Despite decades of research, heart failure continues to be a major health problem, as evidenced by a rise in the number of hospitalizations for heart failure, the number of deaths attributed to heart failure, and the ever-increasing costs associated with its care. Hydrogen sulfide (H2S) is a recently identified, endogenous, gaseous signaling molecule that modulates diverse physiological signals and protects the myocardium during ischemia/reperfusion. Recent clinical evidence suggests that circulating H2S levels are decreased in patients with heart failure and that the severity of heart failure is inversely correlated with H2S bioavailability. In the present study, we examined the effects of H2S deficiency and H2S therapy on the severity of cardiac hypertrophy and heart failure in mice after transverse aortic banding. Mice deficient in a key H2S-generating enzyme exhibited exacerbated heart failure, whereas mice with cardiac-restricted overexpression of this enzyme were protected against heart failure. We also demonstrated that a novel H2S donor significantly attenuates adverse left ventricular modeling and preserves left ventricular function when administered before the onset of heart failure. Key actions of H2S involved reductions in oxidative stress and myocardial fibrosis, coupled with increased myocardial capillary density. Additional experiments determined that the cardioprotective effects of H2S therapy were mediated via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability. Together, these findings further support the emerging concept that H2S therapy may be of clinical importance in the treatment of cardiovascular disease and may have a practical clinical use.
H2S Protects Against Pressure Overload–Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase
Abstract
Background—
Cystathionine γ-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.
Introduction
Cardiac hypertrophy is independently related to cardiovascular events and death.1 Pressure overload initially induces hypertrophy to preserve cardiac function (compensated cardiac hypertrophy), whereas sustained pressure overload and pathological cardiac hypertrophy lead to cardiac remodeling with cardiac dilatation and loss of contractile function.2
Clinical Perspective on p 1127
Historically, hydrogen sulfide (H2S) has been considered a poisonous gas that contributes to morbidity and mortality in various industrial settings.3 However, H2S has recently emerged as a critical physiological gaseous signaling molecule that is produced enzymatically in all mammalian species at low micromolar levels via the action of cysteine metabolic enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfutransferase.4 Recently, our laboratory has demonstrated that H2S protects against acute myocardial ischemia/reperfusion (I/R) injury via antiapoptotic effects mediated by phosphatidylinositol-3-kinase/Akt, protein kinase C, and extracellular signal-regulated kinase 1/2 pathway, as well as antioxidant actions via the activation and translocation of Nuclear-factor-E2-related factor-2 to the nucleus, including an increase in antioxidant response element-related antioxidants.5 Moreover, previous experimental studies suggest that H2S augments angiogenesis under ischemic conditions both in vitro and in vivo.6–8 H2S also exhibits potent antiinflammatory actions9 and modulates mitochondrial respiration in part by reversible inhibition of cytochrome c oxidase.10
Recent studies provide strong evidence that H2S derived from CSE modulates cardioprotection in the setting of myocardial I/R injury. Specifically, cardiac-restricted overexpression of CSE results in increased H2S bioavailability and cardioprotection in response to both acute myocardial I/R injury10 and ischemia-induced heart failure.11 In contrast, genetic deficiency of CSE significantly attenuates H2S, bioavailability and results in exacerbated myocardial I/R injury.12,13 Recent clinical evidence suggests that total plasma sulfide is negatively related to the severity of congestive heart failure and that low plasma sulfide predicts a higher mortality.14 The precise role of H2S in the pathogenesis of pressure-induced cardiac hypertrophy has not yet been established. Specifically, it is currently unknown whether myocardial or circulating levels of H2S are altered and what, if any, role CSE plays in the progression of cardiac hypertrophy and heart failure in the setting of transverse aortic constriction (TAC).
To clarify these issues, we measured myocardial and blood levels of free H2S and the H2S metabolite sulfane sulfur in a murine model of pressure overload–induced cardiac hypertrophy and failure. In addition, we investigated the effects of both genetic deficiency and overexpression of CSE and the effects of exogenous H2S therapy on cardiac pathology in the setting of TAC-induced heart failure.
Methods
Experimental Animals
CSE-deficient (KO) mice (C57/Sv129 background) and cardiac-restricted (α-myosin heavy chain) CSE transgenic (Tg) mice (C57BL/6J background) were developed as described.10,12 Male C57BL/6J mice and endothelial nitric oxide (NO) synthase–deficient (eNOS KO) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 8 to 10 weeks of age. All experimental protocols were approved by the Institute for Animal Care and Use Committee at Emory University School of Medicine and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 86-23, revised 1996) and to federal and state regulations.
TAC Protocol
To create pressure overload, the thoracic aorta was tied between the right inominate and the left carotid arteries against a 27-gauge needle with 7-0 silk suture followed by removal of the needle. Echocardiography was performed with a VisualSonics Vevo-2100 ultrasound system as previously described.15
H2S Donor
A recently developed, orally active H2S-releasing compound (SG-1002) was provided by Sulfagenix (Cleveland, OH). The chemical structure and description are given below. SG-1002 was administered to mice in the diet to achieve dosages of 20 mg·kg−1·d−1 in C57BL/6J mice or 40 mg·kg−1·d−1 in CSE KO mice at 1 week before the TAC procedure and was continued for up to 12 weeks after TAC. In addition, some C57BL/6J mice receiving the SG-1002 diet were placed on the control diet at 1 or 3 weeks after TAC.
Measurement of H2S and Sulfane Sulfur
H2S and sulfane sulfur levels were measured in heart and blood according to previously described methods.16
Western Blot Analysis
Western blot analysis was performed as described previously.15
Myocardial Measurement of NO Metabolites
Nitrite (NO2−) analysis of cardiac tissue was performed as previously described.15
Serum Measurements of Vascular Endothelial Growth Factor and Brain Natriuretic Peptide
Serum levels of vascular endothelial growth factor (VEGF; VEGF ELISA kit, R&D Systems) and brain natriuretic peptide (BNP; BNP enzyme immunoassay kit, Phoenix Pharmaceuticals, Inc) were determined by ELISA at 6 and/or 12 weeks after TAC.
Cardiac Mitochondrial Respiration Assay
Myocardial mitochondria were isolated and mitochondrial respiratory capacity was assessed as previously described.16
8-Isoprostane Assay
Concentrations of 8-isoprostane in the plasma and heart were determined by 8-isoprostane enzyme immunoassay kit according to the manufacturer’s instructions (Cayman Chemicals).
Cardiac cGMP
Concentrations of cGMP in the heart were determined with a commercially available kit according to the manufacturer’s instructions (Abcam).
Histology and Immunohistochemistry
Hearts were collected at the indicated times, fixed in 10% buffered formalin, embedded in paraffin, and stained with Masson trichrome and Picrosirius Red (to detect fibrosis). Digital images were analyzed with ImageJ. Immunohistochemistry was performed to visualize vascular density with a commercially available kit (Blood Vessel Staining Kit, Millipore). Primary antibody against CD31 (abcam; 1:50) was used. Digital images were obtained with a microscope at a magnification of ×400. CD31-positive vessel numbers were counted with ImageJ, and vessel number per 1 mm2 was calculated to evaluate the number of vessels per field.
Powder X-Ray Diffraction and Mass Spectrometry
To determine the precise chemical structure of SG-1002, we performed a combination of powder x-ray diffraction and mass spectrometry. Both powder x-ray diffraction and powder diffraction experiments were performed with a Bruker D8 DIFFRAC powder diffractometer (Co K-alpha radiation) with a VANTECH detector in θ-theta mode. The samples were microcrystalline powders. Theta-theta scans were performed with a step width of 0.01° and scan time of 1 second per step. Analysis was done with the Bruker-AXS EVA software package by comparing the diffraction pattern with known diffraction patterns filed in the PDF-2 2006 database.
For mass spectrometry, water was added to the sample, and the supernatant was analyzed with static nanospray on a Thermo Scientific LTQ Fourier Transform Ultra mass spectrometer. A nanospray voltage of −1.5 kV was used to ionize the sample. The ions were analyzed in the Fourier transform–ion cyclotron resonance portion of the instrument with the resolution set to 100 000 at 400 m/z. Data were acquired with Xcaliber software from Thermo Scientific.
Statistical Analysis
All data are expressed as mean±SEM. Statistical significance was evaluated with an unpaired Student t test for comparison between 2 means and 1-way or 2-way ANOVA for comparison among ≥3 means with Prism 5 (GraphPad Software Inc). For the ANOVA, if a significant result was found, the Tukey (1-way ANOVA) or Bonferroni (2-way ANOVA) test was used as the post hoc analysis. Kaplan-Meier survival curves were compared by use of a log-rank (Mantel-Cox) test. For all data, a value of P<0.05 denotes statistical significance.
Results
Sulfide Levels Decrease After TAC-Induced Heart Failure
We examined the effects of TAC-induced heart failure on the myocardial expression of the 3 known H2S-producing enzymes and the levels of circulating and myocardial sulfide levels at 6 weeks of TAC. Our analysis revealed that the expression of CBS was unaltered (Figure 1A and 1B). However, CSE expression was upregulated in the vehicle mice compared with the sham mice (P<0.001; Figure 1A and 1B), whereas myocardial 3-mercaptopyruvate sulfutransferase expression was significantly downregulated compared with sham levels (P<0.01; Figure 1A and 1B). Interestingly, free H2S and sulfane sulfur levels were significantly lower in the blood (P<0.01) and heart (P<0.001) of TAC+vehicle mice compared with sham-operated control mice (Figure 1E–1H).
CSE Deficiency Exacerbates Cardiac Dysfunction After TAC
To investigate the role of endogenous H2S in pressure overload, we performed TAC surgery in CSE KO mice and evaluated cardiac structure and function using echocardiography. Initially, we confirmed that CSE KO mice exhibited lower free H2S and sulfane sulfur levels in the blood and heart compared with WT mice (P<0.05; Figure I in the online-only Data Supplement). CSE KO mice exhibited significantly greater cardiac enlargement and pulmonary edema at 12 weeks after TAC compared with WT mice (Figure 2A). CSE KO mice exhibited significant left ventricular (LV) cavity dilatation, as seen by increases in LV end-systolic diameter and LV end-diastolic diameter, and exhibited exacerbated cardiac dysfunction from 3 to 12 weeks after TAC compared with WT mice (Figure 2B and 2C and Figure IIA in the online-only Data Supplement). Despite the increased cardiac structure and functional changes in the CSE KO mice, no statistically significant difference in mortality was observed after TAC compared with the WT mice (Figure IIIA in the online-only Data Supplement).
Myocardial Overexpression of CSE Attenuates Cardiac Dysfunction After TAC
Overexpression of CSE has been shown to increase H2S production in the heart without altering CBS expression.10 We also confirmed no alterations in cardiac CBS expression in cardiac-specific CSE transgenic mice (CS-CSE Tg), but CS-CSE Tg mice exhibited less 3-mercaptopyruvate sulfutransferase expression compared with WT mice (Figure IV in the online-only Data Supplement). We next examined whether overexpression of CSE specifically within the cardiac myocyte would attenuate cardiac hypertrophy and/or dysfunction after TAC using CS-CSE Tg mice. CS-CSE Tg mice exhibited significantly less cardiac enlargement and pulmonary edema, as assessed by the ratio of heart and lung weights to tibia length (mg/cm) compared with WT controls (Figure 2D). Furthermore, echocardiography analysis revealed that CS-CSE Tg mice exhibited less cardiac dilatation and dysfunction from 6 to 12 weeks after TAC (Figure 2E and 2F and Figure IIB in the online-only Data Supplement). Again, no statistically significant difference in mortality was observed between the 2 groups (Figure IIIB in the online-only Data Supplement).
Administration of Exogenous H2S Prevents Cardiac Enlargement, Preserves LV Function, and Reduces Fibrosis After TAC
Next, we examined the effects of administration of oral H2S therapy on pressure overload–induced cardiac hypertrophy and dysfunction in wild-type C57BL/6J mice. For these experiments, we administered SG-1002 (20 mg·kg−1·d−1) in the chow. SG-1002 (Figure 3A) is a novel sulfur-containing compound consisting primarily of α-sulfur (≈92%) with lesser amounts of long-chain sodium polythionates (≈8%). Our initial studies found that SG-1002 treatment partially restored free H2S and significantly restored sulfane sulfur levels in the blood (P<0.05 versus TAC+vehicle; Figure 1C and 1D) and heart (P<0.05 versus TAC+vehicle; Figure 1E and 1F) after TAC. Gross morphological analysis at 12 weeks after TAC revealed that hearts from vehicle mice enlarged to a greater extent compared with hearts from SG-1002–treated mice (Figure 3B). This was confirmed by ratios of heart weight to tibia length, which showed that the hearts of both vehicle- and SG-1002–treated mice were significantly increased compared with those of sham mice at 6 and 12 weeks after TAC (P<0.001; Figure 3C). However, SG-1002–treated mice showed a significantly less increase compared with vehicle mice (P<0.001). In addition, SG-1002–treated mice displayed significantly less pulmonary edema compared with vehicle mice (Figure 3D). Moreover, we evaluated circulating BNP levels as an indication of heart failure severity after TAC. BNP levels increased significantly (P<0.01) in vehicle mice at 6 and 12 weeks compared with sham mice, but SG-1002 treatment significantly inhibited BNP (P<0.01 versus TAC+vehicle) levels after TAC (Figure 3E). Echocardiography analysis revealed that SG-1002 treatment prevented cardiac dilatation (P<0.01 versus TAC+vehicle; Figure 3F and Figure IIC in the online-only Data Supplement) and cardiac contractile dysfunction (P<0.001 versus TAC+vehicle; Figure 3G) from 6 to 12 weeks after TAC. Histological analysis of Masson trichrome– and Picrosirius Red–stained sections at 12 weeks after TAC revealed extensive areas of intermuscular and perivascular fibrosis in hearts from TAC+vehicle mice (P<0.01 versus sham; Figure 4). Although fibrosis was evident in the sections taken from TAC+SG-1002 hearts, it was significantly less compared with the TAC+vehicle hearts (P<0.001 for Masson trichrome and P<0.01 for Picrosirius Red). Finally, SG-1002–treated mice exhibited improved, but not statistically significantly improved, survival rate compared with vehicle mice (80% versus 61%; P=0.23; Figure IIIC in the online-only Data Supplement).
Further analysis revealed that the administration of SG-1002 to CSE KO mice slightly, but not significantly, increased free H2S levels in the blood and heart, whereas administration of SG-1002 significantly increased sulfane sulfur levels in both the blood (P<0.001) and the heart (P<0.05) compared with CSE KO mice fed a control diet (Figure I in the online-only Data Supplement). The administration of SG-1002 also completely diminished LV cavity dilatation in CSE KO mice compared with CSE KO mice fed a control diet (P<0.05; Figure 2B). Interestingly, SG-1002–treated CSE KO mice maintained cardiac ejection fraction after TAC compared with not only control diet–fed CSE KO mice but also WT mice at 12 weeks after TAC (P<0.001 versus CSE KO+TAC and P<0.05 versus WT+TAC; Figure 2C). However, no statistically significant difference in mortality was observed between the CSE KO groups (Figure IIIA in the online-only Data Supplement).
Together, these results indicate that endogenous H2S bioavailability is markedly attenuated in heart failure after pressure overload even though CSE and CBS expression levels are maintained or upregulated. Moreover, augmentation of H2S levels by genetic or pharmacological approaches prevents the transition from compensated to decompensated cardiac hypertrophy.
Withdrawal of SG-1002 Leads to Development of Cardiac Dilatation and Dysfunction
Experiments were then conducted to determine how withdrawal of SG-1002 from the chow would affect the development of cardiac dilatation and dysfunction after TAC. For these experiments, we administered SG-1002 in the chow for 1 week and then subjected different groups of mice to 6 weeks of TAC: group 1 mice received SG-1002 in the chow for 6 weeks after TAC; group 2 mice received SG-1002 in the chow for 1 week after TAC and then received normal chow for 5 weeks; and group 3 mice received SG-1002 in the chow for 3 weeks after TAC and then received normal chow for 3 weeks. Echocardiography analysis revealed that withdrawal of SG-1002 after 1 week of TAC resulted in a larger increase in LV end-systolic diameter and end-diastolic diameter and a larger decrease in ejection fraction at 6 weeks of TAC compared with the nonwithdrawal group (P<0.01 versus SG-1002; Figure VA–VC in the online-only Data Supplement). Withdrawing SG-1002 at 3 weeks of TAC resulted in a nonsignificant increase in both of these parameters at 6 weeks of TAC compared with the nonwithdrawal group. These data indicate that the withdrawal of SG-1002 early after the onset of pressure overload does not prevent the development of cardiac dilatation and dysfunction, suggesting that the benefits of SG-1002 are achieved when the diet is maintained throughout the follow-up period.
H2S Therapy Augments VEGF-Akt-eNOS-NO Signaling After TAC
The serine/threonine kinase Akt regulates cardiac growth, myocardial angiogenesis, and survival in cardiac myocytes.17 We investigated whether SG-1002 treatment activated Akt phosphorylation in the heart after TAC (Figure 5). Representative Western blots for Akt phosphorylation status in the heart at 6 weeks after TAC are shown in Figure 5A. SG-1002 treatment did not alter total Akt expression in the heart (Figure 5B) but significantly increased the expression of phosphorylated Akt at threonine residue 308 (Akt-PThr308; P<0.001) and serine residue 473 (Akt-PSer473) compared with vehicle mice (P<0.001; Figure 5C). We next investigated whether SG-1002 treatment upregulated VEGF, a potent angiogenic and cytoprotective cytokine in the myocardium. At 6 weeks after TAC, SG-1002–treated mice showed significantly greater VEGF protein expression levels in the heart (P<0.01 versus sham and P<0.05 versus TAC+vehicle; Figure 5D) but not in the systemic circulation (Figure VIA in the online-only Data Supplement). Further analysis revealed that 6 weeks of TAC caused a significant decrease in the number of CD31+ vessels in the hearts of vehicle-treated mice (P<0.001 versus sham; Figure 4A and 4D). However, treatment with SG-1002 significantly attenuated the observed vessel dropout (P<0.05 versus TAC+vehicle).
NO generated from eNOS is known to promote vascular and myocardial cell cytoprotection during ischemic conditions.18 To investigate the potential involvement of eNOS in SG-1002–induced cardioprotection after TAC, the expression and the phosphorylation status of eNOS at serine residue 1177 (eNOS-PSer1177) were assessed by Western blot analysis in the hearts of sham, vehicle, and SG-1002–treated mice. There were no differences in total eNOS expression in the heart among all groups (Figure 5E and 5F). However, the eNOS activation site (eNOS-PSer1177) exhibited significantly greater phosphorylation after SG-1002 compared with sham and TAC+vehicle mice (P<0.01; Figure 5E and 5F). Furthermore, SG-1002 treatment increased cardiac nitrite and cGMP levels after TAC compared with sham mice (P<0.05; Figure 5G and 5H), which is indicative of increased NO bioavailability after H2S therapy. We also investigated myocardial expression of both neuronal NOS and inducible NOS in mice subjected to TAC that received either vehicle or SG-1002 (Figure VIB–VID in the online-only Data Supplement). Neuronal NOS expression in the both vehicle- and SG-1002–treated mice tended to be higher than in sham mice but did not reach statistical significance. Interestingly, inducible NOS expression in the TAC+vehicle group was upregulated compared with the sham group (P<0.01), but SG-1002 mice diminished this upregulation (P<0.01 versus TAC+vehicle). We next investigated whether eNOS was critical for the protection afforded by SG-1002. Mice deficient in eNOS (eNOS KO) were subjected to TAC and administered normal chow (eNOS KO+TAC+vehicle) or SG-1002 (eNOS KO+TAC+SC-1002). Analysis at 6 weeks after TAC revealed that hearts from both groups of mice were enlarged compared with hearts of sham mice, as evidenced by an increase in the ratios of heart weight to tibia length (Figure 6A) and an increase in circulating BNP levels (Figure 6B). Echocardiography analysis also revealed that both groups of mice displayed cardiac dilatation and cardiac contractile dysfunction from 3 to 6 weeks after TAC (P<0.01 versus baseline; Figure 6C and 6D and Figure IID in the online-only Data Supplement). Importantly, SG-1002 did not reduce cardiac enlargement or cardiac dilatation or improve cardiac contractile function, indicating that eNOS is critical for SG-1002 to provide its protective effects.
H2S Therapy Attenuates Mitochondrial Respiratory Dysfunction and Oxidative Stress After TAC
Mitochondrial energetic failure is considered one of the central pathological mechanisms in heart failure resulting from cardiac hypertrophy.19,20 Therefore, we investigated the respiratory function of isolated mitochondria obtained from mouse hearts at 6 weeks after TAC. A significant decrease in state 3 respiration rates (P<0.01; Figure VIIA in the online-only Data Supplement) and respiratory control rate (P<0.001; Figure VIIB in the online-only Data Supplement) was observed in the TAC+vehicle mice compared with the sham mice. However, SG-1002 treatment preserved mitochondrial respiratory function compared with TAC+vehicle mice (P<0.05 for state 3 and P<0.01 for respiratory control rate). No difference in state 4 respiration was observed among any of the study groups (Figure VIIA in the online-only Data Supplement).
Mitochondrial dysfunction leads to impaired ATP production and increased reactive oxygen species generation, which can result in increased apoptosis.21 We therefore examined 8-isoprostane levels as a marker of antioxidant deficiency and oxidative stress in both the plasma and heart at 6 weeks after TAC. Both the TAC+vehicle– and TAC+SG-1002–treated mice exhibited higher plasma levels of 8-isoprostane compared with sham mice (P<0.05; Figure VIIC in the online-only Data Supplement). However, TAC+vehicle mice exhibited significantly higher 8-isoprostane levels in the heart compared with sham mice (P<0.001), whereas the administration of SG-1002 attenuated the TAC-induced increase in 8-isoprostane levels (P<0.05 versus TAC+vehicle; Figure VIID in the online-only Data Supplement). Next, we checked cardiac Nox4 expression as another marker of oxidative stress. At 6 weeks after TAC, myocardial NADPH oxidase 4 (Nox4) expression was significantly upregulated in the TAC+vehicle mice compared with sham mice (P<0.01; Figure VIIE in the online-only Data Supplement). However, SG-1002 treatment significantly inhibited the upregulation of Nox4 (P<0.01 versus TAC+vehicle). Additional analysis revealed that SG-1002 treatment resulted in upregulation of the expression of the antioxidant heme oxygenase 1 in the heart after TAC (P<0.01 versus sham and TAC+vehicle; Figure VIIF in the online-only Data Supplement).
Discussion
Previous studies have suggested that both exogenously derived H2S and endogenously derived H2S exhibit potent cytoprotective effects in models of acute myocardial I/R and ischemia-induced heart failure.11,22 However, the role of endogenous H2S in pressure overload–induced heart failure has not been fully elucidated. In the present study, we have identified a number of novel findings about the role of CSE-derived H2S on the severity of heart failure after TAC and have provided important insights into the mechanism by which oral H2S therapy attenuates TAC-induced heart failure.
Recently, a clinical study reported that lower circulating sulfide levels in patients suffering from congestive heart failure correlated negatively with the severity of the disease.14 Although this was a small study with a limited sample size, it provided a preliminary indication that sulfide levels are decreased and may be an important predictor of heart failure severity. The present study provides several lines of evidence to support this idea. First, we provide data that this is mirrored in an experimental model of pressure overload–induced heart failure, as evidenced by the finding that myocardial and circulating levels of free H2S and sulfane sulfur are significantly reduced after TAC. Second, we have clearly demonstrated that a deficiency in endogenous H2S results in an exacerbation of cardiac dysfunction after TAC, whereas genetic overexpression of CSE and increased H2S bioavailability significantly preserved LV function. Finally, long-term oral administration of a novel sulfur-containing compound that augments H2S levels provides protection against the adverse remodeling associated with TAC by increasing circulating and cardiac sulfide levels. Although the mechanisms responsible for the heart failure–induced decline in sulfide levels are currently not known, this finding strongly suggests that a deficiency of H2S may contribute to the pathophysiology and progression of heart failure. These findings also suggest that increasing the bioavailability of H2S with an oral H2S-donating agent significantly preserves cardiac function in the setting of heart failure.
One of the main findings of the present study is that administration of SG-1002 significantly preserved cardiac function after TAC. Given that H2S is a physiological gas that freely diffuses into multiple intracellular compartments, it can be postulated that H2S targets multiple pathological cascades simultaneously. In this study, we demonstrated that SG-1002 treatment activated a VEGF-Akt-eNOS-NO-cGMP signaling pathway at 6 weeks after the induction of TAC (a time point when cardiac hypertrophy and LV dysfunction are significant). VEGF is a very potent angiogenic and cytoprotective cytokine. Givvimani et al23 previously reported that sodium hydrogen sulfide in drinking water augmented angiogenesis by increasing VEGF expression and inhibition of antiangiogenic factors (angiostatin and endostatin). The serine/threonine protein kinase Akt regulates cardiac growth, glucose metabolism, contractile function, and cell death and is an important factor for VEGF-mediated angiogenesis.24 In the present study, we found that SG-1002 prevented TAC-induced decrease in CD31+ vessels. Given that VEGF and Akt were increased in the SG-1002–treated hearts, one could argue that it is possible that SG-1002 is inducing angiogenesis.25,26 On the other hand, this observation could be a result of less injury in these hearts. Regardless of the mechanism, the data suggest that SG-1002 prevents vessel dropout after TAC. Therefore, the activation of VEGF and Akt by SG-1002 could contribute to the observed attenuation of cardiac dysfunction by regulating hypertrophy, cell death, and/or vascular density.27,28 Additional studies are certainly warranted to investigate the mechanisms by which SG-1002 activates VEGF and to determine whether SG-1002 induces angiogenesis.
It has been generally thought that H2S and NO exert their biological effects via independent signaling pathways. Recent experimental evidence suggests that there is crosstalk between the H2S and NO signaling pathways that could provide synergistic and additional regulatory effects. For example, H2S upregulates NO production in endothelial cells through the activation of eNOS in an Akt-dependent manner.29 Likewise, NO has been shown to enhance the production of H2S from vascular tissue,30 and more recently, Coletta et al31 demonstrated that NO and H2S are mutually required for the control of vascular function and angiogenesis. Therefore, another major finding of the present study is that exogenous H2S therapy very potently activates eNOS and increases NO bioavailability within the myocardium. This finding is important for 2 reasons: It further corroborates the evidence that there is crosstalk between the H2S and NO signaling pathways under in vivo pathological conditions, and it provides strong evidence that H2S-mediated cardioprotection depends at least in part on increased bioavailability of NO in an in vivo model of disease. Furthermore, the finding that SG-1002 does not provide protection in mice deficient in eNOS indicates that the activation of eNOS by SG-1002 is required for the observed protective effects against TAC.
In terms of its effects on hypertrophy, NO produced from eNOS has been shown to have antihypertrophic effects in the heart as evidenced by the findings that eNOS KO mice have hypertension and cardiac hypertrophy32 and exhibit exacerbated cardiac dysfunction resulting from pressure overload–induced hypertrophy compared with WT mice.33 Moreover, cardiac-specific overexpression of eNOS prevents isoproterenol-induced cardiac hypertrophy.34 However, in sharp contrast, Takimoto et al35 suggested that pressure overload results in eNOS uncoupling, resulting in increased myocardial oxidant production and exacerbated cardiac function. However, physicians have been successfully using drugs that are able to activate eNOS, (ie, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and β-blockers) to treat heart failure.36 Therefore, controversy remains in regard to the utility and effectiveness of NO-based therapies in the treatment of heart failure; further investigation is warranted to resolve these issues. Additionally, both NO and H2S are known to increase heme oxygenase 1 levels, an enzyme that produces carbon monoxide.5,37 This suggests that the activation of one of the endogenously produced gases can lead to the activation of the other 2 gases. Under these conditions, the 3 gases have the ability to synergize to produce antiapoptotic, antioxidant, antiinflammatory, and antihypertrophic effects, which ultimately can lead to cardioprotection.
An increase in oxidative stress and/or a deficiency in the endogenous antioxidant reserve can also cause contractile dysfunction.38 The cardioprotective effects of H2S against myocardial I/R are mediated by antioxidant signaling.5 In addition, H2S directly scavenges reactive oxygen species in vitro.39 Therefore, endogenous H2S may directly and/or indirectly contribute to modulation of oxidative stress in the setting of pressure overload–induced hypertrophy. Here, we demonstrate that H2S attenuates the TAC-induced increase in oxidative stress, as evidenced by the finding that SG-1002 decreases cardiac 8-isoprostane levels. In terms of mechanism, we found that SG-1002 attenuates the TAC-induced upregulation of Nox4, a member of the NADPH oxidase family that is a major source of reactive oxygen species–related cardiac dysfunction in the setting of pressure overload.40 We also found that SG-1002 upregulated the expression of heme oxygenase 1 and preserved mitochondrial respiratory function. Because mitochondrial respiratory dysfunction in the heart leads to metabolic remodeling, deficit cardiac energetics, and increased oxidative stress,19,41 the preserved mitochondrial respiratory function observed in the present study could be an additional mechanism to explain the inhibition of oxidative stress by H2S after TAC.
The findings of the present study indicate that preserving sulfide levels during the development of pressure overload–induced heart failure preserves cardiac function and prevents the transition from compensated to decompensated cardiac hypertrophy. Furthermore, the present study indicates that administration of a novel oral H2S donor facilitates these protective effects by activating a VEGF-Akt-eNOS-NO-cGMP signaling pathway, significantly increasing NO bioavailability (Figure 7). This cardioprotective signaling cascade ultimately results in the inhibition of oxidative stress, attenuation of cardiac fibrosis, prevention of vessel dropout, preservation of mitochondrial respiration, and preservation of LV function. Our study suggests that endogenously produced H2S plays an important role in the preservation of cardiac function in heart failure and that oral H2S therapy may be a therapeutic option for the treatment of LV dysfunction in the setting of pressure overload–induced h-ypertrophy.
Acknowledgments
We thank David Polhemus and Claire Pearce for their expert technical assistance during the course of these studies. We are also grateful to John W. Elrod, PhD, for all of his assistance during the course of these studies.
Clinical Perspective
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© 2013 American Heart Association, Inc.
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History
Received: 4 May 2012
Accepted: 30 January 2013
Published online: 7 February 2013
Published in print: 12 March 2013
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Disclosures
Drs Lefer, Elrod, and Gojon and G. Gojon, Jr, are cofounders of Sulfagenix, Inc. Sulfagenix is currently developing H2S–based therapeutics for cardiovascular disease states. In addition, Dr Gojon and G. Gojon, Jr, are listed as inventors on several US patent applications for the use of H2S-based therapeutics for a variety of disease conditions, including cardiovascular diseases. The other authors report no conflicts.
Sources of Funding
This work was supported by grants from the National Heart, Lung, and Blood Institute (National Institutes of Health; 5R01HL092141, 5R01HL093579, 1U24HL 094373, and 1P20HL113452 to Dr Lefer and 5R01HL098481 to Dr Calvert). This work was also supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to Dr Wang. We are also grateful for the generous funding support from the Carlyle Fraser Heart Center of Emory University Hospital Midtown.
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