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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.

T he concept that oxidative stress-a pathologically high level of oxidant species in cells-may drive cardiovascular disease progression has led to many clinical trials of interventions, such as antioxidant vitamins. These, however, failed to show efficacy in reducing disease risk and progression. Part of the reason may be the focus on oxidative stress as detrimental neglected the wider role of redox balance and reactive oxygen species (ROS) and reactive nitrogen species in cellular (patho)physiology. Redox signaling-defined as the specific, usually reversible, oxidation/reduction modification of cellular signaling pathway components by a reactive species 1 -is increasingly appreciated as centrally important in many physiological and pathological processes. The main ROS involved in redox signaling are the superoxide anion (O 2 − ) and the more stable nonradical hydrogen peroxide (H 2 O 2 ) to which it dismutates, whereas more powerful oxidants such as hydroxyl are so reactive they are unlikely to be specific or reversible. Redox signaling also involves reactive nitrogen species such as NO and peroxynitrite, the latter being formed from the reaction of O 2 − with NO. 2In the heart, redox signaling is involved in physiological processes (eg, excitation-contraction coupling [ECC], cell differentiation), homoeostatic and stress response pathways (eg, adaptation to hypoxia/ischemia), and pathology (eg, adverse cardiac remodeling, fibrosis). This review covers recent advances in understanding the regulation of production of signaling ROS, their mechanisms of action and molecular targets in cardiac cells, and their involvement in cardiac physiopathology. We focus mainly on cardiomyocytes but redox signaling in other cells (eg, fibroblasts, endothelial cells), and functional cross talks among these are also important. Reactive nitrogen species-dependent regulation has been reviewed elsewhere. 2 ROS SourcesROS are generated as a by-product of cellular respiration and metabolism or by specialized enzymes that seem to be centrally involved in redox signaling. The signaling effects of ROS are influenced by their site of production, precise species, local concentration, and cell compartment-specific antioxidant pools. Major ROS sources in the heart and other tissues include the mitochondrial electron transport chain (ETC), other mitochondrial and metabolic enzymes, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs), and uncoupled NO synthases (NOS). Mitochondrial ETCElectron leakage from the ETC causes 1-electron reduction of O 2 to O 2 − (instead of reduction to H 2 O). Although considered to be because of an electron leak, such ROS may nevertheless contribute to homeostatic redox signals. ROS levels increase significantly during mitochondrial dysfunction. They can trigger the mitochondrial permeability transition (MPT) and lead to further ROS release-termed ROS-induced ROS release 3 -which propagates and amplifies ROS production and effects. ROS from non-ETC sources (eg, Noxs) may also stimulate ETC-depende...

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for ␣-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of ␤-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.There is now substantial evidence that oxidant species such as H 2 O 2 are produced in a regulated way in cells where they can function as signaling agents (1, 2). We have been studying the post-translational modification of protein cysteinyl thiols, as this is a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. Our strategy has been to search for proteins that are susceptible to a variety of different modes of cysteine oxidation, such as S-thiolation (3, 4), sulfenation (5), and protein-protein disulfide bond formation (6). The rationale is that once we identify proteins with reactive thiols, the possibility that their oxidation has a functional correlate of physiological significance can be investigated. We previously found the RI regulatory subunits of protein kinase A (PKA) 2 form interprotein disulfide dimers during cardiac oxidative stress (6).Here we investigated the potential impact of this disulfide dimer formation on the function of PKA. PKA has two major forms (type I and type II), both of which exist as a tetramer comprising two catalytic and two regulatory subunits. There are two types of regulatory subunits (RI and RII), the presence of which in the PKA holokinase nominally defines the enzyme as type I or II, respectively. Recent studies have shown that the full dissociation of type I PKA in response to cAMP requires the presence of a substrate (7). This substrateinduced sensitization of type I PKA is not a feature of the type II enzyme (8). The regulatory subunits contain N-terminal sequences that are important for protein kinase A anchor protein (AKAP) binding. AKAPs are a diverse group of proteins that are found next to PKA substrate proteins and, thus, function to target PKA (9). Type I PKA is located in the cytosol, whereas type II is not as a result of being primarily bound (targeted) to AKAP proteins that are associated ...

Significance: Oxidants were once principally considered perpetrators of injury and disease. However, this has become an antiquated view, with cumulative evidence showing that the oxidant hydrogen peroxide serves as a signaling molecule. Hydrogen peroxide carries vital information about the redox state of the cell and is crucial for homeostatic regulation during health and adaptation to stress. Recent Advances: In this review, we examine the contemporary concepts for how hydrogen peroxide is sensed and transduced into a biological response by introducing post-translational oxidative modifications on select proteins. Oxidant sensing and signaling by kinases are of particular importance as they integrate oxidant signals into phospho-regulated pathways. We focus on CAMKII, PKA, and PKG, kinases whose redox regulation has notable impact on cardiovascular function. Critical Issues: In addition, we examine the mechanism for regulating intracellular hydrogen peroxide, considering the net concentrations that may accumulate. The effects of endogenously generated oxidants are often modeled by applying exogenous hydrogen peroxide to cells or tissues. Here we consider whether model systems exposed to exogenous hydrogen peroxide have relevance to systems where the oxidant is generated endogenously, and if so, what concentration can be justified in terms of relevance to health and disease. Future Directions: Improving our understanding of hydrogen peroxide signaling and the sensor proteins that it can modify will help us develop new strategies to regulate intracellular signaling to prevent disease.

Macroautophagy (autophagy) is a crucial cellular stress response for degrading defective macromolecules and organelles, as well as providing bioenergetic intermediates during hypoxia and nutrient deprivation. Here we report a thiol-dependent process that may account for impaired autophagy during aging. This is through direct oxidation of key autophagy-related (Atg) proteins Atg3 and Atg7. When inactive Atg3 and Atg7 are protected from oxidation due to stable covalent interaction with their substrate LC3. This interaction becomes transient upon activation of Atg3 and Atg7 due to transfer of LC3 to phosphatidylethanolamine (lipidation), a process crucial for functional autophagy. However, loss in covalent-bound LC3 also sensitizes the catalytic thiols of Atg3 and Atg7 to inhibitory oxidation that prevents LC3 lipidation, observed in vitro and in mouse aorta. Here findings provide a thiol-dependent process for negatively regulating autophagy that may contribute to the process of aging, as well as therapeutic targets to regulate autophagosome maturation.

Abstract-Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H 2 S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood. H 2 S-dependent vasodilation and blood pressure lowering in vivo was mediated by it catalyzing formation of an activating interprotein disulfide within protein kinase G (PKG) Iα. However, this oxidative activation of PKG Iα is counterintuitive because H 2 S is a thiol-reducing molecule that breaks disulfides, and so it is not generally anticipated to induce their formation. This apparent paradox was explained by H 2 S in the presence of molecular oxygen or hydrogen peroxide rapidly converting to polysulfides, which have oxidant properties that in turn activate PKG by inducing the disulfide. These observations are relevant in vivo because transgenic knockin mice in which the cysteine 42 redox sensor within PKG has been systemically replaced with a redox-dead serine residue are resistant to H 2 S-induced blood pressure lowering. Thus, a primary mechanism by which the reductant molecule H 2 S lowers blood pressure is mediated somewhat paradoxically by the oxidative activation of PKG. (Hypertension. 2014;64:1344-1351.)

A complete elucidation of blood pressure homeostasis is important because its dysregulation commonly results in hypertension, increasing the risk of kidney injury, myocardial infarction, heart failure, and stroke. Three principal pathways control vasodilation and blood pressure lowering, including nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). EDHF is largely absent in conduit vessels, but in resistance vessels, which are the principal regulators of blood pressure, it is a prevalent and perhaps the predominant mechanism controlling vasodilation. [1][2][3][4] NO formation is stimulated by shear stress and circulating factors such as bradykinin, acetylcholine, and adenosine. The ability of NO to stimulate vessel relaxation is extensively characterized and involves its interaction with the heme center of guanylate cyclase, stimulating the catalytic ability of the enzyme to convert guanosine-5´-triphosphate to the second messenger cGMP. cGMP transduces many of the biological effects of NO by directly binding to and stimulating the activity of cGMP-dependent protein kinase, also known as protein kinase G (PKG). PKG activation induces substrate phosphorylation in vascular smooth muscle cells, resulting in blood vessel vasodilation by decreasing intracellular Ca 2+ and myofilament Ca 2+ sensitivity, thereby attenuating myosin actin crossbridge cycling.In addition to the classic NO-cGMP pathway, PKG can also be activated by an oxidation mechanism during which the homodimer complex forms an interprotein disulfide. 5 The disulfide forms in the N-terminus of PKG1α, which is held together by a leucine zipper, with structural studies confirming that Cys42 on each chain closely aligns to explain the susceptibility to oxidation. Oxidation to the disulfide state is sufficient in itself to enable PKG catalytic activity. Classic activation increases PKG V max , whereas disulfide activation increases the kinase affinity for substrate. H 2 O 2 or related oxidants contribute to EDHF-dependent vasodilation of resistance vessel. [6][7][8][9][10][11] This is at least, in part, attributed to EDHF-induced oxidation of PKG1α. PKG oxidation contributes to basal blood pressure as transgenic "redox-dead" Cys42Ser PKG1α knock-in mice Abstract-Protein kinase G (PKG) is activated by nitric oxide (NO)-induced cGMP binding or alternatively by oxidantinduced interprotein disulfide formation. We found preactivation with cGMP attenuated PKG oxidation. 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blockade of cGMP production increased disulfide PKG to 13±2% and 29±4% of total in aorta and mesenteries, respectively. This was potentially anomalous, because we observed 2.7-fold higher NO levels in aorta than mesenteries; consequently, we had anticipated that ODQ would induce more disulfide in the conduit vessel. ODQ also constricted aorta, whereas it had no effect on mesenteries. Thus, mesenteries, but not aorta, can compensate for loss of NO-cGMP by recruiting disulfide activation of PKG. Mechanistically, t...