MBNL overexpression rescues cardiac phenotypes in a myotonic dystrophy type 1 heart mouse model

Abstract

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the dystrophia myotonica protein kinase (DMPK) gene. The expanded CUG repeat RNA (CUGexp RNA) transcribed from the mutant allele sequesters the muscleblind-like (MBNL) family of RNA-binding proteins, causing their loss of function and disrupting regulated pre-mRNA processing. We used a DM1 heart mouse model that inducibly expresses CUGexp RNA to test the contribution of MBNL loss to DM1 cardiac abnormalities and explored MBNL restoration as a potential therapy. AAV9-mediated overexpression of MBNL1 and/or MBNL2 significantly rescued DM1 cardiac phenotypes including conduction delays, contractile dysfunction, hypertrophy, and misregulated alternative splicing and gene expression. While robust, the rescue was partial compared with reduced CUGexp RNA and plateaued with increased exogenous MBNL expression. These findings demonstrate that MBNL loss is a major contributor to DM1 cardiac manifestations and suggest that additional mechanisms play a role, highlighting the complex nature of DM1 pathogenesis.

Keywords: Arrhythmias; Cardiovascular disease; Genetic diseases; Genetics; Therapeutics.

Figure 1. Exogenous MBNL1 and MBNL2 are overexpressed in ventricles and atria of CUG960 +dox mice by AAV9 delivery.

(A) Diagram of the pAAV vectors used to express control tdTomato protein (pAAV-tdTomato), 3xFLAG-MBNL1, and 3xMYC-MBNL2. (B) Diagram of the experimental design including time points and assays. (C) Cardiac ventricular and atrial protein expression was evaluated by Western blotting. n = 3 animals per cohort.

Figure 2. Cardiac conduction intervals were rescued by overexpression of MBNL1 and/or MBNL2.

(A) QRS (left) and QTc (right) intervals were determined by surface ECG recordings in anesthetized CUG960 mice in response to dox induction with different treatments. On/off dox (+/–) animals were taken off dox at the time of AAV9 delivery and served as a control for complete recovery. Data on QRS and QTc intervals from MHCrtTA +dox control animals at 8 weeks of age are indicated by a dashed line (28). n ≥13 per cohort. Data represent the mean ± SEM and were analyzed using 2-way ANOVA followed by Tukey’s multiple-comparison test. (B) Percent rescue of QRS (left) and QTc (right) intervals was calculated using the average ECG parameters from the last time point (21 weeks of age). n ≥13 per cohort. Black lines represent the significant differences of corresponding groups compared with +dox or tdTomato controls; brown lines represent the significant differences between the corresponding groups and +/–dox and –dox controls. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparison test.

Figure 3. Heart weight and echocardiographic parameters induced by CUG_exp RNA show significant or trending rescue with MBNL1 and/or MBNL2 overexpression.

(A) Heart weight was normalized to tibia length. (B and C) Left ventricle anterior wall (LVAW) thickness, (D and E) left ventricle internal diameter (LVID), (F and G) LV volume, (H) ejection fraction (EF), and (I) fractional shortening (FS) were determined by M-mode Echo. n ≥13 per cohort. All animals were analyzed at the 21-week time point. Data represent the mean ± SEM and were analyzed by ordinary 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. d, end of diastole; s, end of systole.

Figure 4. CUG960 +dox mice with MBNL overexpression show colocalization of epitope-tagged MBNL1 and MBNL2 with nuclear CUG_exp RNA foci.

RNA FISH using a (CAG)₅ locked nucleic acid probe targeting CUG_exp RNA combined with IF for (A) FLAG tag and (B) MYC tag in the ventricles of CUG960 +dox mice. The experiments were conducted on 3 animals in each group, and representative images are shown. Scale bars: 10 μm.

Figure 5. MBNL overexpression partially rescues disrupted ventricular splicing events related to cardiac morphology and function.

(A) Alternative splicing signature scores for ventricle samples. The center line indicates the median value, while the upper and lower dashed lines indicate the first and third quartiles. (B) Number and types of alternative splicing events observed in ventricles. IR, intron retention. (C) Percentage of overlapping CE splicing events in each MBNL-overexpressing cohort using the comparison between CUG960 +/–dox and +dox cohorts as a reference. (D) Genes showing differential alternative splicing changes in comparisons of CUG960 –dox mice with +dox mice, as well as in comparisons of all MBNL cohorts to CUG960 +dox cohort were evaluated for enrichment of GO functional terms using the ShinyGO platform. The dashed vertical line: –log₁₀(FDR) = 1.5 (E) Representative polyacrylamide gels showing alternative splicing changes for the candidate genes Scn5A, Tnnt2, and Ryr2 in ventricles of CUG960 +dox mice that underwent different treatments. (F) Quantification of the PSI for each splicing event. n = 3 animals per cohort. Black lines represent the significant differences in the corresponding groups compared with +dox or tdTomato controls; brown lines represent the significant differences between the corresponding groups and +/–dox and –dox controls. E, exon. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test.

Figure 6. MBNL1 and/or MBNL2 overexpression results in partial rescue for widespread DGE in ventricles of CUG960 +dox mice.

(A) Gene signature scores for ventricular samples. The center line represents the median value, while the upper and lower dashed lines indicate the first and third quartiles. (B) Differentially expressed genes displaying at least a 1.5-fold change (adjusted P = 0.05) as compared with expression in CUG960 +dox controls. (C) Percentages of overlapping DGE in each MBNL-overexpressing cohort using the comparison between CUG960 +/-dox and +dox cohorts as a reference. (D) GO functional terms for genes showing DGE changes in the ventricles of mice in all cohorts. Dashed line: –log₁₀(FDR) = 1.5. (E–G) RT-qPCR–based validation of candidate genes showing gene expression changes in ventricles of mice in each cohort. Rpl4 was used as an internal control for normalization. n = 3 animals per cohort. Black lines indicate the significant differences in corresponding groups compared with +dox or tdTomato controls; brown lines indicate the significant differences between the corresponding groups and +/–dox and –dox controls. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ordinary 1-way ANOVA followed by Tukey’s multiple-comparison test. Reg., regulation.