Loss of cadherin-binding proteins β-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis

Abstract

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.

Fig 1

Increased β-catenin association with Cx43 in PG-deficient hearts. (A) Western analysis of WT and PG CKO heart lysates probed for N-cadherin (N-cad), β-catenin (β-cat), and Cx43. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as a loading control. (B) Heart lysates were immunoprecipitated with anti-Cx43 antibody and subsequently immunoblotted for β-catenin, plakoglobin (PG), N-cadherin, and Cx43. No-antibody IP served as the negative control (−Ab). Note the increased association of β-catenin with Cx43 in PG CKO hearts. **, P < 0.01; ***, P < 0.001.

Fig 2

Sudden death in PG/β-catenin DKO mice. (A to D) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) (A and B) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) (C and D) mice were immunostained for plakoglobin (PG) (A and C) and β-catenin (βcat) (B and D) 3 months after Tam administration. (E) Heart lysates from WT (n = 8) and DKO (n = 10) mice were immunoblotted for PG and β-catenin. (F) Kaplan-Meier survival curve shows completely penetrant sudden-death phenotype in the DKO mice (23/23; median survival, 105 days), compared to the survival of PG CKO mice (9/105), β-catenin CKO mice (3/76), and WT controls (3/81) within 6 months after Tam administration. ***, P < 0.001.

Fig 3

Histological analysis of PG/β-catenin DKO hearts. (A to F) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) mice 3 months after Tam administration were stained with H&E (A, B, D, and E) or Masson's trichrome (C and F). Note the extensive fibrosis in the DKO heart (F) compared with the WT heart (C). (G to I) TUNEL analysis of WT and DKO hearts (n ≥ 1,000 nuclei from four different animals per group). (J) Quantification of heart weight/body weight ratios of WT and DKO mice at 3 months after Tam administration (n = 30 for each group). (K) Cardiomyocyte cross-sectional area quantification from histological sections of WT and DKO hearts at 3 months after Tam administration (n ≥ 100 cells from at least five different animals per group). ***, P < 0.001.

Fig 4

ICD expression of adherens junctional proteins in PG/β-catenin DKO hearts. (A to F) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) (A to C) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) (D to F) mice at 3 months after administration of Tam were immunostained for N-cadherin (Ncad) (A and D), αE-catenin (αEcat) (B and E), and αT-catenin (αTcat) (C and F). (G) Heart lysates from WT and DKO mice were immunoblotted for N-cadherin, αE-catenin, and αT-catenin. Note the decreased expression of each adherens junctional component in the DKO hearts compared with the levels in WT hearts (n = 8, WT; n = 10, DKO). ***, P < 0.001.

Fig 5

ICD expression of desmosomal proteins in PG/β-catenin DKO hearts. (A to F) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) (A to C) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) (D to F) mice at 3 months after administration of Tam were immunostained for desmoplakin (DP) (A and D), plakophilin-2 (PKP2) (B and E), and desmoglein-2 (DSG2) (C and F). (G) Heart lysates from WT and DKO mice were immunoblotted for desmoplakin, plakophilin-2, and desmoglein-2. Note the decreased expression of each desmosomal component in the DKO compared with the WT hearts (n = 8, WT; n = 10, DKO). **, P < 0.01; ***, P < 0.001.

Fig 6

Ultrastructural and echocardiographic analysis of PG/β-catenin DKO mice. (A to D) Transmission electron micrographs of LV myocardium from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) (A and B) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) (C and D) hearts at 3 months after administration of Tam (n ≥ 10 fields per heart from three different animals per group). Intercalated discs were readily visualized in the control hearts (A, arrows); in contrast, these structures were absent in the DKO hearts (C). DKO myocytes displayed decreased sarcomere length (double-headed arrow) (D). Bars indicate 2 μm (black) and 500 nm (white). (E and F) Representative M-mode two-dimensional echocardiography displaying LV chamber dilation in DKO compared to WT mice. Mean values of echocardiographic parameters are shown in the table. Data represent means ± standard deviations. Note significant increased LV end-systolic and -diastolic internal dimension (LVID) and volume (LV vol) and reduced LV ejection fraction (EF) and fraction shortening (FS) in DKO mice. N/A, not applicable; NS, not significant; M/F, male/female; bpm, beats per minute; V, velocity.

Fig 7

Inducible and spontaneous ventricular arrhythmias recorded in PG/β-catenin DKO hearts. (A) Representative volume-conducted ECG recordings prior to burst pacing. Note the global low voltage signal in the DKO hearts prior to stimulation. Measurements of ECG recordings revealed increased PR and QRS intervals. (B) Following burst pacing, ventricular fibrillation was only observed in the DKO group (8/11) and not in WT mice (0/9) (P < 0.01). (C) Representative telemetric ECG recordings from a miniaturized transmitter implanted in awake, freely mobile animals captured lethal ventricular arrhythmias in DKO mice (2/3), whereas WT mice showed normal sinus rhythm.

Fig 8

Cx43 expression and protein interactions in single-CKO and DKO hearts. (A to D) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) (A), β-catenin^flox/flox; Cre⁺ (βcat CKO) (B), PG^flox/flox; Cre⁺ (PG CKO) (C), and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) (D) mice were immunostained for Cx43 3 months after Tam administration. (E to H) Representative images of isolated cardiomyocytes from WT (E), β-catenin CKO (F), PG CKO (G), and DKO (H) hearts immunostained for Cx43. Quantitative immunofluorescence microscopy was performed on 20 test areas (ICD regions). (I, J) The average size (I) and area fraction (J) occupied by Cx43-containing clusters for each genotype are shown. (K) Heart lysates were immunoprecipitated with anti-N-cadherin antibody and subsequently immunoblotted for PG, β-catenin (βcat), Cx43, and N-cadherin (Ncad). No-antibody IP served as the negative control (−Ab). Note the significant decrease of the N-cadherin/catenin/Cx43 macromolecular complex in the DKO hearts, whereas the single-CKO hearts maintain the multiprotein complex. (L) Heart lysates from WT, β-catenin CKO, PG CKO, and DKO mice were immunoblotted for dephosphorylated Cx43 (dpCx43) and total Cx43. Note the significant increase in dpCx43 in DKO hearts. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 9

Relationship between cardiomyopathy, gap junction remodeling, and arrhythmic susceptibility in the DKO mice. (A to D) Heart sections from PG^flox/flox/β-catenin^flox/flox; Cre⁻ (WT) and PG^flox/flox/β-catenin^flox/flox; Cre⁺ (DKO) mice were stained with acid fuchsin orange G at 3 (3W) (B), 5 (5W) (C), and 8 (8W) (D) weeks after Tam administration. Note the fibrotic response as early as 3 weeks in DKO hearts (B) compared with WT hearts (A). (E to L) Heart sections were immunostained for N-cadherin (Ncad, red) and connexin43 (Cx43, green) at 3 (F, J), 5 (G and K), and 8 (H and L) weeks, respectively, after Tam administration. Note the decreased localization of both Ncad and Cx43 at the intercalated disc (ID) as early as 3 weeks after Tam administration. Arrowheads denote remaining N-cadherin/Cx43 colocalization at the ID. Ejection fraction (M) and fractional shortening (N) of DKO and WT hearts at 3, 5, 8, and 12 weeks after Tam administration as determined by echocardiography. The incidence of ventricular fibrillation in DKO hearts following burst pacing protocol was not significantly different at 3 (0/4) or 5 (1/5) weeks after Tam administration compared to that in the WT (0/10). However, the percentage of DKO hearts susceptible to induced arrhythmia at 8 (4/5) and 12 (8/11) weeks after Tam administration differed compared to the results for DKO mice at 3 weeks after Tam administration. *, P < 0.05; **, P < 0.01; ***, P < 0.001.