Protective Effects of SIRT2 Inhibition on Cardiac Fibrosis

Abstract

Background: A primary factor in the pathogenesis of aging is oxidative stress, with cardiac inflammation and fibrosis being contributed to by increased oxidative stress as organisms age. Oxidative stress enhances the cardiac fibrotic signaling pathway, with reactive oxygen species inducing cardiac fibrosis through increased expression of the profibrotic factor transforming growth factor-beta 1 (TGF-β1). Furthermore, Wnt/β-catenin signaling pathway is implicated in interstitial fibrosis, which is associated with TGF-β. Sirtuin 2 (SIRT2) is expressed in heart tissue, with protective effects in pathological cardiac hypertrophy. We aimed to investigate the mechanisms of cardiac fibrosis in D-Galactose (D-Gal)-induced accelerated aging, focusing on TGF-β1, β-catenin, and SIRT2.

Methods: A total of 30 young male Sprague-Dawley rats were randomly divided into 4 groups: control group, D-Gal group, D-Gal + 4% dimethyl sulfoxide (DMSO) group, and D-Gal + the SIRT2 inhibitor (AGK2) group. After 10 weeks, the rats were sacrificed, and their hearts were removed. SIRT2 expression levels were measured by western blot and gene expression levels of TGF-β1 and β-catenin by quantitative real-time polymerase chain reaction.

Results: Transforming growth factor-beta 1 (TGF-β1) mRNA expression in heart tissue was higher in the D-Gal group compared to all other groups. β-catenin mRNA expression was higher in the D-Gal group than in the D-Gal + AGK2 group. SIRT2 protein expression was higher in the D-Gal + DMSO group compared to the control group. Sirtuin 2 expression was lower in the D-Gal + AGK2 group compared to the D-Gal and D-Gal + DMSO groups.

Conclusion: Sirtuin 2 inhibition attenuates fibrosis, as evidenced by the downregulation of TGF-β1 and β-catenin. Thus, targeting SIRT2 may represent a potential therapeutic strategy for diseases characterized by cardiac fibrosis in the future.

Figure 1.

Relative mRNA expression of transforming growth factor-beta 1 (A) (TGF-β1) and (B) β-catenin in heart tissue among groups. *Transforming growth factor-beta 1 mRNA expression was significantly higher in the D-Gal group compared to the control, D-Gal + DMSO, and D-Gal + AGK2 groups (P = .04, P = .01, P = .004, respectively). ^#β-catenin mRNA expression was higher in the D-Gal group than the D-Gal + AGK2 group (P = .002).

Figure 2.

Representative western blots for (A) SIRT2 and β-actin, and (B) relative SIRT2 protein expression in heart tissue among groups. *Sirtuin 2 expression was higher in the D-Gal + DMSO group compared to the control group (P = .015). ^#ψSIRT2 expression was lower in the D-Gal + AGK2 group compared to the D-Gal and D-Gal + DMSO groups (P = .039, P = .007, respectively).

Figure 3.

Representative staining micrographs of (A, B) Hematoxylin–Eosin (H&E, ×200) and (C) Masson’s Trichrome (Masson, ×200). The evaluation of (D) cardiomyocyte diameter and (E) fibrotic area. Cardiomyocytes were in a slightly more regular form in the D-Gal + AGK2 group (A). The cardiomyocyte diameter (B, D) and the percentage of fibrotic area (C, E) were *higher in the D-Gal and D-Gal + AGK2 groups compared to the control group (P < .001, P < .001, respectively); ^#lower in the D-Gal + DMSO and D-Gal + AGK2 groups compared to the D-Gal group (P < .001, P < .001, respectively); and ^ψhigher in the D-Gal + AGK2 group compared to the D-Gal + DMSO group (P < .001). No significant differences were observed in cardiomyocyte diameter and percentage of fibrotic area between the control and D-Gal + DMSO groups (P = .165, P = .795, respectively).