Previous studies have demonstrated that hydrogen sulfide (H 2 S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H 2 S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H 2 S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H 2 S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H 2 S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H 2 S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H 2 S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.eNOS uncoupling | myocardial infarction | cystathionase | Cth | nitrite H ydrogen sulfide (H 2 S), historically known for its odorous smell and toxicity at high concentrations, has recently been classified as a physiological signaling molecule with robust cytoprotective actions in multiple organ systems (1-3). H 2 S is produced enzymatically in mammalian tissues by three different enzymes: cystathionine γ-lyase (CSE), cystathionine beta-synthase (CBS), and 3-mercatopyruvate sulfurtransferase (3-MST). CSE, involved in the cysteine biosynthesis pathway, coordinates with L-cystine to produce H 2 S within the vasculature and is known to regulate blood pressure, modulate cellular metabolism, promote angiogenesis, regulate ion channels, and mitigate fibrosis and inflammation (4). Endothelial nitric oxide synthase (eNOS) catalyzes the production of nitric oxide (NO) from L-arginine within the endothelium to regulate vascular tone via cGMP signaling in vascular smooth muscle, mitochondrial respiration, platelet function, inflammation, and angiogenesis. The biological profiles of H 2 S and NO are similar, and both molecules are known to protect cells against various injurious states that result in organ injury. Although H 2 S and NO are thought to modulate independent signaling pathways, there is limited evidence of cross-talk between these two molecules (5, 6).H 2 S therapeutics and endogenous overexpression of CSE have been shown to attenuate ischemia/reperfusion (I/R) injury (7,8). Similarly, NO therapy and eNOS gene overexpression are also protective in ischemic disease states (9). Given the potent antioxidant actions of H 2 S (10, 11) and the effects of exogenous H 2 S therapy on NO bioavailability (5, 8), we investigated the effects of genetic deletion of the cystathionase gene (Cth, i.e., CSE KO) on the regulation of eNOS function and NO bioavailability. ResultsSulfide Levels are Reduced in CSE KO Mice. Whole blood and heart specimens were collected from WT and CSE KO mice to measure H 2 S levels using a high-sensitivity gas chromato...

Introduction Cystathionine gamma-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload-induced heart failure. Methods and Results Transverse aortic constriction (TAC) was performed in wild-type (WT), CSE knockout (KO), and cardiac specific CSE transgenic (CS-CSE Tg) mice. In addition, C57BL/6J or CSE KO mice received a novel–H2S donor (SG-1002). Mice were followed for 12 weeks using echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels following TAC. CSE KO mice exhibited cardiac dilatation and dysfunction significantly greater than WT mice following TAC and CS-CSE Tg mice maintained cardiac structure and function following TAC. H2S therapy with SG-1002 resulted in cardioprotection during TAC via upregulation of the VEGF-Akt-eNOS-nitric oxide-cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. Conclusions Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase (eNOS) and increased NO bioavailability.

Background Trimethylamine N-oxide (TMAO), a gut microbe dependent metabolite of dietary choline and other trimethylamine containing nutrients, is both elevated in the circulation of patients suffering from heart failure (HF) and heralds worse overall prognosis. In animal studies, dietary choline or TMAO significantly accelerate atherosclerotic lesion development in ApoE deficient mice, and reduction in TMAO levels inhibits atherosclerosis development in the LDL receptor knockout mouse. Methods and Results C57BL6/J mice were fed either a control diet, a diet containing choline (1.2%) or a diet containing TMAO (0.12%) starting 3 weeks prior to surgical TAC. Mice were studied for 12 weeks following TAC. Cardiac function and left ventricular structure were monitored at 3-week intervals using echocardiography. Twelve weeks post-TAC myocardial tissues were collected to evaluate cardiac and vascular fibrosis, and blood samples were evaluated for cardiac BNP, choline, and TMAO levels. Pulmonary edema, cardiac enlargement, and left ventricular ejection fraction (LVEF) were significantly (p < 0.05, each) worse in mice fed either TMAO or choline supplemented diets compared to the control diet. In addition, myocardial fibrosis was also significantly greater (p < 0.01, each) in the TMAO and choline groups relative to controls. Conclusions Heart failure severity is significantly enhanced in mice fed diets supplemented in either choline or the gut microbe-dependent metabolite TMAO. The present results suggest that further studies are warranted examining whether gut microbiota and the dietary choline -> TMAO pathway contribute to increased heart failure susceptibility.

Hydrogen sulfide (H2S), known as an important cellular signaling molecule, plays critical roles in many physiological and/or pathological processes. Modulation of H2S levels could have tremendous therapeutic value. However, the study on H2S has been hindered due to the lack of controllable H2S releasing agents which could mimic the slow and moderate H2S release in vivo. In this work we report the design, synthesis and biological evaluation of a new class of controllable H2S donors. Twenty five donors were prepared and tested. Their structures were based on a perthiol template, which was suggested to involve in H2S biosynthesis. H2S release mechanism from these donors was studied and proved to be thiol-dependent. We also developed a series of cell-based assays to access their H2S related activities. H9c2 cardiac myocytes were used in these experiments. We tested lead donors’ cytotoxicity and confirmed their H2S production in cells. Finally we demonstrated that selected donors showed potent protective effects in an in vivo murine model of myocardial ischemia-reperfusion injury, through a H2S related mechanism.

Condit ME, Lefer DJ. 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, 2012. First published March 30, 2012 doi:10.1152/ajpheart.00044.2012.-Diallyl trisulfide (DATS), a polysulfide constituent found in garlic oil, is capable of the release of hydrogen sulfide (H 2S). H2S is a known cardioprotective agent that protects the heart via antioxidant, antiapoptotic, anti-inflammatory, and mitochondrial actions. Here, we investigated DATS as a stable donor of H 2S during myocardial ischemiareperfusion (MI/R) injury in vivo. We investigated endogenous H 2S levels, infarct size, postischemic left ventricular function, mitochondrial respiration and coupling, endothelial nitric oxide (NO) synthase (eNOS) activation, and nuclear E2-related factor (Nrf2) translocation after DATS treatment. Mice were anesthetized and subjected to a surgical model of MI/R injury with and without DATS treatment (200 g/kg). Both circulating and myocardial H 2S levels were determined using chemiluminescent gas chromatography. Infarct size was measured after 45 min of ischemia and 24 h of reperfusion. Troponin I release was measured at 2, 4, and 24 h after reperfusion. Cardiac function was measured at baseline and 72 h after reperfusion by echocardiography. Cardiac mitochondria were isolated after MI/R, and mitochondrial respiration was investigated. NO metabolites, eNOS phosphorylation, and Nrf2 translocation were determined 30 min and 2 h after DATS administration. Myocardial H 2S levels markedly decreased after I/R injury but were rescued by DATS treatment (P Ͻ 0.05). DATS administration significantly reduced infarct size per area at risk and per left ventricular area compared with control (P Ͻ 0.001) as well as circulating troponin I levels at 4 and 24 h (P Ͻ 0.05). Myocardial contractile function was significantly better in DATS-treated hearts compared with vehicle treatment (P Ͻ 0.05) 72 h after reperfusion. DATS reduced mitochondrial respiration in a concentration-dependent manner and significantly improved mitochondrial coupling after reperfusion (P Ͻ 0.01). DATS activated eNOS (P Ͻ 0.05) and increased NO metabolites (P Ͻ 0.05). DATS did not appear to significantly induce the Nrf2 pathway. Taken together, these data suggest that DATS is a donor of H 2S that can be used as a cardioprotective agent to treat MI/R injury. cardioprotection; nitrite; left ventricular function; nitrosothiols; reperfusion injury; endothelial nitric oxide synthase