Honokiol Inhibits Atrial Metabolic Remodeling in Atrial Fibrillation Through Sirt3 Pathway

Abstract

Background and Purpose: Atrial metabolic remodeling plays a critical role in the pathogenesis of atrial fibrillation (AF). Sirtuin3 (Sirt3) plays an important role in energy homeostasis. However, the effect of Sirt3 agonist Honokiol (HL) on AF is unclear. Therefore, the aim of this study is to determine the effect of HL on atrial metabolic remodeling in AF and to explore possible mechanisms. Experimental Approach: irt3 and glycogen deposition in left atria of AF patients were examined. Twenty-one rabbits were divided into sham, P (pacing for 3 weeks), P + H treatment (honokiol injected with pacing for 3 weeks). The HL-1 cells were subjected to rapid pacing at 5 Hz for 24 h, in the presence or absence of HL and overexpression or siRNA of Sirt3 by transfection. Metabolic factors, circulating metabolites, atrial electrophysiology, ATP level, and glycogens deposition were detected. Acetylated protein and activity of its enzymes were detected. Key Results: Sirt3 was significantly down-regulated in AF patients and rabbit/HL-1cell model, resulting in the abnormal expression of its downstream metabolic key factors, which were significantly restored by HL. Meanwhile, AF induced an increase of the acetylation level in long-chain acyl-CoA dehydrogenase (LCAD), AceCS2 and GDH, following decreasing of activity of it enzymes, resulting in abnormal alterations of metabolites and reducing of ATP, which was inhibited by HL. The Sirt3 could regulate acetylated modification of key metabolic enzymes, and the increase of Sirt3 rescued AF induced atrial metabolic remodeling. Conclusion and Implications: HL inhibited atrial metabolic remodeling in AF via the Sirt3 pathway. The present study may provide a novel therapeutical strategy for AF.

Keywords: Honokiol; acetylation; atrial fibrillation; metabolism remodeling; sirt3.

FIGURE 1

Changes of Sirt3 and glycogen in AF patients. (A,B and C) Representative images and statistical results for protein expression of Sirt3 in the atria of patients. (D,E and F) Representative images and statistical results for the accumulation of glycogen in both groups of patients. (G and H) Representative bands and statistical analyzing for protein expression of Sirt3 in patiets. The magnification is ×20. * p < 0.05 vs. SR group, Data from these proteins were normalized to β-actin, n = 6 each group.

FIGURE 2

Electrophysiology detection and assay of glycogen and ATP. (A) AF was induced after rapid-pacing. (B,C) AERP150 ms and AERP200 ms, (D) AF inducibility. n = 6 each group. (E and F) Representative images and statistical results of the glycogen accumulation in the atria of rabbits, the magnification is ×20. (G and H) Statistical analyzes of activity in ATP enzyme and level of ATP. *p < 0.05 vs. sham group,**p < 0.01 vs. sham group, #p < 0.05 vs. P group, n = 6 each group.

FIGURE 3

HL inhibited the remodelling of metabolic factors and acetylation level of key metabolic enzyme in rabbit of AF model. (A) Representative bands of protein expression of Sirt3, AceCS2, Ac-LACD and Ac-AceCS2. (B,C,D,E,F) Quantification analyzing of protein expression of Sirt3, AceCS2, Ac-LACD and Ac-AceCS2. (G and H) Statistical results for ratio of Ac-LCAD/LCAD and Ac-AceCS2/AceCS2. (I) Statistical results for expression of Sirt3 gene.(K) Statistical results for expression of LCAD, CROT, PGC1α,PDH,PDK4, LDHa, AceCS2, NDUFA9, SDHa and GDH genes. Data from these proteins were normalized to β-actin, and genes were normalized to β-actin. *p < 0.05 vs. sham group, **p < 0.01 vs. sham group, ***p < 0.001 vs. sham group; #p < 0.05 vs. P group, ##p < 0.01 vs. P group, n = 6 each group.

FIGURE 4

HL inhibited abnormal expression and acetylation of key metabolic enzyme and restored the activity of these enzymes during AF. (A and B) Representative bands and statistical results of Sirt3, LCAD, AceCS2, GDH, Ac-LCAD, Ac-GDH in pacing-HL-1 cells. Data from these proteins were normalized to β-actin. (C,D and E) Statistical results for activity of LCAD, GDH and AceCS2 in pacing-HL-1cells. *p < 0.05 vs. sham group, #p < 0.05 vs. pacing group, n = 3 each group. (F,G and H) Statiscal results for activity of LCAD, GDH and AceCS2 in all groups of rabbits. n = 5 each group.

FIGURE 5

Sirt3 siRNA up-regulated the acetylation level of metabolic enzymes. (A) Representative bands of Sirt3, pho-AMPKα1, AMPKα1, GDH, Ac-GDH (Ac-gdh), LCAD and Ac-LCAD expression. (B) Statistical results for Sirt3 expression. (C,D) Statistical results for pho-AMPKα1and AMPKα1 expression. (E,F) Statistical results for GDH and Ac-GDH expression. (G,H) Statistical results for LCAD and Ac-LCAD expression. *p < 0.05 vs. control group, **p < 0.05 vs. control group, ***p < 0.05 vs. control group, n = 3 each group.

FIGURE 6

The acetylation level of key metabolic enzymes controlled by Sirt3 pathway in vitro. (A) Representative bands for the protein expression of Sirt3, GDH, Ac-GDH, LCAD, Ac-LCAD, pAMPK, AMPK in the HL-1 cells of all groups. (B,C,E,I,J) Statistical analysis of Sirt3, GDH, LCAD, pAMPK1α and AMPK1α expression. (D,F,G,H) Statistical analysis of Ac-GDH, Ac-LCAD, Ac-GDH/GDH, Ac-LCAD/LCAD. *p < 0.05, **p < 0.01, n = 3 each group.