Adenosine kinase (ADK) inhibition with ABT-702 induces ADK protein degradation and a distinct form of sustained cardioprotection.

Pharmacological inhibition of adenosine kinase (ADK), the major route of myocardial adenosine metabolism, can elicit acute cardioprotection against ischemia-reperfusion (IR) by increasing adenosine signaling. Here, we identified a novel, extended effect of the ADK inhibitor, ABT-702, on cardiac ADK protein longevity and investigated its impact on sustained adenosinergic cardioprotection. We found that ABT-702 treatment significantly reduced cardiac ADK protein content in mice 24-72 h after administration (IP or oral). ABT-702 did not alter ADK mRNA levels, but strongly diminished (ADK-L) isoform protein content through a proteasome-dependent mechanism. Langendorff perfusion experiments revealed that hearts from ABT-702-treated mice maintain higher adenosine release long after ABT-702 tissue elimination, accompanied by increased basal coronary flow (CF) and robust tolerance to IR. Sustained cardioprotection by ABT-702 did not involve increased nitric oxide synthase expression, but was completely dependent upon increased adenosine release in the delayed phase (24 h), as indicated by the loss of cardioprotection and CF increase upon perfusion of adenosine deaminase or adenosine receptor antagonist, 8-phenyltheophylline. Importantly, blocking adenosine receptor activity with theophylline during ABT-702 administration prevented ADK degradation, preserved late cardiac ADK activity, diminished CF increase and abolished delayed cardioprotection, indicating that early adenosine receptor signaling induces late ADK degradation to elicit sustained adenosine release. Together, these results indicate that ABT-702 induces a distinct form of delayed cardioprotection mediated by adenosine receptor-dependent, proteasomal degradation of cardiac ADK and enhanced adenosine signaling in the late phase. These findings suggest ADK protein stability may be pharmacologically targeted to achieve sustained adenosinergic cardioprotection.

Author Correction: S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein ß.

Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein ß (C/EBPß), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPß variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.

Effects of flavoring compounds used in electronic cigarette refill liquids on endothelial and vascular function.

Electronic cigarette refill liquids are commercially provided with a wide variety of flavoring agents. A recent study suggested that several common flavors may scavenge nitric oxide (NO) and cause endothelial dysfunction. It was the aim of the present study to investigate the effects of these flavors on NO/cyclic GMP-mediated signaling and vascular relaxation. We tested the flavoring agents for effects on Ca2+-induced cGMP accumulation and NO synthase activation in cultured endothelial cells. NO scavenging was studied with NO-activated soluble guanylate cyclase and as NO release from a NO donor, measured with a NO electrode. Blood vessel function was studied with precontracted rat aortic rings in the absence and presence of acetylcholine or a NO donor. Cinnamaldehyde inhibited Ca2+-stimulated endothelial cGMP accumulation and NO synthase activation at ≥0.3 mM. Cinnamaldehyde and diacetyl inhibited NO-activated soluble guanylate cyclase with IC50 values of 0.56 (0.54-0.58) and 0.29 (0.24-0.36) mM, respectively, and caused moderate NO scavenging at 1 mM that was not mediated by superoxide anions. The other compounds did not scavenge NO at 1 mM. None of the flavorings interfered with acetylcholine-induced vascular relaxation, but they caused relaxation of pre-contracted aortas. The most potent compounds were eugenol and cinnamaldehyde with EC50 values of ~0.5 mM. Since the flavors did not affect endothelium-dependent vascular relaxation, NO scavenging by cinnamaldehyde and diacetyl does not result in impaired blood vessel function. Although not studied in vivo, the low potency of the compounds renders it unlikely that the observed effects are relevant to humans inhaling flavored vapor from electronic cigarettes.

Role of the ubiquitin-proteasome system in cardiac dysfunction of adipose triglyceride lipase-deficient mice.

Systemic deletion of the gene encoding for adipose triglyceride lipase (ATGL) in mice leads to severe cardiac dysfunction due to massive accumulation of neutral lipids in cardiomyocytes. Recently, impaired peroxisome proliferator-activated receptor α (PPARα) signaling has been described to substantially contribute to the observed cardiac phenotype. Disturbances of the ubiquitin-proteasome system (UPS) have been implicated in numerous cardiac diseases including cardiomyopathy, ischemic heart disease, and heart failure. The objective of the present study was to investigate the potential role of UPS in cardiac ATGL deficiency. Our results demonstrate prominent accumulation of ubiquitinated proteins in hearts of ATGL-deficient mice, an effect that was abolished upon cardiomyocyte-directed overexpression of ATGL. In parallel, cardiac protein expression of the ubiquitin-activating enzyme E1a, which catalyzes the first step of the ubiquitination cascade, was significantly upregulated in ATGL-deficient hearts. Dysfunction of the UPS was accompanied by activation of NF-κB signaling. Moreover, the endoplasmic reticulum (ER)-resident chaperon protein disulfide isomerase was significantly upregulated in ATGL-deficient hearts. Chronic treatment of ATGL-deficient mice with the PPARα agonist Wy14,643 improved proteasomal function, prevented NF-κB activation and decreased oxidative stress. In summary, our data point to a hitherto unrecognized link between proteasomal function, PPARα signaling and cardiovascular disease.

Endothelial dysfunction in adipose triglyceride lipase deficiency.

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4-5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~2.5-fold upregulation of soluble guanylate cyclase activity and a ~2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

Efficient nitrosation of glutathione by nitric oxide.

Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg(2+). These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin.

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH(4)) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH(4) was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N(G)-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH(4)/BH(2) levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH(4) oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH(4) depletion and without observable consequence on eNOS function.

Mechanisms underlying activation of soluble guanylate cyclase by the nitroxyl donor Angeli's salt.

Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMP-dependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC(50) approximately 10 nM, maximum at 1 microM) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of <10 microM AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC(50) approximately 50 nM and approximately 16 microM, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of

Inactivation of soluble guanylate cyclase by stoichiometric S-nitrosation.

Dysfunction of vascular nitric oxide (NO)/cGMP signaling is believed to contribute essentially to various cardiovascular disorders. Besides synthesis and/or bioavailability of endothelial NO, impaired function of soluble guanylate cyclase (sGC) may play a key role in vascular dysfunction. Based on the proposal that desensitization of sGC through S-nitrosation contributes to vascular NO resistance ( Proc Natl Acad Sci U S A 104: 12312-12317, 2007 ), we exposed purified sGC to dinitrosyl iron complexes (DNICs), known as potent nitrosating agents. In the presence of 2 mM GSH, DNICs stimulated cGMP formation with EC(50) values of 0.1 to 0.5 microM and with an efficacy of 70 to 80% of maximal activity measured with 10 microM 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO). In the absence of GSH, the efficacy of DNICs was markedly reduced, and sGC stimulation was counteracted by the inhibition of both basal and DEA/NO-stimulated cGMP formation at higher DNIC concentrations. Inactivation of sGC was slowly reversed in the presence of 2 mM GSH and associated with stoichiometric S-nitrosation of the protein (2.05 +/- 0.18 mol S-nitrosothiol per mol of 143-kDa heterodimer). S-Nitrosoglutathione and sodium nitroprusside caused partial inhibition of DEA/NO-stimulated sGC that was prevented by GSH, whereas nitroglycerin (0.3 mM) had no effect. Our findings indicate that nitrosation of two cysteine residues in sGC heterodimers results in enzyme inactivation. Protection by physiologically relevant concentrations of GSH (10 microM to 3 mM) suggests that S-nitrosation of sGC may contribute to vascular dysfunction in inflammatory disorders associated with nitrosative and oxidative stress and GSH depletion.

Vascular tolerance to nitroglycerin in ascorbate deficiency.

AIMS: Nitroglycerin (GTN) acts through release of a nitric oxide (NO)-related activator of soluble guanylate cyclase in vascular smooth muscle. Besides enzymatic GTN bioactivation catalysed by aldehyde dehydrogenase, non-enzymatic reaction of GTN with ascorbate also results in the formation of a bioactive product. Using an established guinea pig model of ascorbate deficiency, we investigated whether endogenous ascorbate contributes to GTN-induced vasodilation. METHODS AND RESULTS: Guinea pigs were fed either standard or ascorbate-free diet for 2 or 4 weeks prior to measuring the GTN response of aortic rings and isolated hearts. The effects of ascorbate on GTN metabolism were studied with purified mitochondrial aldehyde dehydrogenase (ALDH2) and isolated mitochondria. Ascorbate deprivation led to severe scorbutic symptoms and loss of body weight, but had no (2 weeks) or only slight (4 weeks) effects on aortic relaxations to a direct NO donor. The EC(50) of GTN was increased from 0.058 +/- 0.018 to 0.46 +/- 0.066 and 5.5 +/- 0.9 microM after 2 and 4 weeks of ascorbate-free diet, respectively. Similarly, coronary vasodilation to GTN was severely impaired in ascorbate deficiency. The potency of GTN was reduced to a similar extent by ALDH inhibitors in control and ascorbate-deficient blood vessels. Up to 10 mM ascorbate had no effect on GTN metabolism catalysed by purified ALDH2 or liver mitochondria isolated from ascorbate-deficient guinea pigs. CONCLUSION: Our results indicate that prolonged ascorbate deficiency causes tolerance to GTN without affecting NO/cyclic GMP-mediated vasorelaxation.

Role of myocardial nitric oxide in diabetic ischemia-reperfusion dysfunction: studies in mice with myocyte-specific overexpression of endothelial nitric-oxide synthase.

We investigated the role of nitric oxide (NO) in myocardial ischemia-reperfusion injury of diabetic mice with myocyte-specific overexpression of endothelial NO synthase (NOS). Four weeks after diabetes induction with streptozotocin (blood glucose approximately 29 mM), isolated isovolumic heart function and cellular NO metabolites in response to brief normothermic ischemia-reperfusion were determined. Under normoxic conditions transgenic (TG) hearts from nondiabetic and diabetic animals generated less left-ventricular developed pressure compared with wild-type (WT) control hearts, and this abnormality was unaffected by NOS inhibition. During ischemia, the rise in end-diastolic pressure was less in the TG than WT group of nondiabetic hearts, whereas the transgene had no effect in the diabetic group. Similarly, the transgene also improved reperfusion systolic and diastolic function in nondiabetic but not in diabetic hearts. NOS inhibition worsened reperfusion function in diabetic hearts. Postischemic nitrite and cGMP formation were higher in nondiabetic TG than WT hearts, but in diabetic hearts cGMP was no longer elevated. The formation of reactive oxygen species (superoxide and peroxynitrite) during early reperfusion, measured by electron spin resonance spectroscopy, was similar in nondiabetic WT and TG hearts, but it was significantly higher in diabetic TG hearts. Stimulating endogenous NO production with 10 microM bradykinin more strongly reduced myocardial O(2) consumption in diabetic TG than diabetic WT hearts perfused in normoxia, whereas there was no difference after ischemia-reperfusion. Thus, providing additional endogenous NO is sufficient to protect nondiabetic hearts against ischemia-induced injury, but for a similar protection in diabetic hearts, effective scavenging of reactive oxygen species is also important.

Cardioprotective effects of atrasentan, an endothelin-A receptor antagonist, but not of nitric oxide in diabetic mice with myocyte-specific overexpression of endothelial nitric oxide synthase.

1. We investigated the roles of nitric oxide (NO) and endothelin-1 (ET-1) in organ dysfunction in diabetic mice with normal genotype (wild-type, WT) or myocyte-specific overexpression of endothelial NO synthase (eNOS) (transgenic, TG) after chronic oral treatment with the endothelin-A (ETA) receptor antagonist atrasentan. 2. Mice were rendered diabetic by injection of 200 mg kg-1 streptozotocin (STZ). Experimental groups were: untreated WT diabetic (n=9), untreated TG diabetic (n=9), atrasentan-treated WT diabetic (n=9), atrasentan-treated TG diabetic (n=8) and the four corresponding nondiabetic groups (n=5). Atrasentan was administered orally via drinking water at 3 mg kg-1 per day over 28 days. All diabetic mice developed similar hyperglycaemia (27-30 mmol l-1). 3. Atrasentan treatment significantly improved left ventricular systolic and diastolic function in response to exogenous norepinephrine, but there were no differences between genotypes. 4. Atrasentan antagonized the diabetic impairments in endothelium-dependent coronary relaxation and thromboxane-receptor mediated aortic constriction. Further, it improved cardiac and renal oxidant status as evident from reduced tissue malondialdehyde levels. 5. Atrasentan reduced diabetic urine flow, proteinuria and plasma creatinine levels, but creatinine clearance was not significantly altered. 6. These results suggest that in experimental type 1 diabetes, blocking ETA receptors ameliorates myocardial, coronary and renal function and improves tissue oxidant status, whereas raising myocardial NO levels has neither beneficial nor deleterious effects on diabetic cardiomyopathy in this transgenic model.

In vivo administration of D-arginine: effects on blood pressure and vascular function in angiotensin II-induced hypertensive rats.

OBJECTIVE: We tested the hypothesis that D-arginine (D-Arg), which is not a substrate for nitric oxide synthase but scavenges reactive oxygen in vitro, is protective in vivo. METHODS: Rats were made hypertensive by administering angiotensin II (Ang II) (0.7mg kg(-1) per day) for 7 days (Ang II group). Two other groups additionally received either 3 mmol D-Arg (Ang II + D-Arg group) or vitamin C (1g) (Ang II + Vit C group) per day. Sham-operated animals served as controls (n = 6-9). Systolic blood pressure was monitored daily and cardiovascular function determined ex vivo at 7 days. RESULTS: Ang II raised systolic blood pressure to 184mmHg, the increase was slightly attenuated by D-Arg treatment (-17mmHg; P < 0.05 versus Ang II alone) and prevented by Vit C. Acetylcholine-induced coronary relaxation was impaired in the Ang II group (P < 0.05 versus sham), the impairment was no different in the Ang II + D-Arg group, but prevented by Vit C. Likewise, Vit C but not D-Arg ameliorated reperfusion endothelium-dependent relaxation. However, in aortic rings D-Arg slightly improved acetylcholine relaxation (P < 0.05). Oxidative stress load estimated in plasma with thiobarbituric acid reactive substance was higher in the Ang II than the sham group, Vit C abolished the increase, but D-Arg was without effect. CONCLUSION: D-Arg is weakly antihypertensive in vivo and ameliorates aortic, but not coronary endothelium-dependent relaxation ex vivo. Because D-Arg had no effect on plasma oxidant status, this protection appears to be independent of reactive oxygen scavenging activity.

Effect of endothelin antagonism on contractility, intracellular calcium regulation and calcium regulatory protein expression in right ventricular hypertrophy of the rat.

We have documented the effects of long-term endothelin receptor antagonism on intracellular Ca2+ regulation and Ca2+ regulatory protein expression in rat hearts with right ventricular hypertrophy without signs of heart failure. Rats were given either a single injection of monocrotaline (50 mg/kg, n=9) resulting in pulmonary hypertension-induced myocardial hypertrophy, or monocrotaline followed by daily administration of the endothelin subtype-A receptor antagonist 2-benzo(1,3)dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl-)-4-oxobut-2-enoate-Na (PD 155080, 50 mg/kg) over 9 weeks (n=8). Hearts from saline-injected rats served as controls (n=9). Monocrotaline-treated animals developed marked right-sided hypertrophy without fibrosis as evident from hydroxyproline measurements, systolic contractility was increased, fully compensating for the increased afterload, but diastolic function was impaired as evident from protracted relaxation and slowed diastolic intracellular Ca2+ handling (measured by aequorin bioluminescence). In hypertrophic hearts, quantitative immunoblotting analyses showed increased levels both of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phosphorylated phospholamban, along with decreased levels of total phospholamban, which is in line with strengthened right ventricular systolic function. PD 155080 reversed abnormalities in Ca2+ handling, although SERCA and phospholamban protein levels were not altered (P=not significant versus monocrotaline group). Thus, endothelin-A receptor antagonism attenuates right ventricular remodeling and improves myocardial Ca2+ handling, but has no discernable effect on elevated expression of SERCA and phospholamban observed in hypertrophic hearts. These data indicate that the hypotensive action of PD 155080 is independent of its effects, if any, on SERCA and its regulation.