Progressive cardiomyopathy with intercalated disc disorganization in a rat model of Becker dystrophy

Abstract

Becker muscular dystrophy (BMD) is an X-linked disorder due to in-frame mutations in the DMD gene, leading to a less abundant and truncated dystrophin. BMD is less common and severe than Duchenne muscular dystrophy (DMD) as well as less investigated. To accelerate the search for innovative treatments, we developed a rat model of BMD by deleting the exons 45-47 of the Dmd gene. Here, we report a functional and histopathological evaluation of these rats during their first year of life, compared to DMD and control littermates. BMD rats exhibit moderate damage to locomotor and diaphragmatic muscles but suffer from a progressive cardiomyopathy. Single nuclei RNA-seq analysis of cardiac samples revealed shared transcriptomic abnormalities in BMD and DMD rats and highlighted an altered end-addressing of TMEM65 and Connexin-43 at the intercalated disc, along with electrocardiographic abnormalities. Our study documents the natural history of a translational preclinical model of BMD and reports a cellular mechanism for the cardiac dysfunction in BMD and DMD offering opportunities to further investigate the organization role of dystrophin in intercellular communication.

Keywords: Becker Muscular Dystrophy; Connexins; Dilated Cardiomyopathy; Heart Failure; Tmem65.

Figure 1. Reduction and disorganization of the dystrophin-associated protein complex in muscles of Dmd-edited rat models.

(A) Schematic diagram of a portion of the Dmd gene showing specific deletion of exons 45–47 and exon 52. (B) Western blot analysis for dystrophin from TA and heart of 6-month-old rats using the DYS1 antibody. (C, D) Quantification of Western blot analysis on TA (C) and heart (D). One-way ANOVA, n = 3. (E) Representative immunostaining of dystrophin in TA at 6 months of age. Scale bar 20 µm. (F) Normalized fluorescent signal intensity of dystrophin at 6 months of age. One-way ANOVA, n = 4 for TA and n = 3 for diaphragm and heart. (G) Immunostaining of β-dystroglycan (green), nNOS (red), and nNOS merge with laminin (white) in TA sampled from 6-month-old rats. Scale bar 10 μm. (H) Normalized fluorescent signal intensity of β-dystroglycan from (G). One-way ANOVA, n = 4. (I) Western blot analysis for nNOS in TA muscle extracts at 6 months. (J) Quantification of (I). One-way ANOVA, n = 3. (K, L) qRT-PCR quantification of Nos1 (K), mir34c and mir708 (L) expression. One-way ANOVA, n = 3. In (C, D, F, H, J–L), data are represented as mean ± SD. Source data are available online for this figure.

Figure 2. Histological and functional defects in skeletal muscle of BMD and DMD rats.

(A) H&E (top panel) and Sirius red (bottom panel) staining of TA cross-sections. Scale bar 20 μm. (B) Cross-sectional area (CSA) of TA from WT, BMD and DMD rats at 6 months of age. DMD were significantly different from WT: $, p ≤ 0.0001; #, p = 0.02; &, p = 0.007. Two-way ANOVA, n = 3. (C) CSA of TA from WT, BMD, and DMD rats at 12 months of age. DMD were significantly different from WT: $, p = 0.05; #, p = 0.001 and &, p = 0.03. Two-way ANOVA, n = 3. (D) Fibrosis quantification referred to (A). One-way ANOVA, n = 5 for 6 months and n = 6 for 12 months. (E) Representative immunostaining of anti-rat IgG (purple) and laminin (red) at 6 months. Scale bar 20 µm. (F) Quantification of the number of IgG-positive myofibers. One-way ANOVA, n = 3. (G) Representative immunostaining of eMHC (green) and laminin (red) at 6 months. Scale bar 20 µm. (H) Quantification of the number of eMHC-positive myofibers. One-way ANOVA, n = 3. (I) Corrected maximal force by grip test. One-way ANOVA, n = 4–5. (J) Corrected force maintenance index (FMI) expressed as the percentage of force maintenance after the first grip measurement. One-way ANOVA, n = 4–5. In (B–D, F, H–J), data are represented as mean ± SD. Source data are available online for this figure.

Figure 3. Normal numbers of skeletal muscle stem cells with intact myogenic capacity in BMD rats.

(A) Representative images of PAX7 (white), Ki-67 (red) and laminin (green). Scale bar 10 μm. (B) Quantification of the number of PAX7-positive MuSCs. One-way ANOVA, n = 3. (C) Percentage of activated (Ki-67+). One-way ANOVA, n = 3. (D) Immunostaining for PAX7 and MYOD (white), Ki-67 (red) and Hoechst (blue) on cultured myoblasts. Scale bar 10 μm. (E) Percentage of proliferative (Ki-67+) myoblasts. One-way ANOVA, n = 3–4. (F) Percentage of MYOG-positive cells. One-way ANOVA, n = 4–5. (G) Representative images of MYOG (purple), sarcomeric myosin heavy chain (MHC, green) and Hoechst (blue) on differentiated cultured cells. Scale bar 20 μm. (H) Fusion index referred to (G). One-way ANOVA, n = 4–5. In (B, C, E, F, H), data are represented as mean ± SD. Source data are available online for this figure.

Figure 4. Histological and functional evaluation of the diaphragm in BMD and DMD rats.

(A) H&E (upper panel) and Sirius red (lower panel) staining. Scale bar 20 μm. (B) Fibrosis quantification. One-way ANOVA, n = 5–6. (C) Representative immunostaining of eMHC (green) and laminin (red) in at 6 months of age. Scale bar 20 μm. (D) Quantification of the number of eMHC-positive fibers. One-way ANOVA, n = 4. (E–G) Representative ventilatory flow profiles of 12-month-old WT (E), BMD (F), DMD (G). (H–J) Peak expiratory flow (PEF) in ml/s (H), Peak inspiratory flow (PIF) in ml/s (I), minute volume (MV) in ml/min (J). One-way ANOVA, n = 4. In (B, D, H–J), data are represented as mean ± SD. Source data are available online for this figure.

Figure 5. Hypertrophic remodeling and electrical abnormalities in the heart of BMD and DMD rats.

(A) H&E (upper panel) and Sirius red (lower panel) staining. Scale bar 20 µm. (B) Fibrosis quantification. One-way ANOVA, n = 3 for 6 months and n = 6 for 12 months. (C) Representative immunostaining for laminin. Scale bar 20 µm. (D) Quantification of cardiomyocytes size. One-way ANOVA, n = 3. (E) Representative ECG signals from WT, BMD and DMD at 7–8 months of age under basal conditions (upper panels) and after an isoproterenol challenge (lower panels). The isoelectric line and the P-QRS-T wave legend are shown. The QTp interval is indicated in BMD rat tracings. (F, G) Heart rate (HR) in bpm (F) and the QTpc in ms (G), before (−) and after (+) injection of isoproterenol (ISO). One-way ANOVA, n = 6. In (B, D, F, G), data are represented as mean ± SD. Source data are available online for this figure.

Figure 6. Abnormal lateralization of TMEM65 and connexin-43 proteins at the ICDs in BMD and DMD rats.

(A) UMAP of resident cell populations clusters, identified according to specific markers (Fig. EV2). (B) Gene ontology analysis of down- and up-regulated genes. (C) Heat map showing differentially expressed genes by snRNAseq. (D) TMEM65 immunostaining in left ventricles of 12-month-old rats. Scale bar 20 µm. (E, F) Quantification of TMEM65 intensity (E) and TMEM65-positive area (F) represented as the mean from biological triplicates. One-way ANOVA, n = 3–4. (G–I) NCAD (red) and Cx43 (green) immunofluorescence in WT (G), BMD (H), and DMD (I) left ventricles. Scale bar 5 μm. (J) TEM of the cardiomyocytes of WT, BMD and DMD left ventricles (scale bar 1 µm) at 6 months of age, with magnified images (scale bar 300 nm) of intercalated disks (ICDs), highlighted by yellow rectangles and lines. Asterisks show thick filaments in abnormal fold regions for BMD and DMD samples, compared to WT. (K) Quantifications of ICD amplitude average in µm of WT, BMD, and DMD left ventricles at 6 months of age. Quantified ICD amplitude is marked by arrows in (J). One-way ANOVA, n = 3. In (E, F, K), data are represented as mean ± SD. Source data are available online for this figure.

Figure 7. Progressive development of a dilated cardiopathy with reduced ejection fraction in BMD and DMD rats.

(A) Representative M-mode images of right and left ventricle (LV) at 12 months. Contractile asynchrony is indicated by an arrow. (B–F) Echocardiographic data for LV internal diameter at diastole (LVIDd, B), LV free wall thickness at diastole (LVFWd, C), aorta diameter (Ao, D), LV fractional shortening (LVFS, E) and LV ejection fraction (LVEF, F). One-way ANOVA, n = 4–6 at 7 months and n = 6–11 at 12 months. (G) Individual LVFWd and LVEF values for BMD rats whose LVFWd value is less than or equal to the last quartile of LVFWd values in WT rats (≤2.8 mm; the 2 lowest values in the WT group are indicated by gray symbols). An ejection fraction below 50% is considered mild dysfunction, progressing to severe dysfunction below 30%. One-way ANOVA, n = 4–6 at 7 months and n = 6–10 at 12 months. In (B–F), data are represented as mean ± SD. Source data are available online for this figure.

Figure EV1. Dystrophin immunofluorescence staining.

(A) Localization of dystrophin epitopes recognized by the antibodies used in the study (NCL-DYS1 and NCL-DYS2). The BMD deletion of exons 45–47 at the level of the nNOS binding site is highlighted. ABD, Actin-binding domain; CR, Cysteine rich domain; CT, C-terminal domain; R, rod spectrin-like repeats. (B, C) Representative images of DYS1 (red), DYS2 (purple), and Laminin (cyan) immunostaining on TA (B) and heart (C) of WT, BMD, and DMD rats at 11 months. Scale bar 20 μm. (D, E) Quantification of dystrophin intensity detected by DYS1 (D) and DYS2 (E) antibodies in both TA and heart of WT, BMD, and DMD rats at 11 months. One-way ANOVA, n = 3, corresponding to the number of independent rats. Data are represented as mean ± SD.

Figure EV2. snRNAseq analysis and validations.

(A) Violin plots showing the expression of Myh7 as marker of cardiomyocytes, Dcn for fibroblasts and fibro-endothelial cells, the latest marked also by the expression of Cdh5, Lyz2 for monocytes, Mrc1 for macrophages, Rgs5 for pericytes, Ccl5 for T and B cells, Ccl21 for lymphatic cells, Npr3 for endothelial cells and Cdh19 for neuronal cells. (B) Heat map of expression of differentially expressed genes identified from snRNA-seq. (C) UMAP representation of WT, BMD, and DMD snRNAseq datasets. (D) Proportions of cells (%) identified per sample. (E) CD68 immunostaining on left ventricles from WT, BMD, and DMD rats at 11 months of age. Scale bar 100 μm. (F) RT-qPCR analysis of Nppa, Nppb, Myh6, Tmem65, Gja1, and Ryr2 on ventricle tissue RNA extraction from 12-month-old WT, BMD, and DMD rats. One-way ANOVA, n = 3–4, corresponding to the number of independent rats. Data are represented as mean ± SD.