Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy

Abstract

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal β-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and β-catenin. A pharmacological activator of the WNT/β-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and β-catenin, and evidence for targeted activation of the WNT/β-catenin pathway as a potential treatment for this disease.

Keywords: Arrhythmias; Cardiology; Cardiovascular disease; Cell Biology; Genetic diseases.

Figure 1. Deceased proband harboring AnkB loss-of-function p.Glu1458Gly variant exhibits arrhythmogenic cardiomyopathy.

(A) Surface 12-lead ECG. (B) Loop recording demonstrating nonsustained polymorphic VT. (C) Cardiac magnetic resonance short-axis image revealing moderate biventricular dilation; the yellow ring highlights focal “crinkling” of the subtricuspid region of the right ventricular free wall consistent with the “accordion sign.” (D) Delayed enhancement imaging revealed scarring in the right ventricular (RV) free wall in a short-axis view (arrows) and (E) the left ventricular (LV) lateral wall (arrow) in a long-axis view. (F) Mid-transverse section of the autopsied heart revealing severe right ventricular dilation and wall thinning as well as concentric hypertrophy of the left ventricle. Histology of the (G) right ventricle showing severe fibrofatty infiltration of the free wall in association with extensive ventricular interstitial fibrosis and (H) left ventricle showing moderate hypertrophy with focal areas of fibrofatty muscular infiltration and widespread interstitial fibrosis. Scale bars: 160 μm (G) and 60 μm (H).

Figure 2. Deceased proband possessing AnkB loss-of-function p.Met1988Thr variant exhibits arrhythmogenic cardiomyopathy.

(A) Apical transverse section of the explanted heart of the deceased AnkB-p.Met1988Thr proband revealing fatty infiltration and localized thinning of the anterior and lateral aspects of the right ventricular free wall. Scale bar: 1 cm. (B) Open view of the right ventricle revealing moderate dilation. Scale bar: 1 cm. Histology of the anteroapical right ventricular free wall at (C) ×1 and (D) ×40 original magnification, revealing marked fatty infiltration of the myocardium with patchy interstitial fibrosis and myocardial disorganization. Scale bars: 5 mm (C) and 300 μm (D). (E) Four-lead surface ECG of paternal cousin following a treadmill test, revealing VT.

Figure 3. Cardiomyocyte-specific deletion of AnkB results in ventricular remodeling, cardiac arrhythmias, and reduced survival.

Immunoblotting for AnkB (A) in heart tissue from Ank2-cKO (n = 4) and Ank2^fl/fl (n = 5) mice (complete immunoblots are shown in Supplemental Figure 3). (B and C) Quantification of (B) left ventricular ejection fraction of Ank2^fl/fl mice (n = 4) and Ank2-cKO mice (n = 9) and (C) right ventricular fractional shortening in Ank2^fl/fl (n = 6) and Ank2-cKO (n = 7) mice. (D) Kaplan-Meier survival curves for Ank2^fl/fl (n = 36) and Ank2-cKO (n = 69) mice. (E and F) Representative ECGs for Ank2-cKO mice following injection of 2.0 mg/kg epinephrine. Arrows denote (E) VT and (F) trigeminy. (G) Heart weight/tibia length (HW/TL) ratios of Ank2^fl/fl (n = 8) and Ank2-cKO (n = 9) mice. (H and I) Representative Masson’s trichrome–stained whole slide views of (H) Ank2^fl/fl and (I) Ank2-cKO and (J) magnified Ank2^fl/fl and (K) Ank2-cKO hearts. Images are representative of 4 hearts from mice of each genotype. Scale bars: 2.5 mm (H and I) and 25 μm (J and K). Data represent the mean ± SEM. Statistical significance for A–C and G was determined using a 2-tailed parametric t test at 95% CI and a log-rank test for D.

Figure 4. Ank2-cKO murine hearts show misclocalization of β-catenin.

Representative IF images of (A) desmin and N-cadherin, (B) plakoglobin and α-actinin, (C) connexin-43 and α-actinin, (D) desmoplakin and α-actinin, and (E) AnkB and β-catenin in Ank2^fl/fl and Ank2-cKO cardiac cryoslices. Staining was completed in tissue from 3 hearts for each stain, per genotype, with 3 images taken per heart. Scale bars: 20 μm.

Figure 5. AnkB and β-catenin are molecular partners.

(A and B) Representative images of (A) plakoglobin and β-catenin staining from control and AnkB p.Met1988Thr human right ventricular tissue, and (B) β-catenin staining of control and AnkB p.Glu1458Gly human right ventricular tissue. Scale bars: 20 μm. (C) Co-IP assay of AnkB IgG and β-catenin in human ventricular lysate. (D) GST-pulldown assay of in vitro–translated AnkB MBD and β-catenin in mouse cardiac lysate (complete images are shown in Supplemental Figure 8). The binding experiments were replicated 3 times. (E) Representative immunoblot of β-catenin and (F) quantification of full-length β-catenin in Ank2^fl/fl (n = 4) and Ank2-cKO (n = 5) mouse cardiac lysate (complete images are shown in Supplemental Figure 8). Data represent the mean ± SEM. Statistical analysis for F was performed using a 2-tailed parametric t test at 95% CI. Binding assays were completed in triplicate.

Figure 6. GSK3-β inhibition is sufficient to prevent cardiac remodeling associated with cardiac deletion of AnkB.

(A) Diagrammatic representation of GSK3-βi prevention study in Ank2^fl/fl and Ank2-cKO mice. Echocardiograms were performed at the time points indicated by arrows. (B) Heart weight/tibia length ratios. n = 3. (C) Levels of p–β-catenin by IB of Ank2-cKO cardiac tissue lysates. n = 3. (D and E) Ejection fraction and fractional shortening at baseline. Ejection fraction (F) and fractional shortening (G) after 8 weeks of drug therapy in vehicle- and GSK3-βi–treated Ank2^fl/fl and Ank2-cKO mice in the prevention study. n = 5 (D and E) and n = 9 (F and G). (H) Representative Masson’s trichrome–stained heart sections from vehicle-treated Ank2^fl/fl mice. (I) Ank2^fl/fl GSK-βi–treated, (J) Ank2-cKO vehicle-treated, and (K) Ank2-cKO GSK3-βi–treated mice after 8 weeks of drug therapy in the prevention study. Scale bars: 25 μm (I–K). Images are representative of 2 hearts from mice of each genotype and treatment condition. Data represent the mean ± SEM. Statistical analysis for C was done with a 2-tailed parametric t test at 95% CI. Statistical analysis for B and D–G was performed with a 2-way ANOVA followed by Tukey’s post hoc test.

Figure 7. GSK3-β inhibition is sufficient to reverse cardiac remodeling associated with cardiac deletion of AnkB.

(A) Diagrammatic representation of the GSK3-βi rescue study involving Ank2^fl/fl and Ank2-cKO mice. Echocardiograms were performed at the time points indicated by arrows. Ejection fraction (B) and fractional shortening (C) at 12 weeks of age and ejection fraction (D) and fractional shortening (E) after 4 weeks of drug therapy in vehicle- and GSK3-βi–treated Ank2^fl/fl and Ank2-cKO mice. n = 3 (B–E). (F and G) Myocyte cross-sectional area in Ank2^fl/fl (n = 3 for each treatment) and Ank2-cKO (n = 3 for each treatment) mice following treatment with vehicle or GSK3-βi. (H–K) Representative cross-sectional images of Ank2^fl/fl and Ank2-cKO hearts following vehicle and GSK3-βi therapy. Images are representative of 3 hearts from mice of each genotype and treatment condition. Scale bars: 25 μm. Data represent the mean ± SEM. Statistical analysis for B–F was performed with a 2-way ANOVA followed by Tukey’s post hoc test.