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.

Figures

Figure 1

Heart failure reduces sulfide levels in mice. (A-B) Representative immunoblots and densitometric analysis of cystathionine gamma lyase (CSE), cystathionine beta synthase (CBS), and 3-mercaptopyruvate sulfutransferase (3-MST) in the hearts of Sham, TAC+Vehicle, and TAC+SG-1002 treated mice at 6 weeks of TAC. (C-D) Circulating levels of free H2S and sulfane sulfure after 6 weeks of pressure overload-induced heart failure (TAC) in groups of mice maintained on a standard chow (TAC+Vehicle) or maintained on a chow containing the H2S donor SG-1002 (TAC+SG-1002, 20 mg/kg/day). (E-F) Myocardial levels of free H2S and sulfane sulfur in the experimental groups. Results are expressed as mean ± SEM. Numbers in bars represent the sample size. **p<0.01 and ***p<0.001 vs. Sham.

Figure 2

Deficiency of cystathionine gamma lyase (CSE) exacerbates cardiac dysfunction following TAC, whereas overexpression of CSE attenuates cardiac dysfunction. (A) Myocardial weights (mg/cm) and lung weights (mg/cm) expressed as ratio of tibia length at 12 weeks following TAC in wild-type (WT+TAC) mice, CSE deficient (CSE KO+TAC) mice, and CSE KO mice treated with SG1002 (CSE KO+TAC+SG-1002). (B) LV end-systolic diameter (LVESD in mm) and (C) LV ejection fraction (%) following TAC. (D) Myocardial weights (mg/cm) and lung weights (mg/cm) expressed as ratio of tibia length at 12 weeks of TAC in wild-type (WT+TAC) and cardiac specific CSE transgenic mice (CS-CSE Tg+TAC). (E) LVESD and (F) LV ejection fraction from 1 week to 12 weeks of TAC in WT and CS-CSE Tg mice. Results are expressed as mean ± SEM. †p<0.05, ‡p<0.01 and #p<0.001 vs. WT. *p<0.05, **p<.01, and ***p<0.001 vs. Baseline.

Figure 3

Exogenous H2S therapy prevents cardiac dilatation and dysfunction following TAC. (A) Chemical Structure of SG-1002. (B) Representative heart pictures of Sham, Vehicle (TAC+Vehicle), and SG-1002 (TAC+SG-1002) treated mice at 12 weeks of TAC. (C) The ratio of heart weight to tibia lengths. (D) The ratio of lung weight/tibia lengths. (E) Circulating BNP levels (ng/ml) at 6 and 12 weeks of TAC. (F) LVESD and (H) ejection fraction from 1 week to 12 weeks TAC. Results are expressed as mean ± SEM. *p<0.05 and ***p<0.001 vs. Baseline.

Figure 4

H2S attenuates the intermuscular and perivascular fibrosis following TAC. (A) Representative photomicrographs of Masson’s Trichrome, Picrosirius Red, and CD31 stained heart sections depicting intermuscular and perivascular fibrosis and vascular density in hearts from Sham, TAC+Vehicle, and TAC+SG-1002 treated mice at 6 weeks of TAC. (B) Summary of fibrosis area as % of the LV as calculated from Masson’s Trichrome sections. (C) Summary of fibrosis area as % of the LV calculated from the Picrosirius Red sections. (D) Summary of CD31+ vessels per area (mm2). Results are expressed as mean ± SEM. **p<0.01, and ***p<0.001 vs. Sham.

Figure 5

H2S upregulates Akt phosphorylation, VEGF expression, and activates the eNOS-NO pathway following TAC. (A) Representative immunoblots and densitometric analysis of (A-C) total Akt, Akt-PSer473, and Akt-PThr308, (D) VEGF (E-F) total eNOS and eNOS-PSer1177 in hearts from Sham, TAC+Vehicle, and TAC+SG-1002 at 6 weeks of TAC. (G) Nitrite and (H) cGMP levels in the hearts of the experimental groups at 6 weeks of TAC. Results are expressed as mean ± SEM.

Figure 6

SG-1002 does not provide protection in eNOS deficient mice. (A) Myocardial weights expressed as ratio of tibia length and (B) circulating BNP levels at 6 weeks of TAC in eNOS deficient (eNOS KO) sham, eNOS KO mice subjected to TAC (eNOS KO+TAC) and eNOS KO mice treated with SG-1002 (eNOS KO+TAC+SG-1002). (C) LVESD and (D) ejection fraction from 1 week to 6 weeks TAC. Results are expressed as mean ± SEM. **p<0.01 and ***p<0.001 vs. eNOS KO sham or Baseline.

Figure 7

Schematic diagram highlighting the proposed mechanism by which cystathionine gama lyase (CSE) or exogenous hydrogen sulfide protects the heart following transverse aortic constriction (TAC). Our data suggest (CSE) or hydrogen suflide donor therapy with SG-1002 activates vascular endothelial growth factor (VEGF) and subsequently phosphorylates Akt. Akt activation results in phosphorylation and activation of eNOS. Following eNOS activation nitric oxide (NO) and nitrite (NO2) bioavailability are increased in conjunction with increases in myocardial cGMP. These molecular signals result in reduced myocardial oxidative stress and injury, improvements in mitochondrial respiration, and decreased cardiac fibrosis. Ultimately, these cytoprotective actions prevent the transition from compensated to decompensated heart failure and left ventricular (LV) ejection fraction is preserved.