Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias

Abstract

Aim: Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human.

Methods and results: Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations.

Conclusion: IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.

Keywords: Cardiac conduction system; Sudden cardiac death; Ventricular fibrillation.

Figure 1

Arrhythmia development in Irx3^−/− and Irx3^+/− mice. (A) Representative ambulatory telemetric electrocardiogram recordings in homozygous Irx3^−/− mice. Upper panel shows transient atrio-ventricular block. Arrows indicate P waves without following ventricular excitations. Lower panel shows spontaneous non-sustained ventricular tachycardias. Solid lines indicate the timing with non-sustained ventricular tachycardias. Reverse triangles show ventricular premature contractions. (B) Comparison of frequency of atrio-ventricular block, ventricular tachyarrhytmias, and ventricular tachycardias in ambulatory telemetric electrocardiography in WT (n = 10) and Irx3^−/− (n = 9) mice. Ventricular tachyarrhytmias was defined as consecutive ventricular premature contractions more than couplets, and ventricular tachycardias as consecutive ventricular premature contractions more than triplets. Statistical analysis was done with Fisher's exact test. (C) Representative ambulatory telemetric electrocardiogram recordings in heterozygous Irx3^+/−. Upper panel shows transient atrio-ventricular block. Arrows indicate P waves without following ventricular excitations. Lower panel shows spontaneous non-sustained ventricular tachycardias. Solid lines indicate the timing with non-sustained ventricular tachycardias. Reverse triangles show ventricular premature contractions. (D) Transient atrio-ventricular block induced by isoproterenol infusion in Irx3^−/− mice. Arrows represent P waves without following ventricular excitation. (E) Comparison of frequency of atrio-ventricular block, ventricular tachyarrhytmias, and ventricular tachycardias after isoproterenol infusion in WT (n = 10) and Irx3^−/− (n = 9) mice. Statistical analysis was done with by Fisher's exact test. (F) Non-sustained ventricular tachycardias induced by isoproterenol infusion in Irx3^+/− mice. Solid lines indicate the timing with non-sustained ventricular tachycardias. (G) Representative electrocardiogram recordings during swimming in Irx3^−/− mice. Reverse triangles show ventricular premature contractions. (H) Comparison of frequency of atrio-ventricular block, ventricular tachyarrhytmias, and ventricular tachycardias during swimming in wild-type (n = 10) and Irx3^−/− (n = 9) mice. Statistical analysis was done with Fisher's exact test. (I) Representative electrocardiogram recordings within 24 h after surgical creation of myocardial infarction in Irx3^−/− mice. Dotted lines indicate the timing with bigeminy, and solid lines indicate the timing with ventricular tachycardias. (J) Comparison of frequency of atrio-ventricular block, ventricular tachyarrhytmias, and ventricular tachycardias within 24 h after myocardial infarction in wild-type (n = 6) and Irx3^−/− (n = 6) mice. Statistical analysis was done with Fisher's exact test. (K) Incidence of ventricular premature contractions within 24 h after surgical creation of myocardial infarction in wild-type (n = 6) and Irx3^−/− (n = 6) mice. Statistical analysis was done with Mann–Whitney U test.

Figure 2

Ex vivo optical epicardial mapping and arrhythmia development in Irx3^−/− mice. (A) Representative optical epicardial mapping in wild-type and Irx3^−/− mice in basal condition. (B) Representative optical epicardial mapping in wild-type and Irx3^−/− mice after isoproterenol application. In Irx3^−/− mice, epicardial breakthrough occurs from the base of the right ventricle, and propagates to the apex; the propagation of depolarization became markedly slow. (C) Arrhythmias observed in wild-type and Irx3^−/− mice after isoproterenol application. In Irx3^−/− mice, atrio-ventricular block and atrio-ventricular block with non-sustained ventricular tachycardias occurred. In wild-type mice, only sinus tachycardia occurred. Reverse triangles indicate atrial action potential without following ventricular action potential. Solid bar indicates non-sustained ventricular tachycardias. (D) Comparison of frequency of atrio-ventricular block, ventricular tachyarrhytmias, and ventricular tachycardias after isoproterenol injection in wild-type (n = 7) and Irx3^−/− (n = 6) mice. Statistical analysis was done with Fisher's exact test.

Figure 3

Family pedigrees and surface electrocardiograms in patients without SCN5A mutation. (A) Pedigree of the Family #1 with R421P IRX3 mutation. An arrow indicates the proband. (B) Pedigree of the Family #2 with P485T IRX3 mutation. An arrow indicates the proband. (C) Surface electrocardiogram of the proband in the Family #1 with R421P IRX3 mutation. Electrocardiogram showed coved type ST elevation in V1 and V2 (black arrows), and saddle-back type ST elevation in V3 (grey arrow).

Figure 4

Irx3 mutations were less effective in up-regulation of Cx40 and Scn5a. (A) Homology of human IRX3 and murine Irx3. Amino acids conserved between human IRX3 and mouse Irx3 are shown by white letters in black box. Two missense mutation sites found in ventricular fibrillation patients in this study are shown by reverse triangles. (B and C) Effects of adenoviral infection with Irx3 into HL-1 cells on the expression of Cx40 (B) and Scn5a (C). The expression of Cx40 and Scn5a was normalized to that of Irx3. Adenoviral infection with wild-type Irx3 increased the expression of Cx40 and Scn5a. Up-regulation of Cx40 and Scn5a was significantly less in R426P (n = 6) and P491T (n = 6) infection than in wild-type Irx3 infection (n = 6). The data are presented actual plots beside the box whisker plot in these and following figures. (D and E) Effects of adenoviral infection with Irx3 into neonatal murine ventricular myocytes on the expression of Cx40 (D) and Scn5a (E). The expression of Cx40 and Scn5a was normalized to that of Irx3. Adenoviral infection with wild-type Irx3 into neonatal murine ventricular myocytes increased the expression of Cx40 and Scn5a. Up-regulation of Cx40 and Scn5a was significantly less in R426P (n = 6) and P491T (n = 6) infection than in wild-type Irx3 infection (n = 6). (F and G) Effects of transfection of HL-1 cells with Irx3 in pcDNA3 vector on the expression of Cx40 (F) and Scn5a (G). The expression of Cx40 and Scn5a was normalized to that of Irx3. Transfection of HL-1 cells with wild-type Irx3 increased the expression of Cx40 and Scn5a. Up-regulation of Cx40 and Scn5a was significantly less in R426P (n = 6) and P491T (n = 6) infection than in wild-type Irx3 transfection (n = 6).