Loss of muscleblind-like 1 results in cardiac pathology and persistence of embryonic splice isoforms

Abstract

Cardiac dysfunction is a prominent cause of mortality in myotonic dystrophy I (DM1), a disease where expanded CUG repeats bind and disable the muscleblind-like family of splice regulators. Deletion of muscleblind-like 1 (Mbnl1(ΔE2/ΔE2)) in 129 sv mice results in QRS, QTc widening, bundle block and STc narrowing at 2-4 months of age. With time, cardiac function deteriorates further and at 6 months, decreased R wave amplitudes, sinus node dysfunction, cardiac hypertrophy, interstitial fibrosis, multi-focal myocardial fiber death and calcification manifest. Sudden death, where no end point illness is overt, is observed at a median age of 6.5 and 4.8 months in ~67% and ~86% of male and female Mbnl1(ΔE2/ΔE2) mice, respectively. Mbnl1 depletion results in the persistence of embryonic splice isoforms in a network of cardiac RNAs, some of which have been previously implicated in DM1, regulating sodium and calcium currents, Scn5a, Junctin, Junctate, Atp2a1, Atp11a, Cacna1s, Ryr2, intra and inter cellular transport, Clta, Stx2, Tjp1, cell survival, Capn3, Sirt2, Csda, sarcomere and cytoskeleton organization and function, Trim55, Mapt, Pdlim3, Pdlim5, Sorbs1, Sorbs2, Fhod1, Spag9 and structural components of the sarcomere, Myom1, Tnnt2, Zasp. Thus this study supports a key role for Mbnl1 loss in the initiation of DM1 cardiac disease.

Figure 1. Mbnl1^ΔE2/ΔE2 mice demonstrate a reduced life-span.

(a). The wild type (Mbnl1^+/+) allele (i), targeting construct (ii), Mbnl1^loxE2lox allele (iii) and Mbnl1^ΔE2 allele (iv) are shown. (b). Southern blot analysis of wild-type and targeted 129 sv ES cell DNA digested with EcoRV and analyzed with a probe indicted in Panel a. The image shown is cropped. The full-length gel is shown in Supplementary figure S1a. (c). RT-PCR analysis of Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 heart RNA using primers located in Mbnl1 exon 2. Gapdh was amplified in parallel as an internal control. The images shown are cropped and full-length gels are shown in Supplementary figures S1b and c. (d). Western blot analysis of Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 heart protein lysates using anti-Mbnl1, anti-Mbnl2 and anti-α-tubulin antibodies. The images shown are cropped and the full-length gels are shown in supplementary figures S1d–f. (e). Kaplan-Meyer survival curves are shown. A total of 51 Mbnl1^ΔE2/ΔE2 mice (22 males, 29 females) and a corresponding number of male and female wild-type 129 sv mice were used in this study. Sacrificed mice are indicated by vertical bars. There is a statistically significant difference in the survival curves between Mbnl1^ΔE2/ΔE2 and Mbnl1^+/+ mice (χ²₍₁₎ = 85.7, p < 0.00001). The survival curves between Mbnl1^ΔE2/ΔE2 males and Mbnl1^ΔE2/ΔE2 females is not statistically significant (χ²₍₁₎ = 2.8, p < 0.10). (f, g). The recorded causes of death for the male (f) and female (g) Mbnl1^ΔE2/ΔE2 mice are indicated.

Figure 2. Mbnl1^ΔE2/ΔE2 mice show QRS, QTc widening, bundle block and STc shortening at 2 and 4 months of age.

(a & b). Relative electrocardiogram interval values at 2 and 3–4 months of age for male (2 months: n = 3; 3–4 months: n = 3) and female (2 months: n = 3; 3–4 months: n = 5) Mbnl1^+/+ and male (2 months: n = 3; 3–4 months: n = 3) and female (2 months: n = 3; 3–4 months: n = 5) Mbnl1^ΔE2/ΔE2 mice are shown. Mice underwent light isoflurane anesthesia at an inducing dosage of 3% isoflurane in oxygen, which was reduced to 1–1.5% isoflurane in oxygen when measurements were recorded. As mouse ECG waveform shapes are different from human, QRS duration values in this study include the Tri (transient re-entry current) wave, as it is part of the ventricular depolarization phase. 80–125 beats were analyzed for each mouse. p-values were calculated using the Student's t-test with significance set at p ≤ 0.05. p values ≤0.05 are indicated in red. (c, d). Shown are representative electrocardiogram traces for male (c) and female (d) Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 mice at 3–4 months of age. QRS expansion (cii & dii) and bundle block (ciii & diii) are observed Mbnl1^ΔE2/ΔE2 mice. Bundle block was observed in ~30% of Mbnl1^ΔE2/ΔE2 mice in both age groups. Recorded values for all intervals are shown in Supplementary Fig. S3 & S4 and in Supplementary Tables S1 & S2.

Figure 3. Mbnl1^ΔE2/ΔE2 mice anesthetized with ketamine/xylazine show prolonged QTc intervals, diminished R wave amplitudes and sinus node dysfunction at 6 months of age.

(a). Relative electrocardiogram interval values for male (n = 4) and female (n = 6) Mbnl1^+/+ and male (n = 3) and female (n = 5) Mbnl1^ΔE2/ΔE2 mice at 6 months of age are shown. Mice underwent anesthesia via an intraperitoneal injection of 80 mg/kg ketamine and 8 mg/kg xylazine in saline. QRS durations include the Tri (transient re-entry current) wave, as it is part of the ventricular depolarization phase in mice. As S waves, following the triwaves, were not prominent in Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 mice after ketamine/xylazine treatment QRS intervals were not measured. Beats analyzed for each mouse were male (n = 74) and female (n = 77) Mbnl1^+/+ and male (n = 31) and female (n = 54) Mbnl1^ΔE2/ΔE2 mice. p-values were calculated using the Student's t-test with significance set at p ≤ 0.05. p-values ≤0.05 are indicated in red. (b–d). Representative electrocardiogram traces showing diminished R wave amplitudes in male (b) and female (c) Mbnl1^ΔE2/ΔE2 mice and expanded Tri-waves in female (d) Mbnl1^ΔE2/ΔE2 mice. (e): % Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 mice showing diminished R wave amplitudes. (f, g). Sporadic expansion of RR intervals to ≥200% of the average RR interval was observed in ~30% of the Mbnl1^ΔE2/ΔE2 mice analyzed [male (n = 4) and female (n = 6) Mbnl1^+/+ mice and male (n = 3) and female (n = 5) Mbnl1^ΔE2/ΔE2 mice]. Representative electrocardiogram traces from female Mbnl1^+/+ (f) and Mbnl1^ΔE2/ΔE2 (g) mice are shown. Recorded values for all intervals are shown in Supplementary Fig. S5 and in Supplementary Tables S3.

Figure 4. Mbnl1^ΔE2/ΔE2 mice show ventricular hypertrophy at 6 months of age.

(a,b). Relative values for ultrasound echocardiographic measurements at 2 and 6–7 months of age for male Mbnl1^+/+ (2 months: n = 3; 6–7 months: n = 6) and Mbnl1^ΔE2/ΔE2 mice (2 months: n = 3; 6–7 months: n = 5) are shown. p-values were calculated using Student's t-test with significance set at p ≤ 0.05. Values where p ≤ 0.05 are shown in red. (c). 2-D guided M-Mode images of representative 20 week male Mbnl1^+/+ and Mbnl1^ΔE2/ΔE2 hearts. Ventricular septal thickness and posterior wall thickness are enlarged in the male Mbnl1^ΔE2/ΔE2 hearts. The depth from top to bottom is 7 mm. Time is 100 ms/div. Abbreviations: EDD: End-diastolic dimension (green). ESD: End-systolic dimension (blue). VST: Left ventricular septal thickness (red); PWT: Posterior wall thickness (yellow). Recorded values are shown in Supplementary Fig. S6 and in Supplementary Tables S4 & S5).

Figure 5. Multi-focal calcification and fibrosis in Mbnl1^ΔE2/ΔE2 hearts at 6 months of age.

(a–f). Histological findings in 6 month female Mbnl1^+/+ (a, c, e) hearts (n = 3) and female Mbnl1^ΔE2/ΔE2 (b, d, f) hearts (n = 3) are shown. In Mbnl1^+/+ mice fibrosis and calcifications were not observed ((a): H&E stain ×12.5, (c): H&E stain ×200 and (e): trichrome stain ×400). In the Mbnl1^ΔE2/ΔE2 mice there are multiple foci of calcified myocardial fibers that appear dark blue in the H&E stained sections (arrows) ((b): H&E stain ×40, (d): H&E ×200). The inset shows individual blue dots that likely represent calcification of individual mitochondria (x400). In panel (f), a focus of calcification with interstitial fibrosis (light blue lines in this trichrome stained section, ×400) is shown. Both the calcification and the fibrosis were multifocal, involving less than 1% of the myocardial tissue, and were seen in all the Mbnl1^ΔE2/ΔE2 animals.

Figure 6. Cardiac pathology observed over 2–6 months of age in Mbnl1^ΔE2/ΔE2 mice.

The heart image shown is obtained from ClipArtist.net (http://www.clipartlord.com/free-human-heart-clip-art/).

Figure 7. Mbnl1 depletion results in the persistence of embryonic splice isoforms in adult hearts.

(a). A cardiac RNA network that is misspliced in Mbnl1^ΔE2/ΔE2 hearts is shown. RNAs are categorized by their biological function and Mesh terms. (b–g). Splicing analysis for the indicated RNAs was performed by RT-PCR with E18 Mbnl1^+/+ and 4 months old male Mbnl1^+/+ (n = 3) and male Mbnl1^ΔE2/ΔE2 (n = 3) hearts. Band intensities were quantified by densitometry. Data are standard error of mean (SEM). p-values were calculated using Student's t-test with significance set at p ≤ 0.05. Primer locations, exon numbers and expected band sizes are indicated. The alternatively spliced exons are shown as green boxes. Exon numbers for all genes except Junctate/Asph are annotated based on Refseq from the UCSC genome browser (NCBI37/mm9); Junctate/Asph is annotated based on the study carried out by Dinchuck et al (Dinchuk et al., 2000). Scn5a exon 6 and 6A were distinguished by digestion with SacI. The SacI site within Scn5 exon 6A is indicated in red. Identified novel exons are indicated by asterisks and the sequences of these exons are shown in Supplementary figure S9. The images shown are cropped. The full-length gels are shown in Supplementary figures S10–S15.

Figure 8. Tabulation of splice errors in Mbnl1^ΔE2/ΔE2 hearts.

(a). Splice events examined in this study are tabulated. PSI: percent spliced in; NAS: Alternative splicing not detected by RT-PCR analysis. (b, c). Steady-state Junctin (E1a–5a) and Junctin (E1–5a) mRNA levels were analyzed by qPCR in E18 Mbnl1^+/+ hearts, 4 month male Mbnl1^+/+ (n = 3) and 4 month male Mbnl1^ΔE2/ΔE2 (n = 3) hearts. Exon numbers and primer positions are shown. Data are standard error of mean (SEM). p-values were calculated using Student's t-test with significance set at p ≤ 0.05. Values where p ≤ 0.05 are shown in red. (d). Potential relationships between splice errors and the cardiac pathology observed are indicated. The heart image shown is obtained from ClipArtist.net (http://www.clipartlord.com/free-human-heart-clip-art/).