Chad K. Nicholson

Genetic and Pharmacologic Hydrogen Sulfide Therapy Attenuates Ischemia-Induced Heart Failure in Mice

Background— Hydrogen sulfide (H2S) is an endogenous signaling molecule with potent cytoprotective effects. The present study evaluated the therapeutic potential of H2S in murine models of heart failure. Methods and Results— Heart failure was induced by subjecting mice either to permanent ligation of the left coronary artery for 4 weeks or to 60 minutes of left coronary artery occlusion followed by reperfusion for 4 weeks. Transgenic mice with cardiac-restricted overexpression of the H2S-generating enzyme cystathione &ggr;-lyase (&agr;MHC-CGL-Tg+) displayed a clear protection against left ventricular structural and functional impairment as assessed by echocardiography in response to ischemia-induced heart failure, as well as improved survival in response to permanent myocardial ischemia. Exogenous H2S therapy (Na2S; 100 &mgr;g/kg) administered at the time of reperfusion (intracardiac) and then daily (intravenous) for the first 7 days after myocardial ischemia also protected against the structural and functional deterioration of the left ventricle by attenuating oxidative stress and mitochondrial dysfunction. Additional experiments aimed at elucidating some of the protective mechanisms of H2S therapy found that 7 days of H2S therapy increased the phosphorylation of Akt and increased the nuclear localization of 2 transcription factors, nuclear respiratory factor 1 and nuclear factor-E2-related factor (Nrf2), that are involved in increasing the levels of endogenous antioxidants, attenuating apoptosis, and increasing mitochondrial biogenesis. Conclusions— The results of the present study suggest that either the administration of exogenous H2S or the modulation of endogenous H2S production may be of therapeutic benefit in the treatment of ischemia-induced heart failure.

H2S Protects Against Pressure Overload Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase (eNOS)

Background— Cystathionine &ggr;-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload–induced heart failure. Methods and Results— Transverse aortic constriction was performed in wild-type, CSE knockout, and cardiac-specific CSE transgenic mice. In addition, C57BL/6J or CSE knockout mice received a novel H2S donor (SG-1002). Mice were followed up for 12 weeks with echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels after transverse aortic constriction. CSE knockout mice exhibited significantly greater cardiac dilatation and dysfunction than wild-type mice after transverse aortic constriction, and cardiac-specific CSE transgenic mice maintained cardiac structure and function after transverse aortic constriction. H2S therapy with SG-1002 resulted in cardioprotection during transverse aortic constriction via upregulation of the vascular endothelial growth factor–Akt–endothelial nitric oxide synthase–nitric oxide–cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. Conclusions— Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure, and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability.

Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent

Significance Physiological concentrations of hydrogen sulfide (H2S) exert potent prosurvival actions. We demonstrate that the cytoprotective actions of H2S are mediated in part via a second gaseous signaling molecule, nitric oxide (NO). We found that cystathionine γ-lyase (CSE) KO mice with reduced H2S levels exhibit increased oxidative stress and an exacerbated response to myocardial ischemia/reperfusion injury. CSE KO mice also exhibit reduced levels of NO and reduced NO synthesis via endothelial NO synthase (eNOS). Both oxidative stress and myocardial injury in CSE KO mice were attenuated by exogenous H2S therapy, with increased eNOS function and restoration of NO levels. These findings provide insight into H2S-mediated cytoprotetion and important information regarding the translation of H2S therapy to the clinic. Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

Exercise Protects Against Myocardial Ischemia–Reperfusion Injury via Stimulation of β3-Adrenergic Receptors and Increased Nitric Oxide Signaling: Role of Nitrite and Nitrosothiols

Rationale: Exercise training confers sustainable protection against ischemia–reperfusion injury in animal models and has been associated with improved survival following a heart attack in humans. It is still unclear how exercise protects the heart, but it is apparent that endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) play a role. Objective: To determine the role of &bgr;3-adrenergic receptors (&bgr;3-ARs), eNOS activation, and NO metabolites (nitrite and nitrosothiols) in the sustained cardioprotective effects of exercise. Methods and Results: Here we show that voluntary exercise reduces myocardial injury in mice following a 4-week training period and that these protective effects can be sustained for at least 1 week following the cessation of the training. The sustained cardioprotective effects of exercise are mediated by alterations in the phosphorylation status of eNOS (increase in serine 1177 and decrease in threonine 495), leading to an increase in NO generation and storage of NO metabolites (nitrite and nitrosothiols) in the heart. Further evidence revealed that the alterations in eNOS phosphorylation status and NO generation were mediated by &bgr;3-AR stimulation and that in response to exercise a deficiency of &bgr;3-ARs leads to an exacerbation of myocardial infarction following ischemia–reperfusion injury. Conclusions: Our findings clearly demonstrate that exercise protects the heart against myocardial ischemia–reperfusion injury by stimulation of &bgr;3-ARs and increased cardiac storage of nitric oxide metabolites (ie, nitrite and nitrosothiols).

Hydrogen sulfide and ischemia-reperfusion injury

Gasotransmitters are lipid soluble, endogenously produced gaseous signaling molecules that freely permeate the plasma membrane of a cell to directly activate intracellular targets, thus alleviating the need for membrane-bound receptors. The gasotransmitter family consists of three members: nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H(2)S). H(2)S is the latest gasotransmitter to be identified and characterized and like the other members of the gasotransmitter family, H(2)S was historically considered to be a toxic gas and an environmental/occupational hazard. However with the discovery of its presence and enzymatic production in mammalian tissues, H(2)S has gained much attention as a physiological signaling molecule. Also, much like NO and CO, H(2)Ss role in ischemia/reperfusion (I/R) injury has recently begun to be elucidated. As such, modulation of endogenous H(2)S and administration of exogenous H(2)S has now been demonstrated to be cytoprotective in various organ systems through diverse signaling mechanisms. This review will provide a detailed description of the role H(2)S plays in different model systems of I/R injury and will also detail some of the mechanisms involved with its cytoprotection.

Hydrogen sulfide preconditions the db/db diabetic mouse heart against ischemia-reperfusion injury by activating Nrf2 signaling in an Erk-dependent manner

Hydrogen sulfide (H2S) therapy protects nondiabetic animals in various models of myocardial injury, including acute myocardial infarction and heart failure. Here, we sought to examine whether H2S therapy provides cardioprotection in the setting of type 2 diabetes. H2S therapy in the form of sodium sulfide (Na2S) beginning 24 h or 7 days before myocardial ischemia significantly decreased myocardial injury in db/db diabetic mice (12 wk of age). In an effort to evaluate the signaling mechanism responsible for the observed cardioprotection, we focused on the role of nuclear factor E2-related factor (Nrf2) signaling. Our results indicate that diabetes does not alter the ability of H2S to increase the nuclear localization of Nrf2, but does impair aspects of Nrf2 signaling. Specifically, the expression of NADPH quinine oxidoreductase 1 was increased after the acute treatment, whereas the expression of heme-oxygenase-1 (HO-1) was only increased after 7 days of treatment. This discrepancy was found to be the result of an increased nuclear expression of Bach1, a known repressor of HO-1 transcription, which blocked the binding of Nrf2 to the HO-1 promoter. Further analysis revealed that 7 days of Na2S treatment overcame this impairment by removing Bach1 from the nucleus in an Erk1/2-dependent manner. Our findings demonstrate for the first time that exogenous administration of Na2S attenuates myocardial ischemia-reperfusion injury in db/db mice, suggesting the potential therapeutic effects of H2S in treating a heart attack in the setting of type 2 diabetes.

Nitrite Therapy Improves Left Ventricular Function During Heart Failure via Restoration of Nitric Oxide–Mediated Cytoprotective Signaling

Rationale: Nitric oxide (NO) bioavailability is reduced in the setting of heart failure. Nitrite (NO2) is a critically important NO intermediate that is metabolized to NO during pathological states. We have previously demonstrated that sodium nitrite ameliorates acute myocardial ischemia/reperfusion injury. Objective: No evidence exists as to whether increasing NO bioavailability via nitrite therapy attenuates heart failure severity after pressure-overload–induced hypertrophy. Methods and Results: Serum from patients with heart failure exhibited significantly decreased nitrosothiol and cGMP levels. Transverse aortic constriction was performed in mice at 10 to 12 weeks. Sodium nitrite (50 mg/L) or saline vehicle was administered daily in the drinking water postoperative from day 1 for 9 weeks. Echocardiography was performed at baseline and at 1, 3, 6, and 9 weeks after transverse aortic constriction to assess left ventricular dimensions and ejection fraction. We observed increased cardiac nitrite, nitrosothiol, and cGMP levels in mice treated with nitrite. Sodium nitrite preserved left ventricular ejection fraction and improved left ventricular dimensions at 9 weeks (P<0.001 versus vehicle). In addition, circulating and cardiac brain natriuretic peptide levels were attenuated in mice receiving nitrite (P<0.05 versus vehicle). Western blot analyses revealed upregulation of Akt-endothelial nitric oxide-nitric oxide-cGMP-GS3K&bgr; signaling early in the progression of hypertrophy and heart failure. Conclusions: These results support the emerging concept that nitrite therapy may be a viable clinical option for increasing NO levels and may have a practical clinical use in the treatment of heart failure.

Hydrogen sulfide attenuates high fat diet-induced cardiac dysfunction via the suppression of endoplasmic reticulum stress

Diabetic cardiomyopathy is a significant contributor to the morbidity and mortality associated with diabetes and metabolic syndrome. However, the underlying molecular mechanisms that lead to its development have not been fully elucidated. Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that is critical for the regulation of cardiovascular homeostasis. Recently, therapeutic strategies aimed at increasing its levels have proven cardioprotective in models of acute myocardial ischemia-reperfusion injury and heart failure. The precise role of H2S in the pathogenesis of diabetic cardiomyopathy has not yet been established. Therefore, the goal of the present study was to evaluate circulating and cardiac H2S levels in a murine model of high fat diet (HFD)-induced cardiomyopathy. Diabetic cardiomyopathy was produced by feeding mice HFD (60% fat) chow for 24 weeks. HFD feeding reduced both circulating and cardiac H2S and induced hallmark features of type-2 diabetes. We also observed marked cardiac dysfunction, evidence of cardiac enlargement, cardiac hypertrophy, and fibrosis. H2S therapy (SG-1002, an orally active H2S donor) restored sulfide levels, improved some of the metabolic perturbations stemming from HFD feeding, and attenuated HFD-induced cardiac dysfunction. Additional analysis revealed that H2S therapy restored adiponectin levels and suppressed cardiac ER stress stemming from HFD feeding. These results suggest that diminished circulating and cardiac H2S levels play a role in the pathophysiology of HFD-induced cardiomyopathy. Additionally, these results suggest that H2S therapy may be of clinical importance in the treatment of cardiovascular complications stemming from diabetes.

Thioredoxin 1 Is Essential for Sodium Sulfide–Mediated Cardioprotection in the Setting of Heart Failure

Objective—The aim of this study was to determine whether thioredoxin 1 (Trx1) mediates the cardioprotective effects of hydrogen sulfide (H2S) in a model of ischemic-induced heart failure (HF). Approach and Results—Mice with a cardiac-specific overexpression of a dominant negative mutant of Trx1 and wild-type littermates were subjected to ischemic-induced HF. Treatment with H2S as sodium sulfide (Na2S) not only increased the gene and protein expression of Trx1 in the absence of ischemia but also augmented the HF-induced increase in both. Wild-type mice treated with Na2S experienced less left-ventricular dilatation, improved left-ventricular function, and less cardiac hypertrophy after the induction of HF. In contrast, Na2S therapy failed to improve any of these parameters in the dominant negative mutant of Trx1 mice. Studies aimed at evaluating the underlying cardioprotective mechanisms found that Na2S therapy inhibited HF-induced apoptosis signaling kinase-1 signaling and nuclear export of histone deacetylase 4 in a Trx1-dependent manner. Conclusions—These findings provide novel information that the upregulation of Trx1 by Na2S therapy in the setting of HF sets into motion events, such as the inhibition of apoptosis signaling kinase-1 signaling and histone deacetylase 4 nuclear export, which ultimately leads to the attenuationof left-ventricular remodeling.

DJ-1 protects the heart against ischemia–reperfusion injury by regulating mitochondrial fission

Recent data indicates that DJ-1 plays a role in the cellular response to stress. Here, we aimed to examine the underlying molecular mechanisms mediating the actions of DJ-1 in the heart following myocardial ischemia-reperfusion (I/R) injury. In response to I/R injury, DJ-1 KO mice displayed increased areas of infarction and worsened left ventricular function when compared to WT mice, confirming a protective role for DJ-1 in the heart. In an effort to evaluate the potential mechanism(s) responsible for the increased injury in DJ-1 KO mice, we focused on SUMOylation, a post-translational modification process that regulates various aspects of protein function. DJ-1 KO hearts after I/R injury were found to display enhanced accumulation of SUMO-1 modified proteins and reduced SUMO-2/3 modified proteins. Further analysis, revealed that the protein expression of the de-SUMOylation enzyme SENP1 was reduced, whereas the expression of SENP5 was enhanced in DJ-1 KO hearts after I/R injury. Finally, DJ-1 KO hearts were found to display enhanced SUMO-1 modification of dynamin-related protein 1, excessive mitochondrial fission, and dysfunctional mitochondria. Our data demonstrates that the activation of DJ-1 in response to myocardial I/R injury protects the heart by regulating the SUMOylation status of Drp1 and attenuating excessive mitochondrial fission.