Elevation of RNA-binding protein CUGBP1 is an early event in an inducible heart-specific mouse model of myotonic dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region (3' UTR) of DM protein kinase (DMPK). The key feature of DM1 pathogenesis is nuclear accumulation of RNA, which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of CUG-binding proteins (CUGBPs). Cardiac involvement occurs in more than 80% of individuals with DM1 and is responsible for up to 30% of disease-related deaths. We have generated an inducible and heart-specific DM1 mouse model expressing expanded CUG RNA in the context of DMPK 3' UTR that recapitulated pathological and molecular features of DM1 including dilated cardiomyopathy, arrhythmias, systolic and diastolic dysfunction, and mis-regulated alternative splicing. Combined in situ hybridization and immunofluorescent staining for CUGBP1 and CUGBP2, the 2 CUGBP1 and ETR-3 like factor (CELF) proteins expressed in heart, demonstrated elevated protein levels specifically in nuclei containing foci of CUG repeat RNA. A time-course study demonstrated that colocalization of MBNL1 with RNA foci and increased CUGBP1 occurred within hours of induced expression of CUG repeat RNA and coincided with reversion to embryonic splicing patterns. These results indicate that CUGBP1 upregulation is an early and primary response to expression of CUG repeat RNA.

Figure 1. Generation of bitransgenic mouse expressing expanded CUG RNA in heart.

(A) EpA960 and EpA0 transgene constructs. The spliced mRNA transcripts from the nonrecombined (top) and recombined alleles (bottom) are indicated in blue above the gene diagrams. The mRNAs from the recombined EpA0 and EpA960 alleles are identical except for the presence or absence of the CUG repeats. Primer pairs used for RT-PCR analysis of nonrecombined and recombined alleles are shown. The CMV enhancer and β-actin promoter drive the transcription ubiquitously. Black boxes represent segments of different exons added for splicing. The cassette containing SV40 polyadenylation sites that prevent expression of RNA from downstream gene segments is located between 2 loxP sites. (B) Relative levels of the nonrecombined transgene mRNAs in different tissues from EpA960 and EpA0 transgenic mice using real-time RT-PCR. Results are from 3 mice from each line, with the exception of the muscle sample, taken from 1 mouse in line EpA960/MCM 1332. (C) Relative expression of EpA960(R) and EpA0(R) mRNAs from the recombined alleles after Cre-mediated recombination in heart.

Figure 2. Induced EpA960/MCM mice reproduce functional and histopathological features of DM1.

(A–F) EpA960/MCM and MCM littermates were given tamoxifen (TAM) at the same time. (A and D) H&E stain revealed that EpA960/MCM hearts were dilated compared with hearts of tamoxifen-treated MCM mice. Original magnification, ×2. (B and E) H&E stain revealed that induced EpA960/MCM hearts exhibited hypertrophied cardiomyocytes that focally contained pale, granular cytoplasm compared with MCM hearts. Original magnification, ×40. (C and F) Electron microscopy revealed hypertrophied myocytes with irregular nuclei (N) and abundant mitochondria (m) in EpA960/MCM hearts compared with MCM hearts. Original magnification, ×4,000. (G) Cardiac functional abnormalities in mice expressing EpA960(R) mRNA. Doppler ultrasound was performed before (Pre) and after (Post) tamoxifen administration on EpA960/MCM and MCM littermates and revealed both systolic and diastolic dysfunction in hearts expressing EpA960(R) mRNA. (H) ECG telemetry revealed progressive arrhythmias following induction of expanded CUG RNA expression. The length of the PR interval (thick bar) is indicated; thin bars represent 100 ms.

Figure 3. Misregulated alternative splicing of Tnnt2 and Fxr1h in heart expressing EpA960(R) mRNA but not EpA0(R) mRNA.

(A) Alternative splicing patterns of mouse Tnnt2 exons 4 and 5 and the location of the RT-PCR primers (arrows). (B) Mice expressing EpA960(R) mRNA reverted to the embryonic splicing pattern of Tnnt2. The EpA960/MCM bitransgenic mice from 3 EpA960 lines as well as transgenic EpA960 and MCM mice were given tamoxifen (T) at a dosage of 20 mg/kg/d for 5 consecutive days, and RNA was extracted 1 week after the last injection. EpA960/MCM bitransgenic littermates treated with oil served as mock (M) controls. E18 cardiac tissue exhibited the Tnnt2 embryonic alternative splicing pattern. Percent of mRNAs containing exons 4 and 5 is shown. Asterisks denote a hybrid band of the 2 smallest PCR products. (C) Mice expressing EpA0(R) mRNA exhibited no phenotype and retained adult splicing patterns of Tnnt2 splicing both 1 week and 1 month following tamoxifen injection. (D) Decreased inclusion of exons 15 and 16 of FXR1h in mouse heart expressing EpA960(R) mRNA. Percent of mRNAs containing exons 15 and 16 is shown. (E) Decreased inclusion of exons 15 and 16 of FXR1h was also observed in individuals with DM1 (patient identification number shown in parentheses). Splicing abnormalities were not observed in tissue from unaffected individuals or individuals with non-DM1 forms of dilated cardiomyopathy (DCM).

Figure 4. EpA960(R) mRNA forms foci that colocalize with MBNL1.

In situ hybridization using a Cy3-CAG PNA probe was used to detect RNA foci. DAPI was used for nuclear staining. Heart tissue from the higher-expressing line EpA960/MCM 1323 (A) showed 8- to 10-fold more nuclei containing RNA foci than the lower-expressing line EpA960/MCM 1321 (B). (C) No signal was detected in cardiac tissue from the highest-expressing line using a Cy3-CTG (sense) probe or (D) in MCM mice treated with tamoxifen. (E) Endogenous MBNL1 colocalized with nuclear RNA foci in heart cells expressing EpA960(R) mRNA in line EpA960/MCM 1323. Original magnification, ×40 (A–D); ×63 (E).

Figure 5. CUGBP1 and CUGBP2 protein steady-state levels increase in cardiac tissue expressing EpA960(R) mRNA.

(A) Western blot analysis demonstrated elevated CUGBP1 and CUGBP2 protein expression in cardiac tissues expressing EpA960(R) mRNA (line EpA960/MCM 1323) 1 week following tamoxifen administration compared with tamoxifen-treated MCM littermates. An additional CUGBP2 band, possibly a splice variant, was expressed in some hearts expressing EpA960(R) mRNA. GAPDH was used as loading control. (B) Expression of CELF proteins remained unchanged in cardiac tissues expressing EpA0(R) mRNA (line 4294) 1 week following tamoxifen administration. (C) Nuclei expressing EpA960(R) mRNA foci exhibited elevated CUGBP1 protein expression, as shown by immunofluorescence staining.Arrowheads indicate nuclei containing RNA foci. (D) Higher magnification showed nuclei containing RNA foci also exhibited higher levels of CUGBP1. (E) Endogenous expression of CUGBP1 (arrowheads) in MCM mice given tamoxifen remained low. Nonspecific background in the cytoplasm was the result of mouse IgG. Nuclear CUGBP1 in cells expressing EpA0(R) mRNA (line 4294) remained low (F), as did nuclear CUGBP1 in MCM mice (E), 1 month following tamoxifen administration. (G) Nuclei containing EpA960(R) mRNA foci exhibited elevated expression of CUGBP2 protein. Arrowheads indicate nuclei containing RNA foci. (H) CUGBP2 expression (arrowheads) was low in tamoxifen-treated MCM hearts, with some areas of nonspecific staining in the cytoplasm. (I) Expression of CUGBP1 in the mouse heart at postnatal day 2. Images in E–I were taken using the same exposure time. Original magnification, ×40 (C and E–I); ×63 (D).

Figure 6. Time course demonstrates rapid molecular changes associated with induction of EpA960(R) mRNA.

(A) Tnnt2 splicing changes. Heart tissue was collected from mock and tamoxifen-treated littermates at the indicated time points following a single tamoxifen dose. Inclusion of Tnnt2 exons 4 and 5 was first detected at 12 hours, with increasing exon inclusion at subsequent time points. Asterisk indicates hybrid bands of the 2 smallest PCR products. (B) RNA foci and colocalized MBNL1 foci were detected by 6 hours and with increased intensity at 12 hours. (C) Elevated nuclear staining for CUGBP1 was detected 6 hours following tamoxifen administration compared with tamoxifen-treated MCM and untreated EpA960/MCM mice (0 hours). Arrowheads indicate nuclei containing RNA foci. Original magnification, ×40.