Altered right atrial excitation and propagation in connexin40 knockout mice

Abstract

Background: Intercellular coupling via connexin40 (Cx40) gap junction channels is an important determinant of impulse propagation in the atria.

Methods and results: We studied the role of Cx40 in intra-atrial excitation and propagation in wild-type (Cx40(+/+)) and knockout (Cx40(-/-)) mice using high-resolution, dual-wavelength optical mapping. On ECG, the P wave was significantly prolonged in Cx40(-/-) mice (13.4+/-0.5 versus 11.4+/-0.3 ms in Cx40(+/+)). In Cx40(+/+) hearts, spontaneous right atrial (RA) activation showed a focal breakthrough at the junction of the right superior vena cava, sulcus terminalis, and RA free wall, corresponding to the location of the sinoatrial node. In contrast, Cx40(-/-) hearts displayed ectopic breakthrough sites at the base of the sulcus terminalis, RA free wall, and right superior vena cava. Progressive ablation of such sites in 4 Cx40(-/-) mice resulted in ectopic focus migration and cycle length prolongation. In all Cx40(-/-) hearts the focus ultimately shifted to the sinoatrial node at a very prolonged cycle length (initial ectopic cycle length, 182+/-20 ms; postablation sinus cycle length, 387+/-44 ms). In a second group of experiments, epicardial pacing at 10 Hz revealed slower conduction in the RA free wall of 5 Cx40(-/-) hearts than in 5 Cx40(+/+) hearts (0.61+/-0.07 versus 0.94+/-0.07 m/s; P<0.05). Dominant frequency analysis in Cx40(-/-) RA demonstrated significant reduction in the area of 1:1 conduction at 16 Hz (40+/-10% versus 69+/-5% in Cx40(+/+)) and 25 Hz (36+/-11% versus 65+/-9% in Cx40(+/+)).

Conclusions: This is the first demonstration of intra-atrial block, ectopic rhythms, and altered atrial propagation in the RA of Cx40(-/-) mice.

Figure 1

Representative ECGs from Cx40^+/+ and Cx40^−/− mice. A, Six-lead surface ECGs obtained from conscious Cx40^+/+ and conscious Cx40^−/− mice. B, Lead aVR from 5 different Cx40^+/+ and Cx40^−/− mice. Note prolongation and changed polarity of the P wave. C, Intervals for P, PR, and QRS are shown in volume-conducted ECGs of Langendorff-perfused hearts from Cx40^+/+ and Cx40^−/− mice.

Figure 2

A, Details of the RA epicardium. B and C, Composite mean 1-ms isochronal activation and SD maps from 12 Cx40^+/+ RA during basal rhythm. LA indicates left atrium; LSVC, left superior vena cava; RSVC, right superior vena cava; BB, Bachmann’s bundle; SAN, sinoatrial node; ST, sulcus terminalis; IVC, inferior vena cava; and RAA, RA appendage.

Figure 3

RA activation in Cx40^−/− hearts. A, One-millisecond isochronal activation map in representative RA. An ectopic discharge in the RA free wall results in an altered pattern with prolonged atrial activation time. B, In another RA, the pattern of activation is characterized by extremely delayed activation of an area corresponding to the sinoatrial node. C, Locations of ectopic sites in the RA of 12 Cx40^−/− mice. Abbreviations are defined in Figure 2 legend.

Figure 4

A, Spatial distribution of apparent conduction velocities (CV) over the entire optically mapped area during basal rhythm in both Cx40^+/+ (n=8) and Cx40^−/− mice (n=8). B, Percentage of the area with a given range of apparent conduction velocity. Note larger percentages of slower velocities in the Cx40^−/− mice.

Figure 5

A, RA activation maps and pseudo-ECGs of knockout mouse before (left) and after first (middle) and second (right) ablations. In control, an ectopic discharge (white star) resulted in altered activation. On first ablation (ABL), the ectopic focus migrated, and cycle length (CL) was prolonged. The second ablation shifted the focus to the sinoatrial node (right). B, Cycle length changes induced by consecutive ablations in 4 Cx40^+/+ (left) and 4 Cx40^−/− (right) hearts. Other abbreviations are defined in Figure 2 legend.

Figure 6

A, Representative RA activation maps obtained from Cx40^+/+ and Cx40^−/− mice during epicardial pacing (red arrow) at 10 Hz. The inset indicates the location of the electrodes. B, Apparent conduction velocity (CV) in the 2 genotypes at 10 Hz is shown for each individual experiment. Other abbreviations are defined in Figure 2 legend.

Figure 7

Frequency dependence of RA activation. A, Representative dominant frequency maps of RA paced at 10 Hz (top) and 25 Hz (bottom) in Cx40^+/+ (left) and Cx40^−/− (right) mice. Color bar indicates frequency. At 10 Hz, 1:1 activation occurs throughout the RA in both genotypes. At 25 Hz, the Cx40^+/+ RA follows 1:1; in the Cx40^−/− RA, a breakup of conduction with several lower frequency domains is seen. Single-pixel recordings show complex patters of activation at various sites. B, Percentage of RA following 1:1 is plotted as a function of pacing frequency. Note conduction breakdown between 12 and 16 Hz in Cx40^−/− mice.

Figure 8

A, Western blots for Cx43 in whole heart and atria of wild-type and knockout Cx40 mice. B, Immunofluorescence data from RA in Cx40V^−/− and littermate Cx40^+/+ mice stained for Cx43. C, Representative microelectrode recordings from RA free wall of Cx40^+/+ (left) and Cx40^−/− (right) mice. D, Action potential parameters from 8 Cx40^+/+ and Cx40^−/− mice. RMP indicates resting membrane potential; APA, action potential amplitude; and APD, action potential duration.