Changes in the concentration of oxidants in cells can regulate biochemical signaling mechanisms that control cell function. We have found that guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) functions directly as a redox sensor. The Ialpha isoform, PKGIalpha, formed an interprotein disulfide linking its two subunits in cells exposed to exogenous hydrogen peroxide. This oxidation directly activated the kinase in vitro, and in rat cells and tissues. The affinity of the kinase for substrates it phosphorylates was enhanced by disulfide formation. This oxidation-induced activation represents an alternate mechanism for regulation along with the classical activation involving nitric oxide and cGMP. This mechanism underlies cGMP-independent vasorelaxation in response to oxidants in the cardiovascular system and provides a molecular explantion for how hydrogen peroxide can operate as an endothelium-derived hyperpolarizing factor.

Objective-Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Key Words: blood pressure Ⅲ endothelial function Ⅲ reactive oxygen species Ⅲ vasodilation Ⅲ NADPH oxidase E ndothelial dysfunction, in particular the impairment of endothelium-dependent vasodilatation, is involved in the pathophysiology of hypertension and atherosclerosis. 1 Increased reactive oxygen species (ROS) production contributes to endothelial dysfunction through the inactivation of endothelium-derived nitric oxide (NO) by superoxide and by ROS-dependent modulation of intracellular signaling pathways.

Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes' epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redoxdead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO 2 -OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO 2 -OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO 2 -OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.thiol | cardiovascular

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mM, 30 min) into rat ventricular myocytes, treated them with H 2 O 2 (0.1-10,000 M, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mM H 2 O 2 and declined sharply below basal with 10 mM treatment. Cellwide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H 2 O 2 -induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

Rationale 15-deoxy-Δ-prostaglandin J2 (15d-PGJ2) is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative post-translational modifications that modulate protein and tissue function. Objective To investigate the role of oxidative protein modifications in 15d-PGJ2-mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. Methods and Results Proteomic screening with biotinylated 15d-PGJ2 identified novel vascular targets to which it adducts, most notably soluble Epoxide Hydrolase (sEH). 15d-PGJ2 inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic centre of the hydrolase. Indeed a Cys521Ser sEH ‘redox-dead’ mutant was resistant to 15d-PGJ2-induced hydrolase inhibition.15d-PGJ2 dilated coronary vessels and a role for hydrolase inhibition was supported by two structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ2 and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids (EETs) consistent with their vasodilatory actions. Indeed 14,15 EET alone induced relaxation and 15d-PGJ2-mediated vasodilation was blocked by the EET receptor antagonist 14,15-EEZE. Additionally the coronary vasculature of sEH null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ2. Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ2 adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition sEH null mice failed to vasodilate during hypoxia. Conclusion This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

Rationale: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H 2 O 2 ) ([H 2 O 2 ] mito ). Decreases in [H 2 O 2 ] mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca 2+ ] i ) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O 2 sensor is unknown. Objective: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I’s rotenone binding site, in pulmonary vascular oxygen-sensing. Methods and Results: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H 2 O 2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H 2 O 2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca 2+ ] i . Hypoxia had no effects on [Ca 2+ ] i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H 2 O 2 in PASMC while increasing cytosolic H 2 O 2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H 2 O 2 , prevented hypoxic increases in [Ca 2+ ] i , and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD + ) ratio and decreasing expression of the O 2 -sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III’s Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca 2+ ] i . In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. Conclusions: Ndufs2 is essential for oxygen-sensing and HPV.

The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na+/K+-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 μM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na+/K+-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na+/K+-ATPase activation during reperfusion would attenuate Na+ overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na+/K+-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

Oxidative stress has almost universally and unequivocally been implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress in cells and cardiovascular biology was once considered only in terms of injury, disease and dysfunction. However, it is now appreciated that oxidants are also produced in healthy tissues, and they function as signalling molecules transmitting information throughout the cell. Conversely, when cells move to a more reduced state, as can occur when oxygen is limiting, this can also result in alterations in the function of biomolecules and subsequently cells. At the centre of this 'redox signalling' are oxidoreductive chemical reactions involving oxidants or reductants post translationally modifying proteins. These structural alterations allow changes in cellular redox state to be coupled to alterations in cell function. In this review, we consider aspects of redox signalling in the cardiovascular system, focusing on the molecular basis of redox sensing by proteins and the array of post-translational oxidative modifications that can occur. In addition, we discuss studies utilising proteomic methods to identify redox-sensitive cardiac proteins, as well as those using this technology more broadly to assess redox signalling in cardiovascular disease.