Redox biology of hydrogen sulfide: Implications for physiology, pathophysiology, and pharmacology

Abstract

Hydrogen sulfide (H₂S) has emerged as a critical mediator of multiple physiological processes in mammalian systems. The pathways involved in the production, consumption, and mechanism of action of H₂S appear to be sensitive to alterations in the cellular redox state and O₂ tension. Indeed, the catabolism of H₂S through a putative oxidation pathway, the sulfide quinone oxido-reductase system, is highly dependent on O₂ tension. Dysregulation of H₂S homeostasis has also been implicated in numerous pathological conditions and diseases. In this review, the chemistry and the main physiological actions of H₂S are presented. Some examples highlighting the cytoprotective actions of H₂S within the context of cardiovascular disease are also reported. Elucidation of the redox biology of H₂S will enable the development of new pharmacological agents based on this intriguing new redox cellular signal.

Graphical abstract

Highlights

► Hydrogen sulfide (H₂S) is an endogenously produced cell signaling molecule, which plays a key role in multiple physiological processes including vasodilation, angiogenesis, and inflammation. ► The production and consumption of H₂S in vivo is redox sensitive. ► Dysregulation of H₂S production and consumption contributes to disease pathogenesis. ► Cytoprotective properties of H₂S may be due, in part, to restoration of cellular redox status and upregulation of antioxidant defenses.

1. Introduction

Hydrogen sulfide (H₂S); a toxic gas, is endogenously produced, bioactive, and contributes to numerous physiological functions in mammalian systems. Studies support the possibility that H₂S has therapeutic potential for treating multiple diseases including cardiovascular diseases. For example, experimental animal studies show that H₂S may be effective in treating atherosclerosis and protecting against ischemia-reperfusion injury [1–3]. Interest in the cytoprotective actions of H₂S has grown since the discovery that it can induce a hypometabolic state characterized by decreased O₂ consumption, heart rate, and body temperature in non-hibernating rodents [4]. Although not discussed in this review, H₂S-dependent hypometabolism is an O₂-dependent phenomenon [5]. The proposed mitochondrial and signaling actions of H₂S make this molecule an attractive intervention for preventing and treating diseases and trauma-associated injuries. In this review article, we provide an overview of H₂S redox biology as it relates to the biological and pharmacological actions of this interesting new signaling molecule in mammalian systems.

2. Historical benefits of H₂S

The ancient Greeks, Egyptians, and Romans regularly bathed in natural sulfur springs as treatments for disease [6]. Depending on the microbiota and oxygen content, sulfur springs typically contain H₂S concentrations ranging from 1 to 500 μM [7] with anti-inflammatory, anti-bacterial, vasodilatory, and anti-fungal properties attributed to the sulfur-containing water [8]. Epidemiological studies report that a diet rich in organosulfur species is associated with longevity and decreased morbidity [9]. Members of the Allium genus (garlic and onions), which contain organosulfur compounds have a well-documented history of health benefits [10]. Indeed, garlic-derived compounds such as diallyl trisulfide release H₂S in the presence of cellular reductants like glutathione (GSH) [11]. Populations that consume garlic regularly have low blood pressure, low cholesterol, and less vascular disease [12]. While administration of exogenous sulfur-containing compounds shows strong promise as therapies, H₂S is also endogenously produced in many different human tissues.

3. Endogenous production of H₂S

In the early 1990s, it was discovered that H₂S is enzymatically produced by two cytosolic enzymes; cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) [13,14]. Seminal work of Abe and Kimura showed, for the first time, that H₂S enhances long-term potentiation in the hippocampus [15]. Specifically, they demonstrated that H₂S was produced by CBS and that exogenous H₂S enhanced NMDA receptor-mediated responses. Since then many studies have shown that CBS and CGL are expressed in human tissues with H₂S contributing to physiological and pathophysiological processes (Table 1). In addition to CBS and CGL, there are other enzymes that produce H₂S with several utilizing cysteine as a substrate. The enzyme 3-mercaptopyruvate S-transferase (3-MST) is found in mitochondria and cytosol and produces H2S [16]. Several H2S producing enzymes are pyridoxal-5′-phosphate (PLP) dependent enzymes [17]. Moreover, other sulfur-containing amino acids, such as cystine and homocysteine, can be metabolized to generate H₂S. The enzymatic mechanisms of H₂S production are shown in Fig. 1. Many of these enzymes participate in the cellular sulfur cycle and have multiple enzymatic activities, including H₂S generation. Because the concentration of reduced sulfur species has an effect on many cellular processes [18], the activity of these enzymes is tightly regulated. Much of this regulation is linked to substrate availability [19]. Moreover, these are redox-sensitive enzymes, which exhibit increased activity under oxidative conditions [20]. Considering that H₂S; a reductant, is a product of these enzymes, it is conceivable that enzymatic activity may also be subject to negative feedback regulation. Finally, work by Wang et al. suggests that under certain conditions, such as oxidative stress, H₂S-producing enzymes translocate from the cytosol to mitochondria [21]. This dynamic regulation bolsters the argument that H₂S may function as a redox signaling molecule.

Table 1.

Biological and therapeutic actions of H₂S are O₂-dependent.

Biological context	Low O2	High O2
Inflammation	Anti-inflammatory: IL-10; ↓ IL-6, ICAM	Pro-inflammatory: ↑ NF-κB, TNF-α
Vasoactivity	Vasodilatory: ↑ KATP channel conductance	Vasoconstrictive
Angiogenesis	Pro-angiogenic: VEGF, Hif1-α	No effect
Respiratory inhibition	Higher [H2S] results in more inhibition	O2 acts as an H2S antagonist: ↑SOx
Ischemia-reperfusion	Ischemic tissue has higher [H2S]: mito KATP, ARE genes	Unknown

Fig. 1.

The enzymatic production of H₂S. The two primary enzymes responsible for H₂S production, cystathionine-γ-lyase (CGL) and cystathionine-β-synthase (CBS), are found in the cytosol. CBS catalyzes the first step in H₂S production through the transsulfuration of homocysteine to cystathionine. CGL in an elimination reaction catalyzes the formation of cysteine and α-ketobutyrate. Cysteine is the substrate from which H₂S is directly produced either through elimination (CGL) or β-replacement (CBS). Cysteine amino transferase (CAT) catalyzes the formation of 3-mercaptopyruvate, a substrate for the mitochondrial enzyme 3-mercaptopyruvate-S-transferase (3-MST). 3-MST can directly produce H₂S, albeit at lower levels than CBS and CGL, in mitochondria.

4. Catabolism of H₂S

Several regulated and unregulated non-enzymatic processes participate in H₂S catabolism. These pathways maintain in vivo H₂S concentrations, most likely, in the nM to low μM range. H₂S can react with heme proteins in mitochondria and therefore H₂S can function as a mitochondrial respiratory toxicant [22,23]. Fatal industrial accidents have been documented in individuals exposed to high concentrations of H₂S gas (e.g., >1000 pm) [24]. Therefore, the toxicological profile of H₂S has been well-studied and documented [24]. The mechanism of toxicity is through the binding of H₂S to cytochrome c oxidase (CcO) mediating respiratory inhibition [22]. However, this interaction is complex and poorly understood because H₂S can act as both an inhibitor and an electron donor for CcO [25]. H₂S binds to the oxidized states of the heme a-a₃ binuclear center, resulting in the reduction of the heme molecules [26]. Excess H₂S can also reduce Cu_B [27]. While the stoichiometry may vary, Cooper and Brown reported that 3 molecules of H₂S bind per inhibited CcO [28]. In this inhibitory reaction, H₂S is oxidized to sulfane sulfur and this is coupled to consumption of molecular O₂ [28]. Unlike nitric oxide (^•NO), the inhibition of CcO by H₂S is noncompetitive with O₂ [27,29]. In addition, H₂S can also directly reduce the electron carrier cytochrome c producing the one electron oxidation product, the thiyl radical (^•SH) [28].

Rhodanese, a mitochondrial sulfur transferase enzyme, catalyzes the oxidation of H₂S [30]. It is one part of three enzymatic activities characterized as a major pathway for H₂S catabolism. This pathway consists of a sulfide quinone oxido-reductase (SQR), a sulfur dioxygenase, and the sulfur transferase enzyme rhodanese (Figs. 2 and 3). H₂S reduces the external disulfide on the SQR to form a thiol (RSH) and a perthiol (RSSH). This two electron oxidation of H₂S reduces the FAD prosthetic group, which uses ubiquinone (Q) as an electron acceptor [31] The second sulfur atom on the perthiol is a reactive sulfane (S⁰), which is oxidized by a sulfur dioxygenase enzyme encoded by the gene ETHE1, consuming O₂ and H₂O to form sulfite (SO₃⁻²). While the protein responsible for this enzymatic activity is not known, the ETHE1 gene encoding the protein has been identified. Mutations in this gene cause a buildup of H₂S leading to ethylmalonic encephalopathy [32,33]. Rhodanese then transfers a sulfane sulfur to sulfite to form thiosulfate (S₂O₃⁻²) [34]. This proposed oxidation pathway, in close proximity to CcO, functions as a major clearance pathway of cellular H₂S. H₂S can also be oxidized by non-mitochondrial heme proteins such as hemoglobin (Hb) and myoglobin [35]. H₂S will reduce the ferric iron in met-Hb, restoring the oxygen binding abilities of the protein [36]. At high concentrations of H₂S, sulf-Hb can also be formed from oxy-Hb [37]. While displaying very weak affinity for O₂, sulf-Hb can still deliver O₂, albeit with no cooperativity [38]. As a result, the bioavailability of H₂S, whether in the context of steady state in vivo concentrations or exogenously administered, is dictated by the O₂ concentration. Therefore, O₂ can be considered an H₂S antagonist, accelerating its oxidation and attenuating its biological actions. The effect of O₂ on H₂S concentration is both direct and indirect. The spontaneous reaction of H₂S with O₂, while slow, can cause an appreciable decrease in the H₂S concentration. Thus, tissues with relatively high O₂ concentrations (e.g., alveolar epithelium) may have less H₂S compared to tissues that are in a lower O₂ environment (e.g., centrilobular region of liver). This has implications in pathological states of hypoxia such as ischemia-reperfusion, where the availability, and thus the signaling effects of H₂S may be augmented. Furthermore, O₂ concentration can indirectly affect H₂S concentration through changes in the redox state of heme proteins. Proteins such as Hb will react with H₂S at different rates depending on the redox status of the hemes. For example, H₂S will react more rapidly with met-Hb (Fe⁺³) than with deoxy-Hb (Fe⁺²) [36].

Fig. 2.

Proposed pathways of H₂S removal in mammalian cells. The physiological steady-state concentration of H₂S in vivo is believed to be maintained in the sub-micromolar range. This steady state concentration is established by the production pathways shown in Fig. 1 and the proposed consumption pathways shown within this figure. H₂S will react non-enzymatically with many biomolecules such as reactive oxygen and nitrogen species, electrophilic lipids like 4-hydroxy-2-nonenal, free heme, and disulfide bonds to form a thiol and perthiol. The catabolism of H₂S can also be catalyzed enzymatically by the sulfide quinone oxido-reductase system (SQR) comprised by sulfur dioxygenase, rhodanese, and sulfur quinone reductase.

Fig. 3.

The oxidation of H₂S by the sulfide quinone oxido-reductase system in mitochondria. H₂S reduces the disulfide composed of the vicinal thiols on the sulfide quinone reductase (SQR) forming a thiol and a perthiol. The second sulfur atom on the perthiol, the sulfane sulfur (S⁰), is the substrate for both the sulfur transferase enzyme, rhodanese, and the sulfur dioxygenase enzyme encoded by the gene ETHE1. Rhodanese catalyzes the formation of thiosulfate (S₂O₃⁻²) from sulfite (SO₃⁻²) and S⁰. ETHE1 catalyzes the formation of SO₃⁻². The reduced SQR can then transfer electrons into the ubiquinone (Q) pool, thus coupling the oxidation of H₂S to electron transfer, H⁺ pumping, and ultimately ATP synthesis.

Because H₂S is a nucleophile, it can also react with electrophilic lipids [39]; and the thiolate anion, HS⁻, can also reduce disulfide bonds (Fig. 2) [40]. Indeed, the exfoliation of skin cells in hot sulfur springs is due to H₂S reducing the structural disulfide bonds of cellular junctions in keratinocytes [8]. While this can be harmful at high concentrations, the reduction of external disulfide bonds by H₂S may, in some instances, reverse a deleterious post-translational protein modification. Although still contentious, the S-sulfhydration of cysteine residues may represent an important sink for free H₂S [41]. In theory, H₂S can also reduce higher thiol oxidation states such as S-nitrosothiols and sulfenic acids [42]. H₂S can also be methylated by the cytosolic enzyme thiol-S-methyltransferase to form methane thiol. As with virtually all molecules, H₂S can react with other free radical species, as well as, a number of non-radical reactive oxygen (ROS) and nitrogen (RNS) species (Fig. 2) [43]. Many of the oxidized sulfur species as well as sulfur-centered radicals formed are less reactive than their oxygen-containing counterparts [44]. One of the most important oxidants responsible for the catabolism of H₂S is O₂. In the presence of molecular O₂ and redox active metals, H₂S will spontaneously oxidize [45]. In an oxygenated biological medium, metalloproteins catalyze H₂S oxidation. This makes O₂ tension a critical methodological consideration when conducting biologically relevant experiments.

5. Measurement of H₂S in mammalian samples

As the field of H₂S continues to grow, accurate measurement of H₂S in biological samples is critical for proper understanding of its biochemistry and identifying its key physiological roles. Early studies reported endogenous H₂S concentrations in mammalian tissues to be approximately 160 μM [46]. However, when considering that the K_i of CcO for H₂S is 0.2 μM [28] it is unlikely that free H₂S exists at high μM concentrations. Older methods relied on inducing large pH shifts during sample preparation followed by capturing the released sulfide anions with metals such as silver or zinc [47] and then measuring these complexes by spectrophotometry or chromatography [46]. One problem with these methods is that they liberate multiple acid-labile sulfur species, including those in Fe–S centers, giving artificially high values. Recently, the mono-bromobimane assay has been refined to exclude sulfur species other than free H₂S [47]. As techniques improve, the reported concentrations of H₂S in mammalian samples have decreased from the 100–200 μM range to less than 500 nM [48]. Other techniques, including polarography, have measured H₂S concentrations in mouse blood in the sub-μM range (Stein et al., unpublished data). These lower values are more plausible when considering the inhibitory potential of H₂S for heme-containing proteins, in addition to, parallels between H₂S and other signaling molecules such as ^•NO and carbon monoxide (CO). It is also important to note that whole tissue or cell lysate measurements of H₂S overlook compartmental differences in H₂S concentrations and thus likely underestimate the effective localized concentrations of H₂S in vivo. Because the technology required to accurately measure H₂S on the sub-cellular level is unavailable, it is important to recognize these limitations when considering physiologically relevant levels and reactions of H₂S.

6. Biological roles of H₂S

Like ^•NO and CO, H₂S is produced in many different cell types and can easily diffuse without the need for transporters. Therefore, it is not surprising that H₂S has diverse biological actions (Fig. 4). Important factors that determine the biological actions of H₂S include, but are not limited to, differences in the solubility of H₂S in aqueous vs. lipid phases, proximity of the target to H₂S-detoxifying enzymes, heme redox state, and inter- and intracellular differences in O₂ tension.

Fig. 4.

Interaction of O₂ and H₂S on physiological outcomes. H₂S plays a role in many physiological processes. Additionally, it is capable of acting as a therapeutic agent. The concentration of H₂S, whether endogenously produced or exogenously administered, will dictate the outcome. This can be beneficial at low and intermediate concentrations or harmful at high concentrations. High O₂ can reverse many of the beneficial roles of H₂S seen at lower O₂ concentrations, resulting in, for example, vasoconstriction rather than vasodilation. Additionally, under hypoxic and normoxic conditions, H₂S promotes angiogenesis. However, at higher concentrations of both O₂ and H₂S, an inhibition of cellular proliferation is seen. H₂S has a narrow therapeutic window within which it is cytoprotective. At high concentrations it can be pro-apoptotic and pro-inflammatory. Finally, the larger doses of H₂S necessary to induce a hypometabolic effect, can, if pushed further, result in cardiac and respiratory toxicity.

7. Vasoactivity

One of the first physiological roles that prompted investigators to regard H₂S as the “third gaseous signaling molecule” was vasodilation. In 2001, Zhao et al. showed that H₂S decreased blood pressure in rats in vivo and caused vascular smooth muscle cell (VSMC) relaxation in vitro [49]. H₂S-mediated vasodilation has also been shown in the ileum of the gastrointestinal tract and the vas deferens [50]. Additionally, others have shown that transgenic mice deficient in CBS are chronically hypertensive [51]. H₂S is thought to induce vasodilation by increasing the conductance of adenosine triphosphate (ATP) sensitive potassium channels (K_ATP). Furthermore, the specific molecular targets of H₂S were shown to be cysteine 6 and 26 of the extracellular portion of the rvSUR1 subunit of the K_ATP channel complex [52]. These vicinal thiols form a disulfide bond, which H₂S reduces, increasing channel conductance. However, the vasodilatory effect of H₂S is highly O₂-dependent as supra-physiological levels of O₂ (200 μM) cause H₂S-induced vasoconstriction [53]. Recently, conflicting reports have emerged showing that the contribution of the K_ATP channels to H₂S-induced vasodilation is minimal and that vasodilation is due to metabolic inhibition (i.e., decrease in ATP), intracellular pH changes, and modulation of Cl⁻/HCO⁻₃ channels [54]. There are several methodological differences including O₂ tension, pre-contraction agent, vessel type, and the type of sulfide-based chemical used, which may account for discrepancies among experimental studies. There are also indications that H₂S may act through ^•NO to stimulate vasodilation. Studies suggest that H₂S liberates ^•NO from S-nitrosothiols [55]. Others show that endothelial denudation and nitric oxide synthase (NOS) inhibitors shift the dose–response curve to the right [56]. However, H₂S increases eNOS phosphorylation and subsequent ^•NO production in an Akt-dependent manner [57]. While the mechanism(s) responsible for vasodilation remain unclear, the role of H₂S as a vasodilator is accepted and there is great interest in employing H₂S-releasing agents as therapies to treat hypertension.

8. Angiogenesis

H₂S can cause cell proliferation and migration [58,59]; however, there appears to be a narrow concentration range of the proliferative effect, below which no effect is seen and above which there is anti-proliferation and H₂S cytotoxicity [60]. In cell culture experiments, low micromolar concentrations of H₂S increase endothelial cell number, proto-vessel formation, and cell migration [58]. Chicken chorioallantoic membranes, an in vivo model of angiogenesis, display increased branching and lengthening of blood vessels in response to 48 h incubation with H₂S [59]. Additionally, aortic tissue isolated from transgenic mice lacking CSE, the primary H₂S-producing enzyme in the endothelium, exhibit marked decreases in angiogenesis [59].

The mechanism of H₂S-induced angiogenesis operates through several pathways, including activation of ATP-sensitive potassium (K_ATP) channels [49]. Papapetropoulos et al. showed that treatment of endothelial cells with the K_ATP channel inhibitor glibenclamide reduced cell migration, which was accompanied by decreased H₂S-induced p38 and heat shock protein 27 (Hsp27) phosphorylation [59]. Additionally, H₂S can stimulate angiogenesis through phosphatidylinositol 3-kinase (PI3K) and Akt activation [61]. H₂S can also activate hypoxia inducible factor-1α (HIF-1α) and thus increase expression of vascular endothelial growth factor (VEGF) [62]. Conversely, VEGF-stimulated angiogenesis is suppressed in CSE knockout mice [58].

Endogenous H₂S production is known to be upregulated during wound healing [63]. Topically applied H₂S accelerates wound closure and healing [59]. Angiogenesis is very important in both acute and chronic ischemia as poorly vascularized tissue will lose function and possibly become necrotic. In models of chronic hind limb ischemia, sodium hydrosulfide (NaHS) increased capillary formation and blood flow [64]. Similar results were found in chronically ischemic hearts with improvements in cardiac function following H₂S treatment [65]. These studies indicate that endogenous H₂S is crucial in physiological angiogenesis and that those capabilities can be employed in disease treatment.

9. Inflammation

Several studies report that H₂S is a mediator of inflammation [66–69], while others report that H₂S ameliorates inflammatory sequelae [70–72]. These studies are likely in conflict because different models of inflammation, H₂S production inhibitors, H₂S “donors”, and different O₂ tensions were used for experiments. Using NaHS, several groups have shown that H₂S augments neutrophil migration and adhesion, nociception, and increases endotoxemia [66,67,73]. This is likely because upon application NaHS “releases” all its H₂S instantly, which may be pro-inflammatory in some cases. This is in contrast to the much slower kinetics and magnitude of endogenous H₂S production. Moreover, a burst of H₂S may alter ^•NO homeostasis as well, decreasing its bioavailability through direct reactions and through inhibition of NOS [74]. Regarding endogenous production, some groups report that CSE is upregulated during lipopolysaccharide (LPS)-induced endotoxemia and that pharmacological inhibition of CSE attenuated inflammation [68,69]. Whiteman et al. highlighted differences between using rapid release of high concentrations of H₂S vs. using slow-releasing donor compounds, which more closely mimic endogenous production. They showed that a slow-releasing H₂S donor dose-dependently inhibited LPS-induced inflammation by decreasing the pro-inflammatory mediators IL-1β, IL-6, TNF-α, and PGE₂ [74]. Additionally, they showed an increase in the anti-inflammatory cytokine IL-10 in an NFκB-dependent mechanism [75]. This report is similar to others that show H₂S-dependent inhibition of ^•NO and TNF-α through p38 inhibition; a known downstream target of H₂S [76]. In contrast to [68], which used a non-specific PLP-dependent enzyme inhibitor, propargylglycine (PAG), others have used the CSE inhibitor β-cyanoalanine [70]. Treatment with β-cyanoalanine increased leukocyte adhesion to the endothelium, indicating some off-target effects of PAG and that endogenous H₂S is anti-inflammatory [70]. Like vasodilation, the anti-inflammatory effects of H₂S appear to be K_ATP channel-dependent with glibenclamide reversing the inhibitory effects of H₂S on leukocyte adhesion [71]. Although there are conflicting reports regarding the role of H₂S in inflammation, common themes have emerged that shed light on the reasons for these disparate results. H₂S donor compounds with slow release kinetics that mimic endogenous production appear to ameliorate inflammation. This is contrasted against the apparent pro-inflammatory potential of low purity sulfide salts, which immediately dissociate in solution to rapidly deliver large doses of H₂S. As with vasoactivity, the role of H₂S in inflammation is redox sensitive and this becomes important when impure sulfide salts are used in experiments. In addition to sulfide, these salts contain oxidized sulfur species such as sulfates, sulfenic acid, and sulfonates. In contrast to H₂S, these species can have proinflammatory properties [75]. Additionally, experiments conducted at supra-physiological O₂ tensions may yield misleading results, as high O₂ concentrations increase production of proinflammatory oxidized sulfur species [53,75]. In order to move H₂S from the “bench to the bedside”, comprehensive pharmacological studies are needed, which focus on routes of administration, type of donor compounds used, kinetics and magnitude of H₂S release, timing of the intervention, physiologically and pathologically relevant O₂ tensions, and the type of inflammatory condition being addressed.

10. Hydrogen sulfide and cytoprotection

As mentioned in earlier sections, the therapeutic window for effective H₂S treatment is likely very narrow because H₂S is a potent inhibitor of mitochondrial respiration. Therefore, several pharmacological factors including dose, route of administration, and timing of H₂S exposure must be carefully considered when using H₂S as a therapy. In the following paragraphs, select examples from the literature are presented showing cytoprotective effects of H₂S and other sulfide-based compounds in models of cardiovascular disease.

11. Cardiovascular disease

Studies show decreased plasma levels of H₂S in atherosclerosis models [51,77]. Furthermore, endogenous production of H₂S in sclerotic aortic tissues is impaired due to decreased expression of CGL in VSMCs [78]. Conversely, in cases of H₂S over-production (e.g., Trisomy 21) progression of atherosclerosis is slower [79]. H₂S may slow disease progression of atherosclerosis by inhibiting several elements of foam cell formation. Macrophages incubated with oxidized lipoprotein (oxLDL) show decreased intracellular lipid accumulation when treated with H₂S, whereas inhibiting endogenous H₂S production exacerbates lipid accumulation [80]. Expression of scavenger receptors responsible for oxLDL accumulation was also decreased with H₂S treatment [80]. Using apolipoprotein E knockout mice, H₂S decreases expression of adhesion molecules, thus preventing the recruitment of macrophages into the vascular intima, which is accompanied by a reduction in pro-inflammatory cytokines [2]. In atherosclerotic plaques, VSMCs proliferate and assume a fibrogenic phenotype, contributing to vessel occlusion. H₂S, at high concentrations, prevents VSMC proliferation [81]. Together, these results support the concept that sulfide-based therapies attenuate some of the early metabolic changes that contribute to cardiovascular disease. What is not known is whether H₂S is efficacious in end stage cardiovascular disease.

Considerable work has also focused on the impact sulfide-based therapies have in ameliorating cardiac ischemia-reperfusion injury. Lefer and colleagues have shown that pretreatment with sodium sulfide (Na₂S) before cardiac ischemia-reperfusion reduces infarct size and improves overall cardiac function [3,82,83]. There are several proposed mechanisms underlying this protective effect. First, H₂S up-regulates anti-oxidant response element (ARE) genes by inducing the translocation of Nrf-2 to the nucleus [84]. This increases GSH and expression of antioxidant proteins such as heme oxygenase-1, glutathione S-transferase, and thioredoxin-1 [84]. Second, H₂S acts through Akt-dependent pro-survival pathways to prevent caspase 3 activation and association of pro-apoptotic proteins to mitochondria that are necessary for mitochondrial permeability pore formation and cytochrome c release [85]. The effects of H₂S on the mitochondrion are likely central to overall cardiomyocyte protection [86], which may also involve activation of K_ATP channels [87]. While some groups attribute the cytoprotection to mitochondrial K_ATP channels, others using organelle-specific K_ATP channel blockers show that cytoprotection is due to effects on sarcolemmal channels [88].

Interestingly, daily H₂S administration for several months prevented perivascular fibrosis and subsequent arteriole occlusion in spontaneously hypertensive rats. [89], suggesting that H₂S administration may prevent pathogenic vascular remodeling. H₂S prevents thickening of damaged arterial intima and medial thickening of intramyocardial coronary arterioles [90]. Finally, H₂S induces neovascularization in models of chronic ischemia [64]. This molecular action of H₂S may be highly important following acute cardiac ischemia, as maintenance of a highly vascularized myocardium is required for proper organ function. These select findings, when taken together, demonstrate the promise of H₂S as a treatment for chronic cardiovascular diseases, as well as, in the emergency room for patients suffering from cardiac arrest.

12. Summary

The actions of H₂S described in this review highlight its ability to function in redox biology and cell signaling. With a pK_a of 6.9, both protonated and anion forms of H₂S are present at physiological pH. This allows for the free diffusion of H₂S to all cellular compartments. Because H₂S-producing enzymes play a diverse role in the larger sulfur cycle of the cell, the production of H₂S is dependent on cellular redox status. During conditions of oxidative stress the amount of reduced substrates like cysteine available for H₂S production may be limited. Moreover, steady state H₂S concentrations will be determined by H₂S consumption pathways that are redox sensitive as well. Importantly, O₂ being an antagonist of H₂S will also accelerate H₂S oxidation and inhalation of 100% O₂ is, in fact, a treatment for H₂S gas inhalation. Also highlighted herein, is the impact of O₂ tension on the biological actions and outcomes of H₂S. For example, H₂S-mediated vasodilation is observed at physiological O₂ concentrations, whereas at vasoconstriction occurs at hyperoxia (Fig. 4). These same patterns can also be extended to other biological processes such as inflammation and angiogenesis (Fig. 4). Furthermore, many of the cytoprotective actions of H₂S are triggered by and act to attenuate oxidative stress as exemplified by the ability of H₂S to induce the expression of antioxidant enzymes during myocardial ischemia-reperfusion. In conclusion, investigation of the redox biology of H₂S will not only increase understanding of its role in human physiology, but its potential therapeutic role in pathologies where oxidative stress is central to the disease process.

Authors contributions

AS and SMB wrote the manuscript.