INVESTIGATION OF HNO-INDUCED MODIFICATIONS IN VARIOUS SYSTEMS

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including small organic molecules, peptides, and a protein. At physiological pH and temperature, relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to investigate sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to sulfinamide detection and used this method to explore sulfinamide hydrolysis. Since HNO-derived modifications may depend on local environment, we have also investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative [RS(O)2NHOH] and a thiosulfonate [RS(O)2SR], presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example with tryptophan resulting in N-nitrosotryptophan formation. We have examined the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. HNO has been shown to enhance cardiac sarcoplasmic reticulum Ca2+ cycling independent of the β-adrenergic pathway. In a collaborative project, the effects of HNO on the cardiac proteins, phospholamban (PLN) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) were investigated

Obese mice exposed to psychosocial stress display cardiac and hippocampal dysfunction associated with local brain-derived neurotrophic factor depletion

Introduction: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. Methods: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. Findings: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. Interpretation: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. FUND: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities . M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant\u27Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication

INVESTIGATION OF HNO-INDUCED MODIFICATIONS IN VARIOUS SYSTEMS

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including small organic molecules, peptides, and a protein. At physiological pH and temperature, relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to investigate sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to sulfinamide detection and used this method to explore sulfinamide hydrolysis. Since HNO-derived modifications may depend on local environment, we have also investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative [RS(O)2NHOH] and a thiosulfonate [RS(O)2SR], presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example with tryptophan resulting in N-nitrosotryptophan formation. We have examined the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. HNO has been shown to enhance cardiac sarcoplasmic reticulum Ca2+ cycling independent of the β-adrenergic pathway. In a collaborative project, the effects of HNO on the cardiac proteins, phospholamban (PLN) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) were investigated

Reactivity of Nitroxyl-Derived Sulfinamides

Sulfinamide [RS­(O)­NH₂] formation is known to occur upon exposure of cysteine residues to nitroxyl (HNO), which has received recent attention as a potential heart failure therapeutic. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including a small organic molecule, peptides, and a protein. Although it has generally been assumed that this thiol to sulfinamide modification is irreversible, we show that sulfinamides can be reduced back to the free thiol in the presence of excess thiol at physiological pH and temperature. We have examined this sulfinamide reduction both in peptides, where a cyclic intermediate analogous to that proposed for asparagine deamidation reactions potentially can contribute, and in a small organic molecule, where the mechanism is restricted to a direct thiolysis. These studies suggest that the contribution from the cyclic intermediate becomes more important in environments with lower dielectric constants. In addition, although sulfinic acid [RS­(O)­OH] formation is observed upon prolonged incubations in water, reduction of sulfinamides is found to dominate in the presence of thiols. Finally, studies with the cysteine protease, papain, suggest that the reduction of sulfinamide to the free thiol is viable in a protein environment

Reactivity of C‑Terminal Cysteines with HNO

Nitroxyl (HNO), a potential heart failure therapeutic, is known to target cysteine residues to form sulfinamides and/or disulfides. Because HNO-derived modifications may depend on their local environment, we have investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides at physiological pH and temperature. Our findings indicate that the nature of HNO-derived modifications of C-terminal cysteines is affected by the C-terminal carboxylate. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative (RS­(O)₂NHOH) and a thiosulfonate (RS­(O)₂SR), presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Moreover, these modifications are formed independent of oxygen

Death of an antioxidant brings heart failure with preserved ejection fraction to life: 5-oxoproline and post-ischaemic cardio-renal dysfunction

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Barley (1,3)-β-d-Glucan Dietary Supplementation Prevents Cardiac Dysfunction in Obese Mice with Psychosocial Stress by Attenuating Myocardial Oxidative Stress

Objective: Obesity due to high fat intake and psychosocial stress (PSS) are major determinants of cardiovascular disease (CVD), particularly when combined. Long-term diets enriched with barley (1,3)-β- d-glucan (BBG), an inhibitor of the class I histone deacetylases and an antioxidant, increases post-ischemic survival rate in mice by promoting angiogenesis. However, the impact of a BBG-enriched diet on cardiac dysfunction due to conditions such as high-fat diet (HFD) and PSS, alone or in combination, is unknown. Here, we tested whether supplementing HFD-treated mice with BBG prevents HFD- or HFD/PSS induced cardiac dysfunction by attenuating myocardial oxidative stress. Methods: Controlmale C57BL/6 mice were fed with 3 different diets for 18 wks: 1) standard diet (SD; 10% kcal fromfat; n=9); 2) HFD (58%kcal from fat; n=9); or 3)HFD+3% BBG (HFD+BBG; 58% kcal from fat; n = 9). All mice were then subjected chronic PSS via a daily (10min) encounter with a male intruder (resident/intruder test, fromweek 16 to week 18). Cardiac function was evaluated by echocardiography before and after PSS, whereas left ventricle (LV) tissue was collected at study termination to assess reactive oxygen species (ROS) by emission electron paramagnetic resonance analysis. Results: At week 16, body weight was markedly higher in the HFD than in the SD group (+60.1%, P < 0.001); however, BBG supplementation significantly prevented weight gain (−10% compared with HFD, P < 0.05). The addition of PSS significantly worsened the LV dysfunction in HFD (LV ejection fraction (LVEF), −25%; LV fractional shortening (LVFS), −33% compared with SD (LVEF, −7%; LVFS, −10%) mice. Conversely, the LV function after PSS was significantly improved in HFD + BBG mice (LVEF, +7%; LVFS, +10%) when compared with basal values. HFD and PSS led a marked rise in LV ROS production, as compared with values found in the hearts of mice receiving SD (+76% P < 0.001). However, this increment was significantly attenuated in HFD + BBG mice in which LV ROS emission rose only by 34%. Our study shows that a regular supplementation of 3% wt/vol BBG with diet prevents cardiac functional decay due to the combination of HFD and PSS, likely by countering HFD/PSS-induced myocardial ROS production. These findings may have preventative or therapeutic implications for all CVDs characterized by a cardiac, vascular, or both redox milieu

NMR Detection and Study of Hydrolysis of HNO-Derived Sulfinamides

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS­(O)­NH₂] is unique to HNO. We have applied ¹⁵N-edited ¹H NMR techniques to detect the HNO-induced thiol to sulfinamide modification in several small organic molecules, peptides, and the cysteine protease, papain. Relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS­(O)­OH]. We have investigated sulfinamide hydrolysis at physiological pH and temperature. Studies with papain and a related model peptide containing the active site thiol suggest that sulfinamide hydrolysis can be enhanced in a protein environment. These findings are also supported by modeling studies. In addition, analysis of peptide sulfinamides at various pH values suggests that hydrolysis becomes more facile under acidic conditions