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. 2015 Oct;8(5):1255-64.
doi: 10.1161/CIRCEP.115.002720. Epub 2015 Jul 30.

Mouse Model of Human Congenital Heart Disease: Progressive Atrioventricular Block Induced by a Heterozygous Nkx2-5 Homeodomain Missense Mutation

Rajib Chowdhury  1 , Hassan Ashraf  1 , Michelle Melanson  1 , Yohei Tanada  1 , Minh Nguyen  1 , Michael Silberbach  1 , Hiroko Wakimoto  1 , D Woodrow Benson  1 , Robert H Anderson  1 , Hideko Kasahara  2
Affiliations

Mouse Model of Human Congenital Heart Disease: Progressive Atrioventricular Block Induced by a Heterozygous Nkx2-5 Homeodomain Missense Mutation

Rajib Chowdhury et al. Circ Arrhythm Electrophysiol. 2015 Oct.

Abstract

Background: Heterozygous human NKX2-5 homeodomain (DNA-binding domain) missense mutations are highly penetrant for varied congenital heart defects, including progressive atrioventricular (AV) block requiring pacemaker implantation. We recently replicated this genetic defect in a murine knockin model, in which we demonstrated highly penetrant, pleiotropic cardiac anomalies. In this study, we examined postnatal AV conduction in the knockin mice.

Methods and results: A murine knockin model (Arg52Gly, Nkx2-5(+/R52G)) in a 129/Sv background was analyzed by histopathology, surface, and telemetry ECG, and in vivo electrophysiology studies, comparing with control Nkx2-5(+/+) mice at diverse postnatal stages, ranging from postnatal day 1 (P1) to 17 months. PR prolongation (first degree AV block) was present at 4 weeks, 7 months, and 17 months of age, but not at P1 in the mutant mice. Advanced AV block was also occasionally demonstrated in the mutant mice. Electrophysiology studies showed that AV nodal function and right ventricular effective refractory period were impaired in the mutant mice, whereas sinus nodal function was not affected. AV nodal size was significantly smaller in the mutant mice than their controls at 4 weeks of age, corresponding to the presence of PR prolongation, but not P1, suggesting, at least in part, that the conduction abnormalities are the result of a morphologically atrophic AV node.

Conclusions: The highly penetrant and progressive AV block phenotype seen in human heterozygous missense mutations in NKX2-5 homeodomain was replicated in mice by knocking in a comparable missense mutation.

Keywords: animal models; atrioventricular block; congenital heart defects; genetics; human.

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Conflict of interest statement

Conflict of Interest Disclosures: None.

Figures

Figure 1
Figure 1
Phenotypes associated with Arg52(189)Gly, NKX2-5+/R52,, homeodomain missense mutation. In the kindred, males are denoted by squares, females by circles. Darkened quadrants indicate AVB, ASD, VSD, or tricuspid valve abnormality. Open symbols denote normal genotype and phenotype. Arg52 is positioned at amino acid 189 in human NKX2-5. ASD, atrial septal defect; VSD, ventricular septal defect; TV, tricuspid valve.
Figure 2
Figure 2
Prolongation of PR interval and QRS duration in Nkx2-5+/R52G mice. Representative surface ECG recordings obtained from P1 (A), 4 weeks (B), 7-month-old (C) and 17 month-old (D) control Nkx2-5+/+ (top) and Nkx2-5+/R52G (bottom) mice. (E) PR-interval and QRS duration over time with individual values plotted from control Nkx2-5+/+ (open circle) and Nkx2-5+/R52G (closed circle) mice. The averages of values, and number of mice examined were indicated. (F) PR interval under two different isoflurane dose (1% and 3%) from control Nkx2-5+/+ (open circle) and Nkx2-5+/R52G (closed circle) mice at 4 weeks of age. Signal-averaged ECG waves were utilized for analysis. Summarized data is shown in Table 1. HR, heart rate. Mean ± SE.
Figure 3
Figure 3
AVB displayed in ambulant telemetry ECG recording from 17 month-old Nkx2-5+/R52G mice. (A) Representative telemetry ECG recordings from control Nkx2-5+/+ (top) and Nkx2-5+/R52G (bottom) mice at 17 months of age. Averaged ECG waves from ~ 20 beats at least three different time points were utilized for analysis shown in Table 2. (B) Representative ECG recording of advanced complete AVB demonstrated in Nkx2-5+/R52G mice. HR, heart rate. Mean ± SE.
Figure 4
Figure 4
Advanced 3:1 AVB from Nkx2-5+/R52G mouse (7 months of age) detected shortly after anesthesia for EP study and HCN4-positive AV node vs. penetrating AV bundle. (A) Representative recording of surface and intracardiac recordings (RV or RA) demonstrating 3:1 AVB obtained from Nkx2-5+/R52G mice during EP analysis shortly after induction of isoflurane anesthesia with sinus cycle length of 135 ms. (B) Representative HCN4-stained normal heart tissues of Nkx2-5+/+ at 4 weeks of age including AV node and penetrating AV bundles traced by black dots. (C) Simplified illustration to define AV node vs. penetrating AV bundle with or without direct contact to atrial muscle. HCN, hyperpolarization activated cyclic nucleotide-gated potassium channel. A, right atrial electrogram; V, right ventricular electrogram.
Figure 5
Figure 5
Reduction of AV nodal size composed of smaller cardiomyocytes without apparent increase of fibrosis in 4 weeks or 7 months-old Nkx2-5+/R52G mice. (A) Representative H&E stained heart tissues including AV node and penetrating AV bundles traced in black dots from control Nkx2-5+/+ or Nkx2-5+/R52G mice. (B) Maximum AV nodal area size, calculated AV nodal volume, and nuclear density (number of nuclei × 103/mm2). The numbers of mice and nuclei analyzed were noted. (C) Representative immunofluorescent images of troponin T (red), Nkx2-5 (green) and nuclei (blue) from control Nkx2-5+/+ or Nkx2-5+/R52G mice. AV nodal area is traced by yellow dots. (D) Ratio of Nkx2-5-positive vs. total nuclear number in the AV node. (E) Representative images of picrosirius red-stained tissue sections including AV node from 7-month-old mice. Fibrotic tissues are stained in red and muscle tissues are stained in green. RA, right atrium; AS, atrial septum; IVS, interventricular septum. Mean ± SE.
Figure 6
Figure 6
Unchanged AV nodal size in P1 Nkx2-5+/R52G mice. (A) Representative HCN4 and picrosirius red-stained normal heart tissues of P1 Nkx2-5+/+ including AV node traced by black dots. (B) Representative picrosirius red-stained heart tissues including AV node traced in black dots from control Nkx2-5+/+ or Nkx2-5+/R52G mice. (C) Maximum AV nodal area size (n=7 each). (D) Representative immunofluorescent images of HCN4 (green), actinin (red) and nuclei (blue) from control Nkx2-5+/+ or Nkx2-5+/R52G mice. AV nodal area is traced by yellow dots. Nuclear density (number of nuclei × 103/mm2) is measured from the HCN-positive AV nodal area. RA, right atrium; TV, tricuspid valve; RV, right ventricle; LV, left ventricle; AS, atrial septum; IVS, interventricular septum. Mean ± SE.
Figure 7
Figure 7
Expression of positive- and negative AV nodal, penetrating AV bundle and left bundle branch markers in P1 hearts, and AchE activities in E18.5 developing hearts. (A) Representative images of serial tissue sections positively (+) or negatively (−) stained with HCN4, connexin 40, connexin 43, and Nav1.5 in AV node, penetrating AV bundle (traced by white dots) and left bundle branch (arrowheads) from P1 mice. (B) Representative images demonstrating AchE activity (brown) in left bundle branch and ventricular trabeculations (Purkinje fibers) from serial tissue sectioning of control Nkx2-5+/+ and Nkx2-5+/R52G (+/R52G) hearts, is weaker in Nkx2-5+/R52G hearts. Areas a–d selected on the left are shown enlarged on the right. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; AS, atrial septum; IVS, interventricular septum.
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