Long-Term Dystrophin Replacement Therapy in Duchenne Muscular Dystrophy Causes Cardiac Inflammation

Abstract

Micro-dystrophin replacement gene therapy is currently under clinical trials in Duchenne muscular dystrophy (DMD) patients. However, recent adverse cardiac events has led to serious concerns about this therapeutic intervention. We studied the long-term effect of dystrophin replacement strategies in a severe model of DMD (mdx; utrophin^-/-mice). Although micro-dystrophin remarkably improved survival and cardiac function after 1 year of treatment, we were able to reveal an increased septum thickness, which is the result of cardiac inflammation. Our data warrant consideration that micro-dystrophin replacement therapy may be associated with cardiac inflammation and opens up perspectives for understanding the consequences of using this approach in DMD patients.

Keywords: Duchenne muscular dystrophy; gene therapy; heart; inflammation; micro-dystrophin.

Graphical abstract

Figure 1

AAV-Micro-Dystrophin Treatment Rescue Body Weight Gain and Increase Survival of dKO Mice (A) Schematic representation of the optimized mouse micro-dystrophin MD1 under control of the Sp5.12 promotor. (B) Schematic representation of the double knockout (dKO) systemic treatments with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) and AAV-micro-dystrophin. We injected 80 mg/kg PPMO intravenously into the temporal vein on day 2 and retro-orbitally on day 17. The dKO mice then received retro-orbital injections of 2E+12 vector genomes (corresponding to 1E+14 vector genomes per kg) of AAV2/9-μdystrophin (MD1) under general anesthesia, at the age of 3 weeks. Some of them also received PPMO post-treatment (80 mg/kg by retro-orbital injections) at 8, 12, 16, and 20 weeks of age for the 24-week study or 70 mg/kg at 8, 16-, 24-, 32- and 40-weeks old for the survival study. (C) Over time body weight comparisons between wild-type (WT) mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV), along the course of the disease. Data are means ± SEM of at least 4 mice per group. Statistical significance was determined by repeated 1-way analysis of variance (with Dunnett’s multiple comparison test with WT) (∗∗∗P < 0.001, relative to WT mice). (D) Survival rate of WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV). Groups are composed of at least 8 animals. Survival curves of WT mice and dKO mice treated with P-AAV, P-AAV-P, and AAV-P are superimposed and reached 100%. The other survival curves (P and AAV) are significantly different, log rank test. (Parts of the figure were drawn by using BioRender.com.) AAV = adeno-associated virus.

Figure 2

AAV-Micro-Dystrophin Treatment Improves Cardiac Function in dKO Mice (A) Heart weight-to-body weight ratio calculated from wild-type (WT) mice and double knockout (dKO) mice treated with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. (B) Representative M-mode transthoracic echocardiographic tracings from 52-week-old WT mice and dKO mice treated with PPMO or AAV alone. (C) Bar graphs showing the left ventricular end-diastolic diameters (LVDd) and end-systolic diameters (LVDs) in WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. (D) Bar graphs showing the fractional shortening in WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. (A and B) Data are means ± SEM of at least 8 mice per group except for the PPMO group at 52 weeks, which is means ± SEM of 2 mice 48 weeks old. Significance was determined by 1-way analysis of variance (with Dunnett’s multiple comparison test to WT). (E) Bar graphs showing quantitative real-time polymerase chain reaction analysis of Nppa expression in the hearts of 52-week-old WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV). Of note: the PPMO group includes mice aged 38 to 50 weeks. Data are means ± SEM for at least 5 mice per group, and significance was determined by 1-way analysis of variance with Dunnett’s multiple comparison test to WT. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.

Figure 3

dKO Mice Treated With AAV-Micro-Dystrophin Have Compensatory Cardiac Hypertrophy (A) Schematic significance of h/r ratio modulation. (B) Bar graphs showing the h/r ratio in 24-week-old and 52-week-old wild type (WT) and double knockout (dKO) mice treated with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV). (C) Bar graphs showing the intraventricular end-diastolic thickness (IVSd) and end-systolic thickness (IVSs) in WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV), at weeks 24 and 52. (D) Bar graphs showing the left ventricular posterior wall thickness in diastole (LVPWd) and systole (LVPWs) in WT mice and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV), at weeks 24 and 52. Data are means ± SEM of at least 8 mice per group except for the PPMO group at 52 weeks, which is means ± SEM of 2 mice 48 weeks old. Significance was determined by 1-way analysis of variance when data were normally distributed or Kruskal-Wallis when they were not normally distributed (Dunnett’s comparison with WT). (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (E) Micrographs showing Laminin (blue) and α-actinin (red) staining in hearts from 9-week-old WT and untreated dKO mice (left panel) and 52-week-old WT mice and dKO mice treated with AAV-micro-dystrophin alone (AAV) (right panel). Scale bar: 10 μm. Bar graphs showed the cardiomyocyte area (means ± SEM) quantified with QuPath software. Significance was determined with analysis of variance with multiple comparisons. Abbreviation as in Figure 1.

Figure 4

dKO Mice Treated With AAV-Micro-Dystrophin Show Minor Arrhythmic Events Despite Normal Electrocardiographic and Correct Connexin-43 Localization (A) Bar graphs showing the electrocardiographic data with RR duration (left panel), PR duration (medium panel), and QRS duration (right panel) in wild-type (WT) mice and double knockout (dKO) mice treated with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. Data are means ± SEM of at least 8 mice per group. Significance was determined by 1-way analysis of variance (Dunnett’s multiple comparison with WT). (B) Micrographs showing connexin-43 (green) and desmoplakin (red) staining in hearts from 9-week-old WT and untreated dKO mice (left panel) and 52-week-old WT mice and dKO mice treated with AAV-micro-dystrophin alone (AAV) (right panel). Scale bar: 10 μm. Bar graphs showed connexin-43 and desmoplakin colocalization percentage (means ± SEM) quantified with QuPath software. Significance was determined with the Mann-Whitney test. (C) Graphs showing RR duration over time in 9-week-old WT and untreated dKO mice (left panel) and 52-week-old WT mice and dKO mice treated with AAV-micro-dystrophin alone (AAV) (right panel). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.

Figure 5

AAV-Micro-Dystrophin Treatment Induces Myocardial Inflammation Despite Low Fibrosis (A) Representative micrographs of hematoxylin & eosin–stained sections of hearts from 9-week-old double knockout (dKO) mice, wild-type (WT) mice, and dKO mice treated with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. dKO mice treated with PPMO alone were 38 to 50 weeks old. Scale bars: 100 μm. (B) Representative micrographs of Sirius red–stained sections of hearts from 9-week-old dKO mice, WT mice, and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV) at weeks 24 and 52. dKO mice treated with PPMO alone were 38 to 50 weeks old. Scale bars: 100 μm. (C) Bar graphs showing fibrosis calculated as a percentage of the total area with QuPath software, from Sirius red–stained sections from 52-week-old mice. dKO mice treated with PPMO alone were 38 to 50 weeks old. Data are means ± SEM of at least 5 mice per group, and significance was determined by 1-way analysis of variance with Dunnett’s multiple comparison test to WT. (D) Bar graphs showing quantitative real-time polymerase chain reaction analysis of Col1A2 expression in the hearts of 52-week-old WT and dKO mice treated with PPMO alone (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV). dKO mice treated with PPMO alone were 38 to 50 weeks old. Data are means ± SEM for at least 5 mice per group, and significance was determined by 1-way analysis of variance (Dunnett’s multiple comparison with WT). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.

Figure 6

AAV-Micro-Dystrophin Treatment Induces Inflammatory Pathways Overexpression (A) Principle component analysis (PCA) mapped scatter plot: the global gene expression profiles of the heart from both 52-week-old wild-type (WT) mice and double knockout (dKO) AAV treated mice were analyzed by PCA. The figure represents the first 2 principal components of microarray analysis data (PC1, PC2) in X and Y, respectively, and demonstrated the expression profile of the 5 mice per group (WT blue, AAV orange). (B) Heat map analysis of microarray data showing hierarchical clustering of 1,129 differentially expressed probes between 52-week-old untreated WT and dKO AAV-treated hearts. Each group has 5 mice tested. Red or blue colors indicate differentially up- or down-regulated genes, respectively. (C) Volcano plot of differentially regulated genes between 52-week-old untreated WT and dKO AAV-treated hearts. The mean signals were background corrected and transformed to the log2 scale. Genes with at least 2-fold changes with P < 0.05 at the 95% confidence level were considered significant. (D) Enrichment analysis using hallmark gene sets collection. Data showed only pathways significantly regulated. (E) Gene set enrichment analysis plot (upper panel) and heatmap (lower panel) of hallmark inflammatory response gene expression. (F-I) Bar graphs showing quantitative real-time polymerase chain reaction analysis of CD45 (F), F4/80 (G), CD19 (H), and CD3 (I) expression in the hearts of 52-week-old WT mice and dKO mice treated with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) (P), PPMO+AAV-micro-dystrophin (P-AAV), PPMO+AAV-micro-dystrophin+PPMO (P-AAV-P), AAV-micro-dystrophin+PPMO (AAV-P), and AAV-micro-dystrophin alone (AAV). dKO mice treated with PPMO alone were 38 to 50 weeks old. Data are means ± SEM for at least 5 mice per group, and significance was determined by 1-way analysis of variance with Dunnett’s multiple comparison test to WT. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.

Figure 7

AAV-Micro-Dystrophin Treatment Induces Over-Representation Of Immune Cells (A) T-distributed stochastic neighbor embedding (t-SNE) of the imaging mass cytometry (IMC) dataset. (B) Heat map of average marker expression for each phenotypic cell cluster. Colored bars indicate clusters corresponding to specific cell types as indicated in A. (C) Percentages of different phenotypic cell clusters in untreated wild-type (WT) and AAV-micro-dystrophin–treated double knockout (dKO) heart septum. (D) Heat map of average marker expression for each phenotypic cell cluster. Colored bars indicate clusters corresponding to specific cell types, number of cells in the cluster, and indication of the most prevalent group of mice. (E) IMC images of 52-week-old untreated WT and AAV-micro-dystrophin–treated dKO mice heart septum stained for MHCII (red), CD54 (yellow), Ly6G (cyan), CD146 (magenta), CD31 (gray), DNA (blue), and laminin (green). (F) IMC images of 52-week-old untreated WT and AAV-micro-dystrophin treated dKO mice heart septum stained for CD11b (magenta), CD24 (yellow), CD90 (cyan), and DNA (blue). Abbreviation as in Figure 1.

Figure 8

Cardiac Phenotype Arises From AAV-Micro-Dystrophin Overexpression (A) Bar graphs showing quantitative real-time polymerase chain reaction analysis of Nppa (left panel), F4/80 (midle panel), and CD45 (right panel) expression in the hearts from 22-week-old wild-type (WT) mice and AAV-U7–treated double knockout (dKO) mice, from 24-week-old WT mice and AAV-micro-dystrophin–treated dKO mice, and from 52-week-old WT mice and AAV-micro-dystrophin–treated dKO mice. The mice treated with AAV U7, as reported by Forand et al, represent 30% of the initial population. The remaining 70% succumbed to respiratory arrest. Data are mean ± SEM for at least 5 mice per group, and significance was determined by 1-way analysis of variance (with Tukey’s multiple comparison test). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.

Figure 9

AAV-Micro-Dystrophin Overexpression Alters Cardiac Structure and Function in WT Mice (A) Representative immunoblots showing dystrophin and micro-dystrophin expression in hearts of 24-week-old WT mice treated with AAV-micro-dystrophin. α-Actinin was shown as a loading control. (B) Bar graphs showing the fractional shortening in 24-week-old WT noninjected mince and mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (C) Bar graphs showing the heart weight-to-body weight ratio in 24-week-old WT noninjected mice and mice treated with AAV-backbone, and AAV-micro-dystrophin (AAV-μdys). (D) Bar graphs showing real-time polymerase chain reaction analysis of Nppa in 24-week-old WT and mdx noninjected mice and WT mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (E) Bar graphs showing the h/r ratio in 24-week-old WT noninjected mice and mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (F) Bar graphs showing the left ventricular posterior wall thickness in diastole (LVPWd) and in systole (LVPWs) in 24-week-old WT noninjected mice and mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (G) Bar graphs showing the intraventricular end-diastolic thickness (IVSd) and end-systolic thickness (IVSs) in 24-week-old WT noninjected mice and mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (H) Bar graphs showing real-time polymerase chain reaction analysis of CD3 (T-lymphocytes) and F4/80 (macrophages) in 24-week-old WT and mdx noninjected mice and WT mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (I) Bar graphs showing real-time polymerase chain reaction analysis TNF-α, IL6, IL0, and CD80 in 24-week-old WT and mdx noninjected mice and WT mice treated with AAV-backbone and AAV-micro-dystrophin (AAV-μdys). (B-I) Data are means ± SEM of at least 4 mice per group. Significance was determined by 1-way analysis of variance when data were normally distributed or Kruskal-Wallis when they were not normally distributed. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviation as in Figure 1.