Hydrogen sulfide signaling in mitochondria and disease

Abstract

Hydrogen sulfide can signal through 3 distinct mechanisms: 1) reduction and/or direct binding of metalloprotein heme centers, 2) serving as a potent antioxidant through reactive oxygen species/reactive nitrogen species scavenging, or 3) post-translational modification of proteins by addition of a thiol (-SH) group onto reactive cysteine residues: a process known as persulfidation. Below toxic levels, hydrogen sulfide promotes mitochondrial biogenesis and function, thereby conferring protection against cellular stress. For these reasons, increases in hydrogen sulfide and hydrogen sulfide–producing enzymes have been implicated in several human disease states. This review will first summarize our current understanding of hydrogen sulfide production and metabolism, as well as its signaling mechanisms; second, this work will detail the known mechanisms of hydrogen sulfide in the mitochondria and the implications of its mitochondrial-specific impacts in several pathologic conditions.—Murphy, B., Bhattacharya, R., Mukherjee, P. Hydrogen sulfide signaling in mitochondria and disease.

Hydrogen sulfide (H₂S) is a colorless, poisonous, and corrosive gas that gives rotting eggs their characteristic odor. Structurally, it is the sulfur analog of a water molecule, and it can be oxidized into elemental sulfur, sulfate (SO₄²⁻), thiosulfate (S₂O₃⁻), and sulfur dioxide (SO₂) (1). Ecologically, the sulfur cycle is intricately intertwined with the carbon cycle through the activity of microorganisms known as SO₄²⁻-reducing bacteria (SRBs), which are key for the anaerobic breakdown of organic material. SRBs thrive in a vast array of anoxic habitats, including shallow marine and freshwater sediments, mud volcanoes, and hydrothermal vents. They are unique in their ability to perform anaerobic respiration through utilizing SO₄²⁻ as their terminal electron acceptors rather than oxygen (O₂), generating H₂S as the main respiratory by-product; SRBs can breathe without oxygen (2, 3). They have a symbiotic relationship with aerobic sulfide-oxidizing bacteria, which can oxidize H₂S back into SO₄²⁻ to be continually utilized by SRBs. This is particularly noteworthy given the endosymbiotic hypothesis of mitochondrial origin, which suggests that all mitochondria originate from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea (4). For these reasons, it is not surprising that one of the main targets of H₂S signaling in eukaryotic cells is the mitochondria.

The study of H₂S began in the 1700s after the Italian physician and “father of occupational medicine,” Bernardino Ramazzini, noted the irritating effects of sewer gas on the eyes of sewer workers in his book De Morbis Artificum Diatriba (5). However, it was not until 1775–1776 that the responsible compound was identified as H₂S (6). This discovery catalyzed early research on H₂S as an environmental toxin. Nearly 166 yr later, in 1942, an American biochemist named Vincent Du Vigneaud crudely demonstrated the production of H₂S in mammalian tissue homogenates. However, it was not until 48 yr after that, in 1989/1990, that H₂S was truly considered to be a biologic mediator. During this time, 3 independent groups [Warenycia et al. (7), Goodwin et al. (8), and Savage and Gould (9)] reported the endogenous presence of H₂S in the animal and human brain. Abe and Kimura’s (10) pioneering work in the late 1990s demonstrated the ability of H₂S to act as a neuromodulator and vasorelaxant, confirming H₂S as the third physiologic signaling gas, alongside carbon monoxide (CO) and nitric oxide (NO). Since these landmark studies, the field of sulfide research has grown rapidly, and H₂S signaling has been implicated in several physiologic conditions including, but not limited to, promoting vasorelaxation, altering lipid metabolism, inducing angiogenesis, inhibiting monocyte adhesion, promoting tissue repair, and evading apoptosis (11–16). Moreover, H₂S enzymatic generation and signaling is conserved in many organisms beyond human beings, including other mammals, bacteria, and plants, and may play a protective role in all (17). In a 2005 landmark paper, Blackstone et al. (18) reported that H₂S could induce a reversible suspended animation–like state in mice. They posited that H₂S-mediated induction of suspended animation could have beneficial medical applications, such as following ischemia-reperfusion (I/R) injury or organ preservation following trauma. Another landmark paper by Shatalin et al. (19) suggested that H₂S has a key role in the development of antibiotic resistance in bacteria and showed that inhibition of the H₂S-producing enzymes cystathionine β synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3-MST) rendered many pathogens sensitive to antibiotic treatment. Finally, in plants, H₂S contributes to plant growth and development, plays a key role in the stress response, and may aid adaptation to stressful environments (20). Although H₂S signaling is physiologically far reaching because of its gaseous properties, one of the main intracellular compartments where it exerts its effects in mammalian cells is the mitochondria. This review will summarize the current literature on H₂S production, metabolism, and signaling, and then focus on H₂S signaling in the mitochondria of humans and preclinical models of disease.

HYDROGEN SULFIDE BIOGENESIS AND METABOLISM

Localization of H₂S-producing enzymes: CBS, cystathionine γ lyase, and 3-MST

H₂S is endogenously produced in mammalian tissues through both enzymatic and nonenzymatic pathways. Although the former is generally considered to be the primary source of H₂S, a 2019 report by Yang et al. (21) demonstrated that nonenzymatic H₂S production predominates in all human tissues besides the liver and kidneys. Nonenzymatic generation of H₂S occurs via the reaction of thiol or thiol-containing compounds with other molecules, such as the reduction of dietary inorganic polysulfides by glutathione (GSH) or the hydrolysis of inorganic sulfide salts (i.e., sodium sulfide [Na₂S] or sodium hydrogen sulfide [NaHS] with water) (22–24). Yang et al. (21) reported that cysteine is the preferred substrate for nonenzymatic generation of H₂S and that such a process is catalyzed by iron and vitamin B₆. They emphasize that the nonenzymatic generation of H₂S is underappreciated in the field and suggest that a better understanding of these processes is critical for the treatment of potentially several pathologies. Moreover, H₂S produced by sulfur-reducing gut bacteria has also been reported to supply nonnegligible amounts of H₂S systemically (25).

Enzymatic H₂S production, on the other hand, has been extensively researched and occurs primarily through the actions of 3 enzymes: CBS, cystathionine γ lyase (CSE), and 3-MST. These enzymes are differentially expressed in tissues and can be found in various cellular compartments (26). For example, 3-MST may be more significant for H₂S generation in tissues in which cysteine concentration is high (like the kidney) because it may play a role in sulfur amino acid catabolism (27). 3-MST expression has been reported in human kidney, liver, cardiac, and brain cells (specifically the neurons), with its predominant expression being in the gastrointestinal tract (28). It has also been shown to localize to both the cytosol and mitochondrial cellular compartments (29). CBS is predominantly expressed in the pancreas, male and female reproductive organs, and the astrocytes of the brain (30–34). CBS is also expressed in the liver, but its expression does not appear to be necessary for H₂S production because CBS^−/− mouse livers do not display reduced H₂S levels when compared with wild-type (WT) mouse tissues (35). CSE, on the other hand, is highly expressed in the liver, kidney, neurons, macrophages, and smooth muscle cells, with its highest expression in the liver (36–40). However, CSE expression and activity is generally considered to be negligible in the heart and spleen (1, 41). Furthermore, CBS and CSE exist extracellularly as circulating enzymes, secreted by microvascular endothelial cells and hepatocytes; however, the recent report by Yang et al. (21) suggests circulating H₂S may actually be due to nonenzymatic processes (42). Active 3-MST has been identified in red blood cells, although CBS and CSE expression was undetected (43). Although there is apparent overlap in tissue localization, these enzymes appear to perform nonredundant functions, and one enzyme may be more responsible for a particular tissue’s source of H₂S. Therefore, H₂S signaling may be enzyme and/or tissue specific; this specificity is discussed in later sections.

Our understanding of where these enzymes are located intracellularly is evolving. 3-MST has been reported to be found in both the mitochondria and cytosol, whereas CBS and CSE have been reported to be primarily cytosolic (44). However, our group and others have shown CBS in the mitochondria of both ovarian and colon cancer cells, and CSE can translocate from the cytosol to the mitochondria under conditions of cellular stress, such as increases in intracellular Ca²⁺ levels (45–47). Moreover, CBS and CSE are both capable of undergoing a post-translational modification by small ubiquitin-like modifier (SUMO) protein; SUMO is typically considered to be a nuclear localizing signal (48, 49). Indeed, sumoylation of CBS correlates with its localization to the nucleus and loss of catalytic activity and appears to be driven by levels of its substrates and products in the transsulfuration pathway. The consequences of CSE sumoylation are unknown, but it is hypothesized to be a nuclear localizing signal when nuclear GSH demand is high (49). How these H₂S-producing enzymes are regulated to maintain their spatiotemporal production of H₂S, as well as their crosstalk regulation of one another within mammalian cells, remains underexplored.

CBS and CSE function and regulation

CBS and CSE are key enzymes in the transsulfuration pathway, which fluxes methionine metabolism away from proteinogenesis and toward cysteine production and catabolism (Fig. 1) (50). Primarily, CBS catalyzes the first irreversible step in this pathway by driving the β-replacement reaction of homocysteine with serine to form the asymmetric thioether cystathionine and water. Interestingly, CBS can also carry out this reaction with a homocysteine and cysteine to directly generate cystathionine and H₂S. Nevertheless, serine is believed to be the primary substrate for homocysteine condensation (36, 51). This is because human CBS shows a lower Michaelis constant for serine (2.76 mM) than for cysteine (6.8 mM) (52). The Michaelis constant is defined as the substrate concentration required to reach half the maximum enzymatic rate (V_max); CBS requires less serine to reach half its maximum enzymatic rate compared with cysteine. Additionally, serine is reported to be more abundant within a cell than cysteine (36, 53, 54). It is not known whether or how the substrate preference for CBS can be switched, calling into question the physiologic relevance of homocysteine-cysteine condensation. For these reasons, the main mechanisms by which CBS produces H₂S are via a β-replacement reaction with water and cysteine to form serine and through another β-replacement reaction between 2 cysteines to generate lanthionine (36, 55, 56).

Figure 1.

H₂S generation via the transsulfuration pathway. The transsulfuration pathway plays a central role in sulfur metabolism and redox regulation in cells. It involves the transfer of sulfur from homocysteine to cysteine through an intermediate (cystathionine) and is the only way in which mammals generate cysteine endogenously. Methionine is converted into homocysteine in a reversible 2-step process involving the enzymatic activity of SAM and S-adenosylhomocysteine (S-AdoCys) (not pictured). Homocysteine is then converted to cystathionine through CBS-mediated condensation of homocysteine and serine or cysteine, which can generate H₂O or H₂S, respectively. Cystathionine is then acted upon by CSE to generate cysteine and α-ketobutyrate (not pictured). Cysteine can be further acted upon by CBS, CSE, or 3-MST/cysteine aminotransferase (CAT) to generate H₂S, as well as other byproducts, such as GSH, pyruvate, and lanthionine. Notably, CSE is responsible for the physiologic clearance of homocysteine and can act on it alone to generate H₂S.

The main role of the other H₂S-producing enzyme in the transsulfuration pathway, CSE, is to convert cystathionine into cysteine and α-ketobutyrate. CSE can also produce H₂S by 2 main mechanisms: via an α, β-elimination reaction with cysteine or through a γ-replacement reaction between 2 molecules of homocysteine. Under high homocysteine levels (hyperhomocysteinemia), the latter reaction is dominant and is essential for the rapid clearance of toxic homocysteine from the system (44, 52, 55). Therefore, CBS and CSE activity are key drivers of the transsulfuration pathway, as well as cysteine catabolism and H₂S generation.

CBS is a homotetrameric enzyme of about 63-kDa subunits, and it binds 2 cofactors, pyridoxal 5′-phosphate (PLP) and heme (57). Although it can be regulated at the transcriptional level by nuclear factor (erythroid-derived 2)–like 2 (Nrf2), specific protein (SP)1, and SP3 in response to intrinsic and extrinsic stimuli, it is primarily constitutively expressed and regulated post-translationally (55). The noncatalytic N-terminal heme moiety in CBS acts as a redox sensor and exists in 2 oxidation states: ferric (Fe³⁺) and ferrous (Fe²⁺). Fe²⁺-CBS can bind CO and NO, resulting in the inhibition of its catalytic activity, implicating CBS as a crucial crossroads for all 3 gaseous signaling systems (58). In fact, a CBS variant commonly found in patients with homocystinuria, p.P49L, has altered spectral properties and an increased propensity to bind CO. This inhibition-prone CBS mutant may present a potential pathogenetic mechanism for the development of homocystinuria (59). CO/NO-induced inhibition of CBS can be rescued via exposure to air and the subsequent oxidation and recovery of the Fe³⁺ heme state (43, 58, 60–62). More recently, CBS has been reported to further demonstrate redox sensitivity through a CXXC motif in its central domain, which harbors a redox-active disulfide bond (Cys²⁷²–Cys²⁷⁵) (63). Niu et al. (63) demonstrated that this motif is essential to increase CBS activity and amplify CBS-dependent generation of H₂S in human embryonic kidney (HEK) 293 cells under reductive-stress conditions. Additionally, CBS can be regulated by the allosteric activation of S-adenosylmethionine (SAM), a universal methyl donor (64). The C-terminal regulatory domain of CBS exerts intrasteric regulation through partial inhibition of its own catalytic domain. SAM can bind this C-terminal regulatory domain and induce a conformational change, which clears the way for substrate binding and alleviates this inhibitory effect; SAM binding to CBS enhances the β-replacement reaction of homocysteine with serine 2–5-fold (53, 55, 58, 65). CBS is also modulated by post-translational modifications, such as sumoylation, glutathionylation, and phosphorylation. Sumoylation was the first reported post-translational modification of CBS; Agrawal and Banerjee (49) reported that sumoylation of CBS by human polycomb group protein 2 correlated with its localization to the nucleus and subsequent loss of catalytic activity. Furthermore, Niu et al. (66) later showed that CBS could be S-glutathionylated at Cys³⁴⁶; this modification was induced by oxidative stress in HEK293 cells (challenged with peroxide) and resulted in an ∼3-fold increase in CBS activity. In this way, CBS activity can be propagated by one of the products of the transsulfuration pathway in an indirect positive-feedback mechanism (Fig. 1) (66). Finally, a 2016 report by d’Emmanuele di Villa Bianca et al. (67) demonstrated that activation of muscarinic receptors, M1 and M3, induced phosphorylation of CBS^Ser227 and subsequently enhanced H₂S production in the urothelium, the epithelium that lines much of the urinary tract. Although considered to be constitutively expressed and rarely regulated at the transcriptional level, CBS activity is tightly regulated via its redox-sensitive nature, and post-translational modifications and can be super activated by the universal methyl donor, SAM.

CSE is a homotetramer composed of PLP-bound 45-kDa subunits and is the second H₂S-generating enzyme in the transsulfuration pathway (68). CSE primarily breaks down the CBS by-product cystathionine into cysteine, α-ketobutyrate, and ammonia. Like CBS, CSE can also catabolize cysteine into various other byproducts to generate H₂S (Fig. 1). The PLP-CSE interaction is required for enzymatic activity; the catalytic activity of CSE^N187A, a mutant enzyme that cannot bind PLP, is zero. Unfortunately, we know little about the post-translational regulation of CSE, despite several known phosphorylation sites that may modulate CSE activity. Renga et al. (69) reported that CSE can be phosphorylated, and therefore activated, by the Akt pathway in response to G protein-coupled bile acid receptor 1 (GPBAR1) activation (a bile acid–activated receptor), leading ultimately to H₂S-dependent vasodilation. Another group has shown that CSE can undergo persulfidation (a post-translational modification of cysteine discussed in detail later), but the significance of this modification is unknown (55, 70). Finally, CSE also undergoes sumoylation and the resulting nuclear localization (49). Unlike CBS, CSE is not constitutively expressed within cells; its expression is highly inducible in cells by a range of stimuli including endoplasmic reticulum (ER) stress, oxidative stress, nutrient deprivation, and hyperhomocysteinemia (55). CSE is encoded by the cystathionine gamma lyase (cth) gene, and its transcription is regulated under basal conditions by the transcription factor SP1 (71, 72). In the liver, where CSE is predominately expressed, farnesoid X receptor binds the CSE promotor and induces CSE expression and H₂S production upon activation by bile acid (73). CSE can also be induced by inflammation; in macrophages, LPS and TNF-α stimulation lead to increased CSE expression and H₂S production, as well as subsequent NF-κB persulfidation and recruitment to antiapoptotic gene promoters (74). CSE expression can be additionally induced by oxidative stress because the CSE promoter has been shown to contain a binding site for Nrf2, a master regulator of the oxidative stress response (75). Nrf2 has been reported to have binding sites within the sequences 5′-GTGATCTAGCA-3′ and 5′-ATGAGG CAGCT-3′ for CBS and CSE, respectively, implicating Nrf2 in the observed enhanced expression of both CBS and CSE under conditions of oxidative stress (76, 77). Surprisingly, H₂S can regulate Nrf2 expression as a means of positive feedback, and this is further discussed in the persulfidation signaling section of this review. Nutrient deprivation and amino acid starvation have also been reported to up-regulate CSE expression via activating transcription factor 4 (78). Additionally, hormones can regulate CSE expression; reduced signaling of growth hormone and thyroid stimulating hormone, or depleted growth hormone receptor expression can globally increase CSE and CBS expression via a mechanism involving the hypothalamic-pituitary axis (79). Finally, Nandi et al. (80) recently showed that increasing levels of H₂S itself can decrease CSE expression in HL1 cardiomyocytes. Mechanistically, they determined that H₂S inhibits SP1 activation and CSE transcription, resulting in a loss of CSE expression. Additionally, they showed that increasing homocysteine levels conversely resulted in an increase in CSE expression, but the exact mechanism for this observation remains unknown. Surprisingly, increasing homocysteine and H₂S levels had the opposite effect on CBS expression decreasing and increasing CBS, respectively, indicating an inverse relationship and potential novel feedback mechanism between CBS, CSE, and substrates of the transsulfuration pathway (80). This inverse relationship between CBS and CSE expression has not been adequately explored beyond this study, but it has notably been observed by several independent groups, including our own (12, 45, 46, 81, 82). Further exploration into the regulation and feedback of CBS and CSE is imperative to understanding their seemingly linear, nonredundant functions within the cell. Although CSE can be regulated through similar mechanisms to CBS, unlike CBS, it is predominantly controlled at the transcriptional level in response to cellular stress.

3-MST function and regulation

3-MST is a 33-kDa, cysteine-catabolizing enzyme that exists as either a monomer or an inactive disulfide-linked homodimer. It is primarily located in the mitochondria and cytosol of cells in most mammalian tissues, although its subcellular location seems dependent on its splice variant (29, 83). The human mecaptopyruvate sulfurtransferase (MPST) gene contains 595- and 299-bp exons divided by a 4405-bp intron, and the promoter region has features of a typical housekeeping gene promoter (84). Unlike CBS and CSE, 3-MST is primarily regulated via its redox-sensitive characteristics rather than post-translational modifications or transcriptional regulation. Its homodimer is formed through the redox-sensitive disulfide bond formation between 2 surface cysteines (Cys¹⁵⁴ and Cys²⁶³) of two 3-MST molecules. Three other cysteine residues are present (Cys⁶⁴, Cys²⁴⁷, and Cys²⁵⁴), with Cys²⁴⁷ being within the catalytically active site (85, 86). 3-MST can act as an antioxidant via oxidation of its Cys²⁴⁷ (3-MST-S) to cysteine sulfenate (3-MST-SO) or through oxidant-driven homodimerization. Both of these redox-sensing actions serve as enzymatic switches, thus ablating 3-MST activity; however, enzymatic activity is restored upon reduction by thioredoxin (Trx) of either 3-MST-SO or the disulfide-linked homodimer (83).

3-MST generates H₂S and pyruvate through the breakdown of cysteine in a 2-step manner. First, cysteine aminotransferase converts l-cysteine and α-ketoglutarate to 3-mercaptopyruvate (3-MP) and glutamate. Next, 3-MST transfers the sulfur group from 3-MP to its sulfur-accepting nucleophilic Cys²⁴⁷ to generate a 3-MST–bound persulfide and pyruvate. This MST-persulfide can then react with thiols or be reduced by Trx to generate H₂S (87, 88). Also of note, 3-MST can transfer the sulfur group from 3-MP to a cyanide molecule to form thiocyanate, which is safely metabolized and excreted (83). Because of this, 3-MST activity has inspired the development of a potential sulfanegen for treating cyanide poisoning (89). Like CBS and CSE, 3-MST can generate H₂S through the catabolism of cysteine; however, unlike CBS and CSE, 3-MST regulation is primarily redox driven.

H₂S metabolism

The intracellular levels of H₂S have been reported to range from undetectable to >100 μM, yet the steady-state concentration of H₂S in most tissues is estimated to be in the low nanomolar range (90). Kinetic studies have shown that sulfur fluxes more rapidly into H₂S than to GSH, which, considering the low steady-state concentration of H₂S, indicates a high clearance and/or consumption of H₂S in tissues (91). Indeed, when administered at sublethal doses, H₂S is rapidly oxidized and excreted as S₂O₃⁻ and SO₄²⁻ at a rate that is tissue specific (92, 93). This elimination of H₂S occurs in a tightly regulated enzymatic fashion through a mitochondrial sulfide oxidation pathway, which is associated with the mitochondrial electron transport chain (ETC) at the level of complex III (Fig. 2). Briefly, H₂S is oxidized in the mitochondrial matrix by sulfide quinone oxidoreductase (SQR) to generate a persulfide. This persulfide is further oxidized by ethylmalonic encephalopathy 1 protein (ETHE1), also known as persulfide dioxygenase, to produce sulfite. Sulfite is then further oxidized to SO₄²⁻ by sulfite oxidase, or to S₂O₃⁻ by rhodenase (36). Electrons released by SQR are captured by ubiquinone and transferred to the ETC at complex III, indicating that H₂S oxidation and elimination can promote ATP synthesis.

Figure 2.

H₂S metabolism. H₂S can be metabolized via oxidation or methylation, occurring in the mitochondria and cytoplasm, respectively. However, H₂S oxidation is thought to be the primary clearance pathway. During H₂S oxidation, H₂S is oxidized by SQR, thus forming a persulfide. This persulfide is further oxidized by ETHE1 to generate SO₃²⁻, which is converted to SO₄²⁻ and S₂O₃²⁻ by sulfite oxidase and rhodanese, respectively. SO₄²⁻ and S₂O₃²⁻ are then excreted into the urine and cleared from the body. Electrons released by H₂S oxidation by SQR are captured by ubiquinone and transferred to the ETC at the levels of complex III. During H₂S methylation, H₂S is converted into CH4S and (CH3)2S via S-methyltransferase (TMT). (CH3)2S can also be further metabolized by rhodanese to generate SO₄²⁻, which is excreted as waste through the urine. Cyt c, cytochrome c.

Methylation of H₂S is a secondary mechanism of metabolism and clearance. However, this mechanism is considered to be less important than oxidation and, unlike oxidation, occurs primarily in the cytoplasm of the cell (44). Through this mechanism, H₂S is converted into methanethiol and dimethyl sulfide via thiol S-methyltransferase. Dimethyl sulfide is another substrate of rhodenase, which is oxidized to thiocynate and SO₄²⁻ to be excreted in the urine.

Finally, H₂S can be scavenged by the metalloproteins (methemoglobin, metmyoglobin, and metneuroglobin) by forming sulfheme products or metallo-/disulfide-containing molecules like oxidized GSH (GSSG) (43, 44, 94, 95). This catabolic pathway is particularly interesting when considering the signaling balance of the other bioactive gases, CO and NO, because hemoglobin (Hb) is also a common sink for these gases. Therefore, if this sink is exhausted by any of these gases, the other two are unable to bind, ultimately leading to ineffective quenching and altered target activities.

HYDROGEN SULFIDE SIGNALING

Chemically, H₂S is a weak acid that directly ionizes in aqueous solution and freely diffuses through membranes (96). In biologic systems, H₂S is known to signal through 3 distinct pathways: 1) scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS), 2) binding to and/or reduction of the metal centers of iron-heme proteins, and 3) post-translationally modifying protein cysteine residues in a process known as persulfidation (26).

Redox role of H₂S

Several studies have shown that H₂S can readily scavenge ROS and RNS, like superoxide (⋅O₂), at higher rates than other classic antioxidants, such as cysteine and GSH (97). However, it should be noted that the capacity of H₂S to serve as a physiologic antioxidant is questionable given that its physiologic concentration is much lower than other canonical antioxidants. H₂S is accepted to be in the low nanomolar range, whereas GSH is present in the millimolar range at steady state (26). Nonetheless, exogenous H₂S has demonstrated profound antioxidant and cytoprotective capabilities in physiologic systems exposed to ROS and RNS. Moreover, the small size of H₂S, as well as its ability to freely diffuse across cellular membranes, may make it a more effective antioxidant in its immediate microenvironment than the larger molecule GSH, despite the dramatic concentration differences (98). Even so, it is more readily accepted that the observed antioxidant effects of H₂S are the result of broader indirect signaling rather than direct ROS/RNS quenching. As such, H₂S treatment can exert profound and prolonged antioxidant protection in cells.

One such way H₂S may exert its antioxidant effects is through modulating the expression and activity of classic antioxidants, like GSH and Trx. GSH is a tripeptide made up of glycine, glutamate, and cysteine, one of the major antioxidants in cells and the primary building block in GSH synthesis. As previously described, cysteine is synthesized from methionine through the transsulfuration pathway, wherein the key H₂S-producing enzymes CBS and CSE catalyze each step. Interestingly, several studies have shown that H₂S can promote GSH production and subsequent protection against oxidative stress in the brain, spinal cord, heart, gastrointestinal tract, liver, kidney, and lung (99). This indicates potential positive feedback, whereby the H₂S generated through the synthesis of GSH further promotes GSH production; several studies over the years have demonstrated this feedback. Lu et al. (100) illustrated this potential early on by showing that an H₂S donor, NaHS, promoted glutamate uptake into astrocytes through the enhanced trafficking of glial glutamate transporter [glutamate transporter 1 (GLT-1), also known as excitatory amino acid transporter 2 (EAAT2)], which, in turn, increased the cystine/glutamate antiporter system, which pumps oxidized cystine in from the extracellular matrix. The now intracellular cystine could then be reduced into cysteine and converted to GSH through the concerted actions of 2 enzymes, glutamate-cysteine ligase and GSH synthetase (100). Kimura et al. (101) also described a different means by which H₂S can promote cellular GSH production. They showed that H₂S produced intracellularly diffuses out of the cell to reduce cystine into cysteine in the extracellular space. Cysteine would then be taken up in the cell via a cysteine transporter and utilized for GSH synthesis (101). Jain et al. (102) further reported in 2014 that H₂S was able to increase cellular GSH through the up-regulation of glutamate-cysteine ligase catalytic subunit and modifier subunit. Most recently, however, this same group reported that treatment of C2C12 mouse myotubes with the H₂S-donor NaHS increased the expression of GSH biosynthesis genes and that the silencing of endogenous CSE expression by small interfering RNA attenuated this expression (103). In these ways, H₂S activity may indirectly contribute to redox homeostasis through the modulation of cellular GSH levels.

Trx is a small molecule of ∼12 kDa that contains a cysteine-glycine-proline-cysteine motif in its catalytic site. The 2 cysteine residues are the major sites of Trx oxidation, whereby reduction of ROS is facilitated via thiol-disulfide exchange. Oxidized Trx is reduced by Trx reductase, which is then further reduced by NADPH. Trx activity has been shown to exert intra- and extracellular functions in scavenging ROS to protect against oxidative stress, evade apoptosis, induce gene expression, and promote cellular proliferation (104). One of the earliest reports of H₂S signaling on Trx came in 2008 when Jha et al. (105) demonstrated the protective effect of H₂S against I/R injury in the mouse liver. This H₂S-mediated protection was associated with an improved GSH/GSSG balance, reduced lipid hydroperoxide formation, and an increase in Trx levels (105). A more recent study from 2013 showed that treatment of H₂S as Na₂S not only increased Trx expression in a model of ischemic-induced heart failure but also attenuated high-fat–induced left ventricle remodeling of the heart (106). The cardioprotective effects of H₂S against this model of heart failure were determined to be Trx dependent because mice that expressed a dominant negative mutant of Trx did not respond to Na₂S treatment. Finally, H₂S has also been shown to modulate the expression of Trx-interacting protein (TXNIP) in endothelial cells. TXNIP can bind to and inhibit Trx activity (107). Tian et al. (108) recently reported that endogenous H₂S is necessary for the preservation of endothelial cell function via suppressing the MAPK/TXNIP cascade. CSE^−/− knockout mice exhibited impaired endothelial-dependent relaxation, which was associated with an increase in MAPK phosphorylation and consequent up-regulation of TXNIP. Treatment of these knockout mice with the H₂S donor NaHS rescued these results and restored endothelial function (108). Taken together, one way in which H₂S signaling may contribute to redox homeostasis is through the regulation of classic antioxidants, like GSH and Trx.

As mentioned previously, H₂S readily scavenges ROS/RNS, and such interactions may serve a greater purpose than simple redox balance. Indeed, some studies have shown that H₂S can directly interact with several ROS and RNS, including hypochloric acid (HClO) (which is produced by neutrophils) and peroxynitrite (ONO₂⁻), to not only protect against harmful oxidative stress but also propagate further H₂S signaling and synergy with another signaling gas, NO (26).

Nagy et al. (109) showed that H₂S readily reacts with HClO to form polysulfides, chemical compounds that contain a chain of sulfur atoms. Interestingly, hydrogen polysulfides (H₂S_n) have been recently suggested to have signaling properties of their own, including regulating ion channels, tumor suppressors, and protein kinases (110). Additionally, some effects previously attributed to H₂S signaling, such as persulfidation are now considered to be potentially regulated by H₂S_n rather than directly through H₂S, although the physiologic relevance of this is unclear (50, 111, 112). This is because H₂S cannot react directly with a protein thiolate, because of thermodynamic constraints, but a polysulfide can (26, 111). Indeed, direct incubation of H₂S with proteins that can undergo persulfidation, like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or bovine serum albumin (BSA), was unable to cause persulfide formation (97, 113, 114). However, the internal sulfurs in H₂S_n are more oxidized and can therefore react with cysteine thiols and yield protein persulfidation in vitro (115). However, the physiologic relevance of persulfidation by polysulfides is controversial because persulfides are notoriously unstable and are readily reduced. Therefore, it is unclear how polysulfides survive endogenously when continuously at risk of reduction via intracellular molecules, such as GSH (50).

Of further interest is the interaction of H₂S with ONO₂⁻, a powerful oxidant and inducer of cell death that is formed by the reaction of free radical superoxide (⋅O₂) with the free radical NO (⋅NO) in vivo. Inhibition of ONO₂⁻ formation or promotion of its decomposition has been reported to be cytoprotective in several pathologies, including vascular diseases, I/R injury, circulatory shock, inflammation, pain, and neurodegeneration (116). Thionitrate (HSNO₂), a physiologic NO donor, is generated upon the direct interaction of H₂S with ONO₂⁻ (98). Filipovic et al. (117) first showed this formation of HSNO₂ and consequential promotion of NO signaling under physiologically relevant conditions in cells treated with H₂S. Therefore, H₂S has been shown to not only mitigate the toxicity of ONO₂⁻ through its active scavenging and clearance but also promote NO synergy through the formation of HSNO₂. H₂S can further modulate NO signaling pathways by reacting with S-nitrosothiols to form the small trans-nitrosylating thionitrous acid (HSNO) (117) and by reacting directly with NO to yield the signaling molecule nitroxyl (HNO), which has implications in cardiovascular health (118, 119).

Metalloprotein and H₂S interactions

Another way in which H₂S can exert its effects is through its reduction and/or direct binding of the heme centers of metalloproteins. The mechanism and consequences of these reactions are influenced by several aspects including: 1) the redox state of the heme-iron [(Fe³⁺)-heme or (Fe²⁺)-heme], 2) the environment in the heme pocket, 3) the protonation state of bound sulfide, and 4) the presence or absence of oxygen or reducing agents in solution (1). For example, Fe³⁺-heme can bind H₂S or HS⁻ directly, but the resulting moiety of such interactions is dependent on the residues in the heme pocket. Specifically, if there is a strong presence of basic residues within the pocket that can adequately accept protons from bound H₂S, the resulting sulfide-reacted heme moiety is (Fe³⁺)-heme-HS⁻. On the other hand, if there are several nonpolar residues in the pocket, the fully protonated H₂S interaction with the Fe³⁺-heme is stabilized, yielding (Fe³⁺)-heme-H₂S. A comprehensive review of the chemistry of H₂S heme-iron interactions are beyond the scope of the current review; readers are directed to other articles for additional details (106, 114, 120). This section will continue by examining several reported specific H₂S-metalloprotein interactions and their physiologic consequences.

It is well established that heme proteins can also interact with NO, CO, and O₂, illustrating another layer of crosstalk between the signaling gases. Cytochrome c oxidase (COX), the final structural complex of the ETC, is one such protein that can interact with all 3 signaling gases. Functionally, COX drives the final steps of the ETC by accepting 4 electrons from cytochrome c and transferring them to 1 molecule of oxygen, thereby converting 1 molecular oxygen (O₂) into 2 molecules of water (H₂O). This process of transferring electrons fuels the pumping of 4 protons (H+) across the inner mitochondrial membrane to establish an electrochemical proton gradient, which drives ATP synthesis by ATP synthase. The binding of H₂S to COX has been reported to have a biphasic effect on cellular respiration, promoting it at low levels and completely ablating it at higher levels (121). This effect may be due to the 2 main mechanisms by which H₂S can modulate COX: by binding the Fe³⁺-a₃ and Cu_B subunits of COX, thereby inhibiting its enzymatic activity, or directly donating electrons to (i.e., reducing) COX and ostensibly serving as the only known inorganic substrate of the respiratory chain (122–124). Pietri et al. (125) suggest that the polarity of the environment around the heme a₃ subunit of COX can explain this observed biphasic effect. They posit that the polar environment around the heme a₃ subunit, which contains a tyrosine residue and Cu_B center, promotes heme a₃ reduction to stimulate ETC activity at low H₂S concentrations. However, at higher concentrations, H₂S can directly bind both the a₃ and Cu_B centers, thereby forming stable H₂S-Cu_B and unstable heme H₂S-Fe²⁺ inhibitory moieties. Because of the instability of the heme H₂S-Fe²⁺ inhibitory complex, the reduced activity of COX at moderate levels of H₂S is reversible. In fact, the displacement of H₂S by O₂ can completely restore COX activity at this stage (126). However, if H₂S is in excess, it can push the further reduction of heme a₃ to a stable heme a₃ H₂S-Fe³⁺ inhibitory complex, thereby decreasing heme affinity for O₂ and rendering the inhibition of COX irreversible.

Interestingly, COX is not the only component of the ETC that can interact with H₂S. Vitvitsky et al. (127) reported that the interaction between H₂S and cytochrome c can lead to a plethora of complex signaling outcomes and protective effects. They suggest that the reduction of cytochrome c by H₂S leads to the initial formation of a HS^⋅/S^⋅− radical. This radical can then either interact with a protein thiol to generate a persulfidated protein and superoxide (a process which will be discussed in detail in the next section) or promote further cytochrome c reduction and sulfite/S₂O₃⁻ production. Reduction of cytochrome c by H₂S can adequately promote ETC activity by bypassing complex III, which could lead to an increase in ATP synthesis and an accumulation of reactive sulfur species. Moreover, cytochrome c–mediated protein persulfidation could have major cytoprotective implications in and out of the mitochondria. For example, persulfidation of ATP synthase in the mitochondria would increase ATP synthesis, whereas persulfidation of procaspase 9 by cytoplasmic cytochrome c would abort cellular apoptosis postinitiation. Both scenarios lend credence to the cytoprotective effects of H₂S.

Finally, as mentioned previously, interactions of H₂S with methemoglobin, metmyoglobin, and metneuroglobin can serve as a scavenging mechanism and sink for excess H₂S. Hb is composed of 4 globular chains, all of which harbor an oxygen-binding heme group. Myoglobin and neuroglobin, on the other hand, are monomeric globular proteins with only 1 heme group, both of which have higher affinities for oxygen than Hb (125). Interactions of these heme proteins with H₂S form sulfheme derivatives, although the mechanism by which these are formed remains unknown. These sulfheme products have a much lower affinity for O₂ and cannot be converted back into normal hemeproteins (125, 128–130). Because sulfheme proteins cannot adequately bind O₂, high levels can be potentially toxic (as in the case of sulfhemoglobinemia), although low to moderate concentrations can be tolerated (131). This tolerance can be simply explained due to the physiologic turnover of red blood cells and implicates erythrocyte destruction as another means of H₂S degradation (125, 132). More recently, Potor et al. (133) demonstrated that H₂S can attenuate Hb oxidation and the subsequent lipid peroxidation in atherosclerotic lesions. Oxidized Hb can be both prooxidant and proinflammatory and may play a crucial role in atherogenesis, illustrating another means by which H₂S is cardioprotective.

Persulfidation detection

The third and final way that H₂S can signal is through a process known as persulfidation, whereby a thiol (-SH) is added to a reactive and accessible cysteine (-SSH) within a protein to alter its function. Persulfidation can be identified in many ways, but the most common assay is the modified biotin-switch assay, which was originally designed to identify S-nitrosylation in cell and tissue lysates (Fig. 3A) (134). Simply stated, free cysteines within a protein are blocked via methyl methanethiosulfonate, therefore leaving reactive persulfidated residues alone to be attacked by a biotinylated nucleophile (biotin-HPDP), leaving any disulfide bonds within the protein undisturbed. This biotinylated protein can then be precipitated using streptavidin beads for identification by Western blot and/or mass spectrometry (MS) analysis. Using this method, persulfidation was first identified in 2009 in a report by Mustafa et al. (70), wherein it was suggested that nearly 10–25% of proteins in the liver undergo persulfidation, including actin, tubulin, and GAPDH. This early report showed that persulfidation appeared to enhance cysteine reactivity, whereas S-nitrosylation diminished it. This observed trend of S-nitrosylation and persulfidation acting inversely to one another has held up in many reports following (134). Additionally, this early report identified 2 mechanistic consequences of persulfidation: it enhanced the catalytic activity of GAPDH, and it induced actin polymerization in liver.

Figure 3.

Methods of persulfide detection. A) Thiols are presumed to be selectively blocked by MMTS, which leaves persulfides free to be readily biotinylated by nucleophilic attack. These proteins can then be precipitated on streptavidin beads for analysis; however, this method is chemically problematic. B) In the modified tag-switch assay, both free thiols and persulfides are blocked by methylsulfonyl benzothiazole (MSBT), but the resulting persulfide-mediated mixed aromatic disulfide can selectively react with a cyanoacetic acid–based probe. Differential fluorescence tagging involves the tagging of both thiols and persulfides with a fluorescent maleimide derivative. C) The resulting disulfide from the tagged persulfide can then be reduced by DTT, and fluorescence is lost. D) Persulfidated peptides can be directly identified by MS following persulfide stabilization by maleimide. E) Persulfides can also be stabilized and biotinylated via biotin-tagged maleimide, immobilized on streptavidin beads, and eluted by DTT for detection and analysis. BT, benzothiazole; CN, cyanide; HPDP, pyridyldithiol; Mal, maleimide; MMTS, S-methyl methanethiosulfonate; SMe, methylated sulfur; S-SMe, dithiomethane.

It is worth mentioning that, despite continued wide use in the field, the modified biotin-switch method for persulfide identification is chemically flawed and therefore not considered to be completely selective for persulfides (50, 135). Its predominant flaw is that methyl methanethiosulfonate readily reacts with thiols and persulfides. This is the major hurdle for any method seeking to selectively tag persulfides. Given this flaw, other methods of persulfide identification have been developed, and use of the modified biotin-switch method is discouraged. However, Zhang et al. (113) refined this method and proposed a modified tag-switch assay, which is based on the notion that the disulfide bonds generated from reacting a persulfide with a thiol-blocking reagent are more sensitive to nucleophilic attack than other protein disulfides or thioethers (, 136). They proposed that persulfides could then be tagged using a nucleophile coupled to a reporter molecule following thiol and persulfide blocking. Specifically, they blocked thiols and persulfides using methylsulfonyl benzothiazole and used a biotinylated methyl cyanoacetate to tag the persulfides (Fig. 3B). The selectivity of this method was demonstrated using glutathionylated, sulfenylated, and unmodified BSA (which were untagged and contained intramolecular disulfides) and persulfidated BSA (which was successfully tagged). Two fluorescent cyanoacetic acid derivatives, which are considered to be more specific, have since been developed and now allow for detection of persulfides by fluorescence microscopy or in gels (137). Importantly, sulfenic acids are known to react with cyanoacetic acids, but this issue is easily circumvented by blocking sulfenic acids with dimedone. This method is currently the most selective and sensitive persulfide-tagging method for identification.

Another assay for persulfide tagging exploits the similarity in thiol and persulfide reactivity with electrophiles by labeling both with the fluorescent cyanine 5 (Cy5)-maleimide (74). The resulting R-S-S-maleimideCy5 adduct from the reaction with the protein persulfide is then subsequently reduced using a reducing agent, such as DTT, resulting in a loss of red fluorescent signal (Fig. 3C). Unfortunately, this method of detection is also not without issue. First, the presence of persulfidation is identified by a loss of signal, so it is not easily coupled to methods of proteomic analysis, which require a positive signal, like MS. Next, the ability of Cy5-maleimide to react with amines can also generate a lot of background signal, which could obscure any possible change in signal if persulfides are low in concentration. Finally, it is not clear how reducing agents differentiate between other DTT-reducible residues, like S-nitrosothiols and dilsufides, and persulfides (113).

The final common method of persulfide identification utilizes a proteomics MS analysis approach. Although MS can technically be used to directly identify the presence of persulfides in proteins, the shift in mass due to the addition of a sulfur atom is almost the same as the addition of 2 oxygen atoms (70). This makes it very difficult to differentiate these shifts in whole proteins. Additionally, persulfides are inherently unstable, which further adds to the difficulty of their direct detection by MS. Therefore, methods have been developed to stabilize peptide persulfides prior to MS analysis with N-ethylmaleimide or iodoacetamide (135, 138, 139) (Fig. 3D). A method that also utilizes persulfide trapping and MS analysis is the biotin thiol assay (Fig. 3E). In this assay, biotin maleimide, like Cy5-maleimide, reacts with both thiols and persulfides prior to protein fragmenting. The biotinylated peptides are bound to streptavidin beads, and persulfidated peptides are eluted from the beads via a reducing agent and analyzed via MS. Thiolated peptides, which are not held by a disulfide bond, remain on the beads.

Identified persulfidated proteins

Since the discovery of persulfidation, several other proteins have been shown to undergo this modification. We briefly discuss the following proteins, while leaving several others undescribed: transcription factors NF-κB, SP1, kelch-like ECH-associated protein 1 (Keap1), IFN regulatory factor 1 (IRF-1); enzymes MEK1, protein tyrosine phosphatase 1B (PTP-1b), ATP synthase F1 subunit α (ATP5A1), and lactate dehydrogenase (LDH) A; as well as ion channels such as transient receptor potential (TRP) channels and ATP-sensitive K⁺ (K_ATP) channels.

There are several transcription factors that can be persulfidated. As briefly touched upon in a previous section, the persulfidation of NF-κB, a crucial transcription factor for several prosurvival genes within a cell, is crucial for its antiapoptotic activity (74). Mechanistically, H₂S persulfidates the p65 subunit of NF-kB at Cys³⁸, which promotes binding to its coactivator ribosomal protein S3, therefore enhancing NF-kB transcription activity. Additionally, our group showed that H₂S can persulfidate the transcription factor SP1 in endothelial cells at Cys⁶⁸ and Cys⁷⁵⁵. Persulfidation of these residues stabilizes SP1 and regulates its transcription of VEGF receptor 2 and neurolipin-1, 2 major regulators of endothelial cell function (140). Interestingly, the previously mentioned antioxidant effects of H₂S may be credited to persulfidation because increases in Trx, as well as GSH, may be attributed to impact of H₂S on Nrf2 transcriptional activity. Nrf2 is mostly found inactive in the cytoplasm in a complex with Keap1. However, it has been reported that Keap1 can undergo persulfidation at Cys¹⁵¹, which induces a conformational change in Keap1 and leads to the dissociation of Nrf2 from the complex (141, 142). The newly liberated Nrf2 translocates to the nucleus, where it binds to promoters containing the antioxidant response element sequence and induces antioxidant response element–dependent genes, such as Trx and GSH. Finally, IRF-1, a transcription repressor, has been shown to undergo persulfidation to maintain mitochondrial biogenesis (143). H₂S-induced persulfidation of IRF-1 enhances both its binding to the DNA methyltransferase 3a (Dnmt3a) promoter and the resulting repression of Dnmt3a. Dnmt3a is crucial for the methylation and inhibition of mitochondrial transcription factor A (TFAM) promoter. Therefore, upon IRF-1 persulfidation, Dnmt3a is bound up and unable to methylate TFAM, which enhances mitochondria biogenesis. Clearly, H₂S has far-reaching cellular impacts at the level of protein transcription via regulation of transcription factors.

Cellular enzymatic activity can also be impacted by persulfidation. For example, MEK1 (also known as MAP2K1) has been shown to be persulfidated at Cys³⁴¹ in human endothelial cells and fibroblasts (144). Persulfidation of MEK1 leads to the phosphorylation of ERK1/2 and translocation of ERK1/2 into the nucleus, where it can activate poly [ADP-ribose] polymerase 1 (PARP-1) and induce DNA damage repair. This persulfidation appeared to be crucial because mutations of Cys³⁴¹ inhibited ERK1/2 phosphorylation and attenuated the DNA damage repair response. Moreover, PTP-1b, one of the earliest-identified protein tyrosine phosphatases and classic negative regulator of the insulin signaling pathway, has also been shown to be persulfidated. A 2011 report by Krishnan et al. (145) showed that H₂S-mediated persulfidation of PTP-1b at Cys²¹⁵ inhibited its enzymatic activity. This subsequently resulted in the phosphorylation and activation of downstream ER kinases, which facilitated the restoration of ER homeostasis under conditions of ER stress (145). In this way, persulfidation of PTP-1b may explain how H₂S signaling can protect against ER stress and may therefore be an attractive therapeutic target in several disease states. Another enzyme that has been identified as a target of persulfidation is ATP synthase, which is driven by the proton gradient developed via the ETC. It is located in the inner mitochondrial membrane and is responsible for the generation of ATP by cellular respiration. Módis et al. (146) demonstrated that increasing H₂S levels increased persulfidation of ATP5A1 at 2 cysteine residues (Cys²⁴⁴ and Cys²⁹⁴) in HEK293 and HepG2 cells. ATP synthase mutants containing cysteine-to-serine mutations at both Cys²⁴⁴ and Cys²⁹⁴ had significantly reduced enzymatic activity. Additionally, liver tissues taken from CSE^−/− mice displayed lower ATP5A1 persulfidation and attenuated ATP synthase activity, indicating 1 way in which H₂S signaling can maintain mitochondrial energy production and cellular respiration. Interestingly, H₂S has been suggested to impact mitochondrial respiration beyond just ATP synthase persulfidation, but these additional mechanisms will be discussed in a later section concerning H₂S impacts on mitochondria. Finally, one of the 2 LDH subunits, LDHA, has also been shown to undergo persulfidation (147). LDH is a tetramer that resides at the crossroads of aerobic and anaerobic glycolysis; there are 5 LDH isoenzymes composed of different arrangements of its 2 subunits, LDHA and LDHB. Isoenzymes rich in LDHA tend to assist in pyruvate-to-lactate conversion, whereas isoenzymes rich in LDHB conversely facilitate lactate-to-pyruvate conversion, which can feed into the Krebs cycle and promote oxidative phosphorylation. LDHA expression and activity is particularly pronounced in various cancers, shunting pyruvate away from feeding the Krebs cycle in the mitochondria and promoting the production of lactate and NAD⁺ in the cytosol. This shunting process, known as anaerobic glycolysis, is typically observed in normal cells in hypoxic conditions and is only viable for short amounts of time. However, despite the presence of oxygen, this process has been repeatedly observed in cancer cells (a canonical characteristic of cancer known as aerobic glycolysis or the Warburg effect). Because CBS, H₂S, and LDHA are up-regulated in colon cancer, Untereiner et al. (147) wanted to see whether there was a link between H₂S signaling and LDHA activity. Indeed, they discovered that LDHA activity is increased by H₂S in a dose-dependent manner. Additionally, they showed that LDHA is persulfidated at Cys¹⁶³, which is at least partially responsible for the observed increase in LDHA activity upon treatment with H₂S donor, GYY4137.

The final group of proteins this review will briefly describe are ion channels, specifically TRP channels and K_ATP channels. TRP channels are ion channels located on the plasma membranes of most cells. There are about 30 different kinds of TRP channels, which can mediate a host of sensations like heat, pressure, taste, and pain. The first TRP channel to be identified to undergo persulfidation was reported in 2014 by Liu et al. (148). TRP channel TRPV6 is a membrane Ca²⁺ channel that is expressed in bone marrow mesenchymal stem cells (BMMSCs). Liu et al. (148) demonstrated that BMMSCs produce H₂S as a means to propagate their self-renewal and osteogenic differentiation. They suggested that this self-sustaining process was due to persulfidation of TRPV6 channels at Cys¹⁷² and Cys³²⁹. Indeed, loss of H₂S-dependent TRP cation channel subfamily V member 6 (TRPV6) persulfidation led to aberrant intracellular Ca²⁺ flux, which attenuated PKC/Erk-mediated Wnt/β-catenin signaling, and consequently inhibited osteogenic differentiation of BMMSCs. Although they only looked at the mechanism of TRPV6 in their report, Liu et al. (148) also suggested that several other TRP channels could undergo persulfidation, including TRPV3 and TRPM4, yet there has not been any additional research into the consequence of persulfidation on these channels. However, another group more recently identified another TRP channel, TRPV4, as another target of persulfidation. Naik et al. (149) demonstrated in 2016 that persulfidation stimulates TRPV4 activity in aortic endothelial cells to enhance Ca²⁺ and K⁺ influx and dilate vessels. Finally, the K_ir6.1 subunit of the K_ATP channels was reported to be persulfidated at Cys⁴³ in endothelial cells. Surprisingly, persulfidation led to a reduction in ATP association but an increase in phosphatidylinositol 4,5 bisphosphate binding to K_ir6.1, which resulted in increased K_ATP channel activity and enhanced vasodilation (150). Moreover, both H₂S-mediated K_ATP persulfidation and vasodilation were attenuated in K_ir6.1-Cys⁴³ mutants, suggesting that persulfidation of K_ATP may be the crucial mechanism by which H₂S mediates smooth muscle relaxation and vasodilation.

Persulfidation formation and deformation

Despite the intense interest in H₂S signaling by persulfidation, very little is actually understood about the process by which proteins undergo this modification. Originally, it was believed that thiolate on a protein could react directly with H₂S to form a protein persulfide; however, this reaction is not possible, because of thermodynamic constraints. This lack of reaction was confirmed because incubation of proteins, like GAPDH, with H₂S led to no detectable protein persulfidation (26, 97, 113). There are several working hypotheses to explain how persulfidation can occur (enzymatically or nonenzymatically) (Fig. 4).

Figure 4.

Mechanisms of protein persulfidation. A) H₂S cannot directly act on a free thiol to produce a protein persulfide. B, C) However, H₂S has been shown to directly act on preexisting modifications like S-sulfenylation (B) and S-nitrosylation (C), although reacting H₂S with an S-nitrosothiol residue to generate a protein persulfide has only been demonstrated in silico. It has been reported that H₂S may react with a disulfide bond (D), but the persulfide products are unstable, and the reaction rate is quite slow. E) Finally, persulfidation may not arise from H₂S signaling directly but, rather, through polysulfide (HS_x-, x = 2–7) signaling on a protein cysteine. Reactions in red are impossible, yellow are physiologically questionable, and green are potential true mechanisms.

Reactions with preexisting protein modifications is one proposed means by which proteins can acquire the addition of a thiol group onto its cysteine. For example, S-sulfenylation (S-OH) is an important oxidative post-translational modification that generates sulfenic acids and has been identified in nearly 1000 proteins, including several proteins that have been identified to be persulfidated as well, such as phosphatase and tensin homolog (PTEN) and PTP-1b (151). Sulfenic acids react readily with thiols to generate disulfide bonds and therefore have been suggested to react similarly with H₂S. Indeed, Zhang et al. (113) demonstrated in a 2014 report that GAPDH can form a sulfenic acid that can be acted upon by H₂S to generate a persulfide in its place. Moreover, they were able to generate persulfidated BSA utilizing its relatively stable sulfenic acid, as well as demonstrate that intracellular persulfidation colocalizes with the ER, an organelle that is rich with sulfenic acids. Notably, it is well established that sulfenic acids can be formed on cysteines simply in the presence of oxygen or metal ions in buffers, which can then react readily with H₂S to form S-SH residues. This could offer one explanation as to why it was originally believed that H₂S could directly react with thiols, a process which is now known to be thermodynamically unfavorable (26). As previously described, Vitvitsky et al. (127) demonstrated a means by which the direct interaction of H₂S with cytochrome c generates a reactive HS^⋅/S^⋅− radical, which can tag proteins, such as procaspase 9, with a persulfide and further generate H₂O₂. H₂O₂ can also induce sulfenic acid (S-OH) formation to further propagate persulfidation and H₂S signaling. The researchers posit that such reactivity may be particularly relevant under hypoxic conditions, when H₂S metabolism is inhibited, or under apoptotic conditions, when cytochrome c is released into the cytoplasm.

S-nitrosylation is another important protein post-translational modification that can react directly with H₂S. The reaction of H₂S with S-nitrosothiols can lead to the removal of the -NO group from the protein cysteine to form the smallest S-nitrosothiol, thionitrous acid (HSNO), which can freely diffuse across membranes to propagate NO signaling (117). Although, the generation of a persulfide from this interaction is thermodynamically unfavorable, a 2013 in silico study did indicate that the surrounding molecular environment of the S-NO could feasibly impact the thermodynamics enough to allow for certain proteins to be directly persulfidated (152).

H₂S interaction with an inter- or intramolecular disulfide bond is another attractive means by which H₂S may generate persulfides without the requirement of enzymatic intervention; however, this mechanism’s physiologic relevance is questionable. Francoleon et al. (138) were able to show that GSSH could, in fact, be generated from mixing GSSG and H₂S; however, the product was incredibly unstable. Moreover, Filipovic et al. (117) determined that between the bond energies of GSH disulfide (GSSG) and GSH-persulfide (GSSH), GSSH was lower; therefore indicating that the reaction of H₂S with oxidized thiols is a slow reaction. Additionally, 2 other groups were unable to identify persulfidation of BSA or IgG as a consequence of reacting H₂S with disulfides at physiologically relevant H₂S levels.

Protein persulfidation as an intermediate is a process that has been known to be carried out in several biochemical processes by enzymes known as sulfurtransferases and cysteine desulfurases (153). Two examples of this enzymatic generation of persulfides have already been previously discussed through the examination of 3-MST and SQR activity. Even more interesting, work done by Ida et al. (154) on reactive cysteine persulfides has introduced a new way of thinking of CBS and CSE activity. They suggest that cysteine persulfide (Cys-SSH) arises from the respective CBS and CSE generation and cleavage of Cys-S-S-Cys (cystine) and is the true by-product of the transsulfuration pathway (H₂S being a product of C-SSH decomposition) (154). They found that the generation of the reactive intermediate, Cys-SSH, was sufficient to lead to dramatic increases in per- and polysulfidated proteins rather than H₂S. This offers insight into CBS/CSE/3-MST persulfidation specificity but does not exactly answer why exogenous H₂S or H₂S donors could rescue persulfidation in several studies described above. Nonetheless, such research offers new insights into the complexity of persulfide formation and H₂S signaling.

More intriguing still is that persulfidation may not directly involve H₂S at all but, rather, molecules derived from H₂S, the previously mentioned polysulfides. Polysulfides (HS_x⁻) are products of incomplete H₂S oxidation and can range from x = 2 to x = 7; H₂S₂ is extremely unstable under biologic conditions and readily breaks down into H₂S and elemental sulfur, whereas longer polysulfides are more stable, and HS_x⁻, where x = 4 or 5 are the most abundant species (26). Surprisingly, a 2013 report by Kimura et al. (115) suggested that polysulfides have a 300-times-higher capacity to activate TRP channels than H₂S. Additionally, another group suggested that many experimental H₂S donors, such as NaHS, are contaminated with polysulfides, which may ultimately be responsible for experimentally observed persulfide formations (155). Mechanistically, the sulfane sulfur atoms (sulfur atoms that are linearly covalently bound only to other sulfur atoms) within these polysulfides are believed to attack the free thiol within a protein, thereby leading to persulfidated proteins.

Like other post-translational modifications, cellular mechanisms exist that can remove a persulfide from a protein to shut down the signal. As mentioned previously, Trx is a disulfide oxioreductase, which plays a critical role in redox homeostasis within the body. It has 2 cysteines in its catalytic site that are the primary regions of ROS reduction. However, beyond this redox role, Trx and other Trx-related proteins are also key players for the removal of persulfide modifications (50, 137, 156). Indeed, Trx successfully reduces HAS persulfide and Cys-SSH; auranofin, a Trx reductase inhibitor, increases total levels of intracellular persulfidation (137). Moreover, silencing of Trx-related protein of 14 kDa also was shown to increase intracellular persulfidation, which may be a relevant depersulfidase under conditions in which Trx activity is focused to other systems, like the peroxiredoxin system during conditions of oxidative stress (156). Mechanistically, it is thought that the outer sulfur of the protein persulfide is transferred to the nucleophilic cysteine in Trx, resulting in a transient Trx-SSH, which is rapidly resolved into H₂S and oxidized Trx. Alternatively, the nucleophilic cysteine of Trx may react with the inner sulfur of the persulfide, therefore resulting in the elimination of H₂S and a Trx disulfide complex, which is subsequently resolved. Interestingly, another canonical redox system may also act to depersulfidate proteins. The glutaredoxin system (GSH/GSH reductase/glutaredoxin), which maintains low levels of GSSG to maintain redox homeostasis, has also been reported to reduce persulfides in vitro (156). However, GSH reductase knockout mice did not show any difference in persulfidation when compared with their WT counterparts; the physiologic contribution of the glutaredoxin system to depersulfidation remains unclear.

Although there is still much to learn regarding the specifics of H₂S physiologic signaling, H₂S has promise as a therapeutic agent in a range of disease states. Several were touched upon in earlier sections, but the rest of this review will take a more in-depth look at the role of H₂S in disease and specifically how H₂S effects on the mitochondria may be the central link.

HYDROGEN SULFIDE, MITOCHONDRIA, AND DISEASE

Hydrogen sulfide and mitochondria

The mitochondria are considered one of the primary targets of H₂S signaling, having impacts on mitochondrial respiration, mitochondrion-dependent apoptosis, biogenesis, and morphology (Fig. 5). Like the other signaling gases, NO and CO, H₂S is known to directly impact the activity of the ETC, acting as both a substrate and COX antagonist depending on concentration (122, 157). At concentrations <20 μM, H₂S can actually act as the only inorganic substrate, donating an electron to the ETC via its own metabolism by SQR (158–160). The electrons released from H₂S oxidation by SQR are captured by ubiquinone and transferred to the ETC at complex III to be further propagated down the chain. This activity can stimulate oxidative phosphorylation and ATP production within the cell. In a 2019 report, Libiad et al. (161) demonstrated the importance of this mitochondrial sulfide oxidation pathway in colon cell metabolism. The colon epithelium is harbored in a unique environment in that it is regularly exposed to high levels of H₂S generated from the gut microbiome. Because of this, the sulfide oxidation pathway in colon cells is exquisitely efficient. The researchers report that the sulfide oxidation pathway enzymes are located at the host-microbiome interface and that SQR expression is critical to shield the epithelium from H₂S poisoning and effectively promote oxidative phosphorylation. On the other hand, high levels of H₂S were reported to inhibit the ETC, perturb redox balance, shift metabolism toward reductive carboxylation, and inhibit cellular proliferation (161). Additionally, H₂S generated inside the mitochondria by 3-MST has been reported to be sufficient to enhance mitochondrial electron transport and cellular bioenergetics at low levels in a manner that is SQR-dependent (124). Moreover, a mitochondrion-specific H₂S donor, AP39, which consists of a mitochondrion-targeting motif (triphenylphosphonium) coupled to an H₂S-donating moiety (dithiolethione), was shown to stimulate mitochondrial bioenergetics (162). Furthermore, direct persulfidation and consequential enhancement of ATP synthase is yet another means in which H₂S can stimulate ETC activity within the mitochondria (146).

Figure 5.

Impacts of H₂S on mitochondria. Green arrows indicate persulfidation signaling, blue arrows are antioxidant effects, and red arrows are metalloprotein interactions by H₂S. H₂S can persulfidate a number of transcription factors, such as Nf-KB, IRF-1, and PGC-1a, to promote transcription of several antiapoptotic and mitochondrial biogenesis genes. Moreover, H₂S can increase PGC-1a expression via cAMP/PKA/AMPK activation, which enhances its transcriptional stimulation of mitochondrial biogenesis, as well as coactivates Nrf2, which promotes cellular survival pathways. Nrf2 can also be activated by H₂S through its persulfidation-induced disassociation from its complex with Keap1. Furthermore, H₂S activation of mitoKATP channels via persulfidation has also been shown to directly inhibit apoptosis. Finally, H₂S can prevent apoptosis after its induction via binding to and sequestering the cytochrome c (Cyt c) released into the cytosol from the mitochondria. H₂S can also stimulate mitochondria bioenergetics. Persulfidation of ATP synthase and direct H₂S electron transfer to the ETC via SQR/ubiquinone and COX interactions stimulate ATP production within the mitochondria. Lastly, H₂S production has been shown to maintain mitochondrial fusion and prevent mitochondrial fission. One mechanism by which it can do this is through the redox-sensitive maintenance of the outer mitochondrial membrane fusion protein MFN2.

As H₂S levels increase, its inhibitory effects on COX overpower its stimulatory effects until the complex is completely inhibited. In an in vivo lugworm model, Arenicola marina, complete inhibition is achieved at 50 µM; however, the concentration required for full COX inhibition in other models is dependent on factors such as cell type and cell environment (158, 163). At these levels, H₂S is highly cytotoxic, shutting down ETC activity, decreasing intracellular ATP levels, enhancing ROS generation, and inducing mitochondrial depolarization and apoptosis (158, 164). Moreover, H₂S has been shown to further inhibit ATP production in high doses via up-regulation of uncoupling protein 2, which dissipates the proton gradient to disrupt ATP synthesis, and down-regulation of COX subunits I and II (165). The researchers suggest that through these processes, the cells can adapt to H₂S onslaught. Indeed, up-regulation of uncoupling protein 2 can protect the cells from oxidative stress via reduction of ROS production and down-regulation of COX subunits, which helps to conserve precious energy while ATP synthesis is disrupted.

H₂S can also elicit its effects on mitochondrion-dependent apoptosis. At toxic levels, H₂S induces apoptosis via translocation of Bcl-2 associated x protein (BAX) to the mitochondria, which assists in the formation and opening of the mitochondrial permeability transition pore (mPTP), subsequent release of cytochrome c, activation of proapoptotic proteins (p21), and down-regulation of antiapoptotic proteins [B-cell lymphoma 2 (Bcl-2)] (164, 166, 167). In rat cerebellums with an ETHE1 deficiency, representative of the rare autosomal disorder ethylmalonic encephalopathy, H₂S accumulation led to mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and enhanced mPTP induction (168). Moreover, a 2019 report by George et al. (169) suggested that CSE/H₂S signaling mediated LPS-induced late-apoptotic events in murine macrophages.

However, under certain conditions, and at nontoxic levels, H₂S has actually been demonstrated to play an antiapoptotic role. An early report in 2009 by Hu et al. (170) showed that H₂S attenuated rotenone-induced apoptosis in neuroblastoma cells. The researchers found that H₂S elicited precisely the opposite effects on ΔΨm, cytochrome c release, and BAX levels, respective to earlier reports, thereby explaining the observed antiapoptotic phenotype. They suggested that H₂S achieved this via inhibition of the proapoptotic MAPK pathway, which is mediated by mitochondrial K_ATP, which, as previously established, can directly undergo persulfidation at Cys⁴³ to induce vasorelaxation. Blockage of these channels ablated H₂S-mediated protection, therefore confirming that the cytoprotective role of H₂S is, at least partially, K_ATP dependent. Another study by Kimura et al. (171) also demonstrated the cytoprotective effects of H₂S-dependent K_ATP activation in cortical neuronal cells. Furthermore, a recent report by Wang et al. (15) indicated that persulfidation of mitochondrial ATP5A1 at Cys²⁴⁴ conferred resistance to mitochondrion-mediated apoptosis in the adrenal glands of male mice challenged with LPS as well as CBS^b/− mice (15). Even more recently, Vitvitsky et al. (127) demonstrated a mechanism by which H₂S can directly prevent apoptosis, even after an apoptotic event has been triggered. They suggest that under apoptotic conditions, when cytochrome c is released into the cytoplasm, oxidation of H₂S by cytochrome c can propagate the persulfidation of nearby target proteins, such as procaspase 9. Specifically, they showed that persulfidation of procaspase 9 resulted in its inhibition and consequential inhibition of the caspase cascade and mitochondrion-dependent apoptosis. Finally, H₂S can exert its antiapoptotic effects through direct persulfidation of NF-κB, which enhances its binding to the promoters of several antiapoptotic genes (74).

H₂S has also been shown to regulate mitochondrial biogenesis, or the generation of new mitochondria. This process is controlled by its master regulator, peroxisome proliferator-activated receptor γ 1α (PGC-1α), which coactivates Nrf2, to turn on TFAM and induce the transcription of nuclear-encoded mitochondrial proteins (172). H₂S impacts on Nrf2 and TFAM were already discussed in a previous section of this review, where it was reported to indirectly enhance activity of both via persulfidation of upstream proteins (141–143). Interestingly, 2 more recent reports by Untereiner et al. (173, 174) demonstrated a more direct impact of H₂S signaling on mitochondrial biogenesis. In their first report, they showed that endogenous CSE-dependent H₂S production up-regulated PGC-1α in primary hepatocytes via activation of the cAMP/PKA pathway. Moreover, they reported an increase in PGC-1α activity due to H₂S-driven persulfidation. They postulated that such impacts would enhance mitochondrial biogenesis and went on to confirm this speculation in their following report. They further reported H₂S impact on another regulator of mitochondrial biogenesis, peroxisome proliferator–activated receptor-γ coactivator-related protein (PPRC), which was shown to be a target of persulfidation and whose expression was also regulated by CSE-derived H₂S. Recently, another independent group showed H₂S regulation of mitochondrial biogenesis via AMPK regulation of PGC-1α in primary murine cardiac tissue (175). Oral administration of an active H₂S-releasing prodrug, SG-1002, enhanced cardiac mitochondrial content and localization of PGC-1α to the nucleus in a dose-dependent manner. Perhaps unsurprisingly, lower levels of SG-1002 had a positive influence on PGC-1α activity, whereas higher doses imparted a negative influence. Finally, a 2019 report indicated the propensity of H₂S to maintain mitochondrial biogenesis in osteoblasts challenged with elevated levels of homocysteine, which was able to restore osteoblast function, ATP synthesis, and redox homeostasis (176).

Finally, and most recently, H₂S has been reported to impact mitochondrial morphogenesis, the balance maintained between mitochondrial fission and fusion processes within the cell. Although once thought to be static organelles, mitochondria are now recognized to undergo dynamic fission and fusion processes, which can impact mitochondrial function and spatial placement within the cell (177). These processes are regulated by a family of GTPases known as dynamin-related proteins (Drps). Drp1 is the primary driver of mitochondrial fission, whereas mitofusins 1 and 2 (MFN1/MFN2) drive outer mitochondrial membrane fusion, and optic atrophy 1 (OPA1) drives inner mitochondrial membrane fusion (178–181). Fission 1 (Fis1) regulates Drp1 recruitment to the mitochondria and can further assist in the fission process as well (182). Interestingly, H₂S can also regulate these GTPase expression levels. The first account of H₂S regulation of mitochondrial morphogenesis machineries came from Liu et al. (183) in 2017; they demonstrated that H₂S reduced mitochondrial fragmentation in endothelial cells in a high-glucose/high-palmitate environment by down-regulating phosphorylated Drp1/Drp1 levels, and this in turn prevented ROS accumulation and apoptosis (183). An earlier report by Qiao et al. (184) demonstrated H₂S-driven mitochondrial fusion; utilizing transmission electron microscopy, they showed that the H₂S donor NaHS inhibited mitochondrial fission in a dose- and time-dependent manner in vitro in mouse neuroblastoma cells (neuro-2a). They determined that the impact on mitochondrial morphology was due to an ERK1/2-dependent loss of Drp1 at the mRNA and protein level and that Drp1 overexpression was sufficient to reverse mitochondrial elongation seen in H₂S-treated cells. Two 2018 independent studies further implicated H₂S signaling activity on mitochondrial morphogenesis (185, 186). Both studies observed a sirtuin 3 (SIRT3)-dependent up-regulation in OPA1 and down-regulation in DRP1, whereas Meng et al. (185) also specifically noted an increase in MFN1/MFN2 and decrease in Fis1 expression levels. Both groups noted enhanced mitochondrial function upon H₂S treatment in mouse models of cardiac hypertrophy, which was partly due to its regulation on mitochondrial morphogenesis. A 2018 report by our group further reported the necessity of H₂S promotion of mitochondrial fusion, showing that CBS-dependent H₂S stabilized MFN2 in a redox-sensitive manner in ovarian cancer cells (187). Upon CBS knockdown, mitochondria become hyperfragmented, which leads to an increase in oxidative stress and induces MFN2 degradation through the activation of JNK/HUWE1 pathway. Finally, a 2019 study by Xu et al. (188) on wound healing demonstrated that exogenous H₂S increased OPA1 and decreased Drp1 expression levels, which consequently enhanced ΔΨm, inhibited ROS production, and enhanced wound-healing capacity in skin fibroblasts.

Clearly, H₂S signaling is far reaching, but the importance of its impact on mitochondrial function and mitochondrion-dependent processes cannot be overstated. Mitochondria lie firmly at the crossroads of cell life and death, serving as major redox organelles and a key source of ATP generation. Therefore, it is not surprising that H₂S has shown therapeutic promise in a host of disease states, due in no small part to its regulation of the mitochondria. The following sections will briefly examine the recent and impactful reports of H₂S signaling in several diseases from a mitochondrion-focused perspective. Several reviews exist that focus on the role of H₂S in specific diseases beyond its mitochondrial impact. The reader is encouraged to read these other sources for more in-depth information on H₂S in specific disease states (13, 44, 189–198).

Hydrogen sulfide and cardiovascular disease

Although the role of H₂S as a signaling gas was first demonstrated in the brain, its potential role as a cardioprotective agent was immediately appreciated. This is because it has been shown to be endogenously produced in vascular smooth muscle cells, as well as to induce vasorelaxation in rat aortic tissues, much like another signaling gas, NO (10, 11). Moreover, because of the roles CBS and CSE play in metabolizing homocysteine through the transsulfuration pathway, defective CBS/CSE function has been linked to hyperhomocysteinemia, a medical condition and serious cardiovascular disease risk characterized by excessive levels of homocysteine (>15 µM) in the blood (51, 199). Additionally, homocystinuria, a debilitating multisystem disorder that targets the cardiovascular system as well as connective tissues and the nervous system, is caused by a genetic deficiency of CBS (199). For these reasons, and others, H₂S was considered as a potential cardioprotective agent and potential therapeutic against a multitude of cardiovascular diseases.

Endothelial cells line the interior of blood and lymphatic vessels, and their dysfunction has been suggested to contribute to a host of cardiovascular diseases, including atherosclerosis and hypertension (200, 201). Hydrogen sulfide has been reported to protect endothelial cell function from various stressors (such as hyperglycemic, hyperlipidemic, and high-salt insult) via the mitochondria in several studies. In one such study, Liu et al. (183) reported that rat aortic endothelial cells treated with high glucose and high palmitate (a fatty acid) had significantly less CSE expression and H₂S production. However, treatment of these cells with exogenous H₂S inhibited mitochondrion-dependent apoptosis, which was associated with a reduction in expression of mitochondrial fission proteins, mitochondrial fragmentation, and ROS levels, as well as increased mitophagy activity (183). An earlier study also demonstrated that the mitochondrion-targeted H₂S donors AP39 and AP123 successfully decreased mitochondrial membrane hyperpolarization and ROS production and additionally increased ETC complex III activity and mitochondrial metabolism in endothelial cells challenged by glucose onslaught (202). A high-salt diet, a contributing factor to clinical hypertension, induces vascular endothelial cell apoptosis (203). A 2015 report by Zong et al. (203) demonstrated that high salt decreased expression of CSE in HUVECs and that H₂S may protect endothelial cells from apoptosis by attenuating oxidative stress and maintaining mitochondrial health. Moreover, AP39 pretreatment also protected mitochondrial DNA integrity and bioenergetics in endothelial cells under oxidative stress, thus conferring a cytoprotective effect (162).

Atherosclerosis is defined as the hardening and narrowing of arteries due to the buildup of fatty plaque along the arterial walls. It is the leading risk factor in a host of cardiovascular diseases, including stroke, heart attack, and coronary artery disease, and develops from chronic pathophysiological processes such as vascular inflammation, endothelial dysfunction, smooth muscle cell proliferation and migration, lipoprotein accumulation, macrophage differentiation, and monocyte adhesion (204). Over the last several years, H₂S has been demonstrated to have effects in many of these processes and is therefore considered to be an attractive therapeutic against atherosclerosis. For example, H₂S administration has been shown to inhibit lipid hydroperoxide formation in LDL and protect against oxidized (oxidized LDL) cytotoxicity via inhibition of cytotoxic lipid accumulation and foam cell formation, which are key steps in atherogenesis (14, 142, 205, 206). Moreover, oxidative stress-induced mitochondrial dysfunction and apoptosis in endothelial cells is considered to play a pathogenic role in the development of atherosclerosis (207, 208). Wen et al. (209) demonstrated that CSE-dependent H₂S signaling prevented HUVECs from undergoing apoptosis upon exposure to H₂O₂. This effect was credited to H₂S-mediated protection of mitochondrial function from oxidative damage, as determined by analysis of ATP synthesis, ΔΨm, and cytochrome c release. But H₂S mitigation of atherogenesis goes beyond redox restoration. Mitochondrial DNA damage has been reported as a risk factor independent of ROS to accelerate atherosclerosis by acting on vascular smooth muscle cells and monocytes (210). As previously discussed, MEK1 persulfidation by H₂S induces PARP-1–mediated DNA damage repair (144). A 2018 report by Cheung et al. (211) further elucidated the preventative nature of H₂S against atherosclerosis via persulfidation. They identified 70 persulfidated aortic proteins, 23 of which were localized to the mitochondria, such as superoxide dismutase and medium-chain specific acyl-CoA dehydrogenase, which play key roles in mitochondrial lipid metabolism and redox maintenance. GSH peroxidase, which is key in reducing lipid peroxidation, was also found to be persulfidated, which consequently reduced lipid peroxidation and enhanced antioxidant defense. Finally, H₂S was shown to inhibit macrophage-derived foam cell formation, another key event in atherogenesis, via activation of the K_ATP/ERK1/2 pathway (14).

Untreated atherosclerosis and the resulting blockage of coronary arteries can lead to a life-threatening condition known as acute myocardial infarction, or heart attack. H₂S has been shown to reduce myocardial ischemia injury through protection of mitochondrial function and potentially via mitochondrion-mediated macrophage M2 polarization (212, 213). In the first scenario, rat hearts were perfused and subjected to ischemic conditions both with and without the H₂S donor NaHS. H₂S treatment successfully reduced infarct volumes following ischemia injury (212). The researchers suggested that this protection was mediated by H₂S antioxidant signaling and protection of mitochondrial function. In the latter scenario, the researchers reported reduced pathologic cardiac remodeling following myocardial infarction in WT and CSE^−/− mice treated with H₂S supplementation (213). H₂S treatment significantly shrunk infarct size and mortality, which correlated with an increase in the proresolution M2 subset of macrophages. Interestingly, transplanting NaHS-treated bone marrow–derived macrophages into macrophage-depleted WT and CSE^−/− mice was able to attenuate cardiac damage following myocardial infarction. The researchers determined that this H₂S-dependent M2 polarization was driven by enhanced mitochondrial biogenesis and increased fatty acid oxidation in the macrophages.

Tissue damage caused by the reintroduction of blood into the tissue following periods of ischemia (lack of oxygen/hypoxia) is known as I/R injury, or reoxygenation injury. During the prolonged ischemic period, such as during cardiac arrest, tissues acutely adapt to the hypoxic environment in such a way that upon rapid oxygen reperfusion, oxidative stress and oxidative damage can damage the tissue. In myocardial I/R injury, rapid pH normalization, and Ca²⁺ overload, as well as ROS/RNS onslaught at reperfusion due to loss of proper mitochondrial function, result in the opening of the mPTP and propagation of the apoptotic pathway (214, 215). There is growing evidence that H₂S protects mitochondria during I/R to improve respiration and promote biogenesis. Indeed, treatment of isolated mitochondria with an H₂S donor, Na₂S, induced a greater recovery of posthypoxic respiration than in those mitochondria that went untreated (216). Furthermore, the mitochondrion-targeted H₂S donor, AP39, distributed to rats undergoing I/R at the time of reperfusion, was shown to protect against myocardial I/R injury by inhibiting mPTP in a cyclophilin d–dependent manner (217). AP39 has also shown reproducible protection against I/R in brain and kidney injury (218–220).

Finally, H₂S has also been shown to have promise as a potential therapeutic agent against hypertension and systemic hypertensive damage. Physiologically, although CSE expression is dominant over CBS expression in the vasculature, pharmacological inhibition of CSE by dl-propargylglycine or CBS by aminooxyacetic acid (AOAA) alone was not sufficient to alter blood pressure in rats. However, utilizing both drugs in combination with one another was able to induce hypotension (221). It is worth mentioning that a purely selective pharmacological inhibitor for CBS does not exist on the market. In fact, AOAA, which is commonly used as a CBS inhibitor, actually inhibits both CBS and CSE and surprisingly preferentially inhibits CSE over CBS (222). Therefore, we speculate that perhaps the observed hypotensive phenotype observed by dl-propargylglycine/AOAA treatment was due to intense CSE inhibition rather than CSE/CBS dual inhibition. Regardless, several studies have shown that H₂S deficiency may contribute to the development of hypertension in humans, with reported plasma H₂S levels being lower in patients with grade 2 and 3 hypertension when compared with patients with normal BP (223). Indeed, H₂S intervention in several studies utilizing murine models of hypertension has shown therapeutic promise (224–228).

Hydrogen sulfide and neurodegenerative disease

Before H₂S was assigned any other functional role in humans, it was first established as a neuromodulator (10). All 3 H₂S-generating enzymes have been thoroughly implicated in the CNS, with CBS being the predominant source in astrocytes and CSE/3-MST predominantly in the neurons (192). Therefore, it is not surprising that H₂S signaling has been extensively studied in the brain and neurologic diseases, including Alzheimer’s disease (AD), Parkinson disease (PD), and Huntington’s disease (HD).

The most prevalent neurodegenerative disorder is AD, a devastating condition associated with aging that impacts the cerebral cortex and hippocampus and dramatically impairs an individual’s cognitive function and memory (229). A key characteristic of AD cases is the presence of mutations in the amyloid precursor protein (APP), which undergoes sequential proteolysis by α, β, and γ secretases. Another hallmark of AD is the aggregation of β-amyloid (Aβ), which is formed by the aforementioned dysfunctional processing of APP and Tau proteins, which generate amyloid plaques and neurofibrillary tangles, respectively. Many murine AD models have APP mutations and display pronounced amyloid plaques (230). It has been suggested that soluble Aβ can deregulate H₂S signaling through inhibition of the neuronal cysteine transporter EAAT3/EAAC1, which results in oxidative stress and neurodegeneration (231, 232). This inhibition could impact H₂S generation and GSH production by CBS and CSE acting on cysteine in the brain. Indeed, H₂S levels in the plasma and brain, as well as neuronal expression of SAM, an allosteric activator of CBS, were both diminished in patients with AD when compared with healthy controls (233–235). Treatment of H₂S in the APP/PS1 mouse model of AD has been reported to reduce cognitive impairment as well as mitigate oxidative stress (236). Moreover, H₂S impact on redox homeostasis and mitochondrial biogenesis through Nrf2 activity via Keap1 persulfidation may also offer another explanation of therapeutic protection that H₂S confers against AD, given that NaHS enhances Nrf2 expression (77, 236). Moreover, a recent study reported that H₂S treatment inhibits β-secretase 1 (BACE1), the β secretase that processes APP and is responsible for Aβ generation, via regulation of the PI3K/Akt pathway in the APP/PS1 model of AD (237). Finally, AP39, a mitochondrion-targeted H₂S donor, was shown to enhance H₂S in the mitochondrial intracellular compartments and ameliorate spatial memory defects and inhibit brain atrophy in the APP/PS1 mouse model of AD (238). The researchers surmise that H₂S protection was achieved through the preservation of mitochondrial function and bioenergetic metabolism.

PD is the second most prevalent neurodegenerative disease behind AD, and it is characterized by debilitating motor dysfunction. It affects the substantia nigra of the brain and is associated with misfolding and aggregation of the protein α-synuclein (192). Persulfidation of the E3-ligase, Parkin, has been shown to be important for the clearance of such misfolded proteins, and that dysregulated H₂S signaling has been reported in the disease (239). Indeed, post mortem analysis of patient striata indicated decreased levels of Parkin persulfidation and reduced enzymatic activity. Moreover, H₂S supplementation by pharmacological H₂S donors confers protection against neurodegeneration in cultured cells and animal models of PD. Hu et al. (170) determined that apoptosis induced by rotenone challenge, which is commonly used to model PD in vitro and in vivo, was ameliorated upon NaHs treatment in the human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). They attributed the protective effects of H₂S to maintenance of mitochondrial health via regulation of the ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK_ATP)/p38 and Jnk/MAPK pathway. This same group later demonstrated H₂S protection against PD in 2 mouse models of PD (240). They determined that H₂S impacted neuroinflammation associated with PD by attenuating microglial activation, as well as conferred protection against oxidative stress. Another group treated a mouse model of PD with inhaled H₂S for 8 h/d for 7 d (241). Inhalation of H₂S protected against the PD-induced movement dysfunction and prevented neuronal apoptosis and microglia activation in the nigrostriatal region. Finally, CBS overexpression has also been reported to protect against the 6 hydroxydopamine–induced model of PD (242).

HD, another neurodegenerative disease that affects cognitive and motor function, is characterized by aggregation of the huntingtin protein, which is induced via polyglutamine repeats on the protein (192). Cysteine metabolism and H₂S signaling dysfunction has recently been identified in in vitro and in vivo models of HD, as well as in human HD brains (243). This loss of cysteine and H₂S activity in HD models was attributed to decreased CSE expression due to inhibition of its physiologic transcription factor, SP1, by mutated huntingtin. Unsurprisingly, loss of CSE, cysteine, and H₂S levels contributed to pathophysiological redox imbalance, elevated ROS levels, and impaired stress response. It is worth noting that CSE can be alternatively transcribed by activating transcription factor 4 in response to cellular stressors like amino acid deprivation and ER stress. Interestingly, this pathway has also been reported to be dysregulated and a contributing factor to neuronal death in HD (244). Surprisingly, no study to date has attempted to treat HD with H₂S donor or in models of CSE overexpression. However, H₂S may still be an attractive therapy against HD given that cysteine resupplementation in models of HD have shown therapeutic promise (245). Moreover, our group has shown that persulfidation of SP1 by H₂S can enhance its transcriptional activity, thereby indicating a potential mechanism by which HD may be treated by H₂S (140).

H₂S signaling has wider implications in the field of neuronal health beyond the 3 major neurodegenerative diseases described above. Its regulation of mitoK_ATP channels has been reported to protect against traumatic brain injury in a rat model treated with exogenous H₂S (246). A toxic buildup of H₂S was found in the cerebrospinal fluid of 37 patients with amyotrophic lateral sclerosis when compared with healthy controls. Further examination in in vitro and in vivo models revealed that these enhanced H₂S levels are due to astrocyte and microglia dysfunction and lead to characteristic cell death in motor neurons (54). Additionally, H₂S can restore cognitive function in 2 diabetic murine models (streptozotocin-induced diabetes in rats and db/db mice) by ameliorating ER stress and mitochondrion-dependent apoptosis, respectively, in the hippocampus (247, 248). Finally, CSE expression has been reported to be decreased in patients with spinocerebellar ataxia type 3 (SCA3) (249). SCA3 is a disorder driven by a CAG repeat expansion in the ATXN3 gene, which leads to toxic protein aggregation and consequential tissue damage and impairments in coordination, balance, and movement. Overexpression of CSE in a Drosophila model of SCA3 was reported to be protective against protein aggregation–mediated damage by promoting protein persulfidation, reducing oxidative stress, reducing immune response, and attenuating tissue damage (249).

More broadly, H₂S may also regulate neuronal health via its impacts on mitochondrial morphogenesis. As discussed above, H₂S signaling can alter mitochondrial shape and function through regulation of the mitochondrial morphogenesis GTPases (Drp1, Fis1, MFN2, MFN1, and OPA1). Abnormal mitochondrial morphology has been linked to several neurodegenerative disease states, such as PD, but H₂S signaling and therapy in this context have yet to be examined (250, 251).

Hydrogen sulfide and cancer

The most recent disease of interest in the H₂S therapeutics field is cancer. The impact of H₂S in cancer was first described in 2013 by 2 independent research groups, one [Szabo et al. (46)] working on colorectal cancer and ourselves working on ovarian cancer (45, 46). Szabo et al. (46) were the first to show selective up-regulation of the H₂S-producing enzyme CBS in colon cancer tissues when compared with healthy patient-matched mucosa tissues or the surrounding noncancerous peritumor tissue. CBS was also up-regulated in several colon adenocarcinoma-derived cell lines as compared with the nonmalignant colonic epithelial cell line NCM356 (46). Our group showed that CBS was selectively and significantly up-regulated in several ovarian cancer cell lines and primary epithelial tumor tissues when compared with healthy controls. Moreover, within the patient samples, CBS expression was found to be particularly overexpressed in serous carcinoma (the most commonly diagnosed ovarian cancer type) and higher-grade tumors (45). Both studies surprisingly demonstrated CBS localization in the mitochondria and elucidated the positive effects of CBS/H₂S on mitochondrial bioenergetics and cancer cell malignancy. Our laboratory further showed that CBS expression conferred resistance against platinum-based chemotherapy in ovarian cancer cells. Silencing CBS via small interfering RNA or small-molecule inhibition attenuated cancer cell proliferation, invasion, and migration in colon and ovarian cancer, as well as resensitized ovarian cancer to chemotherapy. Moreover, CBS activation by SAM increased cellular proliferation and mitochondrial bioenergetics in colon cancer cells (252).

Our group has since published 2 more articles elucidating the role of CBS function on lipid metabolism and mitochondrial morphogenesis in ovarian cancer cells. Our 2015 report found that CBS expression was essential to maintain a process known as lipogenesis, or de novo lipid synthesis (12). Elevated lipid generation is recognized as a hallmark of many cancers because an increased lipid content is required for building new lipid membranes in rapidly dividing cells, as well as for oxidation in the mitochondria for ATP synthesis to meet the high energy demand of cancer. We found that CBS silencing greatly attenuated the expression of several key lipogenic enzymes, like fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1), through the repression of their transcription factors sterol regulatory element-binding protein 1 and 2 (SREBP1/2), and inhibited cell migration and invasion as well as tumor growth in vivo. Moreover, SREBP1/2 expression was able to rescue CBS silencing phenotype, implicating CBS regulation of lipid metabolism as essential for ovarian cancer malignancy. Interestingly, H₂S control of mitochondrial morphogenesis may also have implications in cancer because mitochondrial dynamics are strikingly deregulated in cancer (253). Our most recently published study discovered the CBS/H₂S-dependent maintenance of mitochondrial fusion via promotion of redox-sensitive MFN2 stabilization in ovarian cancer (187). Silencing of CBS induced mitochondrial fragmentation and subsequent bioenergetic dysfunction in ovarian cancer cells. Notably, mitochondrial fusion has been shown to be abundant in chemotherapy-resistant ovarian cancer and supportive of cell survival, which can reconcile our initial observation of chemoresistance with our observation of CBS promotion of mitochondrial fusion (254).

Szabo et al. (147, 255, 256) have published several additional studies of H₂S-generating enzymes in colorectal cancer since their initial 2013 report. Phillips et al. (255) notably showed that forced CBS overexpression in an adenoma-like colonic epithelial cell line was sufficient to induce carcinogenesis, during which these “cancer-like” cells develop metabolic and gene signatures that are characteristic of colorectal cancer. CBS overexpression also increased cellular bioenergetics, proliferation, invasion, tumor formation in vivo, and resistance to loss-of-attachment-induced cell death (anoikis). Also, Untereiner et al. (147) reported that H₂S-induced persulfidation of LDHA stimulated mitochondrial respiration in HCT116 colon cancer cells. Another more recent report suggested, strikingly, that acquired resistance to the chemotherapeutic agent 5-FU actually induced CBS up-regulation in HCT116 colon cancer cells, which accompanied a glycolysis-to-oxidative-phosphorylation switch and increased cytochrome p450 enzymes CYP1A2 and CYP2A6, which are the major enzymes involved in drug metabolism (256). Finally, as previously discussed, a report by Libiad et al. (161) reported the importance of the SQR/sulfide oxidation pathway in colon cells. In this same study, they showed that high levels of H₂S had an antiproliferative impact on colon epithelial cells. Colorectal cancer cell lines and tissues were shown to circumvent this attenuation of proliferation by up-regulating the mitochondrial sulfide oxidation pathway enzymes, like SQR (161). Taken together, targeting H₂S biogenesis and/or metabolism may offer a new therapeutic strategy against colorectal cancers.

H₂S signaling and biogenesis have been reported in several other cancer types beyond ovarian and colorectral. Szczesny et al. (257) also demonstrated the up-regulation of all 3 H₂S-producing enzymes (CBS, CSE, and 3-MST) in lung adenocarcinoma. This increase in H₂S production was shown to stimulate mitochondrial DNA repair via persulfidation of exonuclease G, a mitochondrial enzyme that catalyzes the hydrolysis of ester linkages at the 5′ end of a nucleic acid chain and may play a role in apoptosis. Human breast cancer invasion and epithelial-mesenchymal transition has been shown to be inhibited by H₂S sequestration of phosphorylated p38 expression, as well as decreased proliferation, G₀/G₁ phase cell cycle arrest, and increased apoptosis (258). Zhao et al. (259) reported that CSE/H₂S signaling can repress androgen receptor transactivation through direct persulfidation at Cys⁶¹¹ and Cys⁶¹⁴ and can inhibit cellular proliferation in both androgen-dependent and antiandrogen-resistant prostate cancer cell lines. Furthermore, H₂S, as well as CBS, CSE, and 3-MST, was shown to be up-regulated in patient tissues of oral squamous cell carcinoma when compared with surrounding benign oral mucosa, and CBS has been reported as specifically up-regulated in thyroid carcinomas (260, 261). Given that H₂S at elevated, yet nontoxic, levels confers cytoprotection in a host of disease states, it is not surprising that cancer cells would exploit H₂S signaling, especially to achieve therapy resistance.

Hydrogen sulfide and metabolic disease

Diabetes mellitus (DM) is a chronic condition that involves a dysfunction in production and/or utilization of the hormone insulin. Insulin is secreted by β cells from the pancreatic islets to assist the body in the storage and utilization of dietary sugars and fats. After eating, insulin levels spike and stimulate glucose uptake and metabolism in the liver, adipose tissue, and skeletal muscle, where it is metabolized or stored as glycogen for later use. The one exception is in adipose tissue, where glucose is partially metabolized into and stored as fatty acids. DM can occur when the pancreas secretes little to no insulin, or if the body is incapable of responding to insulin signaling. DM itself is not necessarily life threatening; however, it is associated with several other chronic complications, like cardiac myopathy, atherosclerosis, peripheral neuropathy, chronic kidney disease, and stroke (198). H₂S has protective implications in many, if not all, of the previously listed chronic complications (some of which have already been reviewed) primarily through its cytoprotective properties. However, H₂S has also been shown to potentially play a role in the regulation of insulin secretion and sensitivity and may therefore find purpose in the treatment of DM itself, rather than simply the management of symptoms. Although, admittedly, the data are contradictory, this section will briefly focus on a few key studies that elucidate the role of H₂S signaling on insulin secretion and resistance with a small focus on mitochondrial impacts.

H₂S has been shown to be produced in pancreatic β cells primarily through the actions of CBS and CSE, although there have been reported species-specific preferences between the two with little to no evidence of 3-MST activity (262–264). Surprisingly, H₂S signaling has been reported to inhibit insulin secretion from β cells in the pancreas in conditions of hyperglycemia but elicit no effect in the presence of low or no glucose (262, 265, 266). CBS overexpression induced the same effect on insulin secretion in a high-glucose environment, and glucose-stimulated release of insulin was inhibited in CSE^−/− mice when compared with WT (266, 267). Altogether, it appears that endogenous H₂S generated in pancreatic β cells inhibits glucose-dependent release of insulin. The mechanistic explanation for how H₂S achieves this inhibitory effect is somewhat complex. Early hypotheses suggested that H₂S signaling impacted insulin secretion via regulation of K_ATP channels, given that they are targets for persulfidation. Indeed, Yang et al. (268) reported that H₂S could effectively stimulate K_ATP in hyperglycemic conditions but was incapable of such stimulation under normoglycemia. Furthermore, another study reported that NaHS treatment could reduce intracellular [K⁺] and [Ca²⁺] as well as increase membrane potential in nearly all HIT-T15 insulinoma cells, which all indicated K_ATP stimulation (266). However, other mechanisms have been reported as well because H₂S has also been shown to prevent insulin secretion in models that do not rely on K_ATP stimulation, like KCl- and tolbutamide-induced insulin secretion, and was even shown to still inhibit insulin secretion in the presence of a K_ATP opener, suggesting that H₂S may act in a post-K_ATP manner (262). Indeed, another report suggested that H₂S may actually inhibit insulin secretion at the level of voltage-dependent Ca²⁺ channels, which prevents an influx of Ca²⁺ into the cell (the go signal for release of insulin from intracellular stores) (267). Surprisingly, a recent article by Takahashi et al. (269) demonstrated the role of H₂S on proinsulin synthesis and argued that H₂S actually stimulates insulin release. Proinsulin synthesis requires 2-methylthiolation (so-called ms2 modification) of an adenosine residue in lysine-transporting tRNA molecule, a process which has been reported to be tightly regulated by CBS/CSE activity.

Conflicting reports exist with regard to the impact of glucose on H₂S levels and signaling. For example, Yang et al. (268) showed that incubation of INS-1E cells in high-glucose medium for 24 h reduced H₂S production by 46% when compared with cells cultured in normoglycemic conditions, and Zhang et al. (72) demonstrated similar results in INS-1E cells and freshly cultured rat pancreatic islets via CSE inhibition. On the other hand, Kaneko et al. (264) reported that a hyperglycemic environment actually stimulated CSE expression as well as increased H₂S production in isolated mouse islets and cultured MIN6 mouse pancreatic β cells. This discrepancy remains unresolved to date. Moreover, contradictory reports also exist with regard to the pro- and antiapoptotic nature of H₂S on pancreatic β cells. Yang et al. (265) demonstrated that both exogenous H₂S and CSE overexpression decreased cellular viability accompanied by morphologic feature of apoptosis, positive TUNEL staining, and DNA fragmentation. The researchers showed that H₂S-induced apoptosis occurred through p38 MAPK stimulation and ER stress. In contrast, Kaneko et al. (264) showed that NaHS treatment attenuated glucose-stimulated apoptosis in mouse β cells. Taken together with their observation that glucose induced H₂S/CSE expression, the researchers postulate that H₂S/CSE may be up-regulated in hyperglycemic conditions to limit β cell glucotoxicity. This same group reconfirmed their findings in another report 2 years later, demonstrating that NaHS protected isolated mouse pancreatic β cells from apoptosis induced by palmitate, a cytokine mixture of TNF-α, IFN-γ, and IL-1β or hydrogen peroxide (28). In all 3 scenarios, apoptosis was inhibited upon H₂S treatment, as evidenced by reduced DNA fragmentation, TUNEL staining, and caspase activity. The researchers hypothesized that in mouse β cells, which constitutively express CBS, H₂S inhibits insulin under normal conditions. However, under conditions of stress, CSE expression is induced, and CSE-derived H₂S confers cytoprotection. It is worth noting that the discrepancies between studies cannot be attributed to H₂S levels because both groups utilized a similar dose for their respective experiments (50–200 and 100 μM, respectively). Unfortunately, because H₂S regulation of insulin secretion and β cell apoptosis remains controversial, it is unclear whether H₂S signaling contributes to or prevents the development of DM.

H₂S thus far has been suggested to play a therapeutic role in several disease states previously described; however, in the case of DM, it may be disease exacerbating. Yet, again, it remains unclear whether H₂S treatment is helpful or detrimental. It has been reported that H₂S can promote insulin resistance in the liver. NaHS was able to mimic the effects of high insulin/high glucose–induced insulin resistance in hepatocytes and high insulin–induced CSE expression (270). These results indicate that up-regulation of H₂S/CSE contributes to high glucose and high insulin–induced liver insulin resistance. However, in contrast, H₂S promotion of mitochondrial biogenesis suggests a potential therapeutic benefit to patients with DM. Mitochondrial dysfunction contributes greatly to the pathogenesis of insulin resistance. Indeed, reduced mitochondrial content and impaired oxidative phosphorylation have been reported in diabetic mouse models, and human patients and may contribute to pathologic glucose metabolism (271). As previously described, H₂S can elicit its effects for the maintenance and promotion of mitochondria biogenesis. Indeed, CSE expression appears to regulate mitochondrial biogenesis in the liver because CSE^−/− mice had ∼33% lower mitochondrial DNA when compared with WT (174). This loss in mitochondrial content was accompanied by a decrease in biogenesis transcription factors, such as Nrf1/2, PGC-1α, and PPRC. Moreover, NaHS increased ATP synthase, PGC-1α, and PPRC in both WT and CSE^−/− hepatocytes. Additionally, moderate exercise, which is well known to restore insulin sensitivity, has been reported to up-regulate CBS, CSE, and 3-MST levels in the livers of mice kept on a high-fat diet, thereby indicating yet another way H₂S bioavailability may be therapeutically viable in DM (272).

Unfortunately, studies in which in vivo models of insulin resistance are treated with H₂S donor remain just as unclear and contradictory as any other H₂S diabetes study. Intraperitoneal administration of NaHS to adult Wistar rats induced a fleeting increase in blood glucose levels and decreased insulin concentration, indicating H₂S-mediated inhibition of insulin secretion, but did not have an effect on glucose tolerance (273, 274). Moreover, NaHS continuously supplied via osmotic minipump into rats with streptozotocin-induced diabetes did not significantly impact glucose concentration (275). However, in this same study, H₂S protected endothelial cells from hyperglycemic-induced ROS formation via preservation of mitochondrial function. Finally, mice that were fed regular chow developed impaired insulin sensitivity upon treatment with a long-term H₂S donor, GYY4137, but mice that were on a high-fat diet had increased insulin sensitivity upon GYY4137 treatment, indicating that H₂S therapy may potentially only find use in patients with obesity (276).

Despite the multiple studies on the role of H₂S in the development of DM that have occurred over the last decade, much of the data are contradictory, and many issues remain unresolved. Nevertheless, H₂S-based approaches clearly have promise for the mitigation of DM-induced chronic complications, and H₂S study remains a viable candidate in the field of DM treatment.

CONCLUSIONS AND FUTURE DIRECTIONS

This review consolidates recent relevant research on H₂S production, metabolism, regulation, and signaling into one article, specifically highlighting the impact of H₂S signaling on mitochondria (Fig. 6). Several broad conclusions can be drawn:

Figure 6.

Impacts of H₂S signaling on mitochondrial function and disease. H₂S can signal through 3 main mechanisms: via metalloprotein interaction and reduction (A), as a potent antioxidant with implications in cellular redox homeostasis (B), and through a post-translational modification of reactive cysteines known as persulfidation (C). Representative mitochondrial targets of metalloprotein interactions are shown in red, redox regulation in blue, and persulfidation in green. This mitochondrion-specific H₂S signaling has been reported to be cytoprotective by promoting efficient mitochondrial bioenergetics, resisting apoptosis, and increasing mitochondrial biogenesis. Taken together, H₂S cellular protection is implicated in several diseases and may, therefore, be an exciting avenue for developing new targeted therapeutics.

1. H₂S signaling is tightly controlled, yet its apparent enzyme-dependent signaling specificity has not been studied in depth.

2. Persulfidation mechanisms and signaling by polysulfides require more research; understanding these processes better will advance the H₂S field dramatically.

3. H₂S signals in many complex ways, but the mitochondria are an important target of H₂S signaling.

4. At toxic concentrations, H₂S impairs mitochondrial respiration and induces cellular apoptosis via the canonical caspase cascade.

5. Increasing H₂S levels enhance mitochondrial metabolism and function, thus conferring cytoprotection.

6. H₂S donors offer exciting therapeutic opportunities in a variety of disease states, including I/R injury, neurodegenerative diseases, and cancer.

Clearly, a more thorough understanding of H₂S signaling in mitochondria is critical for developing therapeutics against a whole host of disease states. Physiologic H₂S appears to be critical for normal cellular metabolism and redox homeostasis; however, deregulation of H₂S-producing or -metabolizing enzymes can throw cell homeostasis off balance and lead to the development of disease, including neurodegenerative and cardiovascular conditions. Moreover, cancer exploits the cytoprotective nature of H₂S by up-regulating or enhancing H₂S-producing enzyme activity in preclinical models. Therefore, exogenous H₂S treatment or specific H₂S therapeutic targeting may prove valuable in the clinical setting for the treatment of debilitating and potentially deadly diseases.

Mutations in H₂S-producing genes are implicated in cardiovascular and neurovascular diseases, and aberrant H₂S expression is implicated in the pathogenesis of cancer. Recent evidence also indicates that H₂S plays an important role in mitochondrial biogenesis and morphogenesis. However, regulation of H₂S at the molecular, cellular, and organismal levels remains poorly understood. Thus, a critical remaining question is what cues cells rely on to regulate H₂S levels in order to trigger either mitochondrial biogenesis or morphogenesis and to meet the cellular metabolic requirement. Furthermore, the solubility of H₂S in aqueous systems is relatively high, so it may trigger signaling cascades both locally and at a distance. The role of H₂S and H₂S-producing enzymes in regulating the cellular microenvironment, and how that relates to both normal and diseased states, is an exciting area for future exploration.

Glossary

ΔΨm

mitochondrial membrane potential

3-MP

3-mercaptopyruvate

3-MST

3-mercaptopyruvate sulfurtransferase

Aβ

β-amyloid

AD

Alzheimer disease

AOAA

aminooxyacetic acid

APP

amyloid precursor protein

ATP5A1

ATP synthase F1 subunit α

BMMSC

bone marrow mesenchymal stem cell

BSA

bovine serum albumin

CBS

cystathionine β synthase

COX

cytochrome c oxidase

CSE

cystathionine γ lyase

Cy5

cyanine 5

DM

diabetes mellitus

Dnmt3a

DNA methyltransferase 3a

Drp

dynamin-related protein

ER

endoplasmic reticulum

ETC

electron transport chain

ETHE1

ethylmalonic encephalopathy 1 protein

Fe²⁺

ferrous

Fe³⁺

ferric

Fis1

fission 1

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GSH

glutathione

GSSG

oxidized GSH

Hb

hemoglobin

HD

Huntington’s disease

HEK

human embryonic kidney

HSNO₂

thionitrate

I/R

ischemia-reperfusion

IRF-1

IFN regulatory factor 1

K_ATP

ATP-sensitive K⁺

Keap1

kelch-like ECH-associated protein 1

LDH

lactate dehydrogenase

MFN

mitofusin

mPTP

mitochondrial permeability transition pore

MS

mass spectrometry

Nrf2

nuclear factor (erythroid-derived 2)–like 2

ONO₂⁻

peroxynitrite

OPA1

optic atrophy 1

PD

Parkinson’s disease

PGC-1α

peroxisome proliferator-activated receptor γ 1α

PLP

pyridoxal 5′-phosphate

PPRC

peroxisome proliferator–activated receptor-γ coactivator-related protein

PTP-1b

protein tyrosine phosphatase 1B

RNS

reactive nitrogen species

ROS

reactive oxygen species

SAM

S-adenosylmethionine

SCA3

spinocerebellar ataxia type 3

SO₄²⁻

sulfate

SP

specific protein

SRB

SO₄²⁻-reducing bacteria

SQR

sulfide quinone oxidoreductase

SUMO

small ubiquitin-like modifier

TFAM

mitochondrial transcription factor A

TRP

transient receptor potential

Trx

thioredoxin

TXNIP

Trx-interacting protein

WT

wild type

AUTHOR CONTRIBUTIONS

R. Bhattacharya and P. Mukherjee conceived the idea; B. Murphy, R. Bhattacharya, and P. Mukherjee jointly wrote and edited the manuscript; and B. Murphy illustrated the figures.