Emergence of Hydrogen Sulfide as an Endogenous Gaseous Signaling Molecule in Cardiovascular Disease

Abstract

Long recognized as a malodorous and highly toxic gas, recent experimental studies have revealed that hydrogen sulfide (H₂S) is produced enzymatically in all mammalian species including man and exerts a number of critical actions to promote cardiovascular homeostasis and health. During the past 15 years, scientists have determined that H₂S is produced by three endogenous enzymes and exerts powerful effects on endothelial cells, smooth muscle cells, inflammatory cells, mitochondria, endoplasmic reticulum, and nuclear transcription factors. These effects have been reported in multiple organ systems and the vast majority of data clearly indicate that H₂S produced by the endogenous enzymes exerts cytoprotective actions. Recent preclinical studies investigating cardiovascular diseases have demonstrated that the administration of physiological or pharmacological levels of H₂S attenuates myocardial injury, protects blood vessels, limits inflammation, and regulates blood pressure. H₂S has emerged as a critical cardiovascular signaling molecule similar to nitric oxide (NO) and carbon monoxide (CO) with a profound impact on the heart and circulation (Figure 1). Our improved understanding of how H₂S elicits protective actions, coupled with the very rapid development of novel H₂S releasing agents, has resulted in heightened enthusiasm for the clinical translation of this ephemeral gaseous molecule. This review will examine our current state of knowledge regarding the actions of H₂S within the cardiovascular system with an emphasis on the therapeutic potential and molecular crosstalk between H₂S, NO, and CO.

Introduction

Hydrogen Sulfide (H₂S) has traditionally been viewed as an odorous and highly toxic gas devoid of any biological or physiological function. Dating back over 250 million years, H₂S poisoning due to upwelling euxinix bottom water, is postulated to have caused the sudden mass extinction of the Ediacaran fauna¹. In the 18^th century AD, detailed examination of environmental factors and workplace chemical exposures by Bernardino Ramazzini revealed that cesspit workers exposed to toxic levels of H₂S commonly acquired eye inflammation which led to secondary bacterial infection and blindness². H₂S was eventually measured in the brain in 1989 and it quickly emerged as a critically important signaling molecule with widespread physiological actions^{3, 4}. The existence of H₂S in the brain suggested physiological purpose. Cystathionine beta synthase (CBS) is believed to be the critical enzyme that produces H₂S resulting in the modulation of neurological function⁵. H₂S generated by cystathionine gamma lyase (CSE) was next discovered as an important modulator of vasorelaxation in smooth muscle⁶. Following the discovery of H₂S as a potential neurological signaling molecule and a vasorelaxant molecule, the number of publications pertaining to the physiology of H₂S drastically spiked. It was no longer regarded as a toxic modulator of cell death, but a physiologically important and potentially highly salubrious molecule with diverse signaling actions.

Endogenous Synthesis of Hydrogen Sulfide in Mammals

H₂S is produced endogenously via enzymatic activity, non-enzymatic pathways (such as reduction of thiol-containing molecules), and is also released from intracellular sulfur stores (sulfane sulfur) ⁷. In most tissue, CBS and CSE are primarily responsible for the production of H₂S. They separately coordinate with L-cysteine to produce H₂S, L-serine, and ammonium⁸. Although found throughout the body, the discovery in of CBS in the brain led to a consensus that is was the primary H₂S producing enzyme impacting neurological signaling. However, CBS has been identified in tissues throughout the body and is thought to modulate global H₂S generation. More recently, it has been reported that 3-mercaptopyruvate sulfurtransferase (3-MST) is responsible for roughly 90% of H₂S produced in the brain⁹. 3-MST, primarily located in the mitochondria, enzymatically produces H₂S from α-ketoglutarate and L-cysteine via metabolic interactions with cysteine aminotransferase (CAT)⁹. Although 3-MST in the neurons is responsible for much of the brain's H₂S production, CBS is localized in astrocytes suggesting that a portion of the H₂S signaling may be a result of the actions of CBS¹⁰.

Under physiological conditions, H₂S can undergo several catabolic fates. Once deprotonated, HS^- is rapidly oxidized in the mitochondria to form thiosulfate (non-enzymatic conversion), which is ultimately converted to sulfite and sulfate¹¹. H₂S can also be methylated by thiol S-methyltransferase (TMST) to form dimethylsulfide and methanethiol, or it can react with methemoglobin to form sulfermoglobin¹². Similar to the other two gaseous signaling molecules, nitric oxide (NO) and carbon monoxide (CO), H₂S has a very high affinity for hemoglobin, resulting in profound scavenging. One of the major challenges to the study of H₂S under in vivo conditions is the extremely short half-life of this ephemeral molecule; estimated to be between seconds to minutes^{8, 13} (between 12 and 37 hours in air¹⁴)⁸.

Vascular Actions of Endogenous Hydrogen Sulfide

One of the first proposed beneficial physiological effects of H₂S that was reported was its action on vascular tone (i.e., blood pressure regulation) and inflammation¹⁵. H₂S has been widely considered as a potent anti-inflammatory molecule with modest vasodilator actions. One of these effects is its capacity to hinder leukocyte adhesion by inhibition of leukocyte “rolling” and firm adhesion to the endothelium. H₂S has been shown to significantly inhibit the expression of leukocyte adhesion molecules¹⁶. Additionally, H₂S signaling promotes anti-inflammatory action by preventing tissue edema. This finding was shown in rats whereby the administration of an H₂S inhibitor led to edema formation¹⁷. The anti-inflammatory response of H₂S may also be dependent upon the activation of vascular K_ATP channels. Rats treated with a specific K_ATP channel antagonist did not show a reduction in leukocyte adhesion suggesting that the ability of H₂S to modulate adhesion may be dependent on the signaling of this channel¹⁶. H₂S activates K_ATP channels, specifically in the smooth muscle, by increasing whole-cell K_ATP currents to hyperpolarize membrane potentials and increases single-channel activity by enhancing permeability of single K_ATP channels¹⁸.

A somewhat controversial action of H₂S in the circulation is related to the role of the gaseous signaling molecule on vasodilation and blood pressure regulation. There are mixed results in the literature with some studies reporting vasodilatory actions while others report vasoconstrictor effects. Mice with a genetic deletion of CSE, and consequently deficient H₂S production, displayed significant hypertension and diminished endothelial vasorelaxation¹⁹. Other studies reveal that exogenous administration of H₂S can cause vasoconstriction. The discrepancy in these findings appears to depend on the concentration of H₂S, the vascular bed that is studied, and the oxygen tension of the tissue or blood vessel under investigation. When H₂S is held above trace levels, it has been shown to be an effective vasodilator²⁰. Interestingly, it exerted vasodilator effects at an oxygen partial pressure of 30 mmHg, yet acted as a vasoconstrictor at an elevated partial pressure of oxygen of 150 mmHg²¹. It has been suggested that the vasodilator actions of H₂S may be a result of eNOS generated NO promoted by H₂S signaling.

H₂S also has been shown to exert potent pro-angiogenic effect in vascular endothelial cells in the setting of chronic ischemia, while promoting extracellular kinase pathways that promote vessel growth²². Multiple groups have shown that H₂S stimulates endothelial cell proliferation and migration by either further developing current cells or by developing primary endothelial cells^{23, 24}. H₂S participates in vascular endothelial growth factor (VEGF) signaling. CSE^-/- mice exhibited significant reductions in H₂S and growth of endothelial cells in vitro²⁴. However, all pro-angiogenic signaling is not H₂S dependent, since fibroblast growth factor levels were not attenuated in CSE^-/- mice²². The signaling pathways for H₂S-mediated angiogenesis effects are somewhat complex. Exogenous H₂S donors have been shown to activate the AKT pathway, which in turn promotes angiogenesis and tumor development, and enhance phosphorylation of the mitogen activated protein kinase (MAPK) pathway (ERK1/2 and p38)²⁴. These pathways have been shown to regulate RF/6A cells and human umbilical vein endothelial cells, respectively²².

Role of Hydrogen Sulfide in Cardiovascular Physiology and Pathophysiology

Ischemia-Reperfusion Injury in Heart and Brain

H₂S has been extensively examined as a potential therapeutic in the setting of ischemia/reperfusion (I/R) injury in the heart, brain, lungs, and liver. The majority of in vitro and in vivo studies reported thus far have reported beneficial actions of H₂S when administered at physiological or pharmacological concentrations. In the setting of I/R injury, the cytoprotective actions are thought to result from anti-apoptotic, anti-inflammatory, antioxidant, and mitochondrial actions of H₂S. Diallyl trisulfide (DATS), a stable H₂S donor, was administered to mice following acute myocardial ischemia and markedly protected the myocardium²⁵. DATS significantly decreased infarct size and troponin I levels, and improved mitochondrial coupling. Additionally in diabetic mice, H₂S therapy was shown to precondition the myocardium against I/R injury by activating the antioxidant-signaling molecule, Nfr2²⁶. It should be noted that the effective therapeutic range for H₂S releasing agents studied thus far is relatively narrow and the administration of supra-pharmacological levels of H₂S clearly fails to protect and may even exacerbate I/R injury²⁷. In this regard, H₂S therapy is very similar to NO therapy in the setting of I/R injury²⁸.

H₂S has also been reported to be a potent neuroprotective agent. Kimura et al. reported that H₂S protects neurons from oxidative stress by bolstering glutathione levels following I/R in vitro²⁹. To study a more severe cerebral injury model, mice were subjected to 30 minutes of ischemia, and NaHS was administered following 24 hours of reperfusion³⁰. Pro-inflammatory markers, TNF-α and MCP-1, were significantly attenuated, while the anti-inflammatory marker, BcL-2, was enhanced³⁰. In another cerebral ischemic study, H₂S protected against neuronal apoptosis by diminishing infarct volume and activation of caspase-3 in murine neurons³¹.

Heart Failure

Heat failure prevalence has drastically increased, as it has become the primary discharge diagnosis in patients aged 65 years or older³². Following myocardial insult or injury, the left ventricle (LV) undergoes adaptive changes that ultimately transition the ventricle from a compensatory to a decompensated state. LV remodeling includes reactions such as apoptosis, inflammation, oxidative stress, and the development of fibrosis³³. H₂S therapy has recently been shown to ameliorate ischemic-induced heart failure in a murine model system³⁴. Genetic overexpression of CSE in mice resulted in increased H₂S levels and improved left ventricular performance and survival in the setting of ischemic heart failure³⁴. In a hypertension induced heart failure model, it has been clearly demonstrated that H₂S decelerated progression to adverse remodeling of the LV and induced angiogenesis in the myocardium³⁵. Administration of H₂S in the diet during heart failure significantly decreased adverse LV remodeling as compared to the control group³⁶. The transition to decompensated heart failure progresses with a decline in vascular growth³⁷. In a similar heart failure model, NaHS treated mice induced matrix metalloproteinase (MMP-2), which promoted VEGF synthesis and angiogenesis and suppressed anti-angiogenic factors such as MMP-9 and tissue inhibitor of matric metalloproteinase (TIMP-3)³⁸. Increasing myocardial vascularity and perfusion in concert with cardiac myocyte growth is critical to preventing the progression of heart failure and H₂S appears to be a potent pro-angiogenic agent for this indication.

Atherosclerosis

Atherosclerosis is characterized by several pathological events that include endothelial dysfunction, monocyte penetration and conversion into macrophage foam cells, and mired leukocyte rolling along the endothelium. Recent studies have shown that macrophages produce H₂S endogenously and that lipopolysaccharide (LPS), an inflammatory endotoxin, stimulates CSE production of H₂S in macrophages³⁹. Moreover, the H₂S donor, NaHS, inhibited pro-atherogenic oxidized low-density lipoprotein (oxLDL) induced foam cell formation in macrophages⁴⁰. Leukocyte velocity, attachment, and infiltration along the endothelium are key atherosclerotic factors and they were examined following H₂S therapy. Na₂S and NaHS inhibited aspirin and the chemotactic peptide N-formyl-L-leucyl-L-phenylalanine (fMLP) leukocyte adherence in a dose-dependent manner¹⁶. In a further study, deficiency of H₂S (i.e. CSE^-/- mice) promoted leukocyte adhesion and decreased leukocyte velocity and exacerbated leukocyte infiltration, while administration of H₂S donors suppressed leukocyte penetration¹⁶.

Molecular Signaling via Endogenous Hydrogen Sulfide

Similar to NO and CO, the effects of H₂S on the cardiovascular system are mediated via a very diverse array of cellular and molecular signals (Figure 2). Mitochondria are critical for cell survival and energy production. When mitochondrial function is compromised due to hypoxia or an increase reactive oxygen species (ROS), H₂S has been shown to protect mitochondria and ultimately improve respiration and promote biogenesis. This was shown when endogenous stimulation of H₂S production (10-100nM) enhanced mitochondrial electron transport and cellular bioenergetics⁴¹. However, at high concentrations, H₂S is toxic, resulting in inhibition of mitochondrial respiration via direct inhibition of cytochrome c oxidase enzyme by rapid sulfide oxidation, oxygen uptake, and conversion of cytochrome aa₃ into the low spin form^{42, 43}. Isolated murine cardiac mitochondria exposed to 10 μM H₂S were shown to have improved recovery of post-hypoxic respiration rate following 30 minutes of hypoxia²⁷. Mitochondria are unique in that they are critical in the regulation of cell death and apoptosis and much of the cytoprotective actions of H₂S during ischemic states may be a result of potent actions on mitochondria⁴⁴. In vitro, H₂S was shown to attenuate apoptosis in an adenocarcinoma cell line specific to colon cancer by preventing beta-phenyethyl isothiocyanate from inducing cell death⁴⁵. Furthermore, H₂S protects against high-glucose induced cardiomyocyte apoptosis by altering regulatory gene expression⁴⁶. Moreover, following myocardial infarction injury in a murine model, H₂S treated mice displayed significant reductions in apoptosis as evidenced by a decrease in caspase-3 activity TUNEL positive nuclei count²⁷. Another mechanism by which mitochondria modulate cell death (i.e., necrosis) is by the induction of the mitochondrial permeability transition pore (MPTP) in response to oxidative stress, free radicals, and elevated matrix Ca²⁺ count commonly generated in ischemia and reperfusion⁴⁷. The activation of this pore leads to a halt in ATP production and a breakdown of the mitochondria.

Figure 2. Hydrogen Sulfide Mediated Signaling.

H₂S is known to modify proteins, modulate the function of various ion channels, attenuate apoptosis and oxidative stress, and to be a potent modulator of cellular metabolic function. Adapted and modified from King et al.⁷³

A potential mechanism to prevent the induction of MPTP, aside from known inhibitors such as cyclosporine A, could be via potent inhibition of mitochondrial oxidative stress. H₂S has emerged as a very potent anti-oxidant molecule via both direct and indirect actions. Oxidative stress was studied following the introduction of the H₂S donor, sodium hydrosulfide (NaHS), in vitro. In an extracellular cysteine dependent manner, H₂S protected cultured neurons from oxidative stress by increasing glutathione levels instead of acting directly as an antioxidant⁴⁸. Also, Nrf2, a transcription factor, regulates oxidative stress by impacting gene expression of several key enzymes⁴⁹. As Nrf2 breaks its interaction with the repressor cytoplasmic protein Keap-1, Nrf2 translocates to the nucleus and promotes the expression of detoxifying genes such as heme oxygenase 1 (HO-1), superoxide dismutase 1, and catalase^{50, 51}. Daily administration of Na₂S for 7 days increased Nrf2 expression in both cytosolic and nuclear fractions, indicating further antioxidant signaling by H₂S⁵². Furthermore, H₂S was administered to mice exposed to 60 minutes of hepatic ischemia and 5 hours of reperfusion. At both 1-hour and 5-hour reperfusion time points, lipid hydroperoxide levels in hepatic tissue was significantly decreased in the H₂S compared to vehicle treated mice⁵³. H₂S ability to scavenge for oxidants, like hydroperoxide and ROS may also be contributing to its anti-inflammatory actions mentioned earlier.

Crosstalk Between Hydrogen Sulfide and Other Gaseous Signaling Molecules

NO and H₂S share many of the same regulatory roles including vasodilation, promotion of angiogenesis, attenuation of apoptosis, and antioxidant actions. In endothelial cells, NO is synthesized by endothelial nitric oxide synthase (eNOS) and initiates downstream signaling with guanylyl cyclase (GC) to form the second messenger cyclic guanosine 5'-monophosphate (cGMP). Although H₂S and NO exhibit independent signaling, it appears that there is crosstalk between these two molecules in a manner that modulates multiple pathways (Figures 3 and 4). eNOS function is tightly regulated by post-translational modifications (such as the phosphorylation of amino acids such as Ser-1177 and Thr-495) that can enhance or thwart eNOS production of NO^{54, 55}. In a pressure overload murine heart failure, Kondo et al. reported that mice treated with an H₂S donor significantly increased phosphorylation of activation site, eNOS-P^Ser1177 compared to the control group³⁶. This increase in eNOS phosphorylation was accompanied by increased NO production. Mice treated with the H₂S donor, DATS, showed marked increases in plasma nitrite, nitrate, and RXNO levels 30 minutes following injection²⁵. Furthermore, NO can also impact H₂S generation. NO donors have been shown to increase the expression of CSE in isolated aortic smooth muscles cells⁵⁶. There still remains some controversy over crosstalk between H₂S and NO. For example, one group found that eNOS deficiency prevented the ability of H₂S to induce angiogenesis in vivo or in in vitro, suggesting that NO is required for H₂S to have vascular effects⁵⁷. Yet, another group proposed that the pro-angiogenic effect of H₂S is regulated by both an NO-dependent and an independent manner⁵⁸. Once we better understand how these molecules work together, we can begin to build therapeutics that maximize the benefits of both signaling molecules.

Figure 3. Crosstalk Between Exogenous Hydrogen Sulfide (H₂S), Nitric Oxide (NO), and Carbon Monoxide (CO).

Proposed interaction between H₂S and eNOS to increase nitric oxide generation. In addition, H₂S can activate heme oxygenase-1 via genetic upregulation and increased activity to enhance levels of carbon monoxide. Nitric oxide and carbon monoxide can ultimately synergize with H₂S to exert both vascular and cardiac protection during cardiovascular disease states.

Figure 4. Endogenous H₂S Regulation of the eNOS-NO Pathway.

Proposed actions of CSE derived H₂S under normal conditions and in the setting of cardiovascular disease. H₂S is a potent regulator of cellular redox status that limits oxidative stress thereby preserving eNOS function and promoting nitric oxide (NO) production. Diminished endogenous H₂S results in profound oxidative stress, reduced BH₄ levels and dysfunctional eNOS (i.e., eNOS uncoupling). Reduced nitric oxide levels exacerbate cardiovascular pathology.

Far less has been studied regarding the crosstalk between H₂S and carbon monoxide (CO). Endogenously, CO is derived from the breakdown of heme by heme oxygenase (HO) and it can also activate GC which causes an increase in cGMP^{59, 60}. CO also shares many of the same biological effects of NO and H₂S including its apoptotic and anti-inflammatory mechanisms. Zhang et al. demonstrated that exogenous H₂S upregulates the CO system in pulmonary arteries of hypoxic rats⁶¹. More recently, a long lasting H₂S donor was shown to inhibit oxidative stress and increase Nrf2, HO-1, and p-AKT levels more so than its short-acting counterpart⁶², providing some evidence for for H₂S-CO crosstalk.

H₂S Therapeutic Agents and Mutant Mouse Models

Exogenous administration of H₂S or genetic modulation of CSE, CBS, or 3-MST levels are effective means by which the cardiovascular actions of H₂S can be investigated. Numerous H₂S donors with varying chemical and pharmacological properties have emerged as potential therapeutics. Na₂S and NaHS were among the first H₂S releasing agents studied in the cardiovascular system^{27, 48}. These inorganic salts have the advantage of rapidly increasing H₂S concentration within seconds, but they also rapidly decline within tissue and could exert adverse side effects due to rapid increases in H₂S at high concentrations⁶³. Additionally, many of the commercially available formulations of NaHS and Na₂S are highly impure and the impurities elicit toxic effects. Naturally occurring H₂S donors such as diallyl trisulfide (DATS), a polysulfide derived from garlic, have been shown to augment H₂S levels for extended periods of time⁶⁴. Synthetic H₂S releasing compounds have also been developed. SG-1002³⁶ and penicillamine-based donors⁶⁵ are examples of synthesized H₂S donors whose release is more precisely controlled. As novel H₂S releasing agents or H₂S donors develop, these novel agents should ultimately address the clinically relevant issues such as sustained release/half-life, route of administration, tissue specificity, and low toxicity.

The effects of decreased endogenous H₂S production has also been investigated in cardiovascular disease. H₂S enzyme antagonists have been explored to reach the same goal. DL-propargylglycine (PAG), an inhibitor of CSE, exerted a dose-dependent inhibition of sulfide production⁶⁶. However, the inhibition came at the cost of unrealistically high dosages (i.e., 50 mg/kg) and non-specific effects. There remain very few other targeting molecules with high potency and high selectivity, but would be more valuable than knocking out an entire enzyme.

Complete genetic deficiency of CBS (i.e., homozygote knockout mouse) is lethal and limited literature exists examining the genetic deficiency of CBS in heterozygote knockouts. In contrast, a global 3-MST knockout mouse has been developed, but there is a paucity of information regarding genetic deficiency of 3-MST in cardiovascular disease at present. In addition, very little is currently known regarding genetic overexpression of either CBS or 3-MST due to a lack of these transgenic mouse models. However, CSE^-/- and CSE overexpressing transgenic mice have been developed and have been fairly well characterized in terms of cardiovascular disease states. Global CSE^-/- mice show significant reduction in H₂S bioavailability in serum, heart, aorta, and several other tissues¹⁹. Global CSE^-/- mice exhibit pronounced hypertension and reduced vasodilation, indicating CSE derived H₂S is an important mediator of vascular reactivity and blood pressure¹⁹. CSE^-/- mice subjected to myocardial I/R injury⁶⁷ experience a 48% increase in infarct size compared to wild-type mice. Conversely, CSE overexpressing transgenic mice, following myocardial I/R, display a marked reduction in infarction compared to wild-type mice²⁷. In a pressure overload heart failure model, CSE^-/- exhibited exacerbated LV dysfunction, while CSE transgenic mice promoted cardiac structure and function compared to wild-type mice. Cardiac mitochondria isolated from CSE^-/- mice exhibit profound mitochondrial dysfunction³⁶. These data provide clear evidence for the cytoprotective actions of CSE-derived H₂S in various cardiovascular pathologies.

Challenges for the Hydrogen Sulfide Research Field

There are several difficulties that researchers face when studying H₂S in physiological or pathological in vivo systems. It is critical to accurately measure H₂S levels in blood and tissue samples from patients that suffer cardiovascular diseases. The first challenge is the measurement of H₂S and quantifying its bioavailability in vivo. Besides free sulfide, molecule-bound sulfide is also present in biological systems and can be liberated and quantified. One of the most common measurements is by way of the methylene blue method. This assay is conducted under acidic conditions and measures sulfide concentrations in biological samples by releasing acid labile sulfide. A weakness of this method, as well as all colorimetric detection, is that it interferes with other chromophores, resulting in an artifactual signal⁶⁸. Another common mode for measuring H₂S, with sensitivity in the nanomolar range, uses monobromobimane (MBB). This method includes nucleophilic substitution reactions to give a fluorescent sulfide dibrimane (SDB) whose emitted wavelengths can be detected in visible light range detected with HPLC. The limit of detection with this method is 2nM and the SDB product is very stable over time⁶⁹. Likely a more precise measurement of H₂S and sulfane sulfur is through use of a combined gas chromatography-chemiluminescence approach^{70, 71}. This method requires fresh tissue homogenate reacting with buffer for an extended incubation period that releases the gas into headspace. The disadvantage of measuring headspace concentrations that have been incubating for relatively long periods of time make real-time measurements problematic⁷². Another key method of detection is the use of fluorescent probes, but it too faces the challenge of thiol interference that is present in most cellular compartments and biological fluids.

Another challenge for the H₂S field is the development of clinically relevant therapeutic agents to treat CV diseases. Aside from the aforementioned importance of a long acting donor with controlled H₂S release, developing a drug that can specifically target a body system would alleviate unwanted side effects. The mechanisms of site specific delivery remain challenging, however, targeted H₂S delivery to myocardial microvasculature was achieved using ultrasound to release encapsulated H₂S from per flourocarbon filled microbubbles (Wu et al. Circulation 2012;126:A10756 ABSTRACT). Additionally, mitochondria-targeted H₂S donors are in development and contain a mitochondria-targeting moiety aimed to mediate oxidative stress and cell injury (Le Trionnaire et al. Nitric Oxide 2013;31:S57 ABSTRACT). Mastering these issues would drastically advance H₂S research and further translate it into clinical relevance.

Future Directions and Clinical Translation

H₂S has very rapidly emerged as an exciting signaling molecule. This gaseous molecule impacts cells and cellular organelles throughout the body and freely diffuses across cellular membranes resulting in a universal biological impression. Yet, before H₂S therapies can be fully translated to a clinical setting, much more must be accomplished. Mechanistic discovery is underway and much has been accomplished in regards to aforementioned antioxidant and anti-apoptotic signaling. However, a greater depth of knowledge is required to develop effective therapeutics. Specifically, function and signaling relating to the enzymes responsible for the endogenous production of H₂S are worthy of further study. Understanding location and activity of these enzymes in particular disease states would help direct gene therapy or localized drug delivery. Understanding these mechanisms would help identify what tissues can be impacted and what pathological conditions are most responsive to H₂S therapy.

Exploring the relationship and interactions of H₂S with other endogenous gases, specifically NO, could improve clinical translation. H₂S therapy in conjunction with NO donors may augment outcome and bolster cardiovascular response and cellular function. Endogenous production of H₂S was shown to significantly increase the vasorelaxant effect of an NO donor (sodium nitroprusside)⁵⁶. Also, exogenously administered H₂S increased eNOS activation and NO bioavailability^{35, 36}. This indicates that H₂S enhances NO actions in the vasculature. The cooperative or competitive actions of H₂S and NO as they simultaneously interact with proteins in S-sulfhydration and S-nitrosylation reactions are also unknown¹⁴.

Lastly, before making a complete transition to human testing (there are currently 2 cardiovascular H₂S trials on clinicaltrials.gov), well-established, large animal models of cardiovascular disease should be thoroughly investigated as the vast majority of cardiovascular studies have been performed in murine model systems. These murine model systems provide a good foundation, but are lacking in clinical relevance. Examining H₂S actions in an animal model with similar cardiovascular characteristics as humans suffering from CV disease would help verify the safety and efficacy of the drug.

Figure 1. Currently Recognized Gasotransmitters.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are all produced endogenously via enzymes. NO is synthesized by neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS). CO is generated by the heme oxygenase (HO) family of enzymes (HO-1, HO-2, and HO-3). H₂S is synthesized via the actions of cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE or CGL), and 3-mercaptopyruvate sulfur transferase (3MST). These gaseous molecules are produced in very low concentrations ranging from low nM to low µM and are very labile. Adapted and modified from Calvert et al.⁶⁷

Non-Standard Abbreviations and Acronyms

H₂S

hydrogen sulfide

NO

nitric oxide

CO

carbon monoxide

CBS

cystathionine beta synthase

CSE

cystathionine gamma lyase

3-MST

3-mercaptopyruvate sulfurtransferase

CAT

cysteine aminotransferase

TMST

S-methyltransferase

VEGF

vascular endothelial growth factor

MAPK

mitogen activated protein kinase

I/R

ischemia/reperfusion

DATS

diallyl trisulfide

TNF-α

tumor necrosis factor-alpha

MCP-1

monocyte chemotactic protein-1

Bcl-2

B-cell lymphoma 2

LV

left ventricle

MMP-2

matric metalloproteinase-2

TIMP-3

tissue inhibitor of metalloproteinases-3

oxLDL

oxidized low-density lipoprotein

fMLP

N-formyl-L-leucyl-L-phenylalanine

ROS

reactive oxygen species

MPTP

mitochondrial permeability transition pore

NaHS

sodium hydrosulfide

HO-1

heme oxygenase 1

hyc

homocysteine

eNOS

endothelial nitric oxide synthase

(s)GC

(soluble) guanylyl cyclase

cGMP

cyclic guanosine 5'-monophosphate

PAG

DL-propargylglycine

MBB

monobromobimane

SDB

sulfide dibrimane

BH₄

tetrahydrobiopterin