2). In isolated rat ventricular cardiomyocytes, Na₂S has been shown to block L-type Ca²⁺ channels, and in human pluripotent stem cell-derived cardiomyocytes, NaHS has been shown to inhibit slow and rapid delayed rectifier K⁺ channels and hyperpolarization-activated inward current (I_f) (59, 63). Inhalation of H₂S in mice decreased respiratory rate, induced sinus bradycardia, and decreased cardiac output while maintaining stroke volume and mean arterial pressure (MAP) for several hours (61).

In addition to inducing a nonvagally mediated decrease in HR, H₂S can induce changes in respiration that include stimulatory effects at low doses and apnea at higher doses (18, 19, 24, 31). It has been demonstrated that an afferent signal sent from the lungs through the vagus is primarily responsible for modulating the apneic response to H₂S in the rat (3). Inhalation of NaHS has been shown to sensitize rat capsaicin-sensitive lung vagal neurons to chemical and mechanical stimuli by increasing Ca²⁺ transients (21).

It has been reported that ATP-sensitive K⁺ (K_ATP) channel activation is a major mechanism by which H₂S and H₂S donors relax vascular smooth muscle and that other pathways may be involved (9, 13, 26, 68–70). In addition to relaxing vascular smooth muscle and decreasing systemic arterial pressure, it has been reported that H₂S donors can decrease HR and that a methylene blue-sensitive effect on L-type Ca²⁺ channels is a major target for H₂S toxicity (19, 27). Typically, a decrease in systemic arterial pressure will normally induce a baroreceptor-mediated increase in HR. H₂S donor-induced hypotension has been shown to be accompanied by a decrease in HR that was insensitive to parasympathetic blockade by atropine (67).

The present study was undertaken to investigate the effects of the H₂S donor Na₂S on the cardiac electrophysiology of the rat and to determine the relationship between changes in MAP and HR in a cardiac pacing model. In addition, the role of changes in respiratory function in mediating the cardiovascular response to the H₂S donor were investigated in animals with normal spontaneous respiration and in mechanically ventilated animals. The roles of K_ATP channels, a methylene blue-sensitive effect on L-type Ca²⁺ channels, the direct effect of L-type Ca²⁺, and the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel’s effect on I_f in mediating cardiovascular responses to the H₂S donor were investigated. Chronic treatment with NaHS for 5 wk has been shown to significantly reduce MAP in spontaneously hypertensive rats (SHRs), and to a greater degree than in Wistar-Kyoto rats (71). In the last set of experiments, the effect of acute administration of Na₂S in the SHR was investigated. The results of the present study provide electrocardiographic evidence that the major effect of Na₂S is on the sinus node and that decreases in MAP and HR can be dissociated. The present data indicate that changes in respiratory function do not have a major effect in mediating cardiovascular responses to the H₂S donor at the doses used in the present study. Cardiovascular responses to Na₂S are not mediated by K_ATP channel activation, a methylene blue-sensitive effect on L-type voltage-sensitive channels, or the HCN channel. Acute responses to Na₂S appear to be more robust in the SHR than the Sprague-Dawley (S-D) rat.

MATERIALS AND METHODS

All experiments were approved by the Institutional Animal Care and Use Committee of the Tulane University School of Medicine, and all procedures were conducted in accordance with institutional guidelines. Animals were maintained on a 12:12-h light-dark cycle and were provided food and water ad libitum. In these experiments, 64 male S-D rats (275–493 g) and 8 SHRs (291–349 g) were anesthetized with an intraperitoneal injection of thiobutabarbital (Inactin, Sigma-Aldrich, St. Louis, MO) in a dose of 100 mg/kg. Supplemental doses of Inactin were administered intraperitoneally to maintain a uniform level of anesthesia. Animals were placed in the supine position, and body temperature was maintained with the use of Deltaphase Isothermal Pads (Braintree Scientific, Braintree, MA). The trachea was cannulated with a short segment of polyethylene (PE)-240 tubing to maintain a patent airway. In experiments measuring respiratory frequency and airflow amplitude, the tracheal tube was connected to a pneumotachometer (RX237B, Biopac, Santa-Barbara, CA), and the animals spontaneously breathed room air. In experiments where the animal was artificially ventilated, the tracheal tube was connected to a ventilator (model 683 Small Animal Ventilator, Harvard Apparatus, Holliston, MA) and ventilated at a tidal volume of 10 ml/kg at a rate of 70 breaths/min with 100% O₂; decamethonium (Sigma-Aldrich) was administered at a dose of 0.8 mg/kg iv with supplemental doses of 0.4 mg/kg administered as needed to induce neuromuscular blockade. In all other experiments, the animals spontaneously breathed room air enriched with 100% O₂.

A lead II electrocardiogram was measured with 12-mm needle electrodes (EL452, Biopac) placed subcutaneously with the negative electrode placed near the right shoulder, the positive electrode placed to the left of the xyphoid space, and the ground electrode placed near the left shoulder. The left jugular vein was catheterized with PE-50 tubing for the systemic injection of drugs and fluids. The left carotid artery was cannulated with PE-50 tubing, and systemic arterial pressure was measured with a pressure transducer (Namic Perceptor DT, Navilyst Medical, Malborough, MA). For experiments with cardiac pacing, the right jugular was cannulated and a bipolar pacing catheter (REF 401769, St. Jude Medical, St. Paul, MN) was advanced to the right atrium, and the position was confirmed with fluoroscopy (OEC 6600, GE, Boston, MA). The heart was paced with a Grass stimulator (model S88, Grass Medical Instruments, Quincy, MA) and isolation unit at a voltage which was 10–15% above threshold with 5-ms pulses at rates of 347−488 beats/min. Systemic arterial pressure and MAP were obtained by electronic averaging of the pressure signal, and the HR signal was measured from the pressure signal. Hemodynamic, electrocardiographic, and respiratory data were continuously recorded and displayed with a data-acquisition system (MP 100A-CE, Biopac) and stored on a personal computer. Bazett’s formula was used for Q-T correction as follows: QT_c(n)-B = QT/(RR/f)(1/2), where QT_c is the corrected QT interval and f = 150 ms (32). At the conclusion of the experiments, anesthetized animals were euthanized with an intravenous injection of saturated KCl solution, and a bilateral thoracotomy was performed to ensure euthanasia.

Drugs.

Na₂S, diltiazem, decamethonium, and methylene blue (Sigma-Aldrich) were dissolved in 0.9% saline. Glybenclamide (Sigma-Aldrich) was dissolved in 4% glucose and 0.1 N NaOH solution. Ivabradine (Corlanor, Amgen, Thousand Oaks, CA) was suspended in sterile PBS and administered by oral gavage. The dosages used in this study were determined from dosages used in previous studies and pilot experiments in our laboratory (10, 27, 39, 41, 53, 58, 67). New measurement techniques for sulfide pools have been developed, and considerable debate in the literature has focused on ascertaining the true physiological levels of H₂S (15, 34, 44, 64). The rapid clearance of free H₂S from the blood has led to concerns regarding concentrations used in some recent studies (18, 31, 54, 64). Administration of sulfide donors in dosages less than or equal to those in the present study have shown therapeutic benefit in a number of in vivo experimental models (4, 25, 33, 38, 45, 52).

Statistics.

Data are expressed as means ± SE and were analyzed using Student’s t-test for paired and unpaired data; n is the number of animals. A P value of <0.05 was used as the criterion for statistical significance.

RESULTS

The effect of Na₂S in doses of 0.1–0.75 mg/kg iv on MAP, HR, respiratory amplitude, and respiratory frequency are shown in Fig. 1A. Significant decreases in MAP and HR were observed at all doses of Na₂S compared with baseline. Injection of Na₂S in doses of 0.3–0.75 mg/kg iv significantly increased respiratory amplitude, whereas respiratory frequency was increased at all doses compared with baseline. The injections of Na₂S in a dose of 0.75 mg/kg iv were notable in that after periods of increased respiratory amplitude and frequency, transient apneic periods occurred in six of the eight rats (mean duration: 30.73 ± 9.107 s). Records from an experiment illustrating the observed apnea are shown in Fig. 1B.

Fig. 1.

A: bar graphs showing the effect of intravenous injections of Na₂S at doses of 0.1–0.75 mg/kg on mean arterial pressure, heart rate, respiratory frequency, and respiratory amplitude. *P < 0.05 compared with baseline, paired comparison; n = 8. B: records from an experiment showing the effect of intravenous injection of Na₂S at 0.75 mg/kg iv on systemic arterial pressure, heart rate, and respiratory airflow. Transient apnea was observed in six of eight animals (mean duration: 30.73 ± 9.107 s) at the 0.75 mg/kg dose.

Effect of respiration.

To investigate the relationships between changes in respiratory function and cardiovascular responses to the H₂S donor, the decreases in MAP and HR were compared during normal spontaneous respiration and when the rats were ventilated at constant rate and volume with a positive pressure respirator (Fig. 2A). The decreases in HR in response to Na₂S were not significantly different in doses of 0.1–0.75 mg/kg iv, but, interestingly, the decreases in MAP in the response to intravenous injections of Na₂S were significantly increased at the 0.75 mg/kg dose and not significantly different at doses of 0.1–0.5 mg/kg. Records from an experiment illustrating reductions in MAP and HR are shown in Fig. 2B.

Fig. 2.

A: bar graphs comparing the effect of intravenous injections of Na₂S at doses of 0.1–0.75 mg/kg on mean arterial pressure and heart rate during spontaneous breathing and mechanical ventilation. *P < 0.05, paired comparison; n = 6. B: records from an experiment showing the effect of intravenous injection of Na₂S at 0.50 mg/kg on mean arterial pressure and heart rate.

Effect on the electrocardiogram.

The effect of Na₂S on the electrocardiogram in doses of 0.1–0.5 mg/kg iv was investigated and significantly increased RR intervals at the 0.3 and 0.5 mg/kg iv doses but had no significant effect on the PR interval or the QT_c(n)-B interval (Fig. 3A). Records from an experiment showing the baseline electrocardiogram are shown in Fig. 3B; records during the Na₂S-induced bradycardia are shown in Fig. 3C.

Fig. 3.

A: bar graphs showing the effect of intravenous injections of Na₂S on RR, PR, and corrected QT(n)-B [QT_c(n)-B] intervals on the electrocardiogram. *P < 0.05, paired comparison; n = 8. B and C: records from an experiment showing the baseline electrocardiogram (B) and the electrocardiogram during 0.5 mg/kg iv Na₂S-induced bradycardia (C).

Effect of cardiac pacing.

The relationship between the changes in MAP and HR were investigated in experiments in which the HR was paced in both spontaneously breathing (Fig. 4A) and mechanically ventilated rats (Fig. 4B). Intravenous Na₂S administration in doses of 0.1–0.5 mg/kg in both paced spontaneously breathing and paced mechanically ventilated rats produced decreases in MAP that were not different from decreases in MAP in respective unpaced controls, whereas changes in HR were abolished with pacing.

Fig. 4.

Bar graphs showing the effect of cardiac pacing on the decrease in mean arterial pressure and heart rate in response to intravenous injections of Na₂S at doses of 0.1–0.5 mg/kg in spontaneously breathing animals [*P < 0.05, paired comparison; n = 5 (A)] and mechanically ventilated animals [*P < 0.05, paired comparison; n = 5 (B)]. The decreases in mean arterial pressure in response to Na₂S were not different than control responses in the unpaced heart at doses of 0.1–0.5 mg/kg iv. The decreases in mean arterial pressure at doses of 0.1–0.5 mg/kg iv were associated with no change in heart rate in response to the intravenous injections of Na₂S. C: records from an experiment illustrating a decrease in mean arterial pressure in response to a 0.5 mg/kg iv injection of Na₂S in a mechanically ventilated and paced animal.

Effect of glybenclamide.

It has been previously reported that vasorelaxant responses to the H₂S donor are blocked by glybenclamide, suggesting that K_ATP channel activation is a major mechanism by which H₂S relaxes vascular smooth muscle (68). However, glybenclamide did not attenuate responses to the H₂S donors in the intact anesthetized rat (67). In a previous study, glybenclamide was solubilized using organic solvents such as DMSO, which could potentially influence the response (16). In the present study, glybenclamide was solubilized using a glucose-sodium hydroxide vehicle, which had no significant effect on MAP or HR. Rats treated with glybenclamide at a high dose of 20 mg/kg iv demonstrated no significant difference in response to intravenous injections of Na₂S at lower doses compared with control, but the 0.5 mg/kg dose significantly increased MAP and HR responses compared with control (Fig. 5A). Interestingly, this potentiated response after glybenclamide administration was associated with the development of a bigeminal rhythm in seven of seven of the rats (mean duration: 10.3 ± 1.37 s) treated with 0.5 mg/kg iv Na₂S (Fig. 5, B and C).

Fig. 5.

A: bar graphs showing the effect of a high dose of glybenclamide on the decrease in mean arterial pressure and heart rate in response to intravenous injections of Na₂S in doses of 0.1–0.5 mg/kg. *P < 0.05, paired comparison; n = 7. B: records from an experiment illustrating the changes in systemic arterial pressure, mean arterial pressure, heart rate, and electrocardiogram in response to an injection of 0.50 mg/kg iv Na₂S in a glybenclamide-treated animal. C: records from an experiment showing high-resolution electrocardiographic traces showing the baseline sinus rhythm and the bigeminal rhythm when Na₂S was injected at a dose of 0.5 mg/kg iv. A bigeminal rhythm was noted in seven of seven rats (mean duration: 10.3 ± 1.37 s) treated with a dose of 0.5 mg/kg.

Effect of methylene blue, diltiazem, and ivabradine.

It has been previously reported that methylene blue can be used as a rescue treatment for the fatal cardiotoxic effects of NaHS infusion on the heart by an effect on voltage-dependent Ca²⁺ channels in anesthetized rats (27). The effect of methylene blue pretreatment on cardiovascular responses to Na₂S was investigated, and these data are shown in Fig. 6A. Intravenous injection of methylene blue at a single dose of 5 mg/kg or multiple doses of 5 mg/kg did not change the decreases in MAP or HR in response to intravenous injections of Na₂S at doses of 0.1–0.5 mg/kg. Intravenous injection of the L-type voltage-dependent Ca²⁺ channel antagonist diltiazem at a dose of 2 mg/kg did not alter the decreases in MAP or HR in response to intravenous injections of Na₂S (Fig. 6B).

Fig. 6.

A: bar graphs showing the effect of methylene blue at a dose of 5 mg/kg iv on the decreases in mean arterial pressure and heart rate in response to intravenous injections of Na₂S at a dose of 0.1–0.5 mg/kg (paired comparison; n = 7). B: effect of the L-type voltage-dependent Ca²⁺ entry blocking agent diltiazem on the decreases in mean arterial pressure and heart rate in response to intravenous injections of Na₂S at doses of 0.1–0.5 mg/kg (paired comparison, n = 7). C: effect of the hyperpolarization-activated inward current (I_f) channel inhibitor ivabradine on the decrease in mean arterial pressure and heart rate in response of intravenous injections of Na₂S (unpaired comparison; n = 5 control and n = 6 ivabradine).

It has been suggested in the literature that H₂S donors may modulate I_f (63). Treatment with the HCN channel antagonist ivabradine, which modulates I_f at a dose of 10 mg/kg by gavage for 3 days, did not attenuate the decrease in MAP or HR in response to intravenous injections of Na₂S in the anesthetized rat (Fig. 6C).

Effect of Na₂S on SHRs.

The effect of Na₂S at doses of 0.1–0.5 mg/kg iv on MAP and HR in SHRs are shown in Fig. 7. Intravenous injections of the H₂S donor produced dose-dependent significant decreases in MAP and HR in SHRs that were more robust than those observed in S-D rats.

Fig. 7.

Bar graphs showing the effect of intravenous injections of Na₂S at doses of 0.1–0.5 mg/kg on mean arterial pressure (A) and heart rate (B) in spontaneously hypertensive rats (SHRs). *P < 0.05 compared with baseline, paired comparison; n = 8.

DISCUSSION

The present study found that the H₂S donor Na₂S decreases systemic arterial pressure and HR in anesthetized rats and is consistent with previous studies (20, 56, 66, 67). The major findings in the present study indicate that the effects on arterial pressure and HR can be separated by cardiac pacing. In experiments where atrial pacing was used, the Na₂S-induced decrease in MAP was preserved, whereas the decrease in HR was abolished, which suggests that the bradycardic response of Na₂S in the anesthetized rat may be primarily mediated by the sinoatrial node. It has been previously reported that the decrease in HR in response to Na₂S is not blocked by atropine, suggesting that it is not mediated by an increase in vagal tone (67). In the present study, the decrease in HR was associated with no change in the PR or QT_c(n)-B intervals on the electrocardiogram. The results of the electrocardiogram experiments are consistent with the results of experiments with atropine and are also consistent with the hypothesis that the major effects of the H₂S donor Na₂S on HR are mediated by an effect on the sinus node.

It has been previously reported that H₂S can produce respiratory stimulation and, at higher doses, apnea (19). In the present study, intravenous injections of Na₂S at most doses significantly increased respiratory amplitude and significantly increased respiratory rate at all dosages. The 0.75 mg/kg dose was noted to produce transient apnea in some experiments. The Na₂S-induced bradycardia was not different between spontaneously breathing and mechanically ventilated animals, and the decreases in MAP were similar at all doses but the 0.75 mg/kg dose, in which the mechanically ventilated animals exhibited a greater decrease in MAP. These data suggest that the respiratory changes do not play a substantial role in mediating the hypotension and bradycardia observed in the present study.

It has been previously reported that H₂S donors decrease total peripheral resistance in the anesthetized rat, and the mechanism of the vasodilator response to H₂S has been investigated in many studies (9). Although a previous study indicated that the decrease in systemic arterial pressure and HR in response to H₂S donors were not attenuated by the K_ATP channel antagonist glybenclamide, it has been reported that K_ATP channel activation is a major mechanism by which H₂S relaxes vascular smooth muscle (9, 13, 26, 68–70). H₂S donors may also have central effects, and microinjections of NaHS into the nucleus tractus solitarius produced hypotension and bradycadia that was attenuated with prior intranucleus tractus solitarius administration of glybenclamide and the nonselective glutamate antagonist kynurenic acid (49). In an attempt to clarify the role of K_ATP channel activation in mediating responses to H₂S, a different method that does not require the use of organic solvents such as DMSO to prepare glybenclamide solutions was used in the present study. It has been previously reported that systemic administration of glybenclamide may fail to achieve therapeutic levels in the central nervous system, and concerns have been raised about neuronal toxicity in centrally administered H₂S donors (37, 54). The potentiated responses observed at the 0.5 mg/kg dose may be a result of the occurrence of runs of a bigeminal rhythm in the anesthetized rat suggesting a potential interaction of Na₂S and K_ATP channels, although the lack of blockade at the lower dosages in the absence of an arrhythmia suggests that peripheral K_ATP channel activation does not mediate the hypotensive or bradycardic response to the H₂S donor in the anesthetized rat (8, 30). Genetic disruption of the K_ATP channel has been shown to increase susceptibility to catecholamine induced after depolarizations, and it is possible that adrenergic reflexes in response to Na₂S-induced hypotension may have contributed to the observed dysrhythmia noted at the 0.5 mg/kg iv dose (40).

In the development of effective countermeasures against acute H₂S intoxication, studies in the literature have indicated that methylene blue counteracts fatal H₂S infusion-induced cardiac depression by restoring L-type Ca²⁺ channel activity (19, 27, 55). The decreases in arterial pressure and HR in response to intravenous injections of Na₂S were not attenuated by methylene blue at a dose of 5 mg/kg iv, which was used in the previous study, or by repeated injections of methylene blue at this dose (27). These results suggest that the decreases in arterial pressure and HR in response to the H₂S donor Na₂S in the range of doses used in the present study were not mediated by a methylene blue-sensitive mechanism.

It has also been previously reported that changes in L-type Ca²⁺ channel activity may be involved in mediating H₂S toxicity-induced cardiac depression (27). However, in the present study, the L-type Ca²⁺ channel antagonist diltiazem did not attenuate the decreases in systemic arterial pressure or HR in response to Na₂S, suggesting that H₂S-induced hypotension and bradycardia may not involve an effect on L-type Ca²⁺ channels.

Ivabradine is a blocker of the HCN channel that contributes to I_f in the sinus node and decreases HR in patients with heart failure (60). NaHS has been observed to modulate I_f in in vitro studies (63). Treatment with ivabradine at a dose of 10 mg/kg by gavage for 3 days did not alter responses to Na₂S, suggesting that the decrease in HR in response to the H₂S donor is not mediated by activation of the I_f channel in the sinus node of the rat.

In conclusion, the results of the present study show that decreases in systemic arterial pressure and HR in response to intravenous injections of the H₂S donor Na₂S can be dissociated using cardiac pacing to prevent bradycardia. The present data show that changes in respiration do not contribute to the decreases in arterial pressure and HR in that similar decreases were observed during normal spontaneous ventilation and in mechanically ventilated rats. The decreases in systemic arterial pressure and HR could not be blocked by different formulations and high doses of glybenclamide, suggesting that K_ATP channel activation is not a major mechanism mediating hypotensive or bradycardic responses to the H₂S donor in the intact rat model. The cardiovascular responses to Na₂S were not attenuated by methylene blue, ivabradine, or diltiazem, suggesting that a methylene blue-H₂S chemical reaction or that an effect on the HCN channel in the sinus node or L-type Ca²⁺ channels are not involved in mediating responses to the H₂S donor. The hemodynamic responses observed in SHRs were more robust than those in S-D rats. Although this is consistent with chronic studies between Wistar-Kyoto and SHRs, some of the observed difference in the present study may be due to differences between the Wistar-Kyoto and S-D background. Because of the relatively narrow therapeutic window of exogenously administered sulfide donors, future development of slow-release direct or indirect sulfide donors with suitable pharmacokinetics may hold promise in the treatment of many pathological processes.

GRANTS

This work was supported by American Heart Association National Center Scientist Development Grant 14SDG20490359 (to P.V.G.K) and National Institutes of Health Grants NS-094834 (to P.V.G.K) and HL-62000 (to P.J.K).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

K.W.S., B.M.S., and P.K. conceived and designed research; K.W.S. and B.M.S. performed experiments; K.W.S., B.M.S., A.L.C., T.J.C., R.A.C., B.T.G., and P.K. analyzed data; K.W.S., B.M.S., P.V.G.K., E.K.K., T.D.G., and P.K. interpreted results of experiments; K.W.S., B.M.S., A.L.C., T.J.C., and P.K. prepared figures; K.W.S., B.M.S., and P.K. drafted manuscript; K.W.S., B.M.S., A.L.C., T.J.C., R.A.C., B.T.G., P.V.G.K., E.K.K., T.D.G., and P.K. edited and revised manuscript; K.W.S., B.M.S., A.L.C., T.J.C., R.A.C., B.T.G., P.V.G.K., E.K.K., T.D.G., and P.K. approved final version of manuscript.