N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

Abstract

Cardiac-specific deletion of the murine gene (Cdh2) encoding the cell adhesion molecule, N-cadherin, results in disassembly of the intercalated disc (ICD) structure and sudden arrhythmic death. Connexin 43 (Cx43)-containing gap junctions are significantly reduced in the heart after depleting N-cadherin, therefore we hypothesized that animals expressing half the normal levels of N-cadherin would exhibit an intermediate phenotype. We examined the effect of N-cadherin haploinsufficiency on Cx43 expression and susceptibility to induced arrhythmias in mice either wild-type or heterozygous for the Cx43 (Gja1)-null allele. An increase in hypophosphorylated Cx43 accompanied by a modest decrease in total Cx43 protein levels was observed in the N-cadherin heterozygous mice. Consistent with these findings N-cadherin heterozygotes exhibited increased susceptibility to ventricular arrhythmias compared to wild-type mice. Quantitative immunofluorescence microscopy revealed a reduction in size of large Cx43-containing plaques in the N-cadherin heterozygous animals compared to wild-type. Gap junctions were further decreased in number and size in the N-cad/Cx43 compound heterozygous mice with increased arrhythmic susceptibility compared to the single mutants. The scaffold protein, ZO-1, was reduced at the ICD in N-cadherin heterozygous cardiomyocytes providing a possible explanation for the reduction in Cx43 plaque size. These data provide further support for the intimate relationship between N-cadherin and Cx43 in the heart, and suggest that germline mutations in the human N-cadherin (Cdh2) gene may predispose patients to increased risk of cardiac arrhythmias.

Figure 1

N-cadherin and Cx 43 expression in heterozygous mice. A, Quantitative Western blot analysis was performed on heart lysates from wild-type (WT), N-cadherin (N), Cx43 (C), and N-cad/Cx43 compound (N/C) heterozygous adult mice. Blots were probed for N-cadherin, Cx43, and dephosphorylated Cx43 (13-8300). B, Eight independent animals were examined for each genotype and compared with WT. GAPDH signal was used to normalize for loading differences between lanes. The molecular weights for the phosphorylated Cx43 (P) and nonphosphorylated Cx43 (P0) are 44 – 46 kD and 42 kD, respectively. *, P < 0.05.

Figure 2

Ventricular arrhythmia in N-cad/Cx43 compound heterozygous animal. Shown from top to bottom are surface ECG leads I, II and aVF, along with intracardiac recordings from the right atrium (RA) and right ventricle (RV). Episode of ventricular tachycardia induced by programmed stimulation in a N-cad/Cx43 compound heterozygous animal with the pathognomonic characteristic of rapid ventricular beats and atrial dissociation. Programmed stimulation is delivered as a train of impulses at 120 ms (S1) followed by two extrastimulus (S2) and (S3) at 40ms and 20ms respectively. Note sinus rhythm before and after the conclusion of the arrhythmia, for comparison, where the p-wave (P) represents atrial depolarization followed by ventricular depolarization (QRS).

Figure 3

Comparison of Cx43 staining patterns in wild-type and mutant animals. Ventricular myocardium from wild-type (WT), N-cadherin (N-cad −/+), Cx43 (Cx43 −/+), and compound (N-cad/Cx43 −/+) heterozygous adult mice was coimmunostained with Cx43 (red, A–D) and N-cadherin (green, E–H merged). Quantitative immunofluorescence microscopy was performed on Cx43-immunostained sections of ventricular myocardium from four independent animals of each genotype (I). Ten test areas from each animal were examined for 5 and 9 or more contiguous pixels of high signal intensity. The number of Cx43-containing clusters or aggregates, the average size of the Cx43-containing clusters, and the area fraction occupied by Cx43-containing clusters in each category is presented. The error bars represent standard deviation of the mean. WT, wild-type; N, N-cadherin −/+; C, Cx43 −/+; N/C, N-cadherin/Cx43 −/+. *, P<0.05; **, P<0.01; ***, P< 0.001. ns, not significant. The P values for all pairwise comparisons are presented in Supplemental data.

Figure 4

N-cadherin and Cx43 expression in isolated cardiomyocytes. Cardiomyocytes were isolated from adult wild-type (WT) and double mutant (N-cad/Cx43 −/+) animals and coimmunostained for N-cadherin (A,C) and Cx43 (B,D).

Figure 5

Expression of β-catenin and hypophosphorylated Cx43 in isolated cardiomyocytes. Cardiomyocytes were isolated from adult wild-type (WT) and double mutant (N-cad/Cx43 −/+) animals and coimmunostained for β-catenin (A,C) and dephosphorylated Cx43 (13-8300; B,D). Note the overall increase in hypophosphorylated Cx43 at the ICD and lateral side of the cardiomyocyte.

Figure 6

ZO-1 expression in isolated cardiomyocytes. Cardiomyocytes were isolated from adult wild-type (WT), N-cadherin (N-cad −/+), Cx43 (Cx43 −/+), and N-cad/Cx43 compound (N-cad/Cx43 −/+) heterozygous mice and immunostained for the scaffold protein ZO-1 (A–D). ZO-1 was significantly decreased at the ICD of the cardiomyocytes from N-cadherin (64% versus WT; P<0.05; n=8–10 cardiomyocytes) and N-cad/Cx43 compound (50% versus WT; P<0.05; n=8–10 cardiomyocytes) heterozygous mice. No significant change in ZO-1 was found in Cx43 heterozygous mice compared to wild-type (91% versus WT; P>0.05; n=8–10 cardiomyocytes). Note these pictures were assembled from a series of Z-scan images from the bottom to the top surface of the cells providing a complete analysis of ZO-1 protein expression in the cardiomyocytes.

Figure 7

Expression of desmosomal proteins in isolated cardiomyocytes. Cardiomyocytes were isolated from adult wild-type (WT) and double mutant (N-cad/Cx43 −/+) animals and immunostained for plakoglobin (A, C) and desmoplakin (B, D).