Molecular remodeling of Cx43, but not structural remodeling, promotes arrhythmias in an arrhythmogenic canine model of nonischemic heart failure

Abstract

Background: Both gap junctional remodeling and interstitial fibrosis have been linked to impaired electrical conduction velocity (CV) and fatal ventricular arrhythmias in nonischemic heart failure (HF). However, the arrhythmogenic role of the ventricular gap junctional Cx43 in nonischemic HF remains in debate. Here, we assessed this in a newly developed arrhythmogenic canine model of nonischemic HF.

Methods and results: Nonischemic HF was induced in canines by combined aortic valve insufficiency and aortic constriction. Left ventricular (LV) myocardium from HF dogs showed similar pathological changes to that of humans. HF dogs had reduced LV function, widened QRS complexes, and spontaneous nonsustained ventricular tachycardia. CV was measured in intact LV epicardium with high-density grid mapping. Total (Cx43-T) and nonphosphorylated Cx43 (Cx43-NP) and histological interstitial fibrosis were assessed from these mapped LV tissues. Longitudinal CV, which was slowed in HF (49 ± 1 vs. 65 ± 2 cm/s in Ctl), was positively correlated with reduced total junctional Cx43 and negatively correlated with markedly increased junctional Cx43-NP (2-fold) in HF. Cx43 dephosphorylation in HF was associated with enhanced colocalization of PP2A at the level of Cx43. Unchanged action potential upstroke and transverse CV were associated with unaltered Cx43 lateralization and interstitial fibrosis in the nonischemic HF canine LV.

Conclusion: Our unique arrhythmogenic canine model of HF resembles human nonischemic HF (prior to the end stage). Cx43 remodeling occurs prior to the structural remodeling (with lack of fibrosis) in HF and it is crucial in slowed CV and ventricular arrhythmia development. Our findings suggest that altered Cx43 alone is arrhythmogenic and modulation of Cx43 has the anti-arrhythmic therapeutic potential for HF patients.

Keywords: Conduction velocity; Dephosphorylation; Fibrosis; Heart failure; connexin43 remodeling.

Fig. 1.

Canine HF model. A, Aortogram (top) and color-flow echocardiogram (bottom) demonstrating a competent aortic valve pre-AI and severe AI immediately post-AI induction. B, Representative M-mode echo images of LV at baseline and with induction of HF. Summary data for time course of change in LVEDD & LVESD (C) and LV fractional shortening (D) post aortic constriction (AC) N_animal = 20. E, Representative hearts from control dog (left) and HF dog (right). Summary data for HW/BW, N_animal = 9,8 (F), cell length, N_animal = 9,8, n_cell = 65,27 (G) and cell fractional shortening (FS) N_animal = 9,8, n_cell = 65,27 (H) for HF vs controls. **p < 0.01; ***p < 0.001. Each error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used for comparing difference between the two groups. Abbreviations: aortic insufficiency (AI), aortic constriction (AC), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD).

Fig. 2.

Spontaneous arrhythmias in canine HF model. A, Representative ECG from Ctl and HF dog hearts. B, Representative Holter monitor recording in conscious state for HF dog showing frequent PVCs, couplets, and runs of spontaneous nonsustained VT. Insert shows close up of a 16-beat and a 4-beat run of VT. Abbreviations: control (Ctl), heart failure (HF), premature ventricular complex (PVC), ventricular tachycardia (VT).

Fig. 3.

Slow conduction in HF LV. Isochronal maps with CV vector from each recording site for Ctl (A) and HF (B) tissue under drive train (A&B, left). Isochronal maps A and B show labeled recording sites a and b in the direction of CV_L with electrograms from sites a and b (and the peak of the bipolar electrograms) at those respective sites (A&B, right), showing slow CV_L in HF. Summary data for CV_L with drive train stimuli (C) or with premature stimulation (D) and unchanged CV_T (E). N_animal = 9,8, ***p < 0.001. Each data point is the mean value from three technical repeats. Each error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used to compare the difference between the two groups. Abbreviations: control (Ctl), conduction velocity (CV), longitudinal conduction velocity (CV_L), transverse conduction velocity (CV_T), heart failure (HF).

Fig. 4.

Unchanged interstitial collagen deposition and typical AP characteristics in HF dog hearts: A, Trichrome staining in mapped Ctl (left) and HF (right) dog LV Epi tissue. B, Summarized quantification of interstitial collagen in HF vs Ctl. N_animal = 9,8, p = NS. Each quantitative interstitial collagen data point is the mean value from forty histology images of each tissue section. Representative AP’s from Ctl and HF LV epicardium (C) and summarized data for dV/dt_Vmax of phase 0 of AP (D). N_animal = 9,8, p = NS. Each data point is the mean value from three technical repeats. Each error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used to compare the difference between the two groups. Abbreviations: action potential (AP), control (Ctl), heart failure (HF), left ventricle (LV), maximal upstroke velocity (V_max).

Fig. 5.

Reduced Cx43-T is positively correlated with slowed CV_L in HF LV: Confocal images of Cx43 + N-cadherin double staining and Cx43 staining in Ctl (A, B) and HF (C, D) dog tissue used in grid mapping with Cx43 (green), N-cadherin (red) and overlap (yellow). Examples of Cx43_E-E and Cx43_S-S are encircled and labeled in each of the images as well as enlarged images of Cx43_E-E and Cx43_S-S. Quantification of Cx43_E-E (E), Cx43_S-S (F), and lateralization ratio (G) for HF vs Ctl, N_animal = 9,8. Linear regression of Cx43_E-E and CV_L (H) and Cx43_S-S and CV_T (I), N_animal = 9,8. *p < 0.05; ***p < 0.001. Each quantitative immunostaining Cx43 data point (Cx43_E-E or Cx43_S-S) is the mean value from forty confocal images of each tissue section. Each error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used to compare the difference between the two groups. Abbreviations: control (Ctl), longitudinal conduction velocity (CV_L), heart failure (HF), junctional Cx43 (Cx43_E-E), non-junctional Cx43 (Cx43_S-S).

Fig. 6.

Enhanced Cx43-NP is negatively correlated with slowed CV_L by IHC in HF: Confocal images of Cx43-NP + Cx43-T double staining and Cx43-NP staining in Ctl (A, B) and HF (C, D) dog tissue used in grid mapping with Cx43 (red), Cx43-NP (green) and overlap (yellow). An example of Cx43-NP is circled and labeled. E, Quantification of Cx43-NP. F, Linear regression of Cx43-NP and CV_L. N_animal = 9,8, *p < 0.05. Each quantitative Cx43-NP data point is the mean value from forty confocal images of each tissue section. Each error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used to compare the difference between the two groups. Abbreviations: longitudinal conduction velocity (CV_L), non-phosphorylated Cx43 (Cx43-NP), HF (heart failure), IHC (immunohistochemistry).

Fig. 7.

Reduced Cx43-T and enhanced Cx43-NP are associated with increased colocalized PP2A with Cx43 in HF LV: Representative Western blot images for Ctl and HF dog LV (top) and summarized data (bottom) for: Cx43-T and Cx43-NP (A & B), and PP1 (C & D) with GAPDH as controls. Co-immunoprecipitated PP2A with Cx43 for Ctl and HF dog LV (E & F), N_animal = 9,8. *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment had three technical repeats. The error bar indicates the standard error of the mean (SEM) of each experimental group. Mann-Whitney test was used to compare the between the two groups. Abbreviations: non-phosphorylated Cx43 (Cx43-NP), total Cx43 (Cx43-T), heart failure (HF), immuno-coprecipitation (IP), left ventricle (LV).