Focal gap junction uncoupling and spontaneous ventricular ectopy

Abstract

Genetic studies in the mouse have demonstrated that conditional cardiac-restricted loss of connexin43 (Cx43), the major ventricular gap junction protein, is highly arrhythmogenic. However, whether more focal gap junction remodeling, as is commonly seen in acquired cardiomyopathies, influences the propensity for arrhythmogenesis is not known. We examined electrophysiological properties and the frequency of spontaneous and inducible arrhythmias in genetically engineered chimeric mice derived from injection of Cx43-deficient embryonic stem cells into wild-type recipient blastocysts. Chimeric mice had numerous well-circumscribed microscopic Cx43-negative foci in their hearts, comprising approximately 15% of the total surface area as determined by immunohistochemical analysis. Systolic function in the chimeric mice was significantly depressed as measured echocardiographically (19.0% decline in fractional shortening compared with controls, P < 0.05) and by invasive hemodynamics (17.6% reduction in change of pressure over time, P < 0.01). Chimeras had significantly more spontaneous arrhythmic events than controls (P < 0.01), including frequent runs of nonsustained ventricular tachycardia in some of the chimeric mice. However, in contrast to mice with conditional cardiac-resricted loss of Cx43 in the heart, no sustained ventricular tachyarrhythmias were observed. We conclude that focal areas of uncoupling in the myocardium increase the likelihood of arrhythmic triggers, but more widespread uncoupling is required to support sustained arrhythmias.

Fig. 1

Generation and characterization of chimeric connexin43 (Cx43) knockout (KO) mice. Southern blotting (A) of tail DNA samples was performed for the identification of chimeric mice (*). Tail DNA was digested with NcoI, size-fractionated on agarose gels and transferred to a nitrocellulose membrane, which was then probed for the presence of 6.5-kb wild-type allele and 4.3- and 3.4-kb Cx43-null alleles. Immunofluorescent staining for Cx43 in control (B) and chimeric Cx43 KO (C) heart sections shows abundant Cx43 signal at the intercalated discs throughout wild-type hearts. Chimeric hearts, on the other hand, have randomly distributed areas without Cx43 staining juxtaposed to Cx43-expressing regions [demarcation (arrows in C) tends to be rather abrupt in most instances]. Net mean fluorescence (mean fluorescence minus background fluorescence) of Cx43-expressing areas was significantly higher in chimeric hearts than in controls (D). Despite increased expression in Cx43-expressing areas of chimeric hearts, overall Cx43 abundance (representative immunoblots, E) is not significantly elevated in chimeric ventricular lysates compared with controls (F). †P < 0.05.

Fig. 2

Incidence of ventricular arrhythmic events during 24-h monitoring. Total arrhythmic events in chimeric Cx43 KO (●) and control mice (◯) are arranged by time of day from 1 (1 AM) to 24 (12 AM). Chimeric Cx43 KO mice have significantly more arrhythmic events than controls (P < 0.01).

Fig. 3

Chimeric Cx43 KO mice display many different premature ventricular complex (PVC) morphologies. In contrast to the controls, each chimeric mouse showed many different PVC morphologies. Representative examples of PVCs from different days in the same control and chimeric mice are shown, demonstrating a consistent PVC morphology in the control over 6 days of monitoring and several different PVC morphologies in the chimera over the same time span.

Fig. 4

Telemetry recording from a chimeric Cx43 KO mouse. Examples of frequent nonsustained runs of ventricular tachycardia are seen on a telemetry tracing from an ambulatory, unanesthetized chimeric mouse. Cycle lengths of each of the examples of ventricular tachycardia are ~40 ms.