Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study

Abstract

Aims: Anecdotal observations suggest that sub-clinical electrophysiological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC) develop before detectable structural changes ensue on cardiac imaging. To test this hypothesis, we investigated a murine model with conditional cardiac genetic deletion of one desmoplakin allele (DSP ±) and compared the findings to patients with non-diagnostic features of ARVC who carried mutations in desmoplakin.

Methods and results: Murine: the DSP (±) mice underwent electrophysiological, echocardiographic, and immunohistochemical studies. They had normal echocardiograms but delayed conduction and inducible ventricular tachycardia associated with mislocalization and reduced intercalated disc expression of Cx43. Sodium current density and myocardial histology were normal at 2 months of age. Human: ten patients with heterozygous mutations in DSP without overt structural heart disease (DSP+) and 12 controls with supraventricular tachycardia were studied by high-density electrophysiological mapping of the right ventricle. Using a standard S(1)-S(2) protocol, restitution curves of local conduction and repolarization parameters were constructed. Significantly greater mean increases in delay were identified particularly in the outflow tract vs. controls (P< 0.01) coupled with more uniform wavefront progression. The odds of a segment with a maximal activation-repolarization interval restitution slope >1 was 99% higher (95% CI: 13%; 351%, P = 0.017) in DSP+ vs. controls. Immunostaining revealed Cx43 mislocalization and variable Na channel distribution.

Conclusion: Desmoplakin disease causes connexin mislocalization in the mouse and man preceding any overt histological abnormalities resulting in significant alterations in conduction-repolarization kinetics prior to morphological changes detectable on conventional cardiac imaging. Haploinsufficiency of desmoplakin is sufficient to cause significant Cx43 mislocalization. Changes in sodium current density and histological abnormalities may contribute to a worsening phenotype or disease but are not necessary to generate an arrhythmogenic substrate. This has important implications for the earlier diagnosis of ARVC and risk stratification.

Figure 1

(A) Screening of mouse tail DNA by PCR—the floxed DSP allele gives a 410 bp band and the wild-type (WT) allele gives a 320 bp band. (B) Example of a murine surface ECG recording. (C) Example of single premature extrastimulus (S2) causing polymorphic ventricular tachycardia. (RA, right atrial intracardiac electrogram; RV, right ventricular intracardiac electrogram; ECG, surface ECG recording; stim, stimulator output). (D) Activation time curves (AT) normalized to baseline AT for mice by genotype (AT, activation time) (n= 10 WT, n= 11 DSP±). (E) Relative mRNA expression by genotype by RT–PCR in 2-month-old desmoplakin (±) myocytes vs. WT (n= 10 WT, n= 11 DSP±). (F) Electrophysiology of the Na⁺ current using patch–clamp technique: current traces (left), I/V relationships (top right), and electrophysiological parameters (bottom right).

Figure 2

(A) M-mode echocardiogram of a desmoplakin (±) mouse. (B) Histological features of DSP het KO mice (Masson's trichrome stain) illustrating fibrosis in 6-month-old DSP± stained in blue. (C) Oil red O staining in 8-week and 6-month-old Het KO mice illustrating accumulation of fat droplets with age.

Figure 3

Representative immunofluorescence images of murine ventricular myocardium. (A) Cx43, (B) Nav1.5, (C) Plakoglobin (PG), (D) DSP labelling, all 8-week-old mice. (E) Plakoglobin and (F) DSP immunohistochemistry from 6-month-old mice. n=4 per genotype with consistent staining in each mouse.

Figure 4

(A) Isochronal map showing RV activation sequence with steady-state pacing from RV apex at 400 ms cycle length (subject 5 vs. control). PV, pulmonary valve. (B) Mean increase delay according to the region in DSP+ and control groups.

Figure 5

(A) Example of normal and fractionated EGMs analysed using fractionation index algorithm. (B) Fractionation index (FI) map illustrating degree of electrogram fractionation in RV segments. Colour scale represents the FI for sites sampled on the RV endocardium. (C) Histogram illustrating mean FI (no of deflections per EGM) according to the region in DSP+ and control subjects. (D) Mean ARI restitution curves recorded from the apex, RV body, and RVOT in control and DSP+ groups. (E) Mean ARI curves in control and DSP+ subjects in the RV body and (F) RVOT.

Figure 6

Human histology. (A) Expression of N-cadeherin (intercalated disc marker protein), Cx43, and plakoglobin in subjects 5 (with LV impairment on imaging). (B) Immunofluorescence images of Nav1.5 expression in two subjects (8 and 9) without structural changes on cardiac imaging.