Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca2+]c) and sarcoplasmic reticulum (SR) luminal Ca2+ ([Ca2+]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca2+ leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca2+]SR in HF compared to control cells. Both SR Ca2+ leak and [Ca2+]SR were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca2+ leak and decreased [Ca2+]SR in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca2+-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca2+ and was partially normalized by reducing agents through restoring luminal Ca2+ sensitivity oxidation of control RyR2s enhanced their luminal Ca2+ sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.

Coronary vasodilation is impaired in the postischemic heart with a loss of endothelial nitric oxide synthase (eNOS) activity, but the mechanisms underlying ischemia-induced eNOS dysfunction are not understood. For nitric oxide (NO) synthesis, eNOS requires the redox-sensitive cofactor tetrahydrobiopterin (BH 4); however, the role of BH 4 in ischemia-induced endothelial dysfunction remains unknown. Therefore, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac BH 4 concentrations. Ischemia timedependently decreased cardiac BH 4 content with 85, 95, or 97% irreversible degradation after 30, 45, or 60 min of ischemia, respectively. Paralleling the decreases in BH 4, reductions of eNOS activity were seen of 58, 86, or 92%, and NOS-derived superoxide production was greatly increased. Addition of 10 M BH4 enhanced eNOS activity in nonischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. Impaired coronary flow during postischemic reperfusion was improved by BH 4 infusion. Thus, BH4 depletion contributes to postischemic eNOS dysfunction, and BH 4 treatment is effective in partial restoration of endothelium-dependent coronary flow. Supplementation of BH 4 may therefore be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues.ischemia reperfusion injury ͉ nitric oxide ͉ nitric oxide synthase uncoupling ͉ superoxide ͉ vascular function N itric oxide synthase (NOS) converts L-arginine and O 2 to nitric oxide (NO) and L-citrulline. This enzymatic process consumes NADPH and requires Ca 2ϩ /calmodulin, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH 4 ) as NOS cofactors. Endothelial NO synthase (eNOS) contributes to the regulation of vasomotor tone and blood pressure by producing NO that activates soluble guanylate cyclase in vascular smooth muscle, resulting in vasorelaxation (1-3).Endothelial dysfunction is a prognostic marker of cardiovascular disease (4). It has been suggested that limited availability of BH 4 contributes to eNOS dysfunction in hypercholesterolemia, diabetes, atherosclerosis, hypertension, and heart failure (5-9). It was also observed previously that eNOS function is impaired in ischemic hearts (10). In vivo coronary artery occlusion triggers endothelial dysfunction and decreased eNOSdependent vasoreactivity, although reactivity is preserved to exogenous NO (11,12). Endothelial-dependent coronary vasoreactivity is impaired in hearts subjected to periods of global ischemia and reperfusion (10). Endothelium-dependent vasodilators induce a relatively high increase in coronary flow in control hearts or in those made ischemic for short times, but longer periods of ischemia result in an abrupt decline in vasodilatory response.In addition to impairing eNOS-mediated NO formation, BH 4 depletion may have additional detrimental effects in postischem...

Rationale : Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "offtarget" effect of enhancing the enzymatic activity of ACE2 in vitro. Objectives: To evaluate the pharmacological actions of DIZE in experimental models of PH. Methods: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. Measurements and Main Results: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1a, which was corrected by in vitro DIZE treatment. Conclusions: Our results identify a therapeutic potential of DIZE in PH therapy.Keywords: pulmonary hypertension; ACE2; angiogenic progenitor cells; diminazene Pulmonary hypertension (PH) is a life-threatening disease characterized by elevated pressure in the pulmonary arteries and What This Study Adds to the FieldWe show that diminazene, an antitrypanosomal drug, attenuates hemodynamic changes, prevents maladaptive right ventricular remodeling, and enhances pulmonary vasorelaxation in experimental models of PH through activation of ACE2. Furthermore, diminazene improves the functions of APCs obtained from experimental animals and patients with PH. This study identifies a new application for an existing drug, which could be successfully developed for PH therapeutics.

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and N G -methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the K m for L-arginine is 3.14 M with a V max of 0.14 mol mg The biological significance of guanidino-methylated arginine derivatives has been known since the inhibitory actions of N G -methyl-L-arginine (L-NMMA) 3 on macrophage induced cytotoxicity were first demonstrated. It was subsequently realized that these effects were mediated through inhibition of NO release (1). NO has been demonstrated as a critical effector molecule in the maintenance of vascular function (2-4). In the vasculature, NO is derived from the oxidation of L-Arg, catalyzed by the constitutively expressed enzyme, eNOS (5-7). This endothelial-derived NO diffuses from the vascular endothelium into the smooth muscle cell layer where it activates soluble guanylate cyclase leading to smooth muscle relaxation (2-4). In addition to its role in the maintenance of vascular tone, NO helps to maintain the anti-atherogenic character of the normal vascular wall. NO, in concert with various cell signaling molecules, has been demonstrated to maintain smooth muscle cell quiescence and as such, counteracts pro-proliferative agents, specifically those involved in the propagation of athero-proliferative disorders (8 -14). As such, eNOS dysfunction is an early symptom of vascular disease and is manifested through insufficient NO bioavailability. Several potential causes of NO deficiency in disease settings have been proposed. Among these, high circulating levels of the endogenous methylarginine NOS inhibitor asymmetric dimethylarginine (ADMA) has been hypothesized to be of particular importance (15)(16)(17)(18)(19)(20)(21). In neurons and the brain, it has been shown that the methyl arginine L-NMMA is also present, however, the levels of this methylarginine have not been previously considered in studies evaluating vascular dysfunction (22).ADMA and L-NMMA are endogenous inhibitors of NOS and are derived from the proteolysis of methylated arginine residues on various proteins. The methylation is carried out by a group of enzymes referred to as protein-arginine methyltransferase (23). Subsequent proteolysis of proteins containing methylarginine groups leads to the release of free methylarginine into the cytoplasm, and if sufficient levels are reached NO production from NOS would be inhibited (24, 25). To date, six different isoforms of the enzyme have been identified with each

Objective-Fine particulate matter Ͻ2.5 m (PM 2.5 ) has been implicated in vasoconstriction and potentiation of hypertension in humans. We investigated the effects of short-term exposure to PM 2.5 in the angiotensin II (AII) infusion model. Methods and Results-Sprague-Dawley rats were exposed to PM 2.5 or filtered air (FA) for 10 weeks. At week 9, minipumps containing AII were implanted and the responses studied over a week. Mean concentration of PM 2.5 inside the chamber was 79.1Ϯ7.4 g/m 3 . After AII infusion, mean arterial pressure was significantly higher in PM 2.5 -AII versus FA-AII group. Aortic vasoconstriction to phenylephrine was potentiated with exaggerated relaxation to the Rho-kinase (ROCK) inhibitor Y-27632 and increase in ROCK-1 mRNA levels in the PM 2.5 -AII group. Superoxide (O 2 ⅐ Ϫ ) production in aorta was increased in the PM 2.5 -AII compared to the FA group, inhibitable by apocynin and L-NAME with coordinate upregulation of NAD(P)H oxidase subunits p22 phox and p47 phox and depletion of tetrahydrobiopterin. In vitro exposure to ultrafine particles (UFP) and PM 2.5 was associated with an increase in ROCK activity, phosphorylation of myosin light chain, and myosin phosphatase target subunit (MYPT1). Pretreatment with the nonspecific antioxidant N-Acetylcysteine and the Rho kinase inhibitors (Fasudil and Y-27632) prevented MLC and MYPT-1 phosphorylation by UFP suggesting a O 2 ⅐Ϫ -mediated mechanism for PM 2.5 and UFP effects. Conclusions-Short-term air pollution exaggerates hypertension through O 2⅐Ϫ -mediated upregulation of the Rho/ROCK pathway. Key Words: air pollution Ⅲ NADPH oxidase Ⅲ hypertension Ⅲ free radicals Ⅲ Rho/ROCK F ine particulate matter (aerodynamic diameter Ͻ2.5 m, PM 2.5 ) in ambient air has been implicated in the pathogenesis of cardiovascular disease. [1][2][3] Recent studies have suggested that this risk is rapid and occurs within hours to days of exposure to high levels of PM 2.5 . 4 -6 Increases in blood pressure may represent an important mechanism through which PM 2.5 may modulate its effects. Data from recent epidemiological studies from North America and Europe are indeed consistent with this hypothesis and have associated short-term exposure to PM 2.5 with elevations in blood pressure (BP). 7,8 This effect seems to be exaggerated in predisposed individuals, 9 an observation that has also been noted in relation to the association of PM 2.5 with other chronic conditions such as atherosclerosis. 3,6,10 Although the precise mechanisms through which PM 2.5 gains access to the systemic vasculature is still hotly debated, there is increasing evidence that particles in the fine and ultrafine range transgress into the systemic circulation and modulate vascular tone acutely, presumably through reactive oxygen species (ROS)-dependent pathways. 11,12 We hypothesized that short-term (weeks) increases in PM 2.5 levels is associated with an increases in BP and that these responses are exaggerated in a model of angiotensin II (AII)-dependent hypertension through upregulation ...

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Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.