The recent discovery that hydrogen sulfide (H2S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H2S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H2S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemiareperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H2S by cardiac-specific overexpression of cystathionine ␥-lyase (␣-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H2S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H2S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H2S), an endogenous cardioprotective vascular cell signaling molecule. This H2S production, measured in real time by a novel polarographic H2S sensor, is supported by glucosemaintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H2S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the ␣ carbon of the allyl substituent, thereby forming a hydropolysulfide (RSnH), a key intermediate during the formation of H2S. Organic polysulfides (R-Sn-R; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RSnH and H2S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H2S. The vasoactivity of garlic compounds is synchronous with H2S production, and their potency to mediate relaxation increases with H2S yield, strongly supporting our hypothesis that H2S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H2S can be used to standardize garlic dietary supplements.Allium ͉ aorta ͉ polysulfides ͉ red blood cells ͉ vasorelaxation

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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.

DW. Hydrogen sulfide mediates vasoactivity in an O 2-dependent manner. Am J Physiol Heart Circ Physiol 292: H1953-H1960, 2007. First published January 19, 2007 doi:10.1152 doi:10. /ajpheart.01193.2006 has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H 2S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H 2S include ATP-sensitive K ϩ channels, and H2S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O 2 dependent, but this has not been addressed in most studies designed to elucidate the role of H 2S in vascular function. This is important, since H2S reactions can be dramatically altered by the high concentrations of O 2 used in cell culture and organ bath experiments. To test the hypothesis that the effects of H 2S on the vasculature are O2 dependent, we have measured real-time levels of H 2S and O2 in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H2S sensor developed in our laboratory was used to measure H2S levels. Here we report that, in rat aorta, H2S concentrations that mediate rapid contraction at high O2 levels cause rapid relaxation at lower physiological O2 levels. At high O2, the vasoconstrictive effect of H2S suggests that it may not be H2S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H2S to modulate vascular tone in vivo.

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Gap junctional conductance (~) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with COs when extracellular calcium activity is near physiological levels (1.0 mM CaCI 2 added; 470 #M Ca + +). Intracellular calcium concentration (Ca~, measured by fura-2 fluorescence in cell suspensions, was 148 -+ 39 nM (• SEM, n = 6) and intracellular pH (pH), measured with intracellular ion-selective microelectrodes, was 7.05 -+ 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Ca i increased to 515 • 12 nM (n = 6) and pH i decreased to 5.9-6.0 in medium equilibrated with 100% CO s.In air-equilibrated low-calcium medium (no added CaCI~; 2-5 #M Ca+ § Ca i was 61 -+ 9 nM (n s 13) at pH i 7.1. Ca i increased to only 243 • 42 nM (n = 9) at pH i 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pH i 5.9-6.0 in lowcalcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 t~M octanol, an agent which does not significantly affect Ca~.In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO s increased Ca~ to 425 • 35 nM (n = 11) at pH~ 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce g] at pH i 7.0. These results suggest that, although other factors may play a role, H + and Ca ++ act synergistically to decrease g].

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