Obesity-induced activation of NADPH oxidase 2 prolongs cardiac repolarization via inhibiting K+ currents

Figures

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  Fig 1. Basic information of two groups of wild-type mice. A: Photos visually display the obesity phenotype of mice. B: Body weights in control and HFD wild-type mice. C: Fat weight, lean weight, and free body fluid content among the two groups. D: Representative whole heart and left ventricle section stained with hematoxylin and eosin from control and HFD wild-type mice. Scale Bars, 500 μm (top panel) and 50 μm (lower panel). E: Quantification of cardiomyocyte cross-section area of the two groups. F: Representative Masson’s Trichrome stained sections from control and HFD wild-type mice. Scale Bars, 50 μm. G: Scatter plot of collagen content in the ventricular area of the two groups. H: Echocardiograms of the two groups. I: ECG recordings from representative mice of the two groups. QT intervals are indicated below the records. Scattered data of R-R interval (J), QRS width (K), QT interval (L) and QTc (M) in the 2 groups at 20weeks. For A–C, n = 12 animals per group; for D–G, n = 4 animals per group; for H–M, n = 8 animals per group. *p<0.05, **p<0.01, ***p<0.001. HFD, high-fat diet.

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  Fig 2. Ventricular action potential duration (APD) is prolonged in HFD mice. A: The mouse heart perfused through retrograde aortic catheterization. B: The digested single ventricular cardiomyocytes. C: Representative AP tracings in control and HFD mice. D: APD at 20%, 50%, and 90% repolarization. Resting membrane potential (RMP) (E) and action potential amplitude (APA) (F) were not different among control and HFD mice. n = 8–11 cells; three hearts per group. *p<0.05, **p<0.01. HFD, high-fat diet.

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  Fig 3. Peak outward K+ (Ipeak) and fast transient outward K+ (Ito) currents were reduced in HFD mice. A: Outward K+ currents recorded from control (black) and HFD (red) mouse ventricular myocytes were evoked during 300-ms depolarizing voltage steps. B: Voltage relationship (I-V curves) in control and HFD mice. C: Ipeak at +70 mV in control and HFD mice. D: Representative Ito recorded from ventricular myocytes of control (black) and HFD (red) mice. E: Voltage relationship (I-V curves) in the two groups. F: Ito at +70 mV in the two groups. G, H and I: Steady-state activation (SSA), steady-state inactivation (SSI), and recovery from inactivation (RFI) curves of Ito. J: Voltage relationship (I-V curves) of IK,slow in the two groups. K: IK,slow at +70 mV in the two groups. L: Analyzed fast (lower series) and slow (upper series) inactivation time constants (τ values) from currents. M: Representative Western blots of KV4.2, KChIP2 and KV1.5. N: Quantification of KV4.2. O: Quantification of KChIP2. P: Quantification of KV1.5. for A-L, n = 8 cells; three or four hearts per group. for M-P, n = 4 animals per group. *p<0.05, **p<0.01.

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  Fig 4. The consequences of an HFD were not attributed to inward-rectifier K+ current. A: Representative IK1 in the two groups. B: Voltage relationship (I-V curves) in the two groups. C: IK1 at –140 mV in the two groups. D: Representative Western blots of Kir2.1. E: Quantification of Kir2.1. for A-C, n = 8 cells; three hearts per group. for D-E, n = 4 animals per group.

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  Fig 5. HFD causes activation of Nox2. A: Representative Western blots of Nox2 in control and HFD mice. B: Quantification of Nox2. C: Representative Western blots of p22phox, p47phox, p67phox, and Rac1. D: Quantification of p22phox. E: Quantification of p47phox. F: Quantification of p67phox. G: Quantification of Rac1. n = 4 animals per group. *p<0.05, **p<0.01. HFD, high-fat diet.

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  Fig 6. Genetic ablation of Nox2-rescued Ipeak and Ito inhibited by HFD. A: Representative Western blots of Nox2 in KO and WT mice. B: Quantification of Nox2 in KO and WT mice. C: Body weights in Flox and KO mice. D: Representative Ipeak recorded from Flox (grey) and KO (blue) ventricular myocytes of mice from the four groups. E: Voltage relationship (I-V curves) in the four groups. F: Ipeak at +70 mV in the four groups. G: Representative Ito recorded from Flox (grey) and KO (blue) ventricular myocytes of mice from the four groups. H: Voltage relationship (I-V curves) in the four groups. I: Ito at +70 mV in the four groups. J: Analyzed fast (lower series) and slow (upper series) inactivation time constants (τ values) from four groups. For A and B, n = 4 animals per group; for C, n = 12 animals per group; for D-J, n = 8 cells; three or four hearts per group. *p<0.05, **p<0.01, ***p<0.001 vs. KO-Ctrl; #p<0.05, ###p<0.001 vs. Flox-Ctrl. HFD, high-fat diet; KO, knockout.