Cardiac lesions in Duchenne muscular dystrophy model rats with out-of-frame Dmd gene mutation mediated by CRISPR/Cas9 system

Fig. 1.. Immunohistochemical staining of hearts (left ventricles) with anti-dystrophin mouse monoclonal antibody. Dystrophin staining is clearly found especially in the membrane of cardiomyocytes of wild type rats (A, C and E) while there is no staining in the hearts at any age of cDMDR rats (B, D, and F). A: WT at 3 months of age. B: cDMDR at 3 months of age. C: WT at 5 months of age. D: cDMDR at 5 months of age. E: WT at 10 months of age. F: cDMDR at 10 months of age.

Fig. 2.. Histopathological changes in cDMDR hearts. Cardiomyocyte degeneration and necrosis, mononuclear cell infiltration, and fibrosis are observed in all age groups. These changes are found diffusely in the ventricular wall and papillary muscle, and especially prominent in the right ventricular wall and the outer layer of the left ventricular wall. There are, to some degree, differences in the lesions between cDMDR of the same age, but histopathological findings are similar for all cDMDR. There are no apparent histopathological findings indicative of hypertrophic cardiomyopathy or dilated cardiomyopathy. A, B: Left ventricle of cDMDR at 3 months of age. Arrows indicate necrosis of cardiomyocytes. C, D: Left ventricle of cDMDR at 5 months of age. Arrows indicate necrosis of cardiomyocytes. E, F: Left ventricle of cDMDR at 10 months of age. Arrows indicate necrosis of cardiomyocytes. A–F: HE stain.

Fig. 3.. Progression of fibrosis with aging in cDMDR rats. Masson’s-trichrome stain of the heart indicates progression of fibrosis from 3 to 5 and 10 months of age in cDMDR rats (A-C, respectively) but minimal changes in 10-month-old WT rats (D). The percentage of fibrosis area in the overall cross-sectional area of the heart increased with aging (E). Bars indicate Mean ± SE of cDMDR (n = 3/group). X-marks indicate individual values of WT rats at 10 months of age (n = 2). *: p<0.05 (the difference among three age groups of cDMDR). Post-hoc Tukey’s test following one-way ANOVA.

Fig. 4.. Electrocardiographic waveforms of arrhythmias observed in cDMDR

Fig. 5.. Body weight and electrocardiographic waveform components in cDMDR and WT rats at different ages. Greater R-wave amplitudes and shorter PR-intervals in cDMDR rats can be seen, albeit with differences in statistical detection sensitivity between age group caused by the difference in the number of samples per age group. A prolonged QT interval at 3 months of age but a trend toward shorter QT interval at 8 months of age are observed, and a trend toward lower P wave amplitudes at 8 months of age is detected in cDMDR. Mean ± SE, *: p<0.05. Post-hoc Student’s t-test following two-way ANOVA.

Fig. 6.. Left ventricular long-axis cross-sectional imaging (B-Mode) from cDMDR and WT at 9 months of age. Very bright echoed areas (arrows) in the subadventitia and near the papillary muscles indicate tissue sclerosis in cDMDR.

Fig. 7.. Individual data from echo images in cDMDR and WT rats. A: Heart rate; B: Left ventricular wall weight/body weight ratio; C: Stroke volume; D: Left ventricular anterior wall thickness; E: Left ventricular posterior wall thickness; F: Ratio of left ventricular rapid inflow velocity (E-wave) to atrial systolic inflow velocity (A-wave); G: Left ventricular ejection fraction; H: Left ventricular internal diameter fractional shortening; I: Cardiac output. Note that tendency toward worsening of contractile indices (C, G, H, and I) as well as a tendency toward worsening of diastolic function (F) are seen.