1521-0103/358/3/431–440$25.00 http://dx.doi.org/10.1124/jpet.116.235119THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:431–440, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Cardioprotection by H2S Donors: Nitric Oxide-Dependentand ‑Independent Mechanisms

Athanasia Chatzianastasiou, Sofia-Iris Bibli, Ioanna Andreadou, Panagiotis Efentakis,Nina Kaludercic, Mark E. Wood, Matthew Whiteman, Fabio Di Lisa, Andreas Daiber,Vangelis G. Manolopoulos, Csaba Szabó, and Andreas PapapetropoulosGeorge P. Livanos and Marianthi Simou Laboratories, First Department of Pulmonary and Critical Care Medicine, EvangelismosHospital, Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece (A.C., A.P.); Laboratory of Pharmacology,Democritus University of Thrace Medical School, Alexandroupolis, Greece (A.C., V.G.M.); Laboratory of Pharmacology, Faculty ofPharmacy, National and Kapodistrian University of Athens, Greece (S.-I.B., I.A., P.E., A.P.); Neuroscience Institute, CNR, Italy (N.K., F.D.L.);Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom (M.E.W.); University of ExeterMedical School, Exeter, United Kingdom (M.W.); Department of Biomedical Sciences, University of Padova, Padova, Italy (F.D.L.);Center of Cardiology and Center for Thrombosis and Hemostasis, Medical Center of the Johannes Gutenberg University, Mainz,Germany (A.D.); Department of Anesthesiology, University of Texas Medical Branch, Galveston, Texas (C.S.); Center of Clinical,Experimental Surgery & Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece (A.P.)

Received May 11, 2016; accepted June 21, 2016

ABSTRACTHydrogen sulfide (H2S) is a signaling molecule with protectiveeffects in the cardiovascular system. To harness the therapeuticpotential of H2S, a number of donors have been developed. Thepresent study compares the cardioprotective actions of represen-tative H2S donors from different classes and studies their mecha-nisms of action in myocardial injury in vitro and in vivo. Exposure ofcardiomyocytes to H2O2 led to significant cytotoxicity, which wasinhibited by sodium sulfide (Na2S), thiovaline (TV), GYY4137[morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodi-thioate], and AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)-phenoxy)decyl) triphenylphospho-nium bromide]. Inhibition of nitricoxide (NO) synthesis prevented the cytoprotective effects of Na2Sand TV, but not GYY4137 and AP39, against H2O2-inducedcardiomyocyte injury. Mice subjected to left anterior descend-ing 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 phosphorylationof endothelial NOS and vasodilator-associated phosphopro-tein. Both Na2S and AP39 reduced infarct size in mice lackingcyclophilin-D (CypD), a modulator of the mitochondrial permeabil-ity transition pore (PTP). Nevertheless, only AP39 displayed adirect effect on mitochondria by increasing the mitochondrial Ca21

retention capacity, which is evidence of decreased propensity toundergo permeability transition. We conclude that although all theH2S donors we tested limited infarct size, the pathways involvedwere not conserved. Na2S had no direct effects on PTP opening,and its action was nitric oxide dependent. In contrast, the cardio-protection exhibited by AP39 could result from a direct inhibitoryeffect on PTP acting at a site different than CypD.

IntroductionHydrogen sulfide (H2S) is a recently identified signal-

ing molecule with important functions throughout the body

(Szabó, 2007; Li et al., 2011; Paul and Snyder, 2012). Car-diovascular homeostasis relies on the production of ade-quate H2S levels (Wang, 2012; Polhemus et al., 2014).Although H2S can be generated nonenzymatically, the ma-jority of H2S is believed to be produced through the ac-tion of cystathionine g-lyase, cystathionine b-synthase, and3-mercaptopyruvate sulfurtransferase (Kabil and Banerjee,2014; Kimura, 2014). In blood vessels, endogenously producedH2S reduces blood pressure by promoting vasorelaxation(Yang et al., 2008; Bucci et al., 2012), enhances angiogenesisby stimulating endothelial cell proliferation and migration(Papapetropoulos et al., 2009; Coletta et al., 2012), prevents

This work was cofinanced by the EuropeanUnion (European Social Fund – ESF)and Greek national funds through the Operational Program “Education andLifelong Learning” of the National Strategic Reference Framework (NSRF)Research Funding Program: Aristeia 2011 (1436) (to A.P.), funds from the MedicalResearch Council UK (to M.W. and M.E.W.), grants from the Hellenic Institute forthe Study of Sepsis (to A.P.) and from the COST Actions BM1005 (ENOG) andBM1203 (EUROS). M.W., M.E.W., and the University of Exeter have intellectualproperty (patent filings) related to AP39, related compounds and their use.

dx.doi.org/10.1124/jpet.116.235119.

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, Nv-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, permeabilitytransition pore; TTC, 2,3,5-triphenyltetrazolium chloride; TV, thiovaline; VASP, vasodilator-associated phosphoprotein; WT, wild type.

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atherosclerosis development (Mani et al., 2013), and amelio-rates diabetic complications (Wang et al., 2015). In the heart,endogenous H2S limits oxidative stress and reduces myocar-dial injury after ischemia reperfusion (Kondo et al., 2013; Kinget al., 2014).Given the pleiotropic beneficial actions of H2S in the

cardiovascular system, investigators have used pharmaco-logic agents to deliver H2S to prevent organ dysfunction ortreat disease in animal models (Szabó, 2007; Wang, 2012;Wallace and Wang, 2015). In the first era of H2S research, thesodium salts NaHS and sodium sulfide (Na2S) were almostexclusively used to administer H2S (Papapetropoulos et al.,2015). However, these agents suffer a major drawback in thatthey produce H2S instantly in aqueous solutions (Kashfi andOlson, 2013; Zheng et al., 2015). Production of H2S from salts(NaHS or Na2S) cannot be controlled, and H2S is generatedafter 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 usedresult in transiently toxic levels of H2S. Fewer studies haveemployed naturally occurring H2S donors, mainly garlic-derived allyl polysulfides, as an alternative means to deliverH2S (Benavides et al., 2007; Predmore et al., 2012; Panza et al.,2015). The first donor to be synthesized that generates H2S inslow manner, mimicking the low level endogenous production,was GYY4137 [morpholin-4-ium 4methoxyphenyl(morpholino)phosphinodithioate] (Li et al., 2008). Analogs of GYY4137 arenow available, with considerably different rates of Η2Srelease and pharmacologic properties (Whiteman et al.,2015).Salts and GYY4137 exemplify the two extremes in terms of

H2S production rates. Theneed todevelop donorswith improvedproperties for use as research tools and potential therapeuticagents, 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. Watersolubility, oral bioavailability, an intermediate H2S releaserate, and generation of H2S in a controlled fashion (as, forexample, after enzymatic activation), are among the desir-able properties of an “ideal” donor.Two more classes of donors are worth mentioning:

mitochondrial-targeted H2S and hybrid, bifunctional do-nors (Kashfi and Olson, 2013; Le Trionnaire et al., 2014;Szczesny et al., 2014). Representatives of the first class areAP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphospho-nium bromide] and AP123 [hydrox-ythiobenzamide]. In the second class, several molecules carry-ing a H2S donating moiety bound to a known pharmacophorestructure have been reported. A naproxen-based H2S donor(ATB346 [(4-carbamothioylphenyl) 2-(6-methoxynaphthalen-2-yl)propanoate]) is in clinical development as a safer nonsteroi-dal anti-inflammatory agent to treat arthritis (Wallace andWang, 2015). We recently reported on adenine-H2S slow-release hybrids that are effective in reducing infarct sizein vivo (Lougiakis et al., 2016).Apart from the differences in physicochemical properties

and rates of H2S release from donor molecules, differences inthe signaling pathways used by H2S donors have been noted.

We observed that when added to smooth muscle cells NaHSand thioaminoacids enhance cGMP accumulation by inhibit-ing phosphodiesterase activity, but GYY4137 only did so atvery high concentrations (Bucci et al., 2012; Zhou et al., 2012).NaHS can additionally enhance cGMP levels by promotingendothelial nitric oxide synthase (eNOS) phosphorylation andincreasing nitric oxide (NO) production (Bibli et al., 2015). Weand others have observed that the infarct-limiting action ofsulfide 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 cardio-

protection and the lack of comparative mechanistic studies onH2S donors in the context of cardioprotection, herein weselected representative H2S donors (ultrafast, intermediate,slow, and mitochondrial) to assess their ability to rescue themyocardium after ischemia-reperfusion injury in vivo. Fur-thermore, we evaluated the contribution of NO to thebeneficial effects of each donor using in vitro and in vivosystems.

Materials and MethodsReagents. Dulbecco’s modified essential medium (DMEM) and

10% fetal bovine serum (FBS) were obtained from Gibco/ThermoScientific (Waltham, MA). The lactate dehydrogenase (LDH) cytotox-icity assay kit was purchased from Cayman Chemical (via LabSupplies, P. Galanis & Co, Athens, Greece). The following primaryantibodies were obtained from Cell Signaling Technology (Beverly,MA): phospho-eNOS (p-eNOS; S1176), phospho-vasodilator-associatedphosphoprotein (p-VASP), total eNOS, total VASP, b-tubulin, andthe goat anti-rabbit horseradish peroxidase antibody. The Super-Signal chemiluminescence kit was purchased from Thermo ScientificTechnologies (Waltham, MA). H2O2 was obtained from AppliChemGmbH (Darmstadt, Germany). GYY4137, AP39, and thiovaline weresynthesized as previously described elsewhere (Zhou et al., 2012; LeTrionnaire et al., 2014; Alexander et al., 2015). DT2 was obtainedfrom BIOLOG Life Science Institute (Bremen, Germany). CalciumGreen-5N, protease and phosphatase inhibitor cocktail were pur-chased fromThermoFisher Scientific (Waltham,MA). All other reagentsincluding Na2S, NaCl, NaF, EDTA, EGTA, phenylmethylsulfonylfluoride, Nargase, cyclosporine A, L-NAME (Nv-nitro-L-argininemethyl ester), TTC (2,3,5-triphenyltetrazolium chloride), andMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were fromSigma-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 fromAlexander Fleming Institute (Athens, Greece) and CypD KO (samegenetic background as wild-type [WT] mice) were bred in the animalfacility of Universitätsmedizin der Johannes Gutenberg-UniversitätZentrum für Kardiologie I, Labor für Molekulare Kardiologie (Mainz,Germany). Mice were housed in a specific pathogen-free facility at20–25°C and received water and food (regular laboratory animaldiet) ad libitum. All animal procedures were in compliance with theEuropean Community guidelines for the use of experimental ani-mals; experimental protocols were approved by the ethics commit-tee of the Prefecture of Athens or Johannes Gutenberg University(Landesuntersuchungsamt Koblenz).

Cell Culture. The rat embryonic-heart- derived H9c2 cell line wasobtained from the American Type Culture Collection [ATCC] (CRL-1446) (ATCC/LGCStandards, Middlesex, United Kingdom). TheH9c2cells were cultured in DMEM containing 25 mM D-glucose, 1 mMsodium pyruvate, and supplementedwith 10%FBS, 2mM L-glutamine,1% streptomycin (100mg/ml), and 1% penicillin (100U/ml) at pH 7.4 ina 5%CO2 incubator at 37°C. For differentiation, H9c2were seeded andallowed to grow to confluence. The medium was then replaced toDMEM containing 1% FBS with 10 nM all-trans-retinoic acid for

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7 days. Culture of H9c2 myoblasts in low-serum medium andstimulation with 10 nM all-trans-retinoic acid for 7 days resulted inthe appearance of elongated cells connecting at irregular anglesreminiscent of cells with a cardiac phenotype.

In Vitro Oxidative Stress and Anoxia/Reoxygenation. Toinduce oxidative stress injury, H9c2 cells (1.5 � 104 per well) weredifferentiated in 96-well plates. The cells were treated with 500 mMH2O2 in serum-freeDMEMfor 12 hours in a 5%CO2 incubator at 37°C.In the anoxia/reoxygenation (A/R) assay, differentiated H9c2 cells in96-well plates were placed in an anaerobic chamber containing amixture of 95% N2 and 5% CO2 at 37°C for 48 hours. After anoxia, thecells 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 at10,000–15,000 cells per/ well in growth medium. Following oxidativestress injury or anoxia/reoxygenation cell survival was assessed indifferentiated 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 solubili-zation solution (10% Triton-X 100 in acidic 0.1N HCl isopropanol);subsequently, absorbancewasmeasured at 595 nmwith a backgroundcorrection 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, supernatantmedium was collected and centrifuged at 400g for 5 minutes. Cellsupernatant (100 ml) was transferred to a new 96-well assay plate and100 ml of reaction solution containing 1 mMNAD1, 85 mM lactic acid,0.5 mM iodonitrotetrazolium, and LDH diaphorase was added to eachwell. The plate was incubated for 30 minutes at 37°C with gentleshaking. Absorbance was read at 490 nm with a plate reader.

Western Blot Analysis. Frozen ischemic samples were pulver-ized and homogenized with the lysis buffer (1% Triton �100, 20 mMTris pH 7.4–7.6, 150mMNaCl, 50mMNaF, 1mMEDTA, 1mMEGTA,1 mM glycerol phosphatase, 1% SDS, and 100 mM phenylmethylsul-fonyl fluoride, supplemented with protease and phosphatase inhibitor

cocktail). The lysates were centrifuged at 11,000g for 15minutes at 4°C.The supernatants were collected, and the protein concentration wasdetermined based on the Lowry assay. The supernatantwasmixedwitha buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol,0.004% bromophenyl blue, and 0.125 M Tris/HCl. The samples werethen heated at 100°C for 10 minutes and stored at 280°C. An equalamount of protein was loaded in each well and then separated by SDS-PAGE electrophoresis and transferred onto a polyvinylidene difluoridemembrane.

After blocking with 5% nonfat dry milk, the membranes wereincubated overnight at 4°C with primary antibody. The followingprimary antibodies were used: p-VASP (S239), p-eNOS (S1176), totaleNOS, total VASP, and b-tubulin (dilution for all primary antibodieswas 1:1000). Membranes were then incubated with secondary goatanti-rabbit horseradish peroxidase antibody (1:2000) for 2 hours atroom temperature and developed using the Supersignal chemilumi-nesence ECL Western Blotting Detection Reagents (Pierce/ThermoScientific, Rockford, IL). Relative densitometry was determined usinga computerized software package (U.S. National Institutes of HealthImageJ, https://imagej.nih.gov/ij/), and the values for phosphorylatedwere normalized to the values for total proteins, respectively.

Ischemia-Reperfusion Injury Model In Vivo. Male mice wererandomly divided into groups and anesthetized by intraperitonealinjection with a combination of ketamine and xylazine (0.01ml/g, finalconcentrations of ketamine and xylazine, 10 mg/ml and 2 mg/ml,respectively). Anesthetic depth was evaluated by the loss of pedalreflex to toe-pinch stimulus and breathing rate. A tracheotomy wasperformed for artificial respiration at 120–150 breaths/minute. Athoracotomy was then performed between the fourth and fifth ribs,and the pericardium was carefully retracted to visualize the leftanterior descending coronary, which was ligated using a 7-0 Prolenemonofilament polypropylene suture placed 3 mm below the tip of theleft auricle. The heart was allowed to stabilize for 15 minutes beforeligation to induce ischemia. After the ischemic period, the ligaturewasreleased, allowing reperfusion of the myocardium.

Fig. 1. H2S donors protect H9c2 cardio-myocytes from H2O2-induced injury invitro. Differentiated H9c2 cells were ex-posed to vehicle or H2O2 (12 hours; 500 mΜ)in the absence (vehicle) or presence of theindicated concentration of (A) Na2S, (B)thiovaline, (C) GYY4137, or (D) AP39. Inall cases the cellswere pretreated for 1 hourwith the H2S donor before H2O2 exposure.Cells were then incubated with MTT, andformazan production was assessed by mea-suring optical density at 595 nm. Six in-dependent experiments were performed (n =6); for each experiment, measurementswereperformed at least in quadruplicate (i.e.,four wells). *P , 0.05 versus H2O2 vehicle.

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Throughout experiments, body temperature was maintained at37°C6 0.5°C byway of a heating pad andmonitored via a thermocoupleinserted rectally. After reperfusion, the hearts were rapidly excisedfrom mice and directly cannulated and washed with 2.5 ml of saline-heparin 1% for blood removal. We then infused 0.2ml of 1%Evans blue,diluted in distilled water, into the heart. Hearts were kept at220°C for1 hour and then sliced in 1-mm sections parallel to the atrioventriculargroove. The tissues were incubated in 5 ml of 1% TTC phosphate buffer37°C for 15minutes and then fixed in4% formaldehyde overnight. Sliceswere then compressed between glass plates that were 1 mm apart andphotographed with a Cannon Powershot A620 Digital Camera (Canon,Tokyo, Japan) through a Zeiss 459300 microscope (Carl Zeiss LightMicroscopy, Göttingen, Germany) and measured with NIH ImageJsoftware.

The measurements were performed in a blinded fashion. The areasof myocardial tissue at risk and infarcted were automatically trans-formed into volumes. Infarct and risk area volumes were expressed incm3, and the percentage of infarct-to-risk area ratio (%I/R) and of areaat risk to whole myocardial area (% R/A) were calculated.

Experimental Protocol. WT C57BL/6 male mice or CypD KOmale mice were subjected to 30 minutes of regional ischemia of the

myocardium followed by 2 hours of reperfusion with the followinginterventions. The control group (n 5 8) received no further in-tervention; the Na2S group (n 5 8) was administered Na2S as an i.v.bolus dose of 1 mmol/kg at the 20th minute of ischemia; the GYY-4137group (n5 8) was administered GYY-4137 as an i.v. bolus dose of 26.6mmol/kg at the 20th minute of ischemia; the AP39 group (n 5 8) wasadministered AP39 as an i.v. bolus dose of 250 nmol/kg at the 20thminute of ischemia; and the thiovaline group (n5 8) was administeredthiovaline as an i.v. bolus dose of 4 mmol/kg at the 20th minute ofischemia.

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

To inhibit endogenous NO production, mice were given L-NAME at10mg/kg (Bibli et al., 2015) at the 19thminute of ischemia, followed bya H2S donor as described earlier (n 5 8 animals per group). In micereceiving 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 asdescribed earlier (n 5 8 per group). The CypD KO control group (n 58), CypDKO1Na2S group (n5 11), and CypDKO1AP39 group (n59) were treated the same as the WT mice described earlier.

In another series of experiments, C57-Bl/6 were used (6 per groupfor control, Na2S, GYY4137, AP39, and thiovaline). The mice weresubjected to the same interventions up to the 10th minute of reperfu-sion, when tissue samples from the ischemic area of myocardium werecollected, snap-frozen in liquid nitrogen, and stored at 280°C forWestern blot analysis of eNOS and VASP phosphorylation.

Mitochondrial Isolation. C57BL/6 mice weighting 25–30 g wereeuthanized by cervical dislocation, and their hearts were quicklyexcised, 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 addedwith 0.1 mg/ml Nagarse by the use of a glass-Teflon homogenizer. Thehomogenate was diluted in isolation buffer added with 0.2% w/v bovineserum albumin, centrifuged at 500g at 4°C, and filtered through a150-mm mesh. The supernatant was further centrifuged at 8000g toobtain themitochondrial fraction. The pellet was washed with isolationbuffer without bovine serum albumin and centrifuged at 8000g, andthe final pellet was used for protein determination and further assays.

Calcium Retention Capacity Assay. The calcium retentioncapacity (CRC) assay was performed as previously described else-where (Giorgio et al., 2013) to determine the susceptibility of mitochon-dria to undergo permeability transition. Isolated mitochondria werediluted in mitochondrial assay buffer (KCl 137 mM, KH2PO4 2 mM,HEPES 20 mM, EGTA 20 mM, glutamate/malate 5 mM, pH 7.2) at aconcentration of 0.25 mg/ml. Extramitochondrial Ca21 was mea-sured by Calcium Green-5N (1 mM) fluorescence using a Fluoroskan

Fig. 2. Protective effects of H2S donors in anoxia/reoxygenation-inducedcardiomyocyte injury. Differentiated H9c2 cells were cultured eitherunder normal conditions or subjected to anoxia in an anaerobic chamberwith 95%N2 and 5%CO2 at 37°C for 48 hours, followed by 24 hours growthin 95% air and 5% CO2. One hour before the anoxic insult the cells weretreated 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 inquadruplicate (i.e., four wells). *P , 0.05 versus corresponding vehicle;#P , 0.05 versus normoxia vehicle.

Fig. 3. H2S donors abolish LDH releasein response to H2O2 and oxygen depriva-tion/reoxygenation. H9c2 differentiatedcells were treated with H2O2 or exposedto anoxia/reoxygenation as described inFig. 1 and Fig. 2, respectively. Cells wereincubated with the indicated concentra-tion of the H2S donor compound, and LDHactivity wasmeasured in the supernatant.Five to six independent experiments wereperformed (n = 5–6); for each experimentmeasurements were performed in dupli-cate (i.e., two wells). #P , 0.05 versus (A)control, vehicle/vehicle, (B) vehicle/normoxiapanel. *P , 0.05 versus (A) H2O2 vehicle or(B) A48h–R24h vehicle.

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Ascent FL plate reader (Thermo Electron, Waltham, MA). Eachminute, pulses of 10mMCa21were added to the cuvette, up to a pointwhen the accumulated Ca21 was released due to the opening of themitochondrial permeability transition pore (PTP). The experimentswere conducted both in the absence and in the presence of cyclo-sporin A (CsA) (1 mg/ml), a CypD inhibitor. Mitochondria wereexposed to different concentrations of Na2S, GYY4137, or AP39, andtheir calcium retention capacity was determined.

Statistical Analysis. Data are expressed as mean 6 S.E.M.Statistical analysis was determined by using one- or two-way analysisof variance, with Dunnett’s or Bonferroni as a post-test or t testanalysis when appropriate. P , 0.05 was considered statisticallysignificant. GraphPad Prism software (version 4.02; GraphPad Soft-ware, San Diego, CA) was used for all statistical analyses.

ResultsH2S Donors Protect against H2O2-Induced Injury

In Vitro. Initially we determined the effect of different H2Sdonors onH2O2-induced injury. DifferentiatedH9c2 cells wereexposed to increasing concentrations of Na2S, thiovaline (TV),GYY4137, and AP39. In the absence of H2O2 the cells were notaffected by treatment with any of the H2S generating com-pounds (Fig. 1, A–D). Na2S at concentrations over 10 mΜprotected cells fromH2O2 injury (Fig. 1A), as assessed byMTTconversion to formazan.We next tested TV, a H2S donor with intermediate release

rate (Fig. 1B). TV exhibited a biphasic concentration-responsecurve, with 0.01 and 0.1 mM being protective, while 1 mΜ hadno effect. GYY4137, on the other hand, was only effectiveat the highest concentration used (Fig. 1C) whereas theconcentration–response curve of AP39 (Fig. 1D) resembledthat of TV, with injury-limiting effects actions evident at lowconcentrations (1 and 100 nM) and higher concentrations(1 mM) being ineffective.

To confirm our observations in a different model of injuryin vitro, we exposed cells to oxygen deprivation/reoxygenation.All the compounds were used at the concentration affordingmaximal protection against H2O2 injury; in this series ofexperiments, H2S donors were cytoprotective as well (Fig. 2).To evaluate the effect of different donors on cell viability we

measured LDH release in supernatants of cells exposed toH2O2 or oxygen deprivation/reoxygenation. LDH was in-creased in response to the injurious stimuli and H2S donorsprevented this increase (Fig. 3).Role of NO in Cardiomyocyte Protection In Vitro. To

determine the contribution of NO to the mechanism of actionof H2S donors in their protective effect in the H2O2 injurymodel, we treated cells with a NOS inhibitor before H2S donorexposure and evaluated cellular viability using MTT. In-cubation with L-NAME did not significantly increase H2O2-induced cytotoxicity (Fig. 4), but Na2S-induced cytoprotectionwas reversed byNOS inhibition (Fig. 4). By contrast, L-NAMEdid not modulate the effects of GYY4137 and AP39, but theeffects of TV were partially inhibited.Effects of H2S Donors In Vivo: NO-Dependent and

-Independent Effects. To evaluate the cardioprotectiveeffects of Na2S, TV, GYY4137, and AP39 in ischemia-reperfusion in vivo, mice were subjected to 30 minutes ofregional myocardial ischemia by left anterior descendingcoronary artery ligation, followed by 2 hours of reperfusion.All groups had similar risk/all myocardium areas (Fig. 5).We observed that all of the donors inhibited myocardial

injury to a similar extent (Fig. 5). The infarct-to risk area inthe control group was 37.8% 6 3.3%, and it was reduced to17.8%6 1.8%, 14.4%6 1.2%, 19.5%6 1.4%, and 16.5%6 2.3%for Na2S, TV, GYY4137, and AP39, respectively. WhenL-NAME was used before H2S donor administration theprotective effect of Na2S was abolished (Fig. 6), but theresponses to TV, GYY4137, and AP39 remained unaffected.In subsequent experiments, we tested the contribution of

cGMP/PKG in the protective action of Na2S and AP39, asrepresentatives of the NO-dependent and the NO-independent

Fig. 4. NO dependence of the protective effects of different H2S donors inH9c2 subjected to H2O2 injury in vitro. Differentiated H9c2 cells wereexposed to vehicle or H2O2 (12 hours; 500 mΜ) 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 for1 hour with the H2S donor before H2O2 exposure; when L-NAME wasused, this was added 40 minutes before the H2S donor at 100 mΜ. The cellswere then incubated with MTT, and formazan production was assessed bymeasuring optical density at 595 nm. Four independent experiments wereperformed (n = 4); for each experiment, measurements were performed atleast in quadruplicate (i.e., four wells). *P , 0.05 versus corresponding noH2O2/L-NAME; #P , 0.05 versus TV/ H2O2.

Fig. 5. H2S donors attenuate myocardial infarct size after infarct to riskarea (I/R) in vivo. Animals were subjected to 30 minutes of cardiacischemia by left anterior descending occlusion, followed by reperfusion for2 hours. Donors were administered as i.v. bolus 10 minutes beforereestablishing blood flow. The infarct area to area at risk ratio (% I/R) andarea at risk to whole myocardial area (R/A) were calculated as described inMaterials and Methods. n = 8 mice/group; *P , 0.05 versus control.

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H2S donors, respectively.We observed that inhibition of cGMP-dependent protein kinase by DT2 reversed the infarct-limitingeffect of Na2S but not that of AP39 (Fig. 7). In line with thepharmacologic findings described herein, Na2S promoted eNOSphosphorylation on the activator site S1176 in ischemic cardiactissue (Fig. 8). Na2S also increased VASP phosphorylation onSer239, a site phosphorylated by PKG; AP39 altered neitherp-eNOS nor p-VASP levels.Evaluation of the Effects of H2S Donors on Isolated

Heart Mitochondria. Cardioprotective pathways convergeon the mitochondrial PTP, preventing its opening. One of thebest characterized interacting partners of PTP that promotesopening is themitochondrial proteinCypD. Infarct size inCypDKO mice was significantly reduced, in line with what has beenpreviously reported (Baines et al., 2005). Both Na2S and AP39further decreased infarct size, suggesting that the action of bothH2S-generating agents is CypD independent (Fig. 9).To determine the direct effects of H2S donors on mitochon-

dria, we performed in vitro experiments on isolated mouseheart mitochondria. AP39 increased mitochondrial CRC bothin presence and absence of CsA (Fig. 10). By contrast, GYY4137had no effect on mitochondrial CRC, irrespective of thepresence of CsA. Moreover, Na2S did not alter the mito-chondrial susceptibility to permeability transition (data notshown).These observations suggest that AP39 exerts directmitochondrial effects, acting as a PTP desensitizer on a sitedifferent than CypD, whereas GYY4137 and Na2S useupstream signaling pathways to regulate the opening/closingstatus of PTP.

DiscussionH2S has gained a lot of attention as a cytoprotective mol-

ecule in diseases associated with inflammation, apoptosis,

or necrosis (Kimura, 2010; Wang, 2012; Kabil et al., 2014;Wallace and Wang, 2015). Several studies have alreadyestablished its cardioprotective profile (Polhemus and Lefer,2014; Salloum, 2015;Wang et al., 2015). In the vastmajority ofthe cases, H2S salts were used to deliver H2S (Szabó et al.,2011; Salloum, 2015). Herein, we compared some of the mostcommonly used H2S donors in vitro and in vivo.H2S donors inhibited oxidative stress-induced cardiomyo-

cyte toxicity and anoxia/reoxygenation injury in H9c2 cardi-omyocytes. H2S donors reversed the H2O2 injury with a rankorder of potency AP39 . TV . Na2S . GYY4137. AP39preferentially releases H2S in the mitochondria due to itstriphenyl phosphonium group that allows accumulation inthis cellular compartment (Szczesny et al., 2014). AP39, in linewith 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 concentra-tion to 1 mM led to reversal of the protective effect. A similarbell-shaped curve was also noted for thiovaline, the secondmost potent H2S donor used, exerting beneficial effects at10 nM.H2S donors have been shown to behave in an analogousfashion (bell-shaped concentration–response curves) in manyinstances (Szczesny et al., 2014; Hellmich et al., 2015; Ahmadet al., 2016).H2S donors, including diallyl disulfide and S-propargyl-

cysteine, have been shown to rescue cultured cardiomyocytesfrom 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 H2Sdonors have been proposed to exerts their protective effects inthese cultured cell systems include ATP-sensitive potassiumchannel and Akt activation, inhibition of mitogen-activatedprotein kinases pathways (p38, c-Jun N-terminal proteinkinases), and inhibition of endoplasmic reticulum stress, aswell as antioxidant mechanisms (Szabó et al., 2011; Wang,2012; Guo et al., 2013; Salloum, 2015; Wu et al., 2015; Yang

Fig. 6. NO inhibition only blocks the cardioprotective effect of Na2Swithout affecting the responses of other donors. Animals were subjected to30 minutes of cardiac ischemia by left anterior descending occlusion,followed by reperfusion for 2 hours. Animals received L-NAME at the 19thminute of ischemia followed by donor administration. The infarct area toarea 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. 7. PKG inhibition reverses the cardioprotective effect of Na2S but notAP39. Animals were subjected to 30 minutes of cardiac ischemia by leftanterior descending occlusion, followed by reperfusion for 2 hours.Animals received DT2, a PKG-I inhibitor, 10 minutes before sustainedischemia 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 wholemyocardial area (R/A%) were calculated as described in Materials andMethods. n = 8 mice/group; *P , 0.05 versus control.

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et al., 2015). Herein, we determined whether different H2Sdonors have different requirements for NO to exert theireffects. We found that NOS inhibition completely reversed theprotective effects of Na2S and partially those of thiovaline invitro. In line with this finding, Wu et al. (2015) showed thatH2O2 cytotoxicity was associated with decreased expressionand phosphorylation (S1176) of eNOS and that treatmentwithanother sulfide salt (NaHS) increased the ratio of p-eNOS/eNOS. In sharp contrast, to Na2S and thiovaline, the protectiveeffect 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). Onthe other hand, AP39 was shown to protect endothelial cellsexposed to glucose oxidase through antioxidant mechanismsandby preservingmitochondrial 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, orN-mercapto-based agentsand have found them to be protective (Bibli et al., 2015; Zhaoet al., 2015; Lougiakis et al., 2016). In all of the studiesperformed so far a single donor is used. Almost inevitably, the

experimental protocols differ among the studies employingdifferent species, duration of ischemia, different times at whichthe H2S donor was administered, and documented their

Fig. 8. Na2S but not AP39 activatescGMP-PKG pathways in vivo. Mice weresubjected to ischemia for 30 minutes andreperfusion for 10 minutes. Donors wereadministered 10minutes before reestablish-ing blood flow. Ischemic tissues were col-lected, and eNOS phosphorylation (S1176)(A, C) and VASP phosphorylation (S239) (B,D) were determined. Western blots fromrepresentative animals are shown; den-sitometric analysis after normalizationto total protein levels is presented foreach group. n = 6 animals; *P , 0.05versus control.

Fig. 9. Cardioprotection by H2S donors is independent of CypD. WT orCypD KO animals were subjected to 30 minutes of ischemia, followed byreperfusion for 2 hours. The infarct area to area at risk ratio (%I/R) andarea at risk to whole myocardial area (R/A%) were calculated as describedin Materials and Methods. n = 8–11 mice; *P , 0.05 versus control.

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observations regarding cardioprotection at different times (asearly as 2 hours after reperfusion or as long as days/weekslater). It would, therefore, be hard to compare the proposedmechanisms for the H2S donors used in the different studies.A common proposed mechanism among many studies

demonstrating cardioprotection has been the requirementfor 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 thereduction in infarct size and cardioprotective effects of H2Sdonors, we systematically compared four H2S-producingagents: Na2S (an H2S-generating sulfide salt), GYY4137 (aslow H2S releaser), thiovaline (an agent with intermediaterate of H2S release compared with Na2S and GYY4137), andAP39 (a mitochondrial-targeted H2S donor). Among these,thiovaline and AP39 had not been tested before in animalmodels of myocardial ischemia/reperfusion injury. All thecompounds studied inhibited infarct size to a similar degree.However, only in the case of Na2S was the beneficial effectreversed by NOS inhibition. In agreement to this finding,eNOS and VASP phosphorylation, as indexes of enhancedactivity of the NO/cGMP pathway, were only increased inanimals treated with Na2S, not those treated with AP39. Itshould also be noted that responses to sulfide salts weredemonstrated to be NO-dependent in angiogenesis, vaso-relaxation, and cardiac arrest (Minamishima et al., 2009;Coletta et al., 2012). We previously reported that NaHSreduced infarct size through a PKG/phospholamban pathway(Bibli et al., 2015). In agreement to this finding, the infarctsize-reducing effect of Na2S was diminished by PKG-I in-hibition. These results taken together reinforce the notion fora differential requirement of the cGMP/PKG pathway in theaction of exogenously added H2S donor compounds. A similarobservation 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 salvagepathway, and the survivor activating factor enhance-ment pathway (Heusch, 2015). All three pathways con-verge onto the mitochondria, which integrate signals after

ischemia-reperfusion and orchestrate cell survival versusdeath responses (Cohen and Downey, 2011; Sharma et al.,2012). A key event that determines cardiomyocyte fate afterischemia-reperfusion is the opening of the mitochondrial PTP(Bernardi and Di Lisa, 2015). CypD is a mitochondrial matrixisomerase and a key regulator of PTP function (Giorgio et al.,2010). CsA can bind CypD and prevent PTP opening, ame-liorating the effects of reperfusion injury (Hausenloy et al.,2012). To determine whether Na2S and AP39 exert theireffects through CypD, we employed a genetic mouse modellacking CypD. These mice have been shown to exhibit smallercardiac infarcts after ischemia-reperfusion compared withcontrols, a finding that was reproduced in our study (Baineset al., 2005). Both Na2S and AP39 were able to reduce infarctin CypD KO mice, suggesting that the action of H2S isindependent of CypD.PTP inhibition can be obtained also by targeting proteins

other than CypD (Bernardi and Di Lisa, 2015; Kwong andMolkentin, 2015). To elucidate whether H2S donors havedirect effects on mitochondria, we evaluated the Ca21 re-tention capacity of isolated heartmitochondria after a series ofCa21 pulses in the absence or presence of CsA and increasingH2S donor concentrations. In these experiments we observedthat Na2S and GYY4137 did not alter Ca21 uptake bymitochondria, but AP39 significantly increased the mitochon-drial CRC in both the absence and presence of CsA. The effectof AP39 was even potentiated in the presence of CsA, furthersuggesting that AP39 desensitizes pore opening in a CypD-independent manner, confirming our in vivo observationswith CypD KO mice. Thus, AP39 exerts direct mitochondrialeffects while Na2S and GYY4137 rely on intracellular sig-naling to prevent PTP opening and confer cardioprotection.In a study using cultured myocytes, NaHS prevented PTPopening through mitochondrial KATP channels and glyco-gen synthase kinase 3b–regulated pathways (Li et al.,2015), lending support to our hypothesis that sulfide salts,and H2S generated from them, have indirect mitochondrialeffects.We conclude that H2S generated from Na2S, thiovaline,

GYY4137, or AP39 is cytoprotective for cardiomyocytes andreduces infarct size when administered during ischemia. Our

Fig. 10. Direct effects of H2S donors on mitochondria. (A) Calcium retention capacity by mouse heart mitochondria was determined in the presence andabsence of CsA (1 mg/ml). (B) Representative tracing (AP39, 300 nM). n = 3; *P , 0.05 versus vehicle.

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findings reinforce the notion that H2S donors protect againstcardiovascular disease in animal models and could be amena-ble to translation. In spite of containing the same activeprinciple (H2S), the mechanism of action of H2S donors variesconsiderably. Na2S limits infarct size in a NO/cGMP/PKG-dependent pathway, whereas GYY4137, thiovaline, and AP39use predominantly NO-independent pathways. Moreover,unlike GYY4137 and Na2S, AP39 has a direct effect onmitochondria that ismost likely related to its ability to localizeinside this organelle. Selecting the ideal donor for eachpathophysiologic condition would, thus, vary depending onthe deficit observed.Na2Swould be expected to be ineffective ifendothelial dysfunction is present, but it could be used ifNO-regulated pathways remain intact. AP39, on the otherhand, could be used even when upstream intracellularsignaling is compromised due to disease processes. Fu-ture studies should be directed at evaluating H2S donorsin the context of comorbidities that alter cardioprotectivesignaling.

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 themanuscript:Chatzianastasiou,

Andreadou, Whiteman, Di Lisa, Daiber, Manolopoulos, Szabó,Papapetropoulos.

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Address correspondence to: Dr. Andreas Papapetropoulos, Laboratory ofPharmacology, Faculty of Pharmacy, Panepistimiopolis, Zografou, Athens15771, Greece. E-mail: apapapet@pharm.uoa.gr

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