Sacubitril/valsartan attenuates atrial conduction disturbance and electrophysiological heterogeneity with ameliorating fibrosis in mice

Figures

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  Figure 1. Physiological and molecular assessments of murine hearts with pressure overload showed the beneficial effects of SacVal. (A) Representative whole images of murine hearts underwent with sham surgery, TAC surgery (Vehicle; Veh), TAC surgery followed by valsartan (Val) or sacubitril/valsartan (SacVal) treatment. (B) Ventricular weight (left) and left atrial (LA) weight (right) normalized with body weight (n = 13–21). (C) Representative UCG images of M-mode. (D) Measured UCG parameters are shown from left to right, anterior wall thickness, left ventricular diastolic diameter, fractional shortening, and ejection fraction (n = 14–23). (E) Gene expression of Nppa (in atria), Nppb (in ventricles) and Nppc (in atria) (n = 6). (F,G) Gene and protein expression of Mme (encoding neprilysin) (n = 6). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. Error bars, SEM. *P < 0.05; **P < 0.01; ***P < 0.001. TAC, transverse aortic constriction; UCG, ultrasound echocardiography.

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  Figure 2. Electrophysiological assessment and AF inducibility in TAC mice. (A) Representative electrocardiograms of the induction and the termination of AF for each group. (B) P-wave duration (left) and QRS duration (right) (n = 15–17). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. Error bars, SEM. *P < 0.05. The inducibility of AF (C) and the duration of induced AF (D) in each group (n = 17–22). Chi-square test adjusted with the Benjamini-Hochberg procedure was performed. *P < 0.05. Veh, vehicle; Val, valsartan; SacVal, sacubitril/valsartan; AF, atrial fibrillation; TAC, transverse aortic constriction.

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  Figure 3. Optical mapping revealed that SacVal ameliorated the APD prolongation and the conduction disturbance. (A) Representative optical image and an atrial isochronal map overlaid. (B) A schema of signal processing to construct CV, APD and AP rise time map. (C) Representative electrophysiological maps of CV, APD90, and AP rise time at PCL 100 ms and 40 ms. APD90, CV, and AP rise time at PCL 100 ms (D–F) and PCL 40 ms (G–I) for each group (n = 7–10). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. *P < 0.05; **P < 0.01. Val, valsartan; SacVal, sacubitril/valsartan; AP, action potential; APD, action potential duration; CV, conduction velocity; PCL, pacing cycle length.

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  Figure 4. Restitution of APD, CV, and AP rise time. The restitution curves for (A) APD90, (B) CV, and (C) AP rise time. Multiple comparisons were performed using a two-way ANOVA with Tuckey's post-hoc test. Data are presented as mean ± SEM. *P < 0.05, †P < 0.01 relative to vehicle. Val, valsartan; SacVal, sacubitril/valsartan; AP, action potential; APD, action potential duration; CV, conduction velocity.

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  Figure 5. Sacval attenuated the enhanced heterogeneity of CV and AP rise time. The rate dependence of the coefficient of variation derived from (A) APD90, (B) CV, and (C) AP rise time maps (n = 6–8). Multiple comparisons within and between groups were performed using one-way and two-way ANOVA respectively, with Tuckey's post-hoc test. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. †P < 0.05 relative to vehicle at PCL 40 ms. Val, valsartan; SacVal, sacubitril/valsartan; AP, action potential; APD, action potential duration; CV, conduction velocity.

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  Figure 6. Sacval ameliorated the fibrotic change against murine cardiac remodeling. (A) Representative images of Masson's trichrome staining. Magnifying power of 40×. Scale bar = 50 µm. (B) Quantification of atrial fibrosis area (%) (n = 6–8). (C) Relative gene expression regarding fibrosis: Tgfb1, Col1a2, and Col3a1 (n = 6). (D) Western blotting images of Ser423/425-phosphorylated and non-phosphorylated SMAD3. (E) Relative protein expression of pSMAD3 to GAPDH (n = 6). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. Error bars, SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Val, valsartan; SacVal, sacubitril/valsartan.

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  Figure 7. BNP and CNP suppressed the activation of primary adult cardiac fibroblasts. (A) The protocol of the in vitro assay. (B) Effects of ANP (left), BNP (middle) or CNP (right) (at 1.0 µM each) against the pro-fibrotic alteration of Ccn2 and Acta2 induced by TGFβ (n = 5–8). (C) Additional effect of LBQ657 (50 µM) on BNP (left) or CNP (right) (5.0 µM each) (n = 6–7). (D) Antagonizing actions of P19 (1.0 µM) on CNP (1.0 µM) added with LBQ657 (10 µM) (n = 6). (E) Effects of AP811 (1.0 µM) on CNP (1.0 µM) with LBQ657 (10 µM) (n = 7). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. Error bars, SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; TGFβ, transforming growth factor β.