Emerging role of hydrogen sulfide-microRNA crosstalk in cardiovascular diseases

Abstract

Despite an obnoxious smell and toxicity at a high dose, hydrogen sulfide (H₂S) is emerging as a cardioprotective gasotransmitter. H₂S mitigates pathological cardiac remodeling by regulating several cellular processes including fibrosis, hypertrophy, apoptosis, and inflammation. These encouraging findings in rodents led to initiation of a clinical trial using a H₂S donor in heart failure patients. However, the underlying molecular mechanisms by which H₂S mitigates cardiac remodeling are not completely understood. Empirical evidence suggest that H₂S may regulate signaling pathways either by directly influencing a gene in the cascade or interacting with nitric oxide (another cardioprotective gasotransmitter) or both. Recent studies revealed that H₂S may ameliorate cardiac dysfunction by up- or downregulating specific microRNAs. MicroRNAs are noncoding, conserved, regulatory RNAs that modulate gene expression mostly by translational inhibition and are emerging as a therapeutic target for cardiovascular disease (CVD). Few microRNAs also regulate H₂S biosynthesis. The inter-regulation of microRNAs and H₂S opens a new avenue for exploring the H₂S-microRNA crosstalk in CVD. This review embodies regulatory mechanisms that maintain the physiological level of H₂S, exogenous H₂S donors used for increasing the tissue levels of H₂S, H₂S-mediated regulation of CVD, H₂S-microRNAs crosstalk in relation to the pathophysiology of heart disease, clinical trials on H₂S, and future perspectives for H₂S as a therapeutic agent for heart failure.

hydrogen sulfide (H₂S) was first discovered in 1777 as a colorless gas with a strong “rotten egg” odor. It was thought to be a toxic substance found in sewer gas, swamp gas, and volcanic discharge. Since the discovery that H₂S reacts to oxyhemoglobin similar to nitric oxide (NO) and carbon monoxide (17), a number of studies have been carried out to understand the biological functions of H₂S. The obnoxious odor and toxicity discouraged the attention of researchers until it was revealed that H₂S may have a possible role as an endogenous neuromodulator (1). Subsequently, a plethora of investigations were performed on potential roles of H₂S in cardiovascular disease (CVD), which revealed that physiological levels of H₂S have a pivotal role in maintaining cardiac function, and an exogenous supply of H₂S has the potential to ameliorate heart failure in rodents. Empirical studies elucidated several potential mechanisms of H₂S-mediated cardioprotection in different models of heart failure (21, 117, 123, 128, 150). However, regulation of H₂S functions during heart failure is not completely understood. Recently, it was demonstrated that miRNA regulates endogenous H₂S production (129, 148). MicroRNAs (miRNAs) are noncoding, regulatory RNAs that modulate gene expression mostly by translational repression (10). Interestingly, H₂S also regulates miRNA transcription. However, the crosstalk between H₂S and miRNAs, and its impact on CVD, is poorly understood. Considering differential expression of miRNAs in CVD (27, 133), H₂S-miRNAs crosstalk may have a crucial role in pathophysiology of heart failure. Therefore, H₂S-miRNA crosstalk is important for understanding H₂S-mediated cardioprotection. The goal of this review is to summarize the advancements made in H₂S-mediated cardioprotection, highlight the inter-regulation of miRNAs and H₂S, and emphasize potential future avenues of H₂S-miRNA crosstalk in CVD, which will provide an impetus for developing H₂S-based therapeutics for heart disease.

Regulation of H₂S in vivo.

H₂S is toxic at high levels; therefore, the production and degradation of H₂S must be tightly regulated in our body to maintain its physiological level. Multiple synthesis and degradation pathways provide a complex interdependence, which could be crucial for restoring the physiological H₂S level (Figs. 1 and 2). Understanding these pathways is indispensable for developing H₂S-based therapeutics.

Fig. 1.

Biosynthesis of hydrogen sulfide (H₂S). H₂S production is catalyzed by cystathionine β synthase (CBS), cystathionine gamma lyase (CSE), and the coupling of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfur transferase (3-MST). CBS and CSE are involved in transsulfuration of homocysteine, which ultimately generates H₂S. Both enzymes can also convert homocysteine into homolanthionine and H₂S, and cysteine into lanthionine and H₂S. CSE converts homocysteine, cystathionine, and cysteine into H₂S and different by-products. 3-MST and CAT are mostly involved in converting 3-mercaptopyruvate into H₂S in mitochondria. The main pathway of H₂S generation is denoted by large print, whereas additional substrates and products by small print.

Fig. 2.

Cellular catabolism of H₂S. In mitochondria, sulfide quinone oxidoreductase (SQR) oxidizes H₂S to glutathione persulfide (GSSH) with GSH as the electron acceptor or directly to thiosulfate (SO₃²⁻) using co-enzyme Q (Co-Q) as the electron acceptor. The enzymes SOD, sulfur transferase (ST), and sulfite oxidase (SO) further oxidize GSSH to thiosulfate or sulfate, which is excreted via the kidneys. Expiration of H₂S through exhaled air and scavenging by methemoglobin to sulfhemoglobin are alternative methods of H₂S catabolism.

H₂S biosynthesis.

H₂S is predominately and primarily produced by three enzymatic pathways, which include cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and the coupling of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfur transferase (3-MST). CBS, CSE, and CAT are pyridoxal 5-phosphate-dependent enzymes. CBS catalyzes the β-replacement of thiosulfides such as homocysteine to cysteine, whereas CSE catalyzes the α- and γ-replacement of cysteine to H₂S. CAT deaminates cysteine to mercaptopyruvate, which is followed by transulfuration catalyzed by the zinc-dependent enzyme 3-MST that results in biosynthesis of H₂S. These pathways are elaborated in several excellent review articles (4, 8, 12, 20, 69, 75, 111, 113, 150) and summarized in Fig. 1. Although CBS and CSE are localized in the cytoplasm, 3-MST is localized mainly in mitochondria but has also been reported in the cytoplasm of vascular endothelial cells (106, 131). The expression levels of H₂S generating enzymes are tissue specific (113). CSE is the most abundant H₂S-generating enzyme in the cardiovascular system (83). Secondary sources of H₂S include the air we breathe and generation of H₂S in the gastrointestinal tract by bacterial flora (44, 52, 91, 92). The multiple pathways for H₂S synthesis (Fig. 1) suggest an important role of H₂S in cell signaling (53, 87, 99, 104).

Catabolism of H₂S.

High levels of H₂S are toxic. H₂S levels are decreased in our body by several catabolic pathways. H₂S is oxidized to thiosulfate and sulfate in the mitochondria of most mammalian tissues; however, the underlying molecular mechanisms and pathways are poorly understood (11, 69). Sulfide quinone oxidoreductase, which is located on the inner mitochondrial membrane, oxidizes H₂S to glutathione persulfide using GSH as the sulfide acceptor (55, 90). Persulfide dioxygenase or rhodanese catalyze the oxidation of glutathione persulfide to thiosulfate and/or sulfide, which is further oxidized to sulfate by sulfite oxidase (90). Conversely, Jackson et al. (63) have shown sulfide quinone oxidoreductase catalyzes the oxidation of H₂S directly to thiosulfate using co-enzyme Q as the electron acceptor and sulfite as the acceptor of sulfane sulfur. Although the intermediate pathways involved in the oxidation of H₂S are not completely understood, sulfate is the primary by-product of sulfide catabolism, which may be excreted in the urine and feces (63, 69, 90). An additional pathway for excretion of H₂S may include expiration by the lungs (61, 142). H₂S may be scavenged by disulfide-containing molecules and red blood cells, where H₂S binds to methemoglobin forming sulfhemoglobin, which is oxidized to thiosulfate (147). The multiple pathways for reducing H₂S levels are presented in Fig. 2.

Physiological level of H₂S.

Physiological levels of H₂S range between 15 nM to 300 μM in vivo (62, 84, 85, 96, 110, 140, 153, 154). The wide range of H₂S levels results from variable detection methods and the tissues analyzed [see review by Liu et al.(96)]. In in vivo conditions (37°C, pH ∼7.4), H₂S is predicted to exist as 14% free H₂S gas, 86% HS⁻, and trace levels of S²⁻ (96, 153). Limitations to measuring H₂S include 1) free H₂S has a short half-life, ranging from 12 to 300 s in various species, and 2) detection methods may release bound sulfur from proteins, resulting in increased H₂S concentrations (61, 96, 142, 153).

Exogenous sulfur donors.

Sodium hydrogen sulfide (NaHS) and sodium sulfide (Na₂S) are the two most commonly used sources of H₂S. They are water soluble and cost efficient. However, a limitation of NaHS and Na₂S is that H₂S is a volatile substance resulting in evaporation from drinking water. Furthermore, they give more of a bolus effect of H₂S instead of a slow, steady release (19). Diallyl disulfide and diallyl trisulfide (DATS) are organic sulfur compounds found in members of the Allium species (garlic, onions, chives, etc.) that act as H₂S donors and have antioxidant, anti-inflammatory, and vasorelaxant properties (14, 94, 116, 132). Slow release sulfide donors such as GYY4137 (88), SG1002 (9, 78, 118), AP39 (138), and S-propargyl-cysteine (58, 149) are potential options for future H₂S supplementation. In fact, SG1002 successfully completed Phase I clinical trial and is beginning Phase II clinical trial for increasing plasma H₂S levels and mitigating heart failure (clinicaltrials.gov; No. NCT01989208 and No. NCT02278276) (118). Because of the volatile nature and short half-life of H₂S, a continuous, low-level H₂S release may provide an extended therapeutic potential than a single bolus using high levels as used in many NaHS and Na₂S studies (19).

CVD and H₂S.

CVD refers to any disease that leads to dysfunction in the heart and vasculature. Although many therapeutic options are available to treat CVD, the World Health Organization reports CVD remains the leading cause of death globally for both men and women, resulting in 17.5 million deaths in 2012 (155). Further research and better treatment options are necessary to reduce the incidence and burden of CVD. Evidence is mounting that H₂S is important in reducing the symptoms associated with CVD. H₂S reduces hypertension (2, 3), improves glucose uptake and metabolism (9, 89), protects against ischemia-reperfusion (I/R) injury (70, 81, 141, 161), and decreases cardiac hypertrophy (9, 73, 93, 97). Further evidence shows that free H₂S levels are reduced in patients with CVD (64, 79, 118), suggesting H₂S supplementation may be a potential therapy for mitigating CVD. However, the underlying molecular mechanisms for H₂S-mediated improvement in cardiac function, or amelioration of cardiac dysfunction in CVD, are not completely understood.

Diabetic cardiomyopathy is mitigated by H₂S.

Diabetes is among the top 10 causes of death in the United States (25a). One of the many complications of this debilitating disease is myocardial dysfunction (95, 152). Diabetes increases the risk of heart failure two- to fourfold as compared with age- and sex-matched nondiabetics (25, 100). Diabetes may effect both the heart and the vasculature or it may lead to left ventricular heart failure independent of coronary artery disease, a condition known as diabetic cardiomyopathy (26, 95). Diabetic cardiomyopathy is the end result of chronic exposure to a hyperglycemic environment. The pathophysiology includes cardiac dysfunction due to increased fibrosis and left ventricular hypertrophy (48). High glucose induces cardiovascular dysfunction by activating the PKC/diacylglycerol pathway, calcium dysregulation, inducing endoplasmic reticulum stress, and deregulating autophagy and apoptosis, which have been shown to be mitigated by H₂S supplementation (9, 80, 89, 156, 164). Furthermore, plasma H₂S levels are decreased in type 2 diabetic patients, high-fat diet–treated mice (type 2 diabetic patients), and streptozotocin-treated rats (9, 64, 66). The decrease in H₂S levels in diabetics and the ability of exogenous H₂S treatment to ameliorate pathological conditions induced by type 1 and type 2 diabetes suggest that H₂S may be a potential novel treatment option for mitigating cardiomyopathy in diabetics.

H₂S mitigates I/R injury.

Ischemia is the restriction of blood to tissues leading to a decrease in oxygen and glucose. Reperfusion injury occurs when blood flow returns to an area following ischemia and can further exacerbate cell damage (23, 146). I/R injury leads to increased cardiomyocyte apoptosis (81). H₂S reduces reactive oxygen species (ROS) associated with I/R injury as well as decreases in cardiomyocyte apoptosis during ischemia (36, 56, 70, 81, 135, 141, 161) (Table 1). A major complication following I/R injury is the occurrence of cardiac arrhythmias (37). H₂S released by α-lipoic acid treatment activates the potassium ATP-sensitive channels (K_ATP channels) (37, 67) and decreases post I/R arrhythmias (37). It was further demonstrated that lower plasma H₂S levels are associated with greater arrhythmia scores following I/R injury in Wistar rats (137). NaHS treatment inhibited the L-type Ca²⁺ channels, activated the K_ATP channels, and increased the action potential duration in these rats, all of which may result in decreased arrhythmias (137). During myocardial I/R injury, delivery of H₂S at the time of reperfusion reduces infarct size and preserves left ventricular function. Moreover, overexpression of cardiac CSE mitigates myocardial I/R injury (39). H₂S may protect the heart from I/R injury by increasing NO bioavailability and signaling (76). These findings suggest that H₂S treatment may provide protective benefits following I/R injury by decreasing ROS and by mitigating cardiac arrhythmia.

Table 1.

Disease models and signaling mechanisms showing the cardiioprotective role of H₂S in cardiovascular diseases

Disease and Role of H2S	Pathways Regulated by H2S	Reference (No.)
Ischemia-reperfusion
Anti-apoptosis	↓miR-1, ↑Bcl-2	Kang et al. (70)
Anti-inflammatory and anti-apoptosis	↓CD11b+Gr-1+ myeloid cells, ↑Bcl-2	Zhang et al. (161)
Anti-inflammatory	↑miR-21, ↓Inflammasome induction	Toldo et al. (141)
Anti-apoptosis	↑Erk1/2 signaling BCl-xL, ↓Bad, GSK3β	Lambert et al. (81)
Anti-apoptosis	↓Na+/H+ exchanger-1, pHi	Hu et al. (56)
Smoke-induced cardiomyopathy
Anti-inflammatory, anti-apoptosis	↓p38, JNK, caspase 3, ↑Bcl-2	Zhou et al. (163)
Antioxidant	↑PI3K/AKT, Nrf2	Zhou et al. (165)
Hyperglycemia
Glucose utilization	↑GLUT1, GLUT4	Liang et al. (89)
Antioxidant	↓p38-MAPK, ERK1/2	Xu et al. (156)
Anti-apoptosis	↑p-AMPK, ↓p-Mammalian target of rapamycin	Wei et al. (151)
Anti-apoptosis Antioxidant	↓p-JNK, p-cJun, NF-κB, ROS	Kuo et al. (80)
Antioxidant, anti-apoptosis, antifibrosis	↓Estrogen receptor ROS, caspase-12, p-JNK, ↓Picrosirius red, ↑GLUT4	Barr et al. (9)
Antioxidant, anti-apoptosis	↓ROS, caspase-3, ↑PI3K/Akt, Nrf2	Tsai et al. (143)
Hypertension
Vasorelaxation	↑KATP channels, ↓Endothelin-1	Sun et al. (136) Zhao et al. (162)
Vasodilation	↑Nitric oxide	Eberhardt et al. (38)

GLUT, glucose transporter; H₂S, hydrogen sulfide; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species.

H₂S is hypotensive.

Hypertension (high blood pressure) is defined as having a systolic blood pressure >140 mm mercury (mmHg) or a diastolic pressure >90 mmHg (107a, 109). Hypertension is a major medical concern affecting ∼30% of the United States population (109). Chronic high blood pressure creates excessive stress on the vasculature, leading to cardiac hypertrophy over time. An initial response to elevated blood pressure is activation of the renin angiotensin aldosterone system (RAAS) (124, 167). However, chronic activation of the RAAS leads to heart failure (98, 167). H₂S inhibits activation of the RAAS in Dahl rats fed with a high-salt diet, mitigating hypertension (59). Furthermore, abrogation of CSE (involved in H₂S biosynthesis) decreases the level of H₂S and induces hypertension in mice (158). H₂S may decrease hypertension by opening voltage-dependent potassium channels, resulting in vasorelaxation and increasing vascular tone through multiple mechanisms (77, 125). H₂S also induces vasorelaxation by opening K_ATP channels (136, 162) and increasing NO production (38). Contrary to vasorelaxation, Na₂S and NaHS increased intracavernosal pressure by opening potassium channels in penile tissue (68), suggesting that H₂S may cause vasoconstriction in other tissues. Therefore, the role of H₂S for vasorelaxation or vasoconstriction depends on different conditions and tissues. Nevertheless, H₂S has multiple mechanisms for reducing hypertension, suggesting a possible therapeutic role for H₂S supplementation in treating hypertension.

Molecular mechanisms of H₂S-mediated cardioprotection.

The cardioprotective effects of H₂S are related to several important signaling and regulatory pathways including antioxidant, anti-apoptotic, antifibrotic, and anti-inflammatory regulation (Fig. 3).

Fig. 3.

Cardioprotective effects of H₂S. The pathways involved in H₂S cardioprotection and the signaling mechanisms that have been shown to be induced (↑) or downregulated/blocked (↓) by H₂S are indicated. The signaling molecules involved in each pathway and their references are included. CTGF, connective tissue growth factor; NOX, NADPH oxidase; SMA, smooth muscle actin; NO, nitric oxide; ROS, reactive oxygen species; ER, endoplasmic reticulum.

Antioxidant effects of H₂S.

ROS refer to small reactive molecules such as superoxide (O₂^.−), hydrogen peroxide (H₂O₂), hydroxyl (OH^.−), and hypochlorite (OCl^.−), which react readily with other cellular molecules (29, 51). ROS are primarily generated as by-products of oxidative phosphorylation within the mitochondrial electron transport chain (29). Healthy mitochondria are vital for the overall health of the heart, because mitochondrial ATP generation via oxidative phosphorylation is essential for energy supply and for survival of cardiomyocytes. The generation and removal of ROS are tightly controlled during normal homeostasis but ROS generation increases during pathological conditions (18, 46). Superoxide radicals activate JNK, which is a stress-activated protein kinase. Induction of JNK leads to the activation of NF-κB, an important transcription factor of many cytokines, resulting in cardiac cell death (80). High glucose (33 mM) treatment increases expression of JNK, NF-κB, and the pro-apoptotic marker caspase-3, which resulted in increased apoptosis in cardiomyocytes (80).

In humans, red blood cells convert organic polysulfide (derived from garlic) into H₂S, and it is facilitated by allyl substituents (14). DATS inhibits apoptosis by preventing caspase-3 activation, blunting phosphorylation of JNK and c-JUN, and inhibiting nuclear translocation of NF-κB (80). H₂S mitigates upregulation of ROS and prevents cell death associated with several disease states including hyperglycemia, hyper-homocysteinemia, smoke exposure, and I/R injury (49, 54, 144, 145, 156, 165).

Nuclear factor erythroid 2 related factor (Nrf2) is a primary regulator for the transcription of antioxidant response elements (72). NaHS (8 μMol·kg⁻¹·day⁻¹) increases Nrf2 levels in cardiomyocytes, resulting in decreased ROS levels in smoke-exposed rats (165). Furthermore, Nrf2 activation by DATS (10 μM) reduces ROS in high glucose–treated cardiomyocytes (143). In both cases, Nrf2 activation is the result of increased PI3K/Akt signaling induced by H₂S supplementation (143, 165). H₂S administration before myocardial ischemia increases the nuclear localization of Nrf2 and protects the ischemic heart from injury (21). These studies suggest that H₂S regulates several signaling molecules to suppress oxidants in the heart (Fig. 3).

H₂S promotes cell survival.

Apoptosis or “programmed cell death” can be a protective mechanism at physiological levels for removing damaged cells. However, in pathological conditions, apoptosis is deregulated, which causes increased cell death and tissue damage. As previously mentioned, ROS induces apoptosis and ROS can be mitigated by the antioxidant properties of H₂S (see H₂S mitigates I/R injury). In addition, H₂S inhibits apoptosis by suppressing autophagy (65, 163). Autophagy is lysosome-mediated degradation and recycling of damaged and/or unnecessary cellular components (84). NaHS treatment suppresses cigarette smoke-induced upregulation of the autophagy inducers: Beclin-1, LC3II, and AMPK (163). H₂S not only downregulates autophagy but it also induces PI3K and GSK3β. PI3K and GSK3β upregulate Nrf2 for cardioprotection (65, 143, 165). In an in vivo I/R model, NaHS (100 μM) increased phosphorylated mTOR complex 2, resulting in inactivation of cell death initiator Bim (Bcl-2 interacting mediator of cell death) for cell survival (166). Conversely, GYY4137 (100 μM) increases phosphorylated AMPK and decreases mTOR, which prevented high glucose-induced cardiac hypertrophy (151).

Members of the MAPK family, p38-MAPK and ERK1/2, are upregulated in response to cellular stress, leading to cardiomyocyte apoptosis (50, 156). p38-MAPK is induced in several disease conditions including hyperglycemia, I/R injury, and hypoxia (16, 49, 50, 134, 156). NaHS (400 μM) inhibits the activation of p38-MAPK in response to the antitumor drug doxorubicin, resulting in decreased apoptosis (50). Furthermore, pretreatment of H9c2 cardiomyocytes with NaHS (400 μM) reduces the cytotoxicity associated with hyperglycemia by inhibiting p38-MAPK and ERK1/2 (156).

These findings demonstrate that H₂S provides cardioprotection by promoting cell survival via suppressing autophagy and inhibiting multiple proteins involved in apoptosis signaling (Fig. 3).

H₂S mitigates pathological cardiac remodeling by inhibiting fibrosis and hypertrophy.

Cardiac fibrosis results from the proliferation of myofibroblasts, leading to increased extracellular matrix deposition in the muscle (34, 47). Excessive fibrosis decreases contractility and cardiac output and is often associated with cardiomyopathy. NaHS (100 μM) reduces fibroblast proliferation in human atrial fibroblast cells (130). Furthermore, it decreases NADPH oxidase 4, a stimulator of cardiac fibrosis (33), resulting in decreased levels of the profibrotic markers α-smooth muscle actin and connective tissue growth factor (114). Oral supplementation of SG1002 (20 mg·kg⁻¹·day⁻¹) reduces fibrosis in a transverse aortic constriction model as assessed by Masson Trichrome and Picrosirius Red staining (78). These findings suggest that H₂S inhibits fibrosis (Fig. 3), and H₂S supplementation has a therapeutic potential for decreasing cardiac fibrosis.

Cardiomyocyte hypertrophy occurs in response to chronic increases in pressure or stress. NaHS (100 μM) prevents phenylephrine- and isoproterenol-induced hypertrophy in cultured cardiomyocytes and in rat hearts. Furthermore, it downregulates mitochondrial ROS production, apoptosis, and improves glucose uptake through upregulation of the glucose transporters Glut-4 and Glut-1 (89, 97). The H₂S donor SG1002 prevents cardiac hypertrophy and cardiac dysfunction by reducing endoplasmic reticulum stress in high-fat diet–fed mice (9). Our laboratory has demonstrated that H₂S treatment mitigates cardiac hypertrophy by upregulating miR-133a (73), an antihypertrophy (24, 41) and antifibrosis (28, 101) microRNA. To increase the levels of miR-133a, H₂S activates myosin enhancer factor-2c (MEF2C), a transcription factor of miR-133a, by releasing MEF2C from MEF2C-HDAC1 complex (inactivated state) (73). These studies suggest that H₂S plays a crucial role in inhibiting cardiac hypertrophy (Fig. 3).

Anti-inflammatory role of H₂S.

H₂S treatments reduce inflammation by multiple mechanisms. NaHS and Na₂S (100 μM/Kg body wt) suppress leukocyte adhesion by activating K_ATP channels in rats injected with aspirin, an inducer of leukocyte adhesion (160). Furthermore, inhibition of CSE reduces H₂S biosynthesis and promotes leukocyte adhesion (160). H₂S also regulates NF-κB, a transcription factor that is induced in stress, apoptosis, and inflammation signaling. NaHS (400 μM) suppresses high glucose-induced increases in NF-κB and the pro-inflammatory cytokine IL-1β in H9c2 cells (156). These studies along with the previously mentioned decrease in inflammation in response to I/R injury (subheading 4.2), suggest that H₂S plays a pivotal role in mitigating inflammation (Fig. 3).

H₂S regulates microRNAs to ameliorate heart failure.

MicroRNAs are small (∼22 nt), noncoding RNAs that regulate mRNA and protein expression through mRNA degradation or translational inhibition (10). MiRNAs are rapidly emerging as therapeutic targets for CVD (22, 103, 108, 122, 126). H₂S regulates miRNA expression (70, 73, 93); however, the interaction between H₂S and miRNAs in CVD is poorly understood. Only a few publications document that miRNAs may regulate enzymes that control H₂S biosynthesis and that H₂S regulates miRNAs. H₂S and miRNA inter-regulations are summarized in Fig. 4.

Fig. 4.

Interactions between H₂S and microRNAs (miRNAs). MiR-21 and miR-22 inhibit specificity protein 1 [SP1, a transcription factor for cystathionine gamma lyase (CSE)], whereas miR-30 directly inhibits CSE, an enzyme responsible for H₂S production, resulting in decreased H₂S levels. Estrogen (E) activates estrogen receptor α (ERα), which binds to SP1 increasing CSE production and H₂S. ERα also inhibits miR-22, which is an inhibitor of SP1. MiR-22 may also inhibit ERα, providing a secondary pathway for reducing CSE expression. H₂S inhibits miRNAs including miR-221 (anti-neovasculogenic miRNA) and induces miR-133a (antihypertrophy and antifibrotic miRNA). H₂S may inhibit and/or induce miR-21, which is a prohypertrophic miRNA but protects the heart during ischemia-reperfusion injury. H₂S and miR-21 have crosstalk as miR-21 regulates H₂S biosynthesis by inhibiting SP1 and CSE. H₂S regulates Bcl-2 (anti-apoptosis) and Akt (anti-oxidant) either directly or via regulating miR-1 and miR-21, respectively. H₂S may have synergistic effect on these pathways by direct regulation and indirect via miRNA regulation. PTEN, phosphatase and tensin homolog.

MiRNAs regulate H₂S biosynthesis.

Three miRNAs, miR-21, miR-22, and miR-30, have been shown to control H₂S biosynthesis by regulating CSE gene expression. MiR-30 family members are upregulated concomitant with downregulation of CSE in the infarct and border zones following myocardial infarction (MI) in rat hearts (129). Luciferase reporter assay and in vivo silencing of miR-30 have confirmed that miR-30 targets CSE and inhibition of miR-30 can protect the MI heart by upregulating CSE (129). Estrogen (E₂) also induces CSE expression through estrogen receptor α, which upregulates transcription of specificity protein-1 (SP1). SP1 directly binds to the promoter region of CSE to upregulate CSE transcription and H₂S biosynthesis (148). Ovariectomized rats have increased levels of myocardial miR-22, which are normalized by E₂ therapy. Furthermore, addition of miR-22 mimic inhibits estrogen receptor α and SP1 and downregulated CSE transcription (148). This suggests that miR-22 modulates H₂S production in females. Therefore, E₂ status and its regulation by miR-22 could be crucial in females with CVD. However, the role of anti-miR-22 treatment on cardiac function in males is unclear. It would be an interesting avenue to assess impact of anti-miR-22 treatment on cardiac CSE and H₂S levels in males. These studies demonstrate that miRNAs play a crucial role in regulating H₂S biosynthesis (Fig. 4).

H₂S regulates miRNAs.

Not only do miRNAs regulate the biosynthesis of H₂S, but H₂S has also been demonstrated to regulate miRNAs. NaHS (100 μM) treatment upregulates cardioprotective miR-133a in primary cultures of neonatal rat cardiomyocytes to inhibit phenylepinephrine-induced cardiomyocyte hypertrophy (93). Furthermore, Na₂S (30 μM) treatment increased miR-133a level to suppress hyperhomocysteinemia-induced cardiomyocyte hypertrophy in HL1 cardiomyocytes (73). These studies suggest that H₂S may have an important role in miR-133a-mediated alleviation of cardiac dysfunction. Because diabetic human heart failure patients have reduced levels of miR-133a (107), the role of H₂S in these hearts will be an interesting area for investigation. Measuring the cardiac levels of H₂S and designing experiments with late-stage heart failure with H₂S supplementation will provide insight on H₂S-mediated cardioprotection in the failing heart.

MiR-1 is a bicistronic transcript with miR-133a and is upregulated in response to I/R stress (70). I/R-induced apoptosis is mediated through upregulation of miR-1 (70). MiR-1 is pro-apoptotic and directly suppresses anti-apoptotic proteins including Bcl-2 (70, 139), heat shock protein 60 (127), and heat shock protein 70 (159). The preconditioning of myocardial I/R with H₂S decreases miR-1-mediated apoptosis (70). Dietary garlic has a cardioprotective effect, which is mediated through generation of H₂S in cardiomyocytes and endothelial cells (45, 74). DATS releases H₂S (120) and downregulates anti-neovasculogenic miR-221 in a dose-dependent manner (31). Because miR-221 is upregulated in patients with coronary artery disease, garlic or H₂S supplementation could be a potential therapeutic strategy for reducing the levels of miR-221 to mitigate the effects of coronary artery disease. The above studies show that H₂S is involved in regulating miRNAs (Fig. 4).

H₂S-miRNA crosstalk.

The interaction of H₂S with miRNA is recently uncovered and is an emerging area of investigation. An example of H₂S-miRNA crosstalk is that H₂S downregulates miR-21 to mitigate phenylephrine-induced cardiomyocyte hypertrophy (93), and miR-21 targets SP1 to decrease CSE transcription and H₂S production (157). Furthermore, increased miR-21 levels and decreased CSE levels are found in placental tissue from high risk pregnancy as compared with normal pregnancy, suggesting that low H₂S level may induce miR-21 and contribute to increased pregnancy complications (32). Perfusion of placental extracts with NaHS improves vasodilation; however, it is unclear whether NaHS perfusion alters miR-21 levels. Although these studies demonstrate that miR-21 may have a negative impact as it reduces H₂S levels, several studies show the beneficial effects of miR-21 in cardiac cells, including inhibiting apoptosis (30, 121), protecting cardiomyocytes from H₂O₂ (30) and I/R damage by suppressing phosphatase and tensin homolog (158). Na₂S (10 μM) induces miR-21 in primary cardiomyocytes and heart tissue. However, it had no effect on miR-21 knockout mice even though it reduces infarct size and attenuates inflammation in response to I/R injury (141). These findings suggest that H₂S-miRNA crosstalk may vary in a context-dependent manner and may have different roles in different disease conditions (Fig. 4). Therefore, more research using different models of heart failure is required to elucidate H₂S-miRNA crosstalk in CVD.

Potential new areas for investigation on H₂S-miRNA crosstalk.

H₂S and miRNA are relatively new research areas in CVD, and there is a lot of potential inter-regulation between H₂S and miRNAs. Although miR-21, miR-22, and miR-30 are demonstrated to modulate CSE gene expression (Fig. 4), no miRNAs are reported that control CBS or 3-MST expression, suggesting a gap in knowledge. There are ample opportunities for investigations on potential miRNAs regulating genes involved in H₂S biosynthesis. Furthermore, many miRNAs have been shown to regulate CVD, yet the effect of H₂S on only a few miRNAs is reported. This provides an avenue to explore the effect of H₂S on crucial miRNAs involved in the regulation of heart failure. For example, miR-24 is upregulated following MI, and it directly inhibits endothelial NO synthase (102). H₂S provides cardioprotective effects through upregulating endothelial NO synthase (76, 78), yet it is unclear whether H₂S suppresses miR-24. Interestingly, H₂S may regulate anti-apoptotic Bcl-2 by either directly upregulating it or by suppressing miR-1, an inhibitor of Bcl-2. Similarly, H₂S may regulate Akt directly or by regulating miR-21 (Fig. 4). Future studies in these areas will elucidate the complex regulatory network of H₂S-miRNA crosstalk in CVD.

H₂S involved in clinical trials.

Clinical trials with SG1002 will be exciting to follow and to see if the increase in H₂S levels translate into significant benefits in treating or preventing heart failure. Results from the initial clinical trial with SG1002 have recently been reported, demonstrating that SG1002 increases H₂S and nitrite levels with few, mild adverse events (118). Furthermore, sulfur donors are being attached to current drug treatments, such as naproxen, improving their anti-inflammatory effectiveness and decreasing gastrointestinal and cardiovascular side effects (13, 19, 35, 42, 105). H₂S has beneficial effects in other organs including neurons, kidney, pancreas, and gastrointestinal and in arthritis (15, 43). This suggests organ-specific delivery systems for H₂S may need to be developed to localize therapeutic effects. As H₂S levels are decreased in diabetics and MI patients, H₂S may be a useful biomarker for CVD. A clinical trial has been recently completed where H₂S was measured as a biomarker for peripheral artery disease (clinicaltrials.gov; No. NCT01407172). Another clinical trial is recruiting to measure H₂S levels in women with CVD (clinicaltrials.gov; No. NCT02180074).

Future directions.

Many mechanisms of H₂S signaling have been elucidated; however, there are still a lot of unknowns on how H₂S levels influence cardiovascular health. As detection methods improve for more accurate measurements of H₂S and for measuring real-time H₂S generation, we will get a better understanding of how cells maintain H₂S balance. H₂S mitigates many pathologies associated with CVD, and the therapeutic benefits of H₂S are currently being explored in other organ systems. For example, H₂S levels are decreased in the neurodegenerative disorders Alzheimer's and Parkinson's diseases (40, 57), and H₂S treatments have mitigated renal dysfunction by many of the same pathways as involved in cardiomyopathy (115).

The inter-regulation of H₂S and miRNAs suggests H₂S may have a greater role in maintaining cellular homeostasis than previously thought. For example, H₂S may directly regulate Bcl-2 and Akt, or it may control these genes by regulating miR-1 and miR-21, respectively. Besides, H₂S may have a synergistic effect using direct regulation and indirect regulation via miRNAs (Fig. 4). More mechanistic studies are required to elucidate the growing role of H₂S on miRNA regulation and its potential to regulate epigenetic modifications. MiRNAs regulate pathological remodeling in the heart and may be important treatment options for mitigating CVD (103). Two miRNAs or anti-miRNA therapies are undergoing clinical trial for non-CVD including anti-miR-122 (Miravirsen) for treatment of Hepatitis C (clinicaltrials.gov; No. NCT01200420) (112) and a miR-34 mimic for the treatment of primary liver cancer and other solid tumors (clinicaltrials.gov; No. NCT01829971). The role of these miRNAs on H₂S or role of H₂S in the regulation of miRNAs is awaiting.

Long noncoding RNAs (lncRNAs), RNAs with >200 nucleotides, are emerging as regulators of genes, miRNAs, and proteins involved in CVD (5, 60, 71, 119). Transcriptome analyses show distinct patterns of lncRNA profiles in heart failure conditions (82, 86), yet lncRNA-mediated regulation of H₂S or any effect of H₂S on lncRNAs is an open area for investigation. Future studies exploring the crosstalk among lncRNAs, miRNAs, and H₂S may elucidate novel regulatory mechanisms for CVD and provide new strategies for treatment of heart failure.

GRANTS

Financial support is from National Heart, Lung, and Blood Institute Grants HL-113281 and HL-116205 (to P. K. Mishra).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

Author contributions: P.K.M. conception and design of review; B.T.H. prepared figures; B.T.H. and P.K.M. drafted manuscript; B.T.H. and P.K.M. edited and revised manuscript; B.T.H. and P.K.M. approved final version of manuscript.