Chelerythrine triggers the prolongation of QT interval and induces cardiotoxicity by promoting the degradation of hERG channels

Figures

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  Figure 1. The effects of CHE and hypoxia on QT interval and action potential duration (APD) in guinea pigs were measured at animal level.A, electrocardiogram of guinea pigs. B and C, effects of CHE and hypoxia on QT/QTc interval in guinea pigs (n = 5). D, representative map of optical mapping of guinea pigs. E and F, effects of CHE and hypoxia on APD50 and APD90 in guinea pigs (n = 5). Shared control and CHE (8 mg kg−1) group with Fig. S2. G, schematic diagram of experimental design. H and I, representative and statistical results of QT interval (n = 5). The data presented here were representative of five independent experiments. Error bars were represented as mean ± SD, ordinary one-way ANOVA was used for significance test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. CHE, chelerythrine.

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  Figure 2. The effects of CHE and hypoxia on hERG channel proteins and currents in HEK293–hERG cells.A, the structure of the drug. B, the effect of CHE on the expression of hERG channel protein. C, the voltage-clamp protocol and representative hERG current traces. D and E, I–V curve of the tail current density was the tail current normalized to the capacitance of each cell and maximal current amplitude of control group (n = 11). E, ∗p < 0.05 compared with their corresponding control (0 μM group), #p < 0.05 compared with their corresponding CHE incubation group (10 μM group). F, voltage-clamp protocol and representative current tracing for steady-state inactivation. G, voltage-clamp protocol and representative current tracing for onset of inactivation (left) and recovery from inactivation (right). H, normalized steady-state inactivation curves (n = 4). I, the time constant for inactivation and recovery (n = 4). The hERG activation and inactivation–voltage relationships were fitted with a Boltzmann function, y = 1/{1 + exp [(V1/2 − V)/k]}, where y is the normalized peak conductance, V1/2 is the half-activation or half-inactivation potential, V is the variable (test) voltage, and k is the…

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  Figure 3. Identify the degradation pathway of hERG channel inhibition by CHE in HEK293–hERG cells and neonatal rat cardiomyocytes (NRCMs).A–C, microscopic representation of hERG channels and ubiquitin molecules. Confocal images of cells stained with hERG antibodies (magenta); confocal laser scanning microscopy images of cells stained by Ubiquitin antibodies (green); 4′,6-diamidino-2-phenylindole (nucleus, blue); scale bar represents 50 μm, n = 3. Proteasome inhibitor MG132 does not affect the (D) hERG ubiquitination (n = 4) and (F) protein content (n = 6) changes and lysosomal inhibitor Baf increased (E) hERG channel ubiquitination modification (n = 4) and restored (G) hERG protein expression levels (n = 6) caused by CHE by Western blot. H, CHE regulates the expression of hERG protein through K63-linked polyubiquitination (n = 4). The data presented here were representative of a minimum of three independent experiments. Error bars were represented as mean ± SD, ordinary one-way ANOVA was used for significance test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. CHE, chelerythrine; hERG, human ether-a-go-go-related gene.

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  Figure 4. The regulation of hERG channel protein expression by histone deacetylase HDAC6 was determined.A and B, colocalization of hERG channels with histone deacetylase HDAC6 and ubiquitin (Ub) molecules. Confocal images of cells stained with HDAC6 and hERG antibodies (top) and confocal images of cells stained with HDAC6 and Ub antibodies (bottom), scale bar represents 50 μm, n = 5. C, the effects of CHE and hypoxia on HDAC6 protein expression (n = 5). Western blot representation and statistical results of (D) hERG protein primary antibody precipitation HDAC6 or (E) HDAC6 protein primary antibody precipitation hERG (n = 3). Microscopic representation of (F) hERG channels and Ub molecules and (G) expression of hERG channel protein knockdown of HDAC6 (n = 6). Confocal images of cells stained with hERG antibodies (magenta); confocal laser scanning microscope image of cells stained with Ub antibodies (orange); GFP-shRNA-HDAC6 (green), scale bar represents 50 μm, n = 3. The data presented here were representative of a minimum of three independent experiments. Error bars were represented as mean ± SD, ordinary one-way ANOVA was used for significance test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.…

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  Figure 5. The effects of CHE and hypoxia on the number and function of lysosomes.A and B, confocal micrograph of hERG channels and lysosome markers. LAMP1 (lysosome marker, left), n = 4, LAMP2 (lysosome marker, right), n = 3, scale bar represents 50 μm. C–E, effect of CHE and hypoxia on lysosome acidity. (Green: Lyso-Tracker Green, Red: Lyso-Tracker Red, scale bar represents 50 μm, n = 6). The data presented here were representative of a minimum of three independent experiments. Error bars were represented as mean ± SD, ordinary one-way ANOVA was used for significance test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. CHE, chelerythrine; hERG, human ether-a-go-go-related gene; LAMP, lysosome-associated membrane protein.

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  Figure 6. The lysosomal degradation mechanism of drug inhibiting hERG channels. hERG, human ether-a-go-go-related gene.