Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model

Abstract

Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.

Figure 1.

Generation, survival and desmoplakin expression analysis of DSP-cKO mice. (A) Schematic representation of breeding strategy to generate DSP-cKO [DSP^flox/flox:MLC2v^Cre(+)] and littermate control [DSP^flox/flox:MLC2v^Cre(−)] mice. (B) Kaplan–Meier survival curve analysis of control (n = 15) and DSP-cKO (n = 15) mice from birth. (C) Ventricular cardiomyocytes were isolated from DSP-cKO and littermate control hearts at 3 weeks of age. Desmoplakin expression in DSP-cKO and control cardiomyocytes was assessed in both total and insoluble protein extracts using specific antibodies to desmoplakin and GAPDH (loading control). (D) Immunofluorescence staining of desmoplakin in cardiac sections from representative DSP-cKO and littermate control mice at 4 weeks of age. Cardiac sections were double-labeled with antibodies against desmoplakin (red) and N-cadherin (green), as well as being counterstained with DAPI nuclear stain (blue). White arrows highlight areas where there is specific loss of desmoplakin within intact intercalated discs in DSP-cKO hearts (denoted by the presence of N-cadherin). Bar represents 9 μm.

Figure 2.

Ultrastructural analysis of DSP-cKO mouse hearts. Representative transmission electron micrographs from the ventricular myocardium of control (A and C) and DSP-cKO (B and D). Red bars highlight the loss of attachment between plasma membranes of the adjacent cells. Red asterisks denote areas with complete breakdown of the intercalated disc structures. Ds, desmosome; FA, fascia adherens junction. Bar represents 500 nm.

Figure 3.

Histological analysis of DSP-cKO mice. (A) Whole-heart and (B) cardiac sections from DSP-cKO and control mice at 4 weeks. Sections were stained for nuclei and cytoplasm with hematoxylin and eosin, respectively. Bars in A and B represent 2 and 0.67 mm, respectively. (C) Masson trichrome stain of DSP-cKO and control mouse heart sections at 4 weeks. Bar represents 100 μm. (D) Oil Red O staining of DSP-cKO and control mouse heart (upper panel: RV free-wall; lower panels: RV myocardium) sections at 6 weeks. Arrowheads and arrows indicate regions of fat deposition. Bar in lower panels represents 100 μm. (E) Schematic representation of the mating strategy used to generate mice with TomRed reporter in the background of DSP^flox/flox:MLC2v^Cre(+) mice for lineage tracing experiments to seek origin of fat deposition. (F) Immunofluorescence staining of perilipin in subepicardial layer of cardiac sections of DSP-cKO mice. (G) Immunofluorescence staining of perilipin in mid-wall region of cardiac sections of DSP-cKO mice. For F and G, cardiac sections were labeled with an antibody against perilipin (green) as well as being counterstained with DAPI nuclear stain (blue). MLC2v-positive cells were identified by their expression of the red fluorescent protein, TomRed. Bar represents 40 μm.

Figure 4.

Magnetic resonance image (MRI) analysis of DSP-cKO mice. (A) Representative MRIs (short axis) of hearts from control and DSP-cKO mice at 8 weeks. Serial short-axis images were used to generate the 3D geometric model of the endocardium. (B) Representative MRIs (long axis) of the hearts from control and DSP-cKO mice at 8 weeks. (C) Table denotes measurements obtained from MRI analysis. BW indicates body weight; HR, heart rate; EDV, end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; and wThick, wall thickness. *P < 0.05, **P < 0.01.

Figure 5.

Surface ECG analysis of DSP-cKO mice. (A) Representative ECG tracings from control (n = 6) and DSP-cKO (n = 6) mice at 4 weeks, using Lead II. Mathematical analysis of PR and QRS intervals from the ECG tracings was performed. Data from 100 ECG tracings per mouse were averaged for analysis. QRS duration is indicated by the dotted line. **P < 0.01, NS, not significant. (B) Representative surface ECG tracings from sedated DSP-cKO and littermate control mice at 4 weeks, using Lead II. Note the PVC (arrows) in DSP-cKO heart. (C) Representative optical maps (activation time plots) from non-paced Langendorff-perfused DSP-cKO and littermate control hearts at 6 weeks. Asterisks denote PVC coming from both left and right ventricles within DSP-cKO hearts. Voltage-sensitive dye shows changes in activation time (note scale for control ranges from 0 to 6 ms and scale for DSP-cKO ranges from 0 to 20 ms). (D) Analysis of PVC per second in control and DSP-cKO mice at 6 weeks at baseline and with exercise in the absence or presence of low and high doses of epinephrine. *P < 0.05.

Figure 6.

Connexin 40 expression in the ventricular conduction system of DSP-cKO hearts and whole-heart optical mapping analysis of DSP-cKO mice following atrial pacing. (A) Cardiac sections from 3-week-old wild-type mice were co-stained with antibodies against desmoplakin (DESMO, red) and HCN4 (green). Nuclei (blue) were identified through DAPI stain as indicated. Arrows denote doubly stained regions from merged images. Bar represents 50 μm. (B) Activation time plots in control and DSP-cKO hearts following atrial pacing. (C) Summary of activation times, APDs and action potential dispersion in control and DSP-cKO hearts following atrial pacing. *P < 0.05. Please note the slowed activation to the RV and increased activation times, APD and APD dispersion in DSP-cKO hearts. (D) Cardiac sections from control (upper panels) and DSP-cKO (lower panels) mice were co-stained with HCN4 (green) and connexin 40 (red). Nuclei (blue) were identified through DAPI. Arrow denotes connexin 40 expression in His bundle (HB) and bundle branch (BB) of WT hearts. Note reduced connexin 40 staining in DSP-cKO heart. Same exposure times were used to detect connexin 40 in control and DSP-cKO hearts. Bar represents 100 μm.

Figure 7.

Whole-heart optical mapping analysis of DSP-cKO mice following epicardial pacing and intercalated disc protein expression in DSP-cKO hearts. (A) Representative activation time plots in control and DSP-cKO hearts at 6 weeks following basal posterior epicardial pacing from the right ventricle. Summary of activation times and negative curvature. Please note irregular wavefront pattern and increased negative curvature in DSP-cKO hearts. *P < 0.05, NS, non-significant. (B) Protein blot analysis of intercalated disc proteins in total protein extracts from control and DSP-cKO mice at 6 weeks. GAPDH was used as a loading control. (C) Protein blot analysis of plakoglobin in insoluble protein extracts from control and DSP-cKO hearts at 6 weeks and immunofluorescence staining of plakoglobin in cardiac cryosections from control and DSP-cKO mice at 6 weeks. Cryosections were double-labeled with antibodies against desmoplakin (green) and plakoglobin (red), as well as being counterstained with DAPI nuclear stain (blue).

Figure 8.

Desmoplakin knock-down leads to electrical conduction block in aligned neonatal mouse ventricular cardiomyocytes (NMVC). (A) Schematic representation of the strategy used to knock-down desmoplakin expression in NMVC isolated from DSP^flox/flox mice. (B) Western blot analysis of intercalated disc protein levels in viral infected cells was performed 4 days post-infection. GAPDH was used as a loading control. (C) Immunofluorescence staining of desmoplakin in non-aligned and aligned NVMC. NVMC were double-labeled with antibodies against desmoplakin (green) and cypher (red), as well as counterstained with DAPI nuclear stain (blue). Bar represents 40 μm. (D) Distances of continuous propagation were measured at longitudinal and transverse angles as well as at an arbitrary angle across the longest distance. (E) Representative activation time plots from control (AdLacZ) and desmoplakin-deficient (AdCre) NVMC 4 days post-infection. Distances of continuous propagation were measured at longitudinal and transverse angles as well as at an arbitrary angle across the longest distance. (F) Quantification of distances of continuous propagation in control and desmoplakin-deficient NVMC. n = 8, *P < 0.05. (G) Quantification of the area of continuous propagation in control and desmoplakin-deficient NVMC. n = 8, *P < 0.05. An area of continuous propagation of <75% of the total area mapped was demonstrative of conduction block.

Figure 9.

Working model for how cardiomyocyte-specific loss of desmoplakin in DSP-cKO mice leads to disease features reminiscent of a biventricular form of human ARVC. Loss of desmoplakin induces ultrastructural defects at the desmosome that elicit biventricular histological, physiological and electrophysiological defects. Electrophysiological defects also occur independent of structural changes at the desmosome, with loss of desmoplakin directly influencing the levels and function of gap junction proteins.