The dose makes the poison: Hydrogen sulfide is an important gasotransmitter for which rapid detection agents are needed. A hydrogen sulfide probe, which allows for fast (within seconds), selective, and quantitative detection in buffer solution, serum, and whole blood is designed, synthesized, and used for detection of hydrogen sulfide (see picture).

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.

Fatty liver disease associated with chronic alcohol consumption or obesity/type 2 diabetes has emerged as a serious public health problem. Steatosis, accumulation of triglyceride in hepatocytes, is now recognized as a critical "first-hit" in the pathogenesis of liver disease. It is proposed that steatosis "primes" the liver to progress to more severe liver pathologies when individuals are exposed to subsequent metabolic and/or environmental stressors or "second-hits". Genetic risk factors can also influence the susceptibility and severity of fatty liver disease. Furthermore, oxidative stress, disrupted nitric oxide (NO) signaling, and mitochondrial dysfunctional are proposed to be key molecular events that accelerate or worsen steatosis and initiate progression to steatohepatitis and fibrosis. This review article will discuss the following topics regarding the pathobiology and molecular mechanisms responsible for fatty liver disease: 1) the "two-hit" or "multi-hit" hypothesis; 2) the role of mitochondrial bioenergetic defects and oxidant stress; 3) interplay between NO and mitochondria in fatty liver disease; 4) genetic risk factors and oxidative stress responsive genes; and 5) the feasibility of antioxidants for treatment.

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

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

Introduction. Mitochondrial damage and disruption in oxidative phosphorylation contributes to the pathogenesis of alcoholic liver injury. Herein, we tested the hypothesis that the hepatoprotective actions of betaine against alcoholic liver injury occur at the level of the mitochondrial proteome. Methods. Male Wister rats were pair-fed control or ethanol-containing liquid diets supplemented with or without betaine (10 mg/mL) for 4-5 wks. Liver was examined for triglyceride accumulation, levels of methionine cycle metabolites, and alterations in mitochondrial proteins. Results. Chronic ethanol ingestion resulted in triglyceride accumulation which was attenuated in the ethanol plus betaine group. Blue native gel electrophoresis (BN-PAGE) revealed significant decreases in the content of the intact oxidative phosphorylation complexes in mitochondria from ethanol-fed animals. The alcohol-dependent loss in many of the low molecular weight oxidative phosphorylation proteins was prevented by betaine supplementation. This protection by betaine was associated with normalization of SAM : S-adenosylhomocysteine (SAH) ratios and the attenuation of the ethanol-induced increase in inducible nitric oxide synthase and nitric oxide generation in the liver. Discussion/Conclusion. In summary, betaine attenuates alcoholic steatosis and alterations to the oxidative phosphorylation system. Therefore, preservation of mitochondrial function may be another key molecular mechanism responsible for betaine hepatoprotection.

Obesity-related pathologies, such as nonalcoholic fatty liver disease, are linked to mitochondrial dysfunction and nitric oxide (NO) deficiency. Herein, we tested the hypothesis that a high-fat diet (HFD) modifies the liver mitochondrial proteome and alters proteins involved in NO metabolism, namely arginase 1 and endothelial NO synthase. Male C57BL/6 mice were fed a control or HFD and liver mitochondria were isolated for proteomics and reactive oxygen species measurements. Steatosis and hepatocyte ballooning were present in livers of HFD mice, with no pathology observed in the controls. HFD mice had increased serum glucose and decreased adiponectin. Mitochondrial reactive oxygen species was increased after 8 weeks in the HFD mice, but decreased at 16 weeks compared with the control, which was accompanied by increased uncoupling protein 2. Using proteomics, 22 proteins were altered as a consequence of the HFD. This cohort consists of oxidative phosphorylation, lipid metabolism, sulfur amino acid metabolism, and chaperone proteins. We observed a HFD-dependent increase in arginase 1 and decrease in activated endothelial NO synthase. Serum and liver nitrate + nitrite were decreased by HFD. In summary, these data demonstrate that a HFD causes steatosis, alters NO metabolism, and modifies the liver mitochondrial proteome; thus, NO may play an important role in the processes responsible for nonalcoholic fatty liver disease.