Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection.

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

Exposure to cigarette smoke abrogates the beneficial effect of ischemic postconditioning.

Cigarette smoking is one of the risk factors for coronary artery disease. Although conditioning decreases infarct size in hearts from healthy animals, comorbidities may render it ineffective. We investigated the effects of cigarette smoke (CS) exposure on intracellular myocardial signaling, infarct size after ischemia-reperfusion, and the potential interference with ischemic conditioning. Exposure of mice to CS increased blood pressure, caused cardiac hypertrophy, and upregulated the nitric oxide synthatse (NOS)/soluble guanylate cyclase (sGC)/cGMP pathway. To test the effect of CS exposure on the endogenous cardioprotective mechanisms, mice were subjected to regional myocardial ischemia and reperfusion with no further intervention or application of preconditioning (PreC) or postconditioning (PostC). Exposure to CS did not increase the infarction compared with the room air (RA)-exposed group. PreC was beneficial for both CS and RA vs. nonconditioned animals. PostC was effective only in RA animals, while the infarct size-limiting effect was not preserved in the CS group. Differences in oxidative stress markers, Akt, and endothelial NOS phosphorylation and cGMP levels were observed between RA and CS groups subjected to PostC. In conclusion, exposure to CS does not per se increase infarct size. The beneficial effect of ischemic PreC is preserved in mice exposed to CS, as it does not affect the cardioprotective signaling; in contrast, PostC fails to protect CS-exposed mice due to impaired activation of the Akt/eNOS/cGMP axis that occurs in parallel to enhanced oxidative stress.

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

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 Ca(2+) 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.

Modulation of poly(ADP-ribose) polymerase-1 (PARP-1)-mediated oxidative cell injury by ring finger protein 146 (RNF146) in cardiac myocytes.

Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD(+)) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.

Using mechanism-based combinations of H2S-donors to maximize the cardioprotective action of H2S.

H2S-donors are cardioprotective in ischemia/reperfusion (I/R) injury. Some H2S-donors exert their beneficial effects in a nitric oxide (NO)-dependent manner, while others act using NO-independent pathways. The aims of the present study were to (i) evaluate whether H2S-donors with distinct pharmacodynamic properties act synergistically in I/R injury and (ii) determine if H2S-donors remain cardioprotective in obese mice. C57BL/6 mice were subjected to 30 min of ischemia followed by 120 min of reperfusion. Donors were administered intravenously at the end of ischemia (Na2S: 1 µmol/kg, GYY4137: 25 µmol/kg, AP39: 0,25 µmol/kg), while the 3-mercaptopyruvate sulfurtransferase (10 mg/kg) inhibitor was given intraperitonially 1 h prior to ischemia. Infarct size was estimated by 2,3,5-triphenyltetrazolium staining, while the area at risk was calculated using Evans blue. All three donors reduced infarct size when administered as a sole treatment. Co-administration of Na2S/GYY4137, as well as Na2S/AP39 reduced further the I/R injury, beyond what was observed with each individual donor. Since inhibition of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase is known to reduce infarct size, we co-administered C3 with Na2S to determine possible additive effects between the two agents. In this case, combination of C3 with Na2S did not yield superior results compared to the individual treatments. Similarly, to what was observed in healthy mice, administration of a H2S-donor (Na2S or AP39) reduced I/R injury in mice rendered obese by consumption of a high fat diet. We conclude that combining a NO-dependent with a NO-independent H2S-donor leads to enhanced cardioprotection and that H2S-donors remain effective in obese animals.

Synergistic Effects of Resistive Breathing on Endotoxin-Induced Lung Injury in Mice.

Introduction: Resistive breathing (RB) is characterized by forceful contractions of the inspiratory muscles, mainly the diaphragm, resulting in large negative intrathoracic pressure and mechanical stress imposed on the lung. We have shown that RB induces lung injury in healthy animals. Whether RB exerts additional injurious effects when added to pulmonary or extrapulmonary lung injury is unknown. Our aim was to study the synergistic effect of RB on lipopolysaccharide (LPS)-induced lung injury. Methods: C57BL/6 mice inhaled an LPS aerosol (10mg/3mL) or received an intraperitoneal injection of LPS (10 mg/kg). Mice were then anaesthetized, the trachea was surgically exposed, and a nylon band of a specified length was sutured around the trachea, to provoke a reduction of the surface area at 50%. RB through tracheal banding was applied for 24 hours. Respiratory system mechanics were measured, BAL was performed, and lung sections were evaluated for histological features of lung injury. Results: LPS inhalation increased BAL cellularity, mainly neutrophils (p < 0.001 to ctr), total protein and IL-6 in BAL (p < 0.001 and p < 0.001, respectively) and increased the lung injury score (p = 0.001). Lung mechanics were not altered. Adding RB to inhaled LPS further increased BAL cellularity (p < 0.001 to LPS inh.), total protein (p = 0.016), lung injury score (p = 0.001) and increased TNFa levels in BAL (p = 0.011). Intraperitoneal LPS increased BAL cellularity, mainly macrophages (p < 0.001 to ctr.), total protein levels (p = 0.017), decreased static compliance (p = 0.004) and increased lung injury score (p < 0.001). Adding RB further increased histological features of lung injury (p = 0.022 to LPS ip). Conclusion: Resistive breathing exerts synergistic injurious effects when combined with inhalational LPS-induced lung injury, while the additive effect on extrapulmonary lung injury is less prominent.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart.

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Orally Bioavailable Enzymatic Inhibitor of CD38, MK-0159, Protects against Ischemia/Reperfusion Injury in the Murine Heart.

CD38 is one of the major nicotinamide adenine dinucleotide (NAD+)- and nicotinamide adenine dinucleotide phosphate (NADP+)-consuming enzymes in mammals. NAD+, NADP+, and their reduced counterparts are essential coenzymes for numerous enzymatic reactions, including the maintenance of cellular and mitochondrial redox balance. CD38 expression is upregulated in age-associated inflammation as well as numerous metabolic diseases, resulting in cellular and mitochondrial dysfunction. Recent literature studies demonstrate that CD38 is activated upon ischemia/reperfusion (I/R), leading to a depletion of NADP+, which results in endothelial damage and myocardial infarction in the heart. Despite increasing evidence of CD38 involvement in various disease states, relatively few CD38 enzymatic inhibitors have been reported to date. Herein, we describe a CD38 enzymatic inhibitor (MK-0159, IC50 = 3 nM against murine CD38) that inhibits CD38 in in vitro assay. Mice treated with MK-0159 show strong protection from myocardial damage upon cardiac I/R injury compared to those treated with NAD+ precursors (nicotinamide riboside) or the known CD38 inhibitor, 78c.

TRPV4 Inhibition Exerts Protective Effects Against Resistive Breathing Induced Lung Injury.

INTRODUCTION: TRPV4 channels are calcium channels, activated by mechanical stress, that have been implicated in the pathogenesis of pulmonary inflammation. During resistive breathing (RB), increased mechanical stress is imposed on the lung, inducing lung injury. The role of TRPV4 channels in RB-induced lung injury is unknown. MATERIALS AND METHODS: Spontaneously breathing adult male C57BL/6 mice were subjected to RB by tracheal banding. Following anaesthesia, mice were placed under a surgical microscope, the surface area of the trachea was measured and a nylon band was sutured around the trachea to reduce area to half. The specific TRPV4 inhibitor, HC-067047 (10 mg/kg ip), was administered either prior to RB and at 12 hrs following initiation of RB (preventive) or only at 12 hrs after the initiation of RB (therapeutic protocol). Lung injury was assessed at 24 hrs of RB, by measuring lung mechanics, total protein, BAL total and differential cell count, KC and IL-6 levels in BAL fluid, surfactant Protein (Sp)D in plasma and a lung injury score by histology. RESULTS: RB decreased static compliance (Cst), increased total protein in BAL (p < 0.001), total cell count due to increased number of both macrophages and neutrophils, increased KC and IL-6 in BAL (p < 0.001 and p = 0.01, respectively) and plasma SpD (p < 0.0001). Increased lung injury score was detected. Both preventive and therapeutic HC-067047 administration restored Cst and inhibited the increase in total protein, KC and IL-6 levels in BAL fluid, compared to RB. Preventive TRPV4 inhibition ameliorated the increase in BAL cellularity, while therapeutic TRPV4 inhibition exerted a partial effect. TRPV4 inhibition blunted the increase in plasma SpD (p < 0.001) after RB and the increase in lung injury score was also inhibited. CONCLUSION: TRPV4 inhibition exerts protective effects against RB-induced lung injury.

Spontaneous Breathing Through Increased Airway Resistance Augments Elastase-Induced Pulmonary Emphysema.

Introduction: Resistive breathing (RB), the pathophysiologic hallmark of chronic obstructive pulmonary disease (COPD), especially during exacerbations, is associated with significant inflammation and mechanical stress on the lung. Mechanical forces are implicated in the progression of emphysema that is a major pathologic feature of COPD. We hypothesized that resistive breathing exacerbates emphysema. Methods: C57BL/6 mice were exposed to 0.75 units of pancreatic porcine elastase intratracheally to develop emphysema. Resistive breathing was applied by suturing a nylon band around the trachea to reduce surface area to half for the last 24 or 72 hours of a 21-day time period after elastase treatment in total. Following RB (24 or 72 hours), lung mechanics were measured and bronchoalveolar lavage (BAL) was performed. Emphysema was quantified by the mean linear intercept (Lm) and the destructive index (DI) in lung tissue sections. Results: Following 21 days of intratracheal elastase exposure, Lm and DI increased in lung tissue sections [Lm (µm), control 39.09±0.76, elastase 62.05±2.19, p=0.003 and DI, ctr 30.95±2.75, elastase 73.12±1.75, p<0.001]. RB for 72 hours further increased Lm by 64% and DI by 19%, compared to elastase alone (p<0.001 and p=0.02, respectively). RB induced BAL neutrophilia in elastase-treated mice. Static compliance (Cst) increased in elastase-treated mice [Cst (mL/cmH2O), control 0.067±0.001, elastase 0.109±0.006, p<0.001], but superimposed RB decreased Cst, compared to elastase alone [Cst (mL/cmH2O), elastase+RB24h 0.090±0.004, p=0.006 to elastase, elastase+RB72h 0.090±0.005, p=0.006 to elastase]. Conclusion: Resistive breathing augments pulmonary inflammation and emphysema in an elastase-induced emphysema mouse model.

The protective role of the 3-mercaptopyruvate sulfurtransferase (3-MST)-hydrogen sulfide (H2S) pathway against experimental osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H2S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development. METHODS: 3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST-/- meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST-/- cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated. RESULTS: 3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST-/- mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes. CONCLUSION: 3-MST-generated H2S protects against joint calcification and experimental OA. Enhancing H2S production in chondrocytes may represent a potential disease modifier to treat OA.

Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase.

RATIONALE: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. RESULTS: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2-3 months old) 3-MST-/- mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST-/- mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST-/- mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST-/- mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST-/- mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. CONCLUSIONS: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy.

Tricyclic indole and dihydroindole derivatives as new inhibitors of soluble guanylate cyclase.

The synthesis of new tricyclic fused indole and dihydroindole derivatives and preliminary results from their in vitro inhibitory activity against soluble guanylate cyclase (sGC) are presented.

Nitroglycerine limits infarct size through S-nitrosation of cyclophilin D: a novel mechanism for an old drug.

AIMS: Nitroglycerine (NTG) given prior to an ischaemic insult exerts cardioprotective effects. However, whether administration of an acute low dose of NTG in a clinically relevant manner following an ischaemic episode limits infarct size, has not yet been explored. METHODS AND RESULTS: Adult mice were subjected to acute myocardial infarction in vivo and then treated with vehicle or low-dose NTG prior to reperfusion. This treatment regimen minimized myocardial infarct size without affecting haemodynamic parameters but the protective effect was absent in mice rendered tolerant to the drug. Mechanistically, NTG was shown to nitrosate and inhibit cyclophilin D (CypD), and NTG administration failed to limit infarct size in CypD knockout mice. Additional experiments revealed lack of the NTG protective effect following genetic (knockout mice) or pharmacological inhibition (L-NAME treatment) of the endothelial nitric oxide synthase (eNOS). The protective effect of NTG was attributed to preservation of the eNOS dimer. Moreover, NTG retained its cardioprotective effects in a model of endothelial dysfunction (ApoE knockout) by preserving CypD nitrosation. Human ischaemic heart biopsies revealed reduced eNOS activity and exhibited reduced CypD nitrosation. CONCLUSION: Low-dose NTG given prior to reperfusion reduces myocardial infarct size by preserving eNOS function, and the subsequent eNOS-dependent S-nitrosation of CypD, inhibiting cardiomyocyte necrosis. This novel pharmacological action of NTG warrants confirmation in clinical studies, although our data in human biopsies provide promising preliminary results.

p38 Inhibition Ameliorates Inspiratory Resistive Breathing-Induced Pulmonary Inflammation.

Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with strenuous contractions of the inspiratory muscles and increased negative intrathoracic pressures that act as an injurious stimulus to the lung. We have shown that IRB induces pulmonary inflammation in healthy animals. p38 kinase is activated in the lung under stress. We hypothesized that p38 is activated during IRB and contributes to IRB-induced pulmonary inflammation. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve. Resistance was connected to the inspiratory port to provoke a peak tidal inspiratory pressure 50% of maximum. Following 3 and 6 h of IRB, respiratory system mechanics were measured and bronchoalveolar lavage (BAL) was performed. Phosphorylated p38, TNF-α, and MIP-2α were detected in lung tissue. Lung injury was estimated histologically. SB203580 (p38 inhibitor) was administered prior to IRB (1 mg kg-1). Six hours of IRB increased phosphorylated p38 in the lung, compared with quietly breathing controls (p = 0.001). Six hours of IRB increased the numbers of macrophages and neutrophils (p = 0.01 and p = 0.005) in BAL fluid. BAL protein levels and lung elasticity increased after both 3 and 6 h IRB. TNF-α and MIP-2α increased after 6 h of IRB (p = 0.01 and p < 0.001, respectively). Increased lung injury score was detected at 6 h IRB. SB203580 administration blocked the increase of neutrophils and macrophages at 6 h IRB (p = 0.01 and p = 0.005 to 6 h IRB) but not the increase in BAL protein and elasticity. TNF-α, MIP-2α, and injury score at 6 h IRB returned to control. p38 activation contributes to IRB-induced pulmonary inflammation.

Cardioprotection by H2S engages a cGMP-dependent protein kinase G/phospholamban pathway.

AIMS: H2S is known to confer cardioprotection; however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo. METHODS AND RESULTS: Anaesthetized rabbits were subjected to myocardial ischaemia (I)/reperfusion (R), and infarct size was determined in control or H2S-exposed groups. The H2S donor sodium hydrosulfide (NaHS, an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, whereas the control peptide TAT or l-nitroarginine methyl ester (l-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor, glibenclamide, partially reversed the effects of NaHS, whereas inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild-type and PLN KO mice underwent I/R. NaHS did not exert cardioprotection in PLN KO mice. Unlike what was observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. CONCLUSIONS: Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific.