Cardioprotective Effects of S-Nitrosothiols in Ischemia- Reperfusion: Role for Mitochondria and Calcium Channels

The most important clinical consequence of coronary disease is acute myocardial infarction caused by an occlusion that limits the irrigation to the heart. Although the gold standard treatment is to restore blood flow, this reperfusion causes inherent damage by increasing the size of the infarcted area primarily through the opening of the mitochondrial permeability transition pore (MPTP). The cardioprotective effect of nitric oxide (NO) has been described to operate through S-nitrosylation of several important proteins in the cardiomyocytes such as the calcium channels RyR2 and the L-type Ca2+ channel and mitochondrial proteins, including the MPTP. In this sense, an attractive strategy to prevent the ischemia-reperfusion damage is to increase the bioavailability of endogenous S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of NO through denitrosylation, which would limit the cardioprotective effect of NO. Although inhibition of GSNOR has been studied in different organs, its effects on myocardial reperfusion have not yet been fully elucidated. In this chapter, we review the pathophysiology underlying myocardial reperfusion injury and the opening of the MPTP along with the cardioprotective role of S-nitrosothiols and the potential role for GSNOR

Nitroso-Redox Crosstalk in Diabetic Cardiomyopathy

Diabetes mellitus is one of the most common chronic diseases worldwide. Diabetic cardiomyopathy (DM) is the deterioration of the myocardial function and morphology produced by the altered glucose metabolism imposed in diabetes. This process of cardiac deterioration involves the generation of oxidative species. In the diabetic heart, several sources contribute to the observed oxidative stress, such as xanthine oxidoreductase (XOR), nicotinamide adenine dinucleotide phosphate (NADPH), nitrogen oxidases (NOX), mitochondria, and uncoupled nitric oxide synthases (NOS). A direct consequence of the increased production of reactive oxygen species (ROS) is NOS uncoupling. This is the aftermath of the oxidation of tetrahydrobioterin (BH4), an essential cofactor for NOS activity. When NOS is uncoupled, its activity is redirected toward the production of superoxide, instead of nitric oxide (NO), further contributing to the oxidative process. This nitroso-redox disarrangement has a direct impact on the excitation-contraction-coupling machinery of the myocyte, in the mitochondrial stability impairing energy production and favoring apoptosis, myocardial fibrosis, ultimately reducing cardiac function. This review focuses on the impact of superoxide sources in the diabetic heart and the pharmacological approaches that are currently under investigation as possible therapeutic tools

Synthesis of New (E)-2-(1H-Indole-3-ylcarbonyl)-3-heteroaryl-acrylonitriles via Microwave-Assisted Knoevenagel Condensation

Given the broad spectrum of biological uses of heteroaryl-acrylonitrile derivatives, it is necessary to find simple methods to synthesize and diversify this family of compounds. We report a stereoselective synthesis of a series of new (E)-2-(1H-indole-3-ylcarbonyl)-3-heteroaryl-acrylonitriles (3a–3i) obtained from 3-(cyanoacetyl)indole and heteroaryl-aldehydes under microwave-assisted Knoevenagel reaction at 300 W of potency and 100°C. The desired derivatives (3a–3i) were obtained with variable yields (30–94%) and time reactions (8–90 min). All the heteroaryl-acrylonitriles were characterized by physicoanalytical techniques such IR, 1H, 13C NMR, and electrospray mass spectrometry

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes

Altered Ca 2+ homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S -nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S -nitrosylation, leading to altered Ca 2+ homeostasis. Diastolic Ca 2+ levels are elevated in NOS1 −/− and NOS1/NOS3 −/− but not NOS3 −/− myocytes compared with wild-type (WT), suggesting diastolic Ca 2+ leakage. Measured leak was increased in NOS1 −/− and NOS1/NOS3 −/− but not in NOS3 −/− myocytes compared with WT. Importantly, NOS1 −/− and NOS1/NOS3 −/− myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1 −/− hearts, RyR2 S -nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca 2+ leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease

S-nitrosylation of cardiac ion channels

Nitric oxide exerts ubiquitous signaling via post-translational modification of cysteine residues, a reaction termed S -nitrosylation. Important substrates of S -nitrosylation that influence cardiac function include receptors, enzymes, ion channels, transcription factors, and structural proteins. Cardiac ion channels subserving excitation-contraction coupling are potentially regulated by S -nitrosylation. Specificity is achieved in part by spatial co-localization of ion channels with nitric oxide synthases (NOS), enzymatic sources of NO in biologic systems, and by coupling of NOS activity to localized calcium/second messenger concentrations. Ion channels regulate cardiac excitability and contractility in millisecond timescales raising the possibility that NO-related species modulate heart function on a beat-to beat basis. This review focuses on recent advances in understanding of NO regulation of the cardiac action potential, and of the calcium release channel ryanodine receptor, which is crucial for the generation of force. S -nitroso (SNO) signaling is disrupted in pathological states in which the redox state of the cell is dysregulated, including ischemia, heart failure, and atrial fibrillation

Impaired S-nitrosylation of the ryanodine receptor caused by xanthine oxidase activity contributes to calcium leak in heart failure

S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca(2+) leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca(2+) leak, improving Ca(2+) handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca(2+) leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications

Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis

Background: The effects of diabetes on the cardiovascular system as well as in the kidney are profound, which include hypertrophy and fibrosis. Diabetes also induces oxidative stress, at least in part due to the uncoupling of nitric oxide synthase (NOS); this is a shift in NO production toward superoxide production due to reduced levels of the NOS cofactor tetrahydrobiopterin (BH4). With this in mind, we tested the hypothesis that BH4 supplementation may prevent the development of diabetic cardiomyopathy and nephropathy. Methods: Diabetes was induced in Balb/c mice with streptozotocin. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin) in drinking water (daily doses of 15 mg/kg/day, during eight weeks) and the other that received only water. A third group of normoglycemic mice that received only water were used as the control. Results: Cardiac levels of BH4 were increased in mice treated with BH4 (p = 0.0019). Diabetes induced cardiac hypertrophy, which was prevented in the group that received BH4 (p < 0.05). In addition, hypertrophy was evaluated as cardiomyocyte cross-sectional area. This was reduced in diabetic mice that received BH4 (p = 0.0012). Diabetes induced cardiac interstitial fibrosis that was reduced in mice that received BH4 treatment (p < 0.05). We also evaluated in the kidney the impact of BH4 treatment on glomerular morphology. Diabetes induced glomerular hypertrophy compared with normoglycemic mice and was prevented by BH4 treatment. In addition, diabetic mice presented glomerular fibrosis, which was prevented in mice that received BH4. Conclusions: These results suggest that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes,, probably by restoring the nitroso–redox balance. This offers a possible new alternative to explore a BH4-based treatment for the organ damage caused by diabetes

Altered Renal Expression of Angiotensin II Receptors, Renin Receptor, and ACE-2 Precede the Development of Renal Fibrosis in Aging Rats

Background: The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. Methods: Intrarenal expression of angiotensin II type 1 (AT1R), type 2 (AT2R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. Results: Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-β expression (7-fold). AT1R /AT2R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (–50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical –40%, medullary –53%) and medullary endothelial nitric oxide synthase (–48%) were decreased in old rats. Conclusion: Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly