Irx3 is highly expressed in the developing and mature His–Purkinje network. (A–D) β-Galactosidase staining of Irx3^tauLacZ/+ embryos at E10 (A), E11 (B), E14 (C), and in adult heart (D). Arrowhead in A shows a ring-like group of cells that is detected in the developing ventricle. (E) Masson's trichrome staining reveals fibrous insulation of the Irx3^tauLacZ+ bundle of His in an adult heart. (F and G) Optical projection tomography (OPT) images of Irx3^tauLacZ expression at E16.5 (F) and postnatal day 3 (P3; G). (H) Immunofluorescence colocalization of Irx3^tauLacZ and Hcn4 in the VCS at P3. (I) Colocalization of Irx3^tauLacZ and Cx40^EGFP in the VCS of Irx3^tauLacZ/+;Cx40^EGFP/+ mice at P14, including bundle branches (I’) and Purkinje fibers (I’’). (J) Adult Irx3^tauLacZ+ bundle branch cells express Cx40 at longitudinal cell borders. (K) Immunostaining of reaggregated cells for Cx40, Cx43, and EGFP E14.5 and P3. (L) Quantitation of gap junctions formed between reaggregated cells. avc, atrioventricular canal; rv, right ventricle; lv, left ventricle; rbb, right bundle branch; lbb, left bundle branch; tb, trabeculae; rvfw, right ventricular free wall; His, bundle of His.

Irx3 is required for normal ventricular activation. (A) Representative six-lead surface ECG tracing shows prolongation of the QRS and abnormal R’ wave in Irx3-null mice. (B) A summary of six-lead and telemetry ECG parameters. n = 8–12; *P < 0.05 vs. wild type. (C) Representative octapolar intracardiac ECG traces illustrating atrial, ventricular, and His-bundle depolarization signals. (D) Quantification of HV prolongation. n = 9; *P < 0.05. (E) Optical mapping results shown in apical four-chamber view and apical right ventricular two-chamber view. Isochrone lines mark areas where depolarization reached 50% intensity in consecutive 0.5-ms intervals. Depolarization proceeds in an apex-to-base direction. Red indicates earliest activation time (ms). Wild type, n = 12; Irx3-null, n = 9. (F) Quantitation of epicardial conduction velocity (CV) and corresponding QRS. We found that 77% of hearts lacking Irx3 had RBBB, slowed epicardial CV, and QRS widening, in comparison with wild-type hearts, and that 23% of Irx3-null hearts that did not show conduction block (NB). Values are mean ± SEM; n = 4–6; *P < 0.05. (G) Quantitation of VCS fiber CV in Irx3^{taulLacZ/tauLacZ};Cx40^EGFP/+ fibers compared with Irx3^+/+;Cx40^EGFP/+ mice. Values are means ± SEM; n = 6–7; *P < 0.05.

Irx3 regulates Cx40 and Cx43 expression in the VCS. (A) Representative Cx40 immunofluorescence images of the right bundle branch and right ventricular free wall Purkinje fibers of wild-type (WT) and Irx3-null mice at 8 wk. (B) Quantitation of Cx40⁺ plaques. (C) qRT-PCR analysis of LCM captured adult VCS cells for Gja5 mRNA in Irx3^{tauLacZ/tauLacZ};Cx40^EGFP/+ vs. Irx3^+/+;Cx40^EGFP/+ hearts. (D) Representative Cx43 immunofluorescence images. Arrowheads show ectopic Cx43⁺ plaques in Irx3::EGFP⁺ proximal bundle branch cells of Irx3-null mice at P5. Grayscale images show intensity mask for Cx43 signal. (E) Plaque intensity was quantified by using ImageJ software. *P < 0.05 vs. WT. (F) Adult heart confocal imaging at 100× magnification shows ectopic Cx43⁺ plaques at conduction-working myocyte and conduction–conduction myocyte borders in the absence of Irx3. (G) Plaque intensity was quantified by using ImageJ software. *P < 0.05 vs. WT. (H) Immunofluorescence for Cx43 (green), N-cadherin (red), and EGFP (blue) of cells reaggregated from the VCS of either WT or Irx3-null mice, each expressing the Irx3::EGFP reporter. (I) Quantitation of Cx43 plaque expression at cell–cell borders. n = 80; *P < 0.05 vs. WT. (J and K) Fluorescent dye spread (white outline) in microinjected proximal right bundle branch cells (marked by Irx3::EGFP⁺). *P < 0.05 vs. WT. rvfw, right ventricular free wall; Pf, Purkinje fibers; LBB, left bundle branch; Endo, endocardium; Epi, epicardium.

Transcriptional regulation of Cx40 and Cx43 gene expression by Irx3. (A) NVMs were infected with the following adenoviral constructs: GFP control (Ad-GFP), Irx3 (Ad-Irx3, Ad-FLAG-Irx3), Irx3 fused to the VP16 activation domain (Ad-VP16-Irx3), or the Engrailed suppressor domain (Ad-EnR-Irx3). (B) Cx40 and Cx43 protein expression. (C) Quantitation of Gja1 and Gja5 mRNA compared with noninfected and GFP-infected cells. (D) Schematic depicting regulation of Gja1 and Gja5 transcription by dominant active and dominant negative forms of Irx3 in infected NVMs. (E) Genome alignment analysis of the Gja1 promoter revealed an evolutionarily conserved element containing a putative Irx3 binding site that overlaps with an Nkx2-5 binding motif (Irx/NKE), immediately upstream of T-box binding elements (TBE). (F) Chromatin immunoprecipitation using ventricles from Irx3^3myc-6his mice shows enrichment of Irx3 at the conserved Irx/NKE site and core promoter, but not at an intergenic region or the Nppa promoter. (G) Coimmunoprecipitation of Irx3 with Nkx2-5 and Tbx5. Su(fu) serves as negative control. (H) Luciferase activity of Gja1–luciferase in cells cotransfected with Irx3 or Nkx2-5 expression constructs. Gja1mut indicates a Gja1–luciferase reporter in which three point mutations were made in the evolutionarily conserved core binding sequence. ECR, evolutionarily conserved region; CP, core promoter; Irx/NKE, evolutionarily conserved binding site for Irx3 and Nkx2-5.