Background Heterozygous human NKX2-5 homeodomain (DNA binding domain) missense mutations are highly penetrant for varied congenital heart defects, including progressive atrioventricular block (AVB) requiring pacemaker implantation. We recently replicated this genetic defect in a murine knock-in model, in which we demonstrated highly penetrant, pleiotropic cardiac anomalies. In this study, we examined postnatal AV conduction in the knock-in mice. Methods and Results A murine knock-in model (Arg52Gly, Nkx2-5+/R52G) in a 129/Sv background was analyzed by histopathology, surface and telemetry ECG, and in vivo electrophysiology studies (EPS), comparing with control Nkx2-5+/+ mice at diverse postnatal stages, ranging from postnatal day 1 (P1) to 17 months. PR-prolongation (1st degree AVB) was present at 4 weeks, 7 months, and 17 months of age but not at P1 in the mutant mice. Advanced AVB was also occasionally demonstrated in the mutant mice. EPS showed that AV nodal function, and right ventricular effective refractory period, were impaired in the mutant mice, while sinus nodal function was not affected. AV nodal size was significantly smaller in the mutant mice compared to 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 AVB phenotype seen in human heterozygous missense mutations in NKX2-5 homeodomain was replicated in mice by knocking-in a comparable missense mutation.

Introduction: Heterozygous human NKX2-5 homeodomain (DNA binding domain) missense mutations are highly penetrant for varied cardiac anomalies and progressive atrioventricular block (AVB). We replicated many elements of the cardiac anomalies in a murine heterozygous knock-in model (Nkx2-5+/R52G, hereafter Nkx2-5-KI) in a 129/Sv background. Hypothesis: We hypothesized that Nkx2-5-KI mice demonstrate progressive AVB during postnatal life similar to human patients. Methods: Nkx2-5-KI mouse model and control Nkx2-5+/+ mice were analyzed by histopathology, surface and telemetry ECG, and in vivo electrophysiology studies (EPS), and compared at postnatal day 1 (P1) to 17 months. We re-evaluated a pedigree of a human family having Arg52(189)Gly mutation in 5 generations. Results: Nkx2-5-KI mice demonstrated PR-prolongation (1st degree AVB) at 4 weeks, 7 months, and 17 months of age but not at P1 (control n=35, mutant n=38). Advanced AVB was occasionally observed. EPS showed that AV node function, and right ventricular effective refractory period were impaired in Nkx2-5-KI mice, while sinus node function was normal. AV node size was significantly smaller in Nkx2-5-KI mice relative to control mice (maximum area size 2.4 ± 0.2 vs. 1.6 ± 0.1 x 10-2 mm2, 36% reduction; calculated volume 1.50 ± 0.1 vs. 1.11 ± 0.03 x 10-4 mm3, 24% reduction, n=6 each) at 4 weeks of age but not P1, similar to age-dependence of PR-prolongation. AV node in mutant mice was composed of smaller cardiomyocytes without fibrosis, suggesting in part, that conduction abnormalities are the result of an AV node that becomes morphologically atrophic during postnatal life. All 6 genotype-positive humans demonstrated AVB (1st, 2nd, and 3rd degree). QRS prolongation was uniquely demonstrated in Nkx2-5-KI mice from the neonatal stage onward but not in human patients, suggesting substantial discordance in ventricular conduction system between mouse and human. Conclusion: Highly penetrant and progressive AVB similar to that seen in humans with heterozygous NKX2-5 homeodomain missense mutations was replicated in mice by knocking-in a comparable missense mutation. The progressive AVB phenotype is likely due to an AV node composed of smaller cardiomyocytes that becomes atrophic during postnatal life.