Hydrogen sulfide alleviates high-salt-stimulated myocardial fibrosis through inhibiting hypoxia-inducible factor-1α

Abstract

Background: Endogenous hydrogen sulfide (H₂S) and its key generating enzyme, cystathionine β-synthase (CBS), prevent vascular remodeling and damage to target organs during the advancement of hypertension induced by a high-salt diet.

Objective: The contribution of the H₂S/CBS pathway to high-salt-induced myocardial fibrosis (MF) was explored, with a focus on the mechanistic involvement of hypoxia-inducible factor-1α (HIF-1α).

Methods: We used primary rat cardiac fibroblasts stimulated with high-salt medium and an MF model induced by a high-salt diet in Dahl salt-sensitive rats. Sodium hydrosulfide (NaHS), a commonly used H₂S donor, was administered in vitro at 100 μmol/L and in vivo at 90 μmol/kg to maintain adequate H₂S levels. An HIF-1α stabilizer, dimethyloxalylglycine (DMOG), was used to maintain the HIF-1α protein level. The H₂S/CBS pathway was followed using Western blotting and a sulfide-sensitive probe. The extent of MF was examined using histological and immunofluorescence staining techniques, including Sirius red and Masson trichrome. We performed Western blot analysis to measure fibrosis-related protein and HIF-1α protein levels.

Results: High-salt exposure reduced H₂S production and downregulated CBS protein expression in cardiac fibroblasts both in vitro and in vivo. In vitro, the H₂S donor inhibited the activation of cardiac fibroblasts triggered by high-salt conditions, while in vivo, it alleviated MF in salt-sensitive rats. From a mechanistic standpoint, high-salt exposure markedly upregulated HIF-1α expression. However, this increase was reversed by pretreatment with H₂S. Furthermore, the HIF-1α stabilizer DMOG blocked the H₂S-induced reduction in HIF-1α protein levels and consequently abolished the antifibrotic effect of H₂S on cardiac fibroblasts exposed to high-salt conditions.

Conclusion: In conclusion, H₂S attenuates high-salt-induced MF by suppressing fibroblast activity and collagen synthesis, potentially via downregulation of HIF-1α.

Keywords: HIF-1α; cardiac fibroblasts; high-salt diet; hydrogen sulfide; myocardial fibrosis.

FIGURE 1

High-salt stimulation downregulated the endogenous H₂S/CBS pathway in cardiac fibroblasts in vitro and in rat myocardial tissues in vivo. (A) H₂S concentration in the supernatant of cardiac fibroblasts detected by the free radical analyzer TBR4100 (n = 7 per group). (B) Western blot analysis of CBS protein expression in cardiac fibroblasts (n = 9 per group). (C) qRT-PCR analysis of CBS mRNA levels in cardiac fibroblasts (n = 9 per group). (D) Western blot analysis of CSE and MPST protein expression in cardiac fibroblasts (n = 6 per group). (E) Western blot analysis of CBS protein expression in the myocardial tissues of Dahl rats (n = 10 per group). Results are expressed as mean ± SD. *P < 0.05; **P < 0.01; ns, not significant. H₂S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; MPST, mercaptopyruvate sulfurtransferase.

FIGURE 2

H₂S significantly attenuated myocardial fibrosis in Dahl rats fed with a high-salt diet in vivo. (A) Schematic representation of the design of the animal experimental process, including the intraperitoneal injection of NaHS and different dietary interventions. (B) Representative Masson trichrome and Sirius red staining. In the Masson trichrome staining, the collagen area is blue, and the collagen area is red in the Sirius red staining. (C) Quantification of Masson trichrome and Sirius red staining (n = 9 per group). Scale bar = 50 μm. (D) Western blot analysis of collagens I and III in the myocardial tissues of Dahl rats (n = 10 per group). Results are expressed as mean ± SD. *P < 0.05; **P < 0.01.

FIGURE 3

H₂S attenuates high salt-induced cardiac fibroblast activation and collagen deposition via suppression of HIF-1α. (A) Western blot analysis of collagen III, α-SMA, and PCNA protein expression (n = 9 per group). (B) qRT-PCR analysis of Col3a1, Acta2, and Pcna mRNA levels in cardiac fibroblasts (n = 9 per group). (C) Western blot analysis of collagen III and α-SMA protein expression in cardiac fibroblasts treated with NaHS for 12 h, 24 h, and 48 h (n = 6 per group). (D) Western blot analysis of HIF-1α protein level in the myocardial tissues of Dahl rats (n = 10 per group). (E,F) Protein (E) and mRNA (F) expression of HIF-1α in cardiac fibroblasts (n = 6 per group). (G) qRT-PCR analysis of HIF1α-targeted key glycolytic enzyme genes in cardiac fibroblasts (n = 9 per group). Results are expressed as mean ± SD. *P < 0.05; **P < 0.01; ns, not significant. HIF-1α, hypoxia-inducible factor-1α; H₂S, hydrogen sulfide.

FIGURE 4

DMOG upregulated HIF-1α protein level, and reversed the inhibitory effect of H₂S on cardiac fibroblast activation and collagen deposition. (A,B) Protein (A) and mRNA (B) expression of HIF-1α in cardiac fibroblasts (n = 9 per group). (C) Representative immunofluorescence images and quantification of collagen I, collagen III, and α-SMA protein levels in cardiac fibroblasts; n = 9; Scale bar = 50 μm. (D) qRT-PCR analysis of Col1a1, Col3a1, and Acta2 mRNA levels in cardiac fibroblasts (n = 9 per group). Results are expressed as mean ± SD. *P < 0.05; **P < 0.01; ns, not significant. DMOG, dimethyloxalylglycine; HIF-1α, hypoxia-inducible factor-1α.

FIGURE 5

H₂S alleviated high salt-induced HIF-1α protein level and collagen deposition in NIH3T3 fibroblasts and primary cardiomyocytes. (A) Western blot analysis of HIF-1α protein level in NIH3T3 fibroblasts (n = 6 per group). (B) Western blot analysis of collagen III, α-SMA, and PCNA protein expression in NIH3T3 fibroblasts (n = 9 per group). (C) Western blot analysis of HIF-1α protein level in primary cardiomyocytes (n = 6 per group). Results are expressed as mean ± SD. *P < 0.05; **P < 0.01. HIF-1α, hypoxia-inducible factor-1α.

FIGURE 6

Schematic diagram. Downregulation of the endogenous H₂S/CBS pathway is an important mechanism underlying high salt-induced MF. High salt downregulates CBS protein expression, subsequently decreasing myocardial H₂S generation, facilitating HIF-1α protein expression, promoting cardiac fibroblast activation and collagen deposition, and ultimately leading to MF. H₂S, hydrogen sulfide; CBS, cystathionine β-synthase.