Decreased WNT/β-catenin signalling contributes to the pathogenesis of dilated cardiomyopathy caused by mutations in the lamin a/C gene

Abstract

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is characterized by cardiac conduction abnormalities and left ventricular systolic dysfunction predisposing to heart failure. Previous cardiac transcriptional profiling of LmnaH222P/H222P mouse, a small animal model of LMNA cardiomyopathy, suggested decreased WNT/β-catenin signalling. We confirmed decreased WNT/β-catenin signalling in the hearts of these mice by demonstrating decreased β-catenin and WNT proteins. This was correlated with increased expression of soluble Frizzled-related proteins that modulate the WNT/β-catenin signalling pathway. Hearts of LmnaH222P/H222P mice also demonstrated lowered expression of the gap junction connexin 43. Activation of WNT/β-catenin activity with 6-bromoindirubin-3'-oxime improved cardiac contractility and ameliorated intraventricular conduction defects in LmnaH222P/H222P mice, which was associated with increased expression of myocardial connexin 43. These results indicate that decreased WNT/β-catenin contributes to the pathophysiology of LMNA cardiomyopathy and that drugs activating β-catenin may be beneficial in affected individuals.

Figure 1.

Altered WNT/β-catenin signalling in hearts of Lmna^H222P/H222P mice and human subjects with LMNA cardiomyopathy. (A) Representative immunoblots showing active and total β-catenin expression in hearts from 3 month-old and 6 month-old male Lmna^H222P/H222P (H222P) and wild type (WT) mice. Each lane contains protein extracts from a different mouse. Gapdh is the loading control. Error bars represent means ± standard errors of means (n = 4) for total β-catenin/gapdh and active β-catenin/total β-catenin relative expression. *P < 0.05, **P < 0.005, ***P < 0.0005. (B) Representative immunoblots showing WNT10b and WNT1, expression in hearts from 6 month-old male Lmna H222P mice compared to Lmna WT mice. Each lane contains protein extracts from a different mouse. Gapdh is the loading control. (C) Representative immunoblot showing total β-catenin expression in isolated cardiomyocytes from 6 month-old male Lmna H222P mice compared to Lmna WT mice. (D) Representative immunoblots showing total β-catenin expression in explanted hearts from control human subjects and human subjects with cardiomyopathy and LMNA point mutations (LMNA patients). Erk1/2 is the loading control (45). Migrations of molecular mass standards in kilodaltons (kDa) are indicated at the right of the blots.

Figure 2.

Increased expression of secreted antagonists of WNT/β-catenin signalling in hearts of Lmna^H222P/H222P mice and human subjects with LMNA cardiomyopathy. (A) Expression of sFRP1, sFRP2, Frbz and Dkk3 mRNA in hearts from 3 month-old and 6 month-old male Lmna^H222P/H222P (H222P) and wild type (WT) mice. Error bars represent means ± standard errors of means (n = 7). *P < 0.05, ***P < 0.0005. (B) Representative immunoblots showing sFRP1 expression in hearts from 6 month-old male H222P and WT mice. (C) Representative immunoblot showing Dkk3 expression in explanted hearts from control human subjects and human subjects with cardiomyopathy and LMNA point mutations (LMNA patients). Erk1/2 is the loading control (45). Migrations of molecular mass standards in kilodaltons (kDa) are indicated at the right of the blots in panels B and C.

Figure 3.

Decreased expression of connexin 43 in hearts of Lmna^H222P/H222P mice. (A) Micrographs showing β-catenin labelling (green) in heart and isolated cardiomyocytes from male Lmna^H222P/H222P (H222P) and wild type (WT) mice. Nuclei are counter-stained blue with 4′,6-diamidino-2-phenylindole (dapi). Scale bar, 25 μm. Insets are a representative area. (B) Micrographs showing N-cadherin labelling (red) in heart and isolated cardiomyocytes from H222P and WT mice. Nuclei are counter-stained with dapi. Scale bar, 25 μm. Insets are a representative area. (C) Micrographs showing connexin 43 labelling (green) in heart from male H222P mice and WT mice. Nuclei are counter-stained with dapi. Scale bar, 25 μm. Insets are a representative area. (D) Immunohistochemical labelling for connexin 43 in heart from H222P and WT mice. Scale bar, 25 μm. Insets are a representative area. (E) Representative immunoblot showing phospho-connexin 43 (ser368) and total connexin 43 expression in hearts from 6 month-old male H222P and WT mice. Migrations of molecular mass standards in kilodaltons (kDa) are indicated at the right of the blots. The bar graph shows connexin 43 relative expression compared to Gapdh (means ± standard errors of means) in hearts from WT (n = 3) and H222P (n = 3) mice. **P < 0.005.

Figure 4.

Activation of WNT/β-catenin signalling using BIO improves left ventricular function and intraventricular conduction in Lmna^H222P/H222P mice. (A) Schematic representation of the treatment protocol of Lmna^H222P/H222P (H222P) with BIO. I.P., intra-peritoneal; q.d., daily. (B) Representative immunoblots showing phospho-GSK3, total GSK3 and total β-catenin expression in hearts from 6 month-old male H222P mice treated with DMSO placebo or BIO (1.25 μg/kg/daily). Migrations of molecular mass standards in kilodaltons (kDa) are indicated at the right of the blots. Error bars represent means ± standard errors of means for total β-catenin/gapdh, p − GSK3/total GSK3 and p − GSK3/total GSK3 relative expression in 20-week-old male H222P mice treated with BIO (n = 4) or DMSO (n = 3). *P < 0.05, **P < 0.005. (C) Representative M-mode transthoracic echocardiographic tracings from 20-week-old male H222P mice treated with DMSO or BIO. Graphs show mean left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and left ventricular fractional shortening (FS) in 20-week-old male H222P mice treated with BIO (n = 5) or DMSO (n = 8). Values for each individual mouse as well as means ± standard errors of means are shown. *P < 0.05, ***P < 0.0005. (D) Representative electrocardiogram tracings from 20-week-old H222P mice treated with DMSO or BIO. Graphs showing mean PR and QRS intervals in 20-week-old H222P mice treated with BIO (n = 5) or DMSO (n = 8). Data are represented as means ± standard errors of means. Values are shown as 25^th to 75^th percentiles of data values. The line in the middle is the median. Whiskers (Tukey method) extend down to the minimum value and up to the maximum value. *P < 0.05.

Figure 5.

Expression and localization of connexin 43 are under WNT/β-catenin signalling regulation in hearts from Lmna^H222P/H222P mice and cultured C2C12 cells. (A) Representative immunoblots showing WNT-1, total β-catenin and connexin 43 expression in hearts from 20-week-old male wild type (WT) and Lmna^H222P/H222P (H222P) mice untreated (-), treated with BIO or with DMSO. (B) Immunohistochemistry for connexin 43 labelling in hearts from H222P mice treated with BIO or with DMSO. Scale bar, 50 μm. (C) Representative immunoblot showing connexin 43 and active β-catenin expression in C2C12 cells treated with BIO to activate Wnt/β-catenin signalling, or IWP2 and LGK974 to inhibit Wnt/β-catenin signalling. Migrations of molecular mass standards in kilodaltons (kDa) are indicated between the blots.