Mechanism and pharmacological rescue of berberine-induced hERG channel deficiency

Abstract

Berberine (BBR), an isoquinoline alkaloid mainly isolated from plants of Berberidaceae family, is extensively used to treat gastrointestinal infections in clinics. It has been reported that BBR can block human ether-a-go-go-related gene (hERG) potassium channel and inhibit its membrane expression. The hERG channel plays crucial role in cardiac repolarization and is the target of diverse proarrhythmic drugs. Dysfunction of hERG channel can cause long QT syndrome. However, the regulatory mechanisms of BBR effects on hERG at cell membrane level remain unknown. This study was designed to investigate in detail how BBR decreased hERG expression on cell surface and further explore its pharmacological rescue strategies. In this study, BBR decreases caveolin-1 expression in a concentration-dependent manner in human embryonic kidney 293 (HEK293) cells stably expressing hERG channel. Knocking down the basal expression of caveolin-1 alleviates BBR-induced hERG reduction. In addition, we found that aromatic tyrosine (Tyr652) and phenylalanine (Phe656) in S6 domain mediate the long-term effect of BBR on hERG by using mutation techniques. Considering both our previous and present work, we propose that BBR reduces hERG membrane stability with multiple mechanisms. Furthermore, we found that fexofenadine and resveratrol shorten action potential duration prolongated by BBR, thus having the potential effects of alleviating the cardiotoxicity of BBR.

Keywords: LQTS; berberine; cardiotoxicity; cavoline-1; hERG; pharmacological rescue.

Figure 1

BBR reduced hERG channel expression by disrupting cav-1 membrane stability. Notes: (A) Cav-1 expression levels in hERG-HEK293 cells under control conditions or 1 μM/10 μM BBR treatment for 24 hours. Cav-1 levels were reduced in a concentration-dependent manner. *P<0.05, ***P<0.001, n=5. (B) Cav-1 expression levels in hERG-HEK293 cells transfected with Ctl-siRNA or Cav-1-siRNA under control conditions or 10 μM BBR treatment for 24 hours. The level of cav-1 was successfully inhibited by using cav-1 specific siRNA. **P<0.01, n=5. (C) hERG channel expression levels in hERG-HEK cells transfected with Ctl-siRNA or Cav-1-siRNA under control conditions or 10 μM BBR treatment for 24 hours. Inhibition ratio of 155 kDa hERG protein by 10 μM BBR was diminished when cav-1 was knocked down. *P<0.05, n=5. Abbreviations: BBR, berberine; Cav-1, caveolin-1; Ctl, control; hERG, human ether-a-go-go-related gene; siRNA, small interfering RNA.

Figure 2

BBR-induced hERG channel deficiency was on account of Phe656 and Tyr652 binding. Notes: HEK293 cells were transfected with WT-hERG, Y652A-hERG, or F656V-hERG. (A) Western blot results and statistics of mutant hERG protein under control condition or 10 μM BBR treatment for 24 hours. Mature WT-hERG protein was inhibited by BBR, while F656V-hERG or Y652A-hERG was not affected. ***P<0.001, n=5. (B–D) Examples and I–V curves of WT-hERG and mutant hERG current under control condition or 10 μM BBR treatment. WT-hERG current was inhibited by BBR, while F656V-hERG or Y652A-hERG current was not affected. *P<0.05, n=10. Abbreviations: BBR, berberine; Ctl, control; hERG, human ether-a-go-go-related gene; F656V, phenylalanine to valine; WT, wild-type; Y652A, tyrosine to alanine.

Figure 3

Pharmacological rescue of hERG protein reduced by BBR. Notes: (A) Resveratrol (10 μM) upregulated 155 kDa hERG expression to normal levels after BBR treatment. *P<0.05 vs control and ^#P<0.05 vs BBR, n=5. (B) Astemizole (5 μM) upregulated 155 kDa hERG expression to normal levels after BBR treatment. *P<0.05 vs control and ^#P<0.05 vs BBR, n=5. (C) Fexofenadine (1 μM) upregulated 155 kDa hERG expression to normal levels after BBR treatment. *P<0.05 vs control and ^#P<0.05 vs BBR, n=5. hERG-HEK293 cells were cultured overnight under control conditions, 10 μM BBR group, co-incubation group, and rescue agent group. Abbreviations: Ast, astemizole; BBR, berberine; Ctl, control; Fex, fexofenadine; hERG, human ether-a-go-go-related gene; Res, resveratrol.

Figure 4

Pharmacological rescue of hERG current inhibited by BBR. Notes: (A) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 10 μM resveratrol. Inhibited hERG current induced by BBR was slightly rescued by resveratrol. *P<0.05 vs control and ^#P<0.05 vs BBR, n=10. (B) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 5 μM astemizole (washout, 1 hour). Inhibited hERG current induced by BBR was not rescued by 5 μM astemizole. **P<0.01 vs control, n=10. (C) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 1 μM fexofenadine. Inhibited hERG current induced by BBR was slightly rescued by 1 μM fexofenadine. *P<0.05 vs control and ^#P<0.05 vs BBR, n=10. hERG-HEK293 cells were cultured overnight under control conditions, 10 μM BBR group, co-incubation group, and rescue agent group. Abbreviations: Ast, astemizole; BBR, berberine; Ctl, control; Fex, fexofenadine; hERG, human ether-a-go-go-related gene; Res, resveratrol.

Figure 4

Pharmacological rescue of hERG current inhibited by BBR. Notes: (A) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 10 μM resveratrol. Inhibited hERG current induced by BBR was slightly rescued by resveratrol. *P<0.05 vs control and ^#P<0.05 vs BBR, n=10. (B) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 5 μM astemizole (washout, 1 hour). Inhibited hERG current induced by BBR was not rescued by 5 μM astemizole. **P<0.01 vs control, n=10. (C) Representative hERG currents and normalized I–V curves of tail current of control, BBR treatment, and rescue with 1 μM fexofenadine. Inhibited hERG current induced by BBR was slightly rescued by 1 μM fexofenadine. *P<0.05 vs control and ^#P<0.05 vs BBR, n=10. hERG-HEK293 cells were cultured overnight under control conditions, 10 μM BBR group, co-incubation group, and rescue agent group. Abbreviations: Ast, astemizole; BBR, berberine; Ctl, control; Fex, fexofenadine; hERG, human ether-a-go-go-related gene; Res, resveratrol.

Figure 5

Reverse effects of resveratrol and fexofenadine on BBR-induced APD prolongation. Notes: (A) Representative APD and statistics of NRVM under control (black line), 10 μM BBR (blue line), 10 μM BBR with 10 μM resveratrol (green line), and 10 μM BBR with 1 μM fexofenadine (red line) treatment. ***P<0.05 vs control and ^###P<0.05 vs BBR, n=6. (B) Representative action potential traces of guinea pig ventricular cardiomyocytes under control (black line), 10 μM BBR (green line), and 10 μM BBR +10 μM resveratrol (red line) treatment. ***P<0.001 vs control and ^##P<0.01 vs BBR, n=4. Abbreviations: APD, action potential duration; BBR, berberine; Ctl, control; Fex, fexofenadine; NRVM, neonatal rat ventricular myocytes; Res, resveratrol.