Skip to main content
NCBI home page
The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2016 Sep;358(3):431–440. doi: 10.1124/jpet.116.235119

Cardioprotection by H2S Donors: Nitric Oxide-Dependent and ‑Independent Mechanisms

Athanasia Chatzianastasiou 1, Sofia-Iris Bibli 1, Ioanna Andreadou 1, Panagiotis Efentakis 1, Nina Kaludercic 1, Mark E Wood 1, Matthew Whiteman 1, Fabio Di Lisa 1, Andreas Daiber 1, Vangelis G Manolopoulos 1, Csaba Szabó 1, Andreas Papapetropoulos 1,✉
PMCID: PMC6047225  PMID: 27342567

Abstract

Hydrogen sulfide (H2S) is a signaling molecule with protective effects in the cardiovascular system. To harness the therapeutic potential of H2S, a number of donors have been developed. The present study compares the cardioprotective actions of representative H2S donors from different classes and studies their mechanisms of action in myocardial injury in vitro and in vivo. Exposure of cardiomyocytes to H2O2 led to significant cytotoxicity, which was inhibited by sodium sulfide (Na2S), thiovaline (TV), GYY4137 [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate], and AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphospho-nium bromide]. Inhibition of nitric oxide (NO) synthesis prevented the cytoprotective effects of Na2S and TV, but not GYY4137 and AP39, against H2O2-induced cardiomyocyte injury. Mice subjected to left anterior descending coronary ligation were protected from ischemia-reperfusion injury by the H2S donors tested. Inhibition of nitric oxide synthase (NOS) in vivo blocked only the beneficial effect of Na2S. Moreover, Na2S, but not AP39, administration enhanced the phosphorylation of endothelial NOS and vasodilator-associated phosphoprotein. Both Na2S and AP39 reduced infarct size in mice lacking cyclophilin-D (CypD), a modulator of the mitochondrial permeability transition pore (PTP). Nevertheless, only AP39 displayed a direct effect on mitochondria by increasing the mitochondrial Ca2+ retention capacity, which is evidence of decreased propensity to undergo permeability transition. We conclude that although all the H2S donors we tested limited infarct size, the pathways involved were not conserved. Na2S had no direct effects on PTP opening, and its action was nitric oxide dependent. In contrast, the cardioprotection exhibited by AP39 could result from a direct inhibitory effect on PTP acting at a site different than CypD.

Introduction

Hydrogen sulfide (H2S) is a recently identified signaling molecule with important functions throughout the body (Szabó, 2007; Li et al., 2011; Paul and Snyder, 2012). Cardiovascular homeostasis relies on the production of adequate H2S levels (Wang, 2012; Polhemus et al., 2014). Although H2S can be generated nonenzymatically, the majority of H2S is believed to be produced through the action of cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase (Kabil and Banerjee, 2014; Kimura, 2014). In blood vessels, endogenously produced H2S reduces blood pressure by promoting vasorelaxation (Yang et al., 2008; Bucci et al., 2012), enhances angiogenesis by stimulating endothelial cell proliferation and migration (Papapetropoulos et al., 2009; Coletta et al., 2012), prevents atherosclerosis development (Mani et al., 2013), and ameliorates diabetic complications (Wang et al., 2015). In the heart, endogenous H2S limits oxidative stress and reduces myocardial injury after ischemia reperfusion (Kondo et al., 2013; King et al., 2014).

Given the pleiotropic beneficial actions of H2S in the cardiovascular system, investigators have used pharmacologic agents to deliver H2S to prevent organ dysfunction or treat disease in animal models (Szabó, 2007; Wang, 2012; Wallace and Wang, 2015). In the first era of H2S research, the sodium salts NaHS and sodium sulfide (Na2S) were almost exclusively used to administer H2S (Papapetropoulos et al., 2015). However, these agents suffer a major drawback in that they produce H2S instantly in aqueous solutions (Kashfi and Olson, 2013; Zheng et al., 2015). Production of H2S from salts (NaHS or Na2S) cannot be controlled, and H2S is generated after pH-dependent dissociation.

In open systems, such as in cultured cells and ex vivo organ bath studies, H2S levels decline rapidly due to volatilization (DeLeon et al., 2012). The typical concentrations of salts used result in transiently toxic levels of H2S. Fewer studies have employed naturally occurring H2S donors, mainly garlic-derived allyl polysulfides, as an alternative means to deliver H2S (Benavides et al., 2007; Predmore et al., 2012; Panza et al., 2015). The first donor to be synthesized that generates H2S in slow manner, mimicking the low level endogenous production, was GYY4137 [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate] (Li et al., 2008). Analogs of GYY4137 are now available, with considerably different rates of Η2S release and pharmacologic properties (Whiteman et al., 2015).

Salts and GYY4137 exemplify the two extremes in terms of H2S production rates. The need to develop donors with improved properties for use as research tools and potential therapeutic agents, led to the synthesis of thioaminoacids (Zhou et al., 2012), arylthioamides (Martelli et al., 2013), N-mercapto (N-SH)-based derivatives(Zhao et al., 2011, 2015), 1,2-dithiole-3-thiones (Caliendo et al., 2010), dithioperoxyanhydrides (Roger et al., 2013), and photoinduced (Zheng et al., 2015) and esterase-sensitive prodrugs (Zheng et al., 2016) as H2S donors. Water solubility, oral bioavailability, an intermediate H2S release rate, and generation of H2S in a controlled fashion (as, for example, after enzymatic activation), are among the desirable properties of an “ideal” donor.

Two more classes of donors are worth mentioning: mitochondrial-targeted H2S and hybrid, bifunctional donors (Kashfi and Olson, 2013; Le Trionnaire et al., 2014; Szczesny et al., 2014). Representatives of the first class are AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphospho-nium bromide] and AP123 [hydroxythiobenzamide]. In the second class, several molecules carrying a H2S donating moiety bound to a known pharmacophore structure have been reported. A naproxen-based H2S donor (ATB346 [(4-carbamothioylphenyl) 2-(6-methoxynaphthalen-2-yl)propanoate]) is in clinical development as a safer nonsteroidal anti-inflammatory agent to treat arthritis (Wallace and Wang, 2015). We recently reported on adenine-H2S slow-release hybrids that are effective in reducing infarct size in vivo (Lougiakis et al., 2016).

Apart from the differences in physicochemical properties and rates of H2S release from donor molecules, differences in the signaling pathways used by H2S donors have been noted. We observed that when added to smooth muscle cells NaHS and thioaminoacids enhance cGMP accumulation by inhibiting phosphodiesterase activity, but GYY4137 only did so at very high concentrations (Bucci et al., 2012; Zhou et al., 2012). NaHS can additionally enhance cGMP levels by promoting endothelial nitric oxide synthase (eNOS) phosphorylation and increasing nitric oxide (NO) production (Bibli et al., 2015). We and others have observed that the infarct-limiting action of sulfide salts is NO-dependent (King et al., 2014; Bibli et al., 2015; Sun et al., 2016).

Given the importance of the NO/cGMP pathway in cardioprotection and the lack of comparative mechanistic studies on H2S donors in the context of cardioprotection, herein we selected representative H2S donors (ultrafast, intermediate, slow, and mitochondrial) to assess their ability to rescue the myocardium after ischemia-reperfusion injury in vivo. Furthermore, we evaluated the contribution of NO to the beneficial effects of each donor using in vitro and in vivo systems.

Materials and Methods

Reagents.

Dulbecco’s modified essential medium (DMEM) and 10% fetal bovine serum (FBS) were obtained from Gibco/Thermo Scientific (Waltham, MA). The lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from Cayman Chemical (via Lab Supplies, P. Galanis & Co, Athens, Greece). The following primary antibodies were obtained from Cell Signaling Technology (Beverly, MA): phospho-eNOS (p-eNOS; S1176), phospho-vasodilator-associated phosphoprotein (p-VASP), total eNOS, total VASP, β-tubulin, and the goat anti-rabbit horseradish peroxidase antibody. The SuperSignal chemiluminescence kit was purchased from Thermo Scientific Technologies (Waltham, MA). H2O2 was obtained from AppliChem GmbH (Darmstadt, Germany). GYY4137, AP39, and thiovaline were synthesized as previously described elsewhere (Zhou et al., 2012; Le Trionnaire et al., 2014; Alexander et al., 2015). DT2 was obtained from BIOLOG Life Science Institute (Bremen, Germany). Calcium Green-5N, protease and phosphatase inhibitor cocktail were purchased from ThermoFisher Scientific (Waltham, MA). All other reagents including Na2S, NaCl, NaF, EDTA, EGTA, phenylmethylsulfonyl fluoride, Nargase, cyclosporine A, L-NAME (Nω-nitro-l-arginine methyl ester), TTC (2,3,5-triphenyltetrazolium chloride), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).

Animals.

Male C57BL/6J and cyclophilin-D (CypD) knockout (KO) mice, 8 to 12 weeks old, were used. C57BL/6J were purchased from Alexander Fleming Institute (Athens, Greece) and CypD KO (same genetic background as wild-type [WT] mice) were bred in the animal facility of Universitätsmedizin der Johannes Gutenberg-Universität Zentrum für Kardiologie I, Labor für Molekulare Kardiologie (Mainz, Germany). Mice were housed in a specific pathogen-free facility at 20–25°C and received water and food (regular laboratory animal diet) ad libitum. All animal procedures were in compliance with the European Community guidelines for the use of experimental animals; experimental protocols were approved by the ethics committee of the Prefecture of Athens or Johannes Gutenberg University (Landesuntersuchungsamt Koblenz).

Cell Culture.

The rat embryonic-heart- derived H9c2 cell line was obtained from the American Type Culture Collection [ATCC] (CRL-1446) (ATCC/LGC Standards, Middlesex, United Kingdom). The H9c2 cells were cultured in DMEM containing 25 mM d-glucose, 1 mM sodium pyruvate, and supplemented with 10% FBS, 2 mM l-glutamine, 1% streptomycin (100 μg/ml), and 1% penicillin (100 U/ml) at pH 7.4 in a 5% CO2 incubator at 37°C. For differentiation, H9c2 were seeded and allowed to grow to confluence. The medium was then replaced to DMEM containing 1% FBS with 10 nM all-trans-retinoic acid for 7 days. Culture of H9c2 myoblasts in low-serum medium and stimulation with 10 nM all-trans-retinoic acid for 7 days resulted in the appearance of elongated cells connecting at irregular angles reminiscent of cells with a cardiac phenotype.

In Vitro Oxidative Stress and Anoxia/Reoxygenation.

To induce oxidative stress injury, H9c2 cells (1.5 × 104 per well) were differentiated in 96-well plates. The cells were treated with 500 μM H2O2 in serum-free DMEM for 12 hours in a 5% CO2 incubator at 37°C. In the anoxia/reoxygenation (A/R) assay, differentiated H9c2 cells in 96-well plates were placed in an anaerobic chamber containing a mixture of 95% N2 and 5% CO2 at 37°C for 48 hours. After anoxia, the cells were incubated under normal growth conditions (95% air and 5% CO2) for an additional 24 hours.

MTT Measurement.

H9c2 cells were seeded in 96-well plates at 10,000–15,000 cells per/ well in growth medium. Following oxidative stress injury or anoxia/reoxygenation cell survival was assessed in differentiated H9c2 cells by using the conversion of MTT to formazan. Cells were incubated with MTT at a final concentration of 0.5 mg/ml, for 2 hours at 37°C. The formazan formed was dissolved in solubilization solution (10% Triton-X 100 in acidic 0.1N HCl isopropanol); subsequently, absorbance was measured at 595 nm with a background correction at 750 nm using a microplate reader.

LDH Measurement.

LDH release was used to detect cytotoxicity/cell death using a commercially available kit. In brief, supernatant medium was collected and centrifuged at 400g for 5 minutes. Cell supernatant (100 μl) was transferred to a new 96-well assay plate and 100 μl of reaction solution containing 1 mM NAD+, 85 mM lactic acid, 0.5 mM iodonitrotetrazolium, and LDH diaphorase was added to each well. The plate was incubated for 30 minutes at 37°C with gentle shaking. Absorbance was read at 490 nm with a plate reader.

Western Blot Analysis.

Frozen ischemic samples were pulverized and homogenized with the lysis buffer (1% Triton ×100, 20 mM Tris pH 7.4–7.6, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM glycerol phosphatase, 1% SDS, and 100 mM phenylmethylsulfonyl fluoride, supplemented with protease and phosphatase inhibitor cocktail). The lysates were centrifuged at 11,000g for 15 minutes at 4°C. The supernatants were collected, and the protein concentration was determined based on the Lowry assay. The supernatant was mixed with a buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenyl blue, and 0.125 M Tris/HCl. The samples were then heated at 100°C for 10 minutes and stored at −80°C. An equal amount of protein was loaded in each well and then separated by SDS-PAGE electrophoresis and transferred onto a polyvinylidene difluoride membrane.

After blocking with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with primary antibody. The following primary antibodies were used: p-VASP (S239), p-eNOS (S1176), total eNOS, total VASP, and β-tubulin (dilution for all primary antibodies was 1:1000). Membranes were then incubated with secondary goat anti-rabbit horseradish peroxidase antibody (1:2000) for 2 hours at room temperature and developed using the Supersignal chemiluminesence ECL Western Blotting Detection Reagents (Pierce/Thermo Scientific, Rockford, IL). Relative densitometry was determined using a computerized software package (U.S. National Institutes of Health ImageJ, https://imagej.nih.gov/ij/), and the values for phosphorylated were normalized to the values for total proteins, respectively.

Ischemia-Reperfusion Injury Model In Vivo.

Male mice were randomly divided into groups and anesthetized by intraperitoneal injection with a combination of ketamine and xylazine (0.01 ml/g, final concentrations of ketamine and xylazine, 10 mg/ml and 2 mg/ml, respectively). Anesthetic depth was evaluated by the loss of pedal reflex to toe-pinch stimulus and breathing rate. A tracheotomy was performed for artificial respiration at 120–150 breaths/minute. A thoracotomy was then performed between the fourth and fifth ribs, and the pericardium was carefully retracted to visualize the left anterior descending coronary, which was ligated using a 7-0 Prolene monofilament polypropylene suture placed 3 mm below the tip of the left auricle. The heart was allowed to stabilize for 15 minutes before ligation to induce ischemia. After the ischemic period, the ligature was released, allowing reperfusion of the myocardium.

Throughout experiments, body temperature was maintained at 37°C ± 0.5°C by way of a heating pad and monitored via a thermocouple inserted rectally. After reperfusion, the hearts were rapidly excised from mice and directly cannulated and washed with 2.5 ml of saline-heparin 1% for blood removal. We then infused 0.2 ml of 1% Evans blue, diluted in distilled water, into the heart. Hearts were kept at −20°C for 1 hour and then sliced in 1-mm sections parallel to the atrioventricular groove. The tissues were incubated in 5 ml of 1% TTC phosphate buffer 37°C for 15 minutes and then fixed in 4% formaldehyde overnight. Slices were then compressed between glass plates that were 1 mm apart and photographed with a Cannon Powershot A620 Digital Camera (Canon, Tokyo, Japan) through a Zeiss 459300 microscope (Carl Zeiss Light Microscopy, Göttingen, Germany) and measured with NIH ImageJ software.

The measurements were performed in a blinded fashion. The areas of myocardial tissue at risk and infarcted were automatically transformed into volumes. Infarct and risk area volumes were expressed in cm3, and the percentage of infarct-to-risk area ratio (%I/R) and of area at risk to whole myocardial area (% R/A) were calculated.

Experimental Protocol.

WT C57BL/6 male mice or CypD KO male mice were subjected to 30 minutes of regional ischemia of the myocardium followed by 2 hours of reperfusion with the following interventions. The control group (n = 8) received no further intervention; the Na2S group (n = 8) was administered Na2S as an i.v. bolus dose of 1 μmol/kg at the 20th minute of ischemia; the GYY-4137 group (n = 8) was administered GYY-4137 as an i.v. bolus dose of 26.6 μmol/kg at the 20th minute of ischemia; the AP39 group (n = 8) was administered AP39 as an i.v. bolus dose of 250 nmol/kg at the 20th minute of ischemia; and the thiovaline group (n = 8) was administered thiovaline as an i.v. bolus dose of 4 μmol/kg at the 20th minute of ischemia.

The doses to be used for each donor were chosen as follows. In preliminary experiments, we administered three different doses of each donor based on literature reports (Li et al., 2008; Szabó et al., 2011; Tomasova et al., 2015). The maximal dose that did not affect blood pressure (data not shown) was used throughout the present series of experiments.

To inhibit endogenous NO production, mice were given L-NAME at 10 mg/kg (Bibli et al., 2015) at the 19th minute of ischemia, followed by a H2S donor as described earlier (n = 8 animals per group). In mice receiving the cGMP-dependent protein kinase-I (PKG-I) inhibitor, DT2 was given at a dose of 0.37 mg/kg i.v. bolus (Bibli et al., 2015) 10 minutes before sustained ischemia, followed by Na2S or AP39 as described earlier (n = 8 per group). The CypD KO control group (n = 8), CypD KO + Na2S group (n = 11), and CypD KO + AP39 group (n = 9) were treated the same as the WT mice described earlier.

In another series of experiments, C57-Bl/6 were used (6 per group for control, Na2S, GYY4137, AP39, and thiovaline). The mice were subjected to the same interventions up to the 10th minute of reperfusion, when tissue samples from the ischemic area of myocardium were collected, snap-frozen in liquid nitrogen, and stored at −80°C for Western blot analysis of eNOS and VASP phosphorylation.

Mitochondrial Isolation.

C57BL/6 mice weighting 25–30 g were euthanized by cervical dislocation, and their hearts were quickly excised, rinsed, and cut in the isolation buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES-Tris, 1 mM EGTA-Tris, pH 7.4). Subsequently, the tissue was homogenized in the isolation buffer added with 0.1 mg/ml Nagarse by the use of a glass-Teflon homogenizer. The homogenate was diluted in isolation buffer added with 0.2% w/v bovine serum albumin, centrifuged at 500g at 4°C, and filtered through a 150-μm mesh. The supernatant was further centrifuged at 8000g to obtain the mitochondrial fraction. The pellet was washed with isolation buffer without bovine serum albumin and centrifuged at 8000g, and the final pellet was used for protein determination and further assays.

Calcium Retention Capacity Assay.

The calcium retention capacity (CRC) assay was performed as previously described elsewhere (Giorgio et al., 2013) to determine the susceptibility of mitochondria to undergo permeability transition. Isolated mitochondria were diluted in mitochondrial assay buffer (KCl 137 mM, KH2PO4 2 mM, HEPES 20 mM, EGTA 20 µM, glutamate/malate 5 mM, pH 7.2) at a concentration of 0.25 mg/ml. Extramitochondrial Ca2+ was measured by Calcium Green-5N (1 µM) fluorescence using a Fluoroskan Ascent FL plate reader (Thermo Electron, Waltham, MA). Each minute, pulses of 10 μM Ca2+ were added to the cuvette, up to a point when the accumulated Ca2+ was released due to the opening of the mitochondrial permeability transition pore (PTP). The experiments were conducted both in the absence and in the presence of cyclosporin A (CsA) (1 µg/ml), a CypD inhibitor. Mitochondria were exposed to different concentrations of Na2S, GYY4137, or AP39, and their calcium retention capacity was determined.

Statistical Analysis.

Data are expressed as mean ± S.E.M. Statistical analysis was determined by using one- or two-way analysis of variance, with Dunnett’s or Bonferroni as a post-test or t test analysis when appropriate. P < 0.05 was considered statistically significant. GraphPad Prism software (version 4.02; GraphPad Software, San Diego, CA) was used for all statistical analyses.

Results

H2S Donors Protect against H2O2-Induced Injury In Vitro.

Initially we determined the effect of different H2S donors on H2O2-induced injury. Differentiated H9c2 cells were exposed to increasing concentrations of Na2S, thiovaline (TV), GYY4137, and AP39. In the absence of H2O2 the cells were not affected by treatment with any of the H2S generating compounds (Fig. 1, A–D). Na2S at concentrations over 10 μΜ protected cells from H2O2 injury (Fig. 1A), as assessed by MTT conversion to formazan.

Fig. 1.

Fig. 1.

Open in a new tab

H2S donors protect H9c2 cardiomyocytes from H2O2-induced injury in vitro. Differentiated H9c2 cells were exposed to vehicle or H2O2 (12 hours; 500 μΜ) in the absence (vehicle) or presence of the indicated concentration of (A) Na2S, (B) thiovaline, (C) GYY4137, or (D) AP39. In all cases the cells were pretreated for 1 hour with the H2S donor before H2O2 exposure. Cells were then incubated with MTT, and formazan production was assessed by measuring optical density at 595 nm. Six independent experiments were performed (n = 6); for each experiment, measurements were performed at least in quadruplicate (i.e., four wells). *P < 0.05 versus H2O2 vehicle.

We next tested TV, a H2S donor with intermediate release rate (Fig. 1B). TV exhibited a biphasic concentration-response curve, with 0.01 and 0.1 μM being protective, while 1 μΜ had no effect. GYY4137, on the other hand, was only effective at the highest concentration used (Fig. 1C) whereas the concentration–response curve of AP39 (Fig. 1D) resembled that of TV, with injury-limiting effects actions evident at low concentrations (1 and 100 nM) and higher concentrations (1 µM) being ineffective.

To confirm our observations in a different model of injury in vitro, we exposed cells to oxygen deprivation/reoxygenation. All the compounds were used at the concentration affording maximal protection against H2O2 injury; in this series of experiments, H2S donors were cytoprotective as well (Fig. 2).

Fig. 2.

Fig. 2.

Open in a new tab

Protective effects of H2S donors in anoxia/reoxygenation-induced cardiomyocyte injury. Differentiated H9c2 cells were cultured either under normal conditions or subjected to anoxia in an anaerobic chamber with 95% N2 and 5% CO2 at 37°C for 48 hours, followed by 24 hours growth in 95% air and 5% CO2. One hour before the anoxic insult the cells were treated with the indicated concentration of Na2S, thiovaline (TV), GYY4137, or AP39. Six independent experiments were performed (n = 6 ); for each experiment, measurements were performed at least in quadruplicate (i.e., four wells). *P < 0.05 versus corresponding vehicle; #P < 0.05 versus normoxia vehicle.

To evaluate the effect of different donors on cell viability we measured LDH release in supernatants of cells exposed to H2O2 or oxygen deprivation/reoxygenation. LDH was increased in response to the injurious stimuli and H2S donors prevented this increase (Fig. 3).

Fig. 3.

Fig. 3.

Open in a new tab

H2S donors abolish LDH release in response to H2O2 and oxygen deprivation/reoxygenation. H9c2 differentiated cells were treated with H2O2 or exposed to anoxia/reoxygenation as described in Fig. 1 and Fig. 2, respectively. Cells were incubated with the indicated concentration of the H2S donor compound, and LDH activity was measured in the supernatant. Five to six independent experiments were performed (n = 5–6); for each experiment measurements were performed in duplicate (i.e., two wells). #P < 0.05 versus (A) control, vehicle/vehicle, (B) vehicle/normoxia panel. *P < 0.05 versus (A) H2O2 vehicle or (B) A48h–R24h vehicle.

Role of NO in Cardiomyocyte Protection In Vitro.

To determine the contribution of NO to the mechanism of action of H2S donors in their protective effect in the H2O2 injury model, we treated cells with a NOS inhibitor before H2S donor exposure and evaluated cellular viability using MTT. Incubation with L-NAME did not significantly increase H2O2-induced cytotoxicity (Fig. 4), but Na2S-induced cytoprotection was reversed by NOS inhibition (Fig. 4). By contrast, L-NAME did not modulate the effects of GYY4137 and AP39, but the effects of TV were partially inhibited.

Fig. 4.

Fig. 4.

Open in a new tab

NO dependence of the protective effects of different H2S donors in H9c2 subjected to H2O2 injury in vitro. Differentiated H9c2 cells were exposed to vehicle or H2O2 (12 hours; 500 μΜ) in the absence (vehicle) or presence of the indicated concentration of sodium sulfide (Na2S), thiovaline (TV), GYY4137, or AP39. In all cases, cells were pretreated for 1 hour with the H2S donor before H2O2 exposure; when L-NAME was used, this was added 40 minutes before the H2S donor at 100 μΜ. The cells were then incubated with MTT, and formazan production was assessed by measuring optical density at 595 nm. Four independent experiments were performed (n = 4); for each experiment, measurements were performed at least in quadruplicate (i.e., four wells). *P < 0.05 versus corresponding no H2O2/L-NAME; #P < 0.05 versus TV/ H2O2.

Effects of H2S Donors In Vivo: NO-Dependent and -Independent Effects.

To evaluate the cardioprotective effects of Na2S, TV, GYY4137, and AP39 in ischemia-reperfusion in vivo, mice were subjected to 30 minutes of regional myocardial ischemia by left anterior descending coronary artery ligation, followed by 2 hours of reperfusion. All groups had similar risk/all myocardium areas (Fig. 5).

Fig. 5.

Fig. 5.

Open in a new tab

H2S donors attenuate myocardial infarct size after infarct to risk area (I/R) in vivo. Animals were subjected to 30 minutes of cardiac ischemia by left anterior descending occlusion, followed by reperfusion for 2 hours. Donors were administered as i.v. bolus 10 minutes before reestablishing blood flow. The infarct area to area at risk ratio (% I/R) and area at risk to whole myocardial area (R/A) were calculated as described in Materials and Methods. n = 8 mice/group; *P < 0.05 versus control.

We observed that all of the donors inhibited myocardial injury to a similar extent (Fig. 5). The infarct-to risk area in the control group was 37.8% ± 3.3%, and it was reduced to 17.8% ± 1.8%, 14.4% ± 1.2%, 19.5% ± 1.4%, and 16.5% ± 2.3% for Na2S, TV, GYY4137, and AP39, respectively. When L-NAME was used before H2S donor administration the protective effect of Na2S was abolished (Fig. 6), but the responses to TV, GYY4137, and AP39 remained unaffected.

Fig. 6.

Fig. 6.

Open in a new tab

NO inhibition only blocks the cardioprotective effect of Na2S without affecting the responses of other donors. Animals were subjected to 30 minutes of cardiac ischemia by left anterior descending occlusion, followed by reperfusion for 2 hours. Animals received L-NAME at the 19th minute of ischemia followed by donor administration. The infarct area to area at risk ratio (%I/R) and area at risk to whole myocardial area (R/A%) were calculated as described in Materials and Methods. n = 8 mice/group; *P < 0.05 versus control.

In subsequent experiments, we tested the contribution of cGMP/PKG in the protective action of Na2S and AP39, as representatives of the NO-dependent and the NO-independent H2S donors, respectively. We observed that inhibition of cGMP-dependent protein kinase by DT2 reversed the infarct-limiting effect of Na2S but not that of AP39 (Fig. 7). In line with the pharmacologic findings described herein, Na2S promoted eNOS phosphorylation on the activator site S1176 in ischemic cardiac tissue (Fig. 8). Na2S also increased VASP phosphorylation on Ser239, a site phosphorylated by PKG; AP39 altered neither p-eNOS nor p-VASP levels.

Fig. 7.

Fig. 7.

Open in a new tab

PKG inhibition reverses the cardioprotective effect of Na2S but not AP39. Animals were subjected to 30 minutes of cardiac ischemia by left anterior descending occlusion, followed by reperfusion for 2 hours. Animals received DT2, a PKG-I inhibitor, 10 minutes before sustained ischemia followed by donor administration at the 20th minute of ischemia. The infarct area to area at risk ratio (%I/R) and area at risk to whole myocardial area (R/A%) were calculated as described in Materials and Methods. n = 8 mice/group; *P < 0.05 versus control.

Fig. 8.

Fig. 8.

Open in a new tab

Na2S but not AP39 activates cGMP-PKG pathways in vivo. Mice were subjected to ischemia for 30 minutes and reperfusion for 10 minutes. Donors were administered 10 minutes before reestablishing blood flow. Ischemic tissues were collected, and eNOS phosphorylation (S1176) (A, C) and VASP phosphorylation (S239) (B, D) were determined. Western blots from representative animals are shown; densitometric analysis after normalization to total protein levels is presented for each group. n = 6 animals; *P < 0.05 versus control.

Evaluation of the Effects of H2S Donors on Isolated Heart Mitochondria.

Cardioprotective pathways converge on the mitochondrial PTP, preventing its opening. One of the best characterized interacting partners of PTP that promotes opening is the mitochondrial protein CypD. Infarct size in CypD KO mice was significantly reduced, in line with what has been previously reported (Baines et al., 2005). Both Na2S and AP39 further decreased infarct size, suggesting that the action of both H2S-generating agents is CypD independent (Fig. 9).

Fig. 9.

Fig. 9.

Open in a new tab

Cardioprotection by H2S donors is independent of CypD. WT or CypD KO animals were subjected to 30 minutes of ischemia, followed by reperfusion for 2 hours. The infarct area to area at risk ratio (%I/R) and area at risk to whole myocardial area (R/A%) were calculated as described in Materials and Methods. n = 8–11 mice; *P < 0.05 versus control.

To determine the direct effects of H2S donors on mitochondria, we performed in vitro experiments on isolated mouse heart mitochondria. AP39 increased mitochondrial CRC both in presence and absence of CsA (Fig. 10). By contrast, GYY4137 had no effect on mitochondrial CRC, irrespective of the presence of CsA. Moreover, Na2S did not alter the mitochondrial susceptibility to permeability transition (data not shown).These observations suggest that AP39 exerts direct mitochondrial effects, acting as a PTP desensitizer on a site different than CypD, whereas GYY4137 and Na2S use upstream signaling pathways to regulate the opening/closing status of PTP.

Fig. 10.

Fig. 10.

Open in a new tab

Direct effects of H2S donors on mitochondria. (A) Calcium retention capacity by mouse heart mitochondria was determined in the presence and absence of CsA (1 µg/ml). (B) Representative tracing (AP39, 300 nM). n = 3; *P < 0.05 versus vehicle.

Discussion

H2S has gained a lot of attention as a cytoprotective molecule in diseases associated with inflammation, apoptosis, or necrosis (Kimura, 2010; Wang, 2012; Kabil et al., 2014; Wallace and Wang, 2015). Several studies have already established its cardioprotective profile (Polhemus and Lefer, 2014; Salloum, 2015; Wang et al., 2015). In the vast majority of the cases, H2S salts were used to deliver H2S (Szabó et al., 2011; Salloum, 2015). Herein, we compared some of the most commonly used H2S donors in vitro and in vivo.

H2S donors inhibited oxidative stress-induced cardiomyocyte toxicity and anoxia/reoxygenation injury in H9c2 cardiomyocytes. H2S donors reversed the H2O2 injury with a rank order of potency AP39 > TV > Na2S > GYY4137. AP39 preferentially releases H2S in the mitochondria due to its triphenyl phosphonium group that allows accumulation in this cellular compartment (Szczesny et al., 2014). AP39, in line with the literature, was the most potent in preventing H2O2-induced toxicity, with 1 nM sufficing to exert its biologic effect (Szczesny et al., 2014). However, increasing AP39 concentration to 1 μM led to reversal of the protective effect. A similar bell-shaped curve was also noted for thiovaline, the second most potent H2S donor used, exerting beneficial effects at 10 nM. H2S donors have been shown to behave in an analogous fashion (bell-shaped concentration–response curves) in many instances (Szczesny et al., 2014; Hellmich et al., 2015; Ahmad et al., 2016).

H2S donors, including diallyl disulfide and S-propargyl-cysteine, have been shown to rescue cultured cardiomyocytes from high glycose injury, doxorubicin-induced toxicity or ROS-triggered injury (Szabó et al., 2011; Guo et al., 2013; Wu et al., 2015; Yang et al., 2015). The mechanisms through which H2S donors have been proposed to exerts their protective effects in these cultured cell systems include ATP-sensitive potassium channel and Akt activation, inhibition of mitogen-activated protein kinases pathways (p38, c-Jun N-terminal protein kinases), and inhibition of endoplasmic reticulum stress, as well as antioxidant mechanisms (Szabó et al., 2011; Wang, 2012; Guo et al., 2013; Salloum, 2015; Wu et al., 2015; Yang et al., 2015). Herein, we determined whether different H2S donors have different requirements for NO to exert their effects. We found that NOS inhibition completely reversed the protective effects of Na2S and partially those of thiovaline in vitro. In line with this finding, Wu et al. (2015) showed that H2O2 cytotoxicity was associated with decreased expression and phosphorylation (S1176) of eNOS and that treatment with another sulfide salt (NaHS) increased the ratio of p-eNOS/eNOS. In sharp contrast, to Na2S and thiovaline, the protective effect of GYY4137 and AP39 remained unaffected by L-NAME, suggesting that these agents exerted NO-independent effects. GYY4137 was shown to protect H9c2 cells from high-glucose-induced cytotoxicity through a AMP-activated protein kinase/mammalian target of rapamycin pathway (Wei et al., 2014). On the other hand, AP39 was shown to protect endothelial cells exposed to glucose oxidase through antioxidant mechanisms and by preserving mitochondrial DNA integrity (Szczesny et al., 2014).

Several in vivo studies have demonstrated that exogenously administered H2S protects against myocardial infarction (Johansen et al., 2006; Calvert et al., 2009, 2010; Szabó et al., 2011; King et al., 2014; Zhang et al., 2014; Lilyanna et al., 2015). Different laboratories have studied NaHS, Na2S, GYY4137, diallyl trisulfide, adenine analogs, or N-mercapto-based agents and have found them to be protective (Bibli et al., 2015; Zhao et al., 2015; Lougiakis et al., 2016). In all of the studies performed so far a single donor is used. Almost inevitably, the experimental protocols differ among the studies employing different species, duration of ischemia, different times at which the H2S donor was administered, and documented their observations regarding cardioprotection at different times (as early as 2 hours after reperfusion or as long as days/weeks later). It would, therefore, be hard to compare the proposed mechanisms for the H2S donors used in the different studies.

A common proposed mechanism among many studies demonstrating cardioprotection has been the requirement for eNOS for the H2S releasing salt to exert its effects (Predmore et al., 2012; Kondo et al., 2013; King et al., 2014; Bibli et al., 2015). To evaluate whether NO is required for the reduction in infarct size and cardioprotective effects of H2S donors, we systematically compared four H2S-producing agents: Na2S (an H2S-generating sulfide salt), GYY4137 (a slow H2S releaser), thiovaline (an agent with intermediate rate of H2S release compared with Na2S and GYY4137), and AP39 (a mitochondrial-targeted H2S donor). Among these, thiovaline and AP39 had not been tested before in animal models of myocardial ischemia/reperfusion injury. All the compounds studied inhibited infarct size to a similar degree. However, only in the case of Na2S was the beneficial effect reversed by NOS inhibition. In agreement to this finding, eNOS and VASP phosphorylation, as indexes of enhanced activity of the NO/cGMP pathway, were only increased in animals treated with Na2S, not those treated with AP39. It should also be noted that responses to sulfide salts were demonstrated to be NO-dependent in angiogenesis, vasorelaxation, and cardiac arrest (Minamishima et al., 2009; Coletta et al., 2012). We previously reported that NaHS reduced infarct size through a PKG/phospholamban pathway (Bibli et al., 2015). In agreement to this finding, the infarct size-reducing effect of Na2S was diminished by PKG-I inhibition. These results taken together reinforce the notion for a differential requirement of the cGMP/PKG pathway in the action of exogenously added H2S donor compounds. A similar observation has been made with regards to vasodilation, where NaHS but not GYY4137 was found to induce PKG-dependent effects (Bucci et al., 2012).

Three main cardioprotective signaling mechanisms have been shown to exist: the NO, the reperfusion injury salvage pathway, and the survivor activating factor enhancement pathway (Heusch, 2015). All three pathways converge onto the mitochondria, which integrate signals after ischemia-reperfusion and orchestrate cell survival versus death responses (Cohen and Downey, 2011; Sharma et al., 2012). A key event that determines cardiomyocyte fate after ischemia-reperfusion is the opening of the mitochondrial PTP (Bernardi and Di Lisa, 2015). CypD is a mitochondrial matrix isomerase and a key regulator of PTP function (Giorgio et al., 2010). CsA can bind CypD and prevent PTP opening, ameliorating the effects of reperfusion injury (Hausenloy et al., 2012). To determine whether Na2S and AP39 exert their effects through CypD, we employed a genetic mouse model lacking CypD. These mice have been shown to exhibit smaller cardiac infarcts after ischemia-reperfusion compared with controls, a finding that was reproduced in our study (Baines et al., 2005). Both Na2S and AP39 were able to reduce infarct in CypD KO mice, suggesting that the action of H2S is independent of CypD.

PTP inhibition can be obtained also by targeting proteins other than CypD (Bernardi and Di Lisa, 2015; Kwong and Molkentin, 2015). To elucidate whether H2S donors have direct effects on mitochondria, we evaluated the Ca2+ retention capacity of isolated heart mitochondria after a series of Ca2+ pulses in the absence or presence of CsA and increasing H2S donor concentrations. In these experiments we observed that Na2S and GYY4137 did not alter Ca2+ uptake by mitochondria, but AP39 significantly increased the mitochondrial CRC in both the absence and presence of CsA. The effect of AP39 was even potentiated in the presence of CsA, further suggesting that AP39 desensitizes pore opening in a CypD-independent manner, confirming our in vivo observations with CypD KO mice. Thus, AP39 exerts direct mitochondrial effects while Na2S and GYY4137 rely on intracellular signaling to prevent PTP opening and confer cardioprotection. In a study using cultured myocytes, NaHS prevented PTP opening through mitochondrial KATP channels and glycogen synthase kinase 3β–regulated pathways (Li et al., 2015), lending support to our hypothesis that sulfide salts, and H2S generated from them, have indirect mitochondrial effects.

We conclude that H2S generated from Na2S, thiovaline, GYY4137, or AP39 is cytoprotective for cardiomyocytes and reduces infarct size when administered during ischemia. Our findings reinforce the notion that H2S donors protect against cardiovascular disease in animal models and could be amenable to translation. In spite of containing the same active principle (H2S), the mechanism of action of H2S donors varies considerably. Na2S limits infarct size in a NO/cGMP/PKG-dependent pathway, whereas GYY4137, thiovaline, and AP39 use predominantly NO-independent pathways. Moreover, unlike GYY4137 and Na2S, AP39 has a direct effect on mitochondria that is most likely related to its ability to localize inside this organelle. Selecting the ideal donor for each pathophysiologic condition would, thus, vary depending on the deficit observed. Na2S would be expected to be ineffective if endothelial dysfunction is present, but it could be used if NO-regulated pathways remain intact. AP39, on the other hand, could be used even when upstream intracellular signaling is compromised due to disease processes. Future studies should be directed at evaluating H2S donors in the context of comorbidities that alter cardioprotective signaling.

Abbreviations

AP39

(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphosphonium bromide

AP123

hydroxythiobenzamide

A/R

anoxia/reoxygenation

ATB346

(4-carbamothioylphenyl) 2-(6-methoxynaphthalen-2-yl)propanoate

CRC

calcium retention capacity

CypD

cyclophilin-D

CsA

cyclosporin A

DMEM

Dulbecco’s modified essential medium

eNOS

endothelial nitric oxide synthase

FBS

fetal bovine serum

GYY4137

morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate

H2S

hydrogen sulfide

KO

knockout

LDH

lactate dehydrogenase

L-NAME

Nω-nitro-l-arginine methyl ester

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Na2S

sodium sulfide

NO

nitric oxide

p-eNOS

phospho-endothelial nitric oxide synthase

p-VASP

phospho-vasodilator-associated phosphoprotein

PKG

cGMP-dependent protein kinase

PTP

permeability transition pore

TTC

2,3,5-triphenyltetrazolium chloride

TV

thiovaline

VASP

vasodilator-associated phosphoprotein

WT

wild type

Authorship Contributions

Participated in research design: Andreadou, Di Lisa, Daiber, Manolopoulos, Szabó, Papapetropoulos.

Conducted experiments: Chatzianastasiou, Bibli, Efentakis, Kaludercic.

Contributed new reagents or analytic tools: Kaludercic, Wood, Whiteman, Di Lisa, Daiber.

Performed data analysis: Chatzianastasiou, Bibli, Efentakis, Kaludercic, Papapetropoulos.

Wrote or contributed to the writing of the manuscript: Chatzianastasiou, Andreadou, Whiteman, Di Lisa, Daiber, Manolopoulos, Szabó, Papapetropoulos.

Footnotes

This work was cofinanced by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) Research Funding Program: Aristeia 2011 (1436) (to A.P.), funds from the Medical Research Council UK (to M.W. and M.E.W.), grants from the Hellenic Institute for the Study of Sepsis (to A.P.) and from the COST Actions BM1005 (ENOG) and BM1203 (EUROS). M.W., M.E.W., and the University of Exeter have intellectual property (patent filings) related to AP39, related compounds and their use.

References

  1. Ahmad A, Olah G, Szczesny B, Wood ME, Whiteman M, Szabo C. (2016) AP39, a mitochondrially targeted hydrogen sulfide donor, exerts protective effects in renal epithelial cells subjected to oxidative stress in vitro and in acute renal injury in vivo. Shock 45:88–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander B, Coles SJ, Fox BC, Khan TF, Maliszewi J, Perry A, Pitak MP, Whiteman M, Wood ME. (2015) Investigating the generation of hydrogen sulfide from the phosphinodithioate slow-release donor GYY4137. MedChemComm 6:1649–1655. [Google Scholar]
  3. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434:658–662. [DOI] [PubMed] [Google Scholar]
  4. Benavides GA, Squadrito GL, Mills RW, Patel HD, Isbell TS, Patel RP, Darley-Usmar VM, Doeller JE, Kraus DW. (2007) Hydrogen sulfide mediates the vasoactivity of garlic. Proc Natl Acad Sci USA 104:17977–17982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernardi P, Di Lisa F. (2015) The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J Mol Cell Cardiol 78:100–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bibli S-I, Andreadou I, Chatzianastasiou A, Tzimas C, Sanoudou D, Kranias E, Brouckaert P, Coletta C, Szabo C, Kremastinos DT, et al. (2015) Cardioprotection by H2S engages a cGMP-dependent protein kinase G/phospholamban pathway. Cardiovasc Res 106:432–442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bucci M, Papapetropoulos A, Vellecco V, Zhou Z, Zaid A, Giannogonas P, Cantalupo A, Dhayade S, Karalis KP, Wang R, et al. (2012) cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation. PLoS One 7:e53319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caliendo G, Cirino G, Santagada V, Wallace JL. (2010) Synthesis and biological effects of hydrogen sulfide (H2S): development of H2S-releasing drugs as pharmaceuticals. J Med Chem 53:6275–6286. [DOI] [PubMed] [Google Scholar]
  9. Calvert JW, Elston M, Nicholson CK, Gundewar S, Jha S, Elrod JW, Ramachandran A, Lefer DJ. (2010) Genetic and pharmacologic hydrogen sulfide therapy attenuates ischemia-induced heart failure in mice. Circulation 122:11–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, Kevil CG, Lefer DJ. (2009) Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res 105:365–374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cohen MV, Downey JM. (2011) Ischemic postconditioning: from receptor to end-effector. Antioxid Redox Signal 14:821–831. [DOI] [PubMed] [Google Scholar]
  12. Coletta C, Papapetropoulos A, Erdelyi K, Olah G, Módis K, Panopoulos P, Asimakopoulou A, Gerö D, Sharina I, Martin E, et al. (2012) Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc Natl Acad Sci USA 109:9161–9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DeLeon ER, Stoy GF, Olson KR. (2012) Passive loss of hydrogen sulfide in biological experiments. Anal Biochem 421:203–207. [DOI] [PubMed] [Google Scholar]
  14. Giorgio V, Soriano ME, Basso E, Bisetto E, Lippe G, Forte MA, Bernardi P. (2010) Cyclophilin D in mitochondrial pathophysiology. Biochim Biophys Acta 1797:1113–1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabó I, et al. (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 110:5887–5892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guo R, Wu K, Chen J, Mo L, Hua X, Zheng D, Chen P, Chen G, Xu W, Feng J. (2013) Exogenous hydrogen sulfide protects against doxorubicin-induced inflammation and cytotoxicity by inhibiting p38MAPK/NFκB pathway in H9c2 cardiac cells. Cell Physiol Biochem 32:1668–1680. [DOI] [PubMed] [Google Scholar]
  17. Hausenloy DJ, Boston-Griffiths EA, Yellon DM. (2012) Cyclosporin A and cardioprotection: from investigative tool to therapeutic agent. Br J Pharmacol 165:1235–1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hellmich MR, Coletta C, Chao C, Szabo C. (2015) The therapeutic potential of cystathionine β-synthetase/hydrogen sulfide inhibition in cancer. Antioxid Redox Signal 22:424–448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heusch G. (2015) Molecular basis of cardioprotection: signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res 116:674–699. [DOI] [PubMed] [Google Scholar]
  20. Johansen D, Ytrehus K, Baxter GF. (2006) Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemia-reperfusion injury—evidence for a role of K ATP channels. Basic Res Cardiol 101:53–60. [DOI] [PubMed] [Google Scholar]
  21. Kabil O, Banerjee R. (2014) Enzymology of H2S biogenesis, decay and signaling. Antioxid Redox Signal 20:770–782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kabil O, Motl N, Banerjee R. (2014) H2S and its role in redox signaling. Biochim Biophys Acta 1844:1355–1366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kashfi K, Olson KR. (2013) Biology and therapeutic potential of hydrogen sulfide and hydrogen sulfide-releasing chimeras. Biochem Pharmacol 85:689–703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kimura H. (2010) Hydrogen sulfide: from brain to gut. Antioxid Redox Signal 12:1111–1123. [DOI] [PubMed] [Google Scholar]
  25. Kimura H. (2014) Production and physiological effects of hydrogen sulfide. Antioxid Redox Signal 20:783–793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. King AL, Polhemus DJ, Bhushan S, Otsuka H, Kondo K, Nicholson CK, Bradley JM, Islam KN, Calvert JW, Tao YX, et al. (2014) Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent. Proc Natl Acad Sci USA 111:3182–3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kondo K, Bhushan S, King AL, Prabhu SD, Hamid T, Koenig S, Murohara T, Predmore BL, Gojon G, Sr, Gojon G, Jr, et al. (2013) H₂S protects against pressure overload-induced heart failure via upregulation of endothelial nitric oxide synthase. Circulation 127:1116–1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kwong JQ, Molkentin JD. (2015) Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab 21:206–214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Le Trionnaire S, Perry A, Szczesny B, Szabo C, Winyard PG, Whatmore JL, Wood ME, Whiteman M. (2014) The synthesis and functional evaluation of a mitochondria-targeted hydrogen sulfide donor, (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)decyl)triphenylphosphonium bromide (AP39). MedChemComm 5:728–736. [Google Scholar]
  30. Li H, Zhang C, Sun W, Li L, Wu B, Bai S, Li H, Zhong X, Wang R, Wu L, et al. (2015) Exogenous hydrogen sulfide restores cardioprotection of ischemic post-conditioning via inhibition of mPTP opening in the aging cardiomyocytes. Cell Biosci 5:43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li L, Rose P, Moore PK. (2011) Hydrogen sulfide and cell signaling. Annu Rev Pharmacol Toxicol 51:169–187. [DOI] [PubMed] [Google Scholar]
  32. Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y, Baskar R, Tan CH, Moore PK. (2008) Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 117:2351–2360. [DOI] [PubMed] [Google Scholar]
  33. Lilyanna S, Peh MT, Liew OW, Wang P, Moore PK, Richards AM, Martinez EC. (2015) GYY4137 attenuates remodeling, preserves cardiac function and modulates the natriuretic peptide response to ischemia. J Mol Cell Cardiol 87:27–37. [DOI] [PubMed] [Google Scholar]
  34. Lougiakis N, Papapetropoulos A, Gikas E, Toumpas S, Efentakis P, Wedmann R, Zoga A, Zhou Z, Iliodromitis EK, Skaltsounis A-L, et al. (2016) Synthesis and pharmacological evaluation of novel adenine-hydrogen sulfide slow release hybrids designed as multitarget cardioprotective agents. J Med Chem 59:1776–1790. [DOI] [PubMed] [Google Scholar]
  35. Mani S, Li H, Untereiner A, Wu L, Yang G, Austin RC, Dickhout JG, Lhoták Š, Meng QH, Wang R. (2013) Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis. Circulation 127:2523–2534. [DOI] [PubMed] [Google Scholar]
  36. Martelli A, Testai L, Citi V, Marino A, Pugliesi I, Barresi E, Nesi G, Rapposelli S, Taliani S, Da Settimo F, et al. (2013) Arylthioamides as H2S donors: l-cysteine-activated releasing properties and vascular effects in vitro and in vivo. ACS Med Chem Lett 4:904–908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Minamishima S, Bougaki M, Sips PY, Yu JD, Minamishima YA, Elrod JW, Lefer DJ, Bloch KD, Ichinose F. (2009) Hydrogen sulfide improves survival after cardiac arrest and cardiopulmonary resuscitation via a nitric oxide synthase 3-dependent mechanism in mice. Circulation 120:888–896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Panza E, De Cicco P, Armogida C, Scognamiglio G, Gigantino V, Botti G, Germano D, Napolitano M, Papapetropoulos A, Bucci M, et al. (2015) Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression. Pigment Cell Melanoma Res 28:61–72. [DOI] [PubMed] [Google Scholar]
  39. Papapetropoulos A, Pyriochou A, Altaany Z, Yang G, Marazioti A, Zhou Z, Jeschke MG, Branski LK, Herndon DN, Wang R, et al. (2009) Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci USA 106:21972–21977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Papapetropoulos A, Whiteman M, Cirino G. (2015) Pharmacological tools for hydrogen sulfide research: a brief, introductory guide for beginners. Br J Pharmacol 172:1633–1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Paul BD, Snyder SH. (2012) H₂S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol 13:499–507. [DOI] [PubMed] [Google Scholar]
  42. Polhemus DJ, Calvert JW, Butler J, Lefer DJ. (2014) The cardioprotective actions of hydrogen sulfide in acute myocardial infarction and heart failure. Scientifica (Cairo) 2014:768607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Polhemus DJ, Lefer DJ. (2014) Emergence of hydrogen sulfide as an endogenous gaseous signaling molecule in cardiovascular disease. Circ Res 114:730–737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Predmore BL, Kondo K, Bhushan S, Zlatopolsky MA, King AL, Aragon JP, Grinsfelder DB, Condit ME, Lefer DJ. (2012) The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability. Am J Physiol Heart Circ Physiol 302:H2410–H2418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roger T, Raynaud F, Bouillaud F, Ransy C, Simonet S, Crespo C, Bourguignon M-P, Villeneuve N, Vilaine J-P, Artaud I, et al. (2013) New biologically active hydrogen sulfide donors. ChemBioChem 14:2268–2271. [DOI] [PubMed] [Google Scholar]
  46. Salloum FN. (2015) Hydrogen sulfide and cardioprotection—mechanistic insights and clinical translatability. Pharmacol Ther 152:11–17. [DOI] [PubMed] [Google Scholar]
  47. Sharma V, Bell RM, Yellon DM. (2012) Targeting reperfusion injury in acute myocardial infarction: a review of reperfusion injury pharmacotherapy. Expert Opin Pharmacother 13:1153–1175. [DOI] [PubMed] [Google Scholar]
  48. Sun J, Aponte AM, Menazza S, Gucek M, Steenbergen C, Murphy E. (2016) Additive cardioprotection by pharmacological postconditioning with hydrogen sulfide and nitric oxide donors in mouse heart: S-sulfhydration vs. S-nitrosylation. Cardiovasc Res 110:96–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Szabó C. (2007) Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov 6:917–935. [DOI] [PubMed] [Google Scholar]
  50. Szabó G, Veres G, Radovits T, Gerő D, Módis K, Miesel-Gröschel C, Horkay F, Karck M, Szabó C. (2011) Cardioprotective effects of hydrogen sulfide. Nitric Oxide 25:201–210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Szczesny B, Módis K, Yanagi K, Coletta C, Le Trionnaire S, Perry A, Wood ME, Whiteman M, Szabó C. (2014) AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro. Nitric Oxide 41:120–130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tomasova L, Pavlovicova M, Malekova L, Misak A, Kristek F, Grman M, Cacanyiova S, Tomasek M, Tomaskova Z, Perry A, et al. (2015) Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels. Nitric Oxide 46:131–144. [DOI] [PubMed] [Google Scholar]
  53. Wallace JL, Wang R. (2015) Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous gasotransmitter. Nat Rev Drug Discov 14:329–345. [DOI] [PubMed] [Google Scholar]
  54. Wang R. (2012) Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 92:791–896. [DOI] [PubMed] [Google Scholar]
  55. Wang R, Szabó C, Ichinose F, Ahmed A, Whiteman M, Papapetropoulos A. (2015) The role of H2S bioavailability in endothelial dysfunction. Trends Pharmacol Sci 36:568–578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wei WB, Hu X, Zhuang XD, Liao LZ, Li WD. (2014) GYY4137, a novel hydrogen sulfide-releasing molecule, likely protects against high glucose-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells. Mol Cell Biochem 389:249–256. [DOI] [PubMed] [Google Scholar]
  57. Whiteman M, Perry A, Zhou Z, Bucci M, Papapetropoulos A, Cirino G, Wood ME. (2015) Phosphinodithioate and phosphoramidodithioate hydrogen sulfide donors, in Chemistry, Biochemistry and Pharmacology of Hydrogen Sulfide (Moore KP, Whiteman M, eds) pp 337–363, Springer International, Cham, Switzerland. [DOI] [PubMed] [Google Scholar]
  58. Wu D, Hu Q, Liu X, Pan L, Xiong Q, Zhu YZ. (2015) Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. Nitric Oxide 46:204–212. [DOI] [PubMed] [Google Scholar]
  59. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, et al. (2008) H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine γ-lyase. Science 322:587–590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yang H, Mao Y, Tan B, Luo S, Zhu Y. (2015) The protective effects of endogenous hydrogen sulfide modulator, S-propargyl-cysteine, on high glucose-induced apoptosis in cardiomyocytes: a novel mechanism mediated by the activation of Nrf2. Eur J Pharmacol 761:135–143. [DOI] [PubMed] [Google Scholar]
  61. Zhang Y, Li H, Zhao G, Sun A, Zong NC, Li Z, Zhu H, Zou Y, Yang X, Ge J. (2014) Hydrogen sulfide attenuates the recruitment of CD11b⁺Gr-1⁺ myeloid cells and regulates Bax/Bcl-2 signaling in myocardial ischemia injury. Sci Rep 4:4774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhao Y, Wang H, Xian M. (2011) Cysteine-activated hydrogen sulfide (H2S) donors. J Am Chem Soc 133:15–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhao Y, Yang C, Organ C, Li Z, Bhushan S, Otsuka H, Pacheco A, Kang J, Aguilar HC, Lefer DJ, et al. (2015) Design, synthesis, and cardioprotective effects of N-mercapto-based hydrogen sulfide donors. J Med Chem 58:7501–7511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zheng Y, Ji X, Ji K, Wang B. (2015) Hydrogen sulfide prodrugs—a review. Acta Pharm Sin B 5:367–377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zheng Y, Yu B, Ji K, Pan Z, Chittavong V, Wang B. (2016) Esterase-sensitive prodrugs with tunable release rates and direct generation of hydrogen sulfide. Angew Chem Int Ed Engl 55:4514–4518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhou Z, von Wantoch Rekowski M, Coletta C, Szabó C, Bucci M, Cirino G, Topouzis S, Papapetropoulos A, Giannis A. (2012) Thioglycine and l-thiovaline: biologically active H₂S-donors. Bioorg Med Chem 20:2675–2678. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Pharmacology and Experimental Therapeutics are provided here courtesy of American Society for Pharmacology and Experimental Therapeutics

RESOURCES