Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias

Abstract

Rationale: Posttranslational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathological gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood.

Objective: To establish whether specific Cx43 phosphorylation events influence gap junction expression and pathological remodeling.

Methods and results: We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or nonphosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathological gap junction remodeling and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electric remodeling, and were highly susceptible to inducible arrhythmias.

Conclusions: These data demonstrate a mechanistic link between posttranslational phosphorylation of Cx43 and gap junction formation, remodeling, and arrhythmic susceptibility.

Figure 1. Electrophoretic mobility of Cx43 mutants

(A) High resolution Western blot analysis of whole cell lysates (WCL) or Triton X-100 insoluble pellets (pellet) prepared from ventricles of mice with the indicated genotypes, probed with polyclonal panCx43 antisera. Wild type Cx43 lysate treated with calf intestine phosphatase (CIP) migrates at P0 and is shown for comparison to various major phosphorylated forms of Cx43 (P1, P2, P3). (B) Western blot analysis of whole cell lysates (upper) or Triton X-100 insoluble pellets (lower). N=3 for each genotype. Quantification is shown to the right. *P<0.05 compared to WT.

Figure 2. Cx43-S3E phospho-mimetic mutation prevents pathologic gap junction remodeling

(A) Representative immunofluorescent staining with panCx43 (Cx43, green) and N-cadherin (N-cad, red) antibodies at baseline (B) and four weeks following transverse aortic constriction (TAC). Scale bar = 10 μm. Magnified views of individual gap junction plaques for each genotype after TAC are shown below. (B) Quantitative analysis of Cx43 and N-cadherin colocalization at baseline (□) and following TAC (■). *P<0.05 compared to S3E, Post-TAC; +P<0.05 compared to WT and S3E.

Figure 3. S3E mice are resistant to structural GJR after chronic pathologic stimuli

(A) Representative immunoblots of whole cell lysates at baseline (B) and after TAC (T), probed with polyclonal panCx43 and GAPDH antibodies. (B) Quantitative analysis of Cx43 in gap junction enriched Triton X-100 insoluble fractions, at baseline and after TAC, normalized to GAPDH. N=3 hearts for each group. *P<0.05, TAC vs same genotype at baseline.

Figure 4. S3E mice are resistant to structural GJR after acute pathologic stimuli

(A) Representative immunoblots of samples probed with polyclonal panCx43, monoclonal non-phosphorylated Cx43 (np-Cx43) and GAPDH antibodies prior to (0 min) and 30 minutes (30min) after imposition of global ischemia. (B) Quantitative analysis of np-Cx43 signal relative to panCx43 signal. N=3 hearts for each group. *P<0.05 compared to S3E at 30 minutes global ischemia.

Figure 5. Functional analysis of isolated-perfused hearts

(A) Representative activation maps and (B) quantification of conduction velocity at baseline and after TAC. *P<0.05 compared to baseline for each genotype; ⁺P<0.05 compared to TAC WT and S3A. (C) Representative APD₅₀ maps. The shadow in each image represents a non-pacing positioning. (D) Quantification of averages values of APD₅₀ dispersion at an S1S2 coupling interval of 95 ms. (E) Average values of APD₅₀ taken at a range of coupling intervals. CV, conduction velocity. *P<0.05. N=6 hearts for each genotype.

Figure 6. S3E mice are resistant to inducible ventricular arrhythmias

Percentage of mice of each genotype with ventricular tachycardia (VT) induced by premature extrastimuli (A) or burst pacing (B). (C) Representative intracardiac and surface electrocardiograms of an S3A mouse during an episode of VT induced by a single extrastimulus. AV dissociation is noted after onset of VT. (D) Representative intracardiac and surface electrocardiograms of an S3E mouse, showing resumption of sinus rhythm after the premature beat. RV-IECG, right ventricular intracardiac electrocardiogram; RA-IECG, right atrial intracardiac electrocardiogram; SECG, surface electrocardiogram; Arrowheads, stimulus artifact; v, ventricular electrogram; a, atrial electrogram. *P<0.05.