Modulation of cardiac gap junction expression and arrhythmic susceptibility

Abstract

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.

Figure 1

Kaplan–Meier survival analysis comparing F1 CKO, O-CKO, and control mice. A, Median survival of O-CKO mice (solid line) was significantly longer than in the F1 CKO mice (dashed line; P<0.0001). B, Despite delayed sudden death in the O-CKO mice (solid line), their lifespan was significantly shorter than that of control littermates (dashed line; P<0.0001).

Figure 2

Connexin expression in O-CKO ventricular lysates. A, Representative lanes comparing Cx43 expression in O-CKO and control ventricular lysates at 25, 35, and 45 days of age demonstrate a progressive decline in Cx43 abundance in the O-CKO hearts. B, Expressed as a percentage of matched controls, Cx43 concentration is significantly lower at all time points in the O-CKO ventricles and decreases with time. *P<0.01 compared with matched controls; **P<0.001 compared with matched controls; ^#P<0.01. C, Representative immunoblots indicating no significant change in Cx40 expression in O-CKO ventricular lysates compared with controls at 45 days. D, Representative immunoblots of atrial lysates demonstrating no significant difference in Cx43 abundance between control and O-CKO samples at 45 days. Blotting for tubulin was performed to indicate relative loading.

Figure 3

Immunofluorescent staining for connexin43 in control and O-CKO mid-ventricular sections. Abundant Cx43 staining (bright green signal) is seen in sections from 25-, 35-, and 45-day-old control ventricles (A to C). In contrast, gradual loss of Cx43 expression is evident in O-CKO ventricular sections from the same time points (D to F). Variable expression of Cx43 is observed in the ventricular myocardium of a 45-day-old O-CKO heart (G to I). Scale bar=200 μm in panels A to F, 100 μm in panels G to I.

Figure 4

Quantification of connexin43 immunofluorescent signal in O-CKO and control heart sections. A, Graph of immunofluorescent clusters (IFC) per 20× field in control (solid bars) and O-CKO hearts (shaded bars) shows a significant decline in both groups with time, whereas O-CKO values are significantly less than those of controls at each time point. B, IFC per 20× field in O-CKO hearts as a percentage of age-matched controls decreases significantly over time. C, Percent area occupied by Cx43 immunofluorescent signal in control (solid bars) and O-CKO hearts (shaded bars) shows a similar relationship to that seen for IFC in (A). D, Percent area of Cx43 signal expressed relative to age-matched controls decreases significantly over time. *P<0.05; **P<0.01.

Figure 5

QRS amplitude progressively decreases in O-CKO mice. Representative electrocardiograms from control and 25-, 35-, and 45-day-old O-CKO mice demonstrate a gradual loss of QRS amplitude in the O-CKO mice. Note that P wave amplitude remains unchanged.

Figure 6

Programmed electrical stimulation in an O-CKO mouse. A, Several beats of nonsustained ventricular tachycardia are induced by a single extrastimulus. S1-S1, 120 ms; S1–S2, 35 ms. B, Sustained ventricular tachycardia with a cycle length of ≈40 ms is induced by double extrastimuli. S1-S1, 120 ms; S1–S2, 35 ms; S2–S3, 35 ms. C, Rapid incessant ventricular tachycardia persists despite attempts at overdrive pacing. D, Within 2 minutes of induction of ventricular tachycardia, the arrhythmia degenerates into coarse ventricular fibrillation.

Figure 7

Connexin43 abundance in inducible and noninducible O-CKO mice. Cx43 abundance as a percentage of control levels in individual O-CKO mice with and without inducible sustained VT is shown. Mean±SEM for each population is indicated. *P=0.001.