Conduction defects and arrhythmias in mdx mice are not associated with a degeneration of the cardiac Purkinje network

Figures

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  FIGURE 1. Progressive cardiac conduction defects in mdx mice. (a) Kaplan-Meier survival plot. The survival curves showed that 27% of the mdx mice (n = 26) died at 12-month-old, whereas all WT survived (n = 9). (b) Measurements of cardiac parameters in WT and mdx mice by electrocardiography. Graphs representing the evolution of the PR-II and QRS-II intervals measured in the same mice over a year show a progressive increase in these parameters in mdx (n = 26) compared to WT (n = 9) mice. (c) Graphs representing heart size measurements in WT (n = 5) and mdx mice (n = 11). Mdx mice present a significantly greater heart length and width compared to WT. *p < 0.1; **p < 0.01; ***p < 0.001.

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  FIGURE 2. Spontaneous ventricular arrhythmic events in mdx mice. (a) Representative tracings from surface ECG measured in lead II in anaesthetized mice before and after ISO (2 mg/kg) IP injection. At baseline, only mdx mice present premature ventricular complexes (PVC, black arrow). After ISO challenge, the cardiac rhythm is accelerated in all mice and PVCs and ventricular tachycardia are mainly observed in mdx mice (VT). (b) Histograms showing that PVCs are more frequent in mdx mice with age. ISO stimulation revealed a greater prevalence of PVC in mdx mice compared to WT at 12 months of age. WT (n = 9) and mdx mice (n = 26).

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  FIGURE 3. No morphological and maturation defects of the Purkinje Fiber network enriched in dystrophin. (a) Whole-mount immunofluorescence with Cx43 antibodies and Cx40-GFP on opened LV from WT and mdx adult mice. On the left image, the GFP fluorescence indicates a similar pattern of the Purkinje Fiber network between mdx and WT mice. Scale bar = 500 µm. Higher magnifications indicated by squares show the normal expression of the gap junction Cx43 at the intercalated discs (IDs) in contractile cardiomyocytes (white arrows) or along the membrane in PF (Yellow arrows) in both mdx and WT mice. Scale bar = 100 µm (WT n = 3; mdx n = 9). (b) Immunofluorescence with Contactin-2, Cx43, DMD and WGA-cy3 antibodies on transversal sections at the mid-ventricular level from WT and mdx mice. While Cntn2 expression, the marker of VCS maturation is similar in WT and mdx mice, DMD is absent in mdx hearts and enriched in their Purkinje fibers compared to other cardiomyocytes in WT. WGA and Cx43 staining at the membrane highlight the cardiac hypertrophy seen in mdx hearts. Scale bar = 50 µm (n = 11).

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  FIGURE 4. Sodium current (INa) densities in cardiac Purkinje fibers and ventricular cardiomyocytes from dystrophin-deficient mdx and wild-type (WT) mice at different animal ages. (a) Representative original whole cell INa traces recorded from Purkinje fibers from 11–13- or 19–21-week-old WT and mdx mice. The pulse protocol to elicit the currents is shown in the inset. (b) Current density-voltage relationships derived from a series of experiments as displayed in a (38 cells, six animals, WT 11–13 weeks; 23 cells, five animals, mdx 11–13 weeks; 48 cells, six animals, WT 19–21 weeks; 34 cells, five animals, mdx 19–21 weeks). The solid lines represent fits with a function given in the methods section. (c) Statistical comparison of current density values at −37 mV between WT and mdx Purkinje fibers from 11–13-week-old mice. In this age range, current densities in mdx Purkinje fibers (−62.6 ± 4.7 pA/pF) were only reduced by trend when compared to WT Purkinje fibers (−74.1 ± 6 pA/pF). (d) Comparison of current density values at −37 mV between WT and mdx Purkinje fibers from 19–21-week-old mice. Here, a significant difference between WT (−72.7 ± 3.3 pA/pF) and mdx (−52.5 ± 3.5 pA/pF) Purkinje…

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  FIGURE 5. Arrhythmogenic cardiomyopathy associated with ventricular dyssynchrony and fibrosis. (a) The cardiac electrical axis calculated from surface electrocardiograms is normally comprised between 0° and 120° in WT while a progressive deviation towards the left is observed with age in mdx mice (WT n = 9; mdx n = 26). (b) WGA staining by immunofluorescence shows the presence of cardiac fibrosis only in mdx mice. Scale bar = 500 µm. (c) Fibrosis was quantified by measuring the percentage of WGA positive area in the LV and is significantly increased in mdx (n = 7) mice in comparison to WT (n = 4)-II (unpaired t-test, **p = 0.0042). (d) Cardiac fibrosis correlates with cardiac axis deviation in mdx mice (n = 11) (r = 0.58; p = 0.06).