Hydrogen sulfide attenuates cardiac dysfunction after heart failure via induction of angiogenesis

Abstract

Background: Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro models and to promote vessel growth in the setting of hindlimb ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donor, diallyl trisulfide, in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis.

Methods and results: Transverse aortic constriction was performed in mice (C57BL/6J; 8-10 weeks of age). Mice received either vehicle or diallyl trisulfide (200 µg/kg) starting 24 hours after transverse aortic constriction and were followed up for 12 weeks using echocardiography. H2S therapy with diallyl trisulfide improved left ventricular remodeling and preserved left ventricular function in the setting of transverse aortic constriction. H2S therapy increased the expression of the proangiogenic factor, vascular endothelial cell growth factor, and decreased the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial NO synthase and the bioavailability of NO. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts.

Conclusions: These results suggest that H2S therapy attenuates left ventricular remodeling and dysfunction in the setting of heart failure by creating a proangiogenic environment for the growth of new vessels.

Keywords: H2S donor; angiogenesis; diallyl trisulfide; endothelial nitric oxide synthase; nitric oxide.

Figures

Figure 1. DATS Increases Sulfide Levels Following TAC

(A) Mice (C57 BL6/J) were subjected to transverse aortic constriction (TAC) surgery and were studied for a period of 12 weeks following TAC. The H2S donor, Diallyl trisulfide (DATS), was initiated at a dose of 200 μg/kg (daily i.p. injection) at 24 hours following TAC surgery. Vehicle-treated mice were administered 1% DMSO. Baseline echocardiography was performed 1-week prior to TAC and thereafter at 1, 3, 6, 9, and 12 weeks following TAC. Additional mice were sacrificed at 6 weeks post TAC. Heart and lung tissue were collected for assessment of cardiac and lung weights, cardiac histology, and myocardial molecular determinations. In addition, blood samples were also collected for measurement of nitric oxide intermediates. Representative gas chromatograph peaks and summary data for (B-D) serum and (E-G) myocardial levels of free H2S and sulfane sulfur after 1 week of TAC in groups of mice injected daily with vehicle or DATS. Results are expressed as mean ± SEM. Numbers in bars represent sample size.

Figure 2. DATS Prevents Adverse Cardiac Remodeling and heart failure after TAC

(A) Inter-ventricular septal wall thickness (IVSd, mm), (B) Left ventricular end-diastolic diameter (LVEDD, mm) (C) Left venricular end-systolic diameter (LVESD, mm) and (D) Left ventricular ejection fraction (LVEF, %) as determined by echocardiography at baseline and from 1 to 12 weeks of TAC. (E) Heart weights (mg/cm) and (F) lung weights (mg/cm) expressed as ratio of tibia length at 6 weeks following TAC. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001 vs. Baseline.

Figure 3. DATS Attenuates Myocardial Fibrosis after TAC

(A) Representative photomicrographs of Picrosirius Red and Masson’s Trichrome stained heart sections depicting perivascular intermuscular fibrosis in hearts from Sham, Vehicle, and DATS treated mice at 6 weeks of TAC. Summary of fibrosis area as % of the LV calculated from (B) Picrosirius Red sections and (C) Masson’s Trichrome sections. Scale bar equals 50-μm. Results are expressed as mean ± SEM.

Figure 4. DATS Augments Myocardial Angiogenic Factors

(A) Representative immunoblots and densitometric analysis for PECAM-1 (CD31) and VEGF-A expression in hearts from Sham, Vehicle, and DATS-treated mice. Representative immunoblots and densitometric analysis for myocardial (B) basic fibroblast growth factor (bFGF) and (C) angiostatin expression in hearts from Sham, Vehicle, and DATS-treated mice. Horizontal solid lines define noncontiguous gel lanes. Results are expressed as mean ± SEM.

Figure 5. DATS Increases Vascular Density after TAC

(A-B) Representative photomicrographs of von Willebrand factor (vWF), CD31, and Ki67 stained heart sections from Sham, Vehicle, and DATS treated mice at 6 weeks of TAC. Staining. (C) Summary of capillary density as determined by von Willebrand staining. (D) Angiogenesis index expressed as a ratio between CD31 and DAPI positive regions. (E) Cellular proliferation determined as the ratio between Ki67 and DAPI positive areas. Results are expressed as mean ± SEM.

Figure 6. DATS upregulates Akt phosphorylation and activates the eNOS-No pathway after TAC

Representative immunoblots and densitometric analysis of (A) phosphorylated Akt at serine residue 473 (Akt-PSer473) and total Akt, (B) phosphorylated AMPK at threonine residue 172 (AMPK-PThr172) and total AMPK, and (C) phosphorylated eNOS at serine residue 1177 (eNOS-PSer1177), phosphorylated eNOS at threonine residue 495 (eNOS-PThr495), and total eNOS in hearts from Sham, Vehicle, and DATS-treated mice at 6 weeks of TAC. (D) Plasma and myocardial nitrite levels (μM) at 6 weeks following TAC. Results are expressed as mean ± SEM.

Figure 7. DATS upregulates GPx-1 and HO-1 after TAC

Representative immunoblots and densitometric analysis of (A) glutamate-cysteine ligase (GCLC) and Glutathione Peroxidase 1 (GPx-1) and (B) heme oxygenase 1 (HO-1) and HO-2 in hearts from Sham, Vehicle, and DATS-treated mice at 6 weeks of TAC. Results are expressed as mean ± SEM.