PMC

Electron micrographs of adult heart sections of α-MHC-LacZ (A) and α-MHC-LacZ-(CTG)₄₀₀TG^high (B) mice are shown. Mitochondrial cristae were disarrayed in a subset of sections viewed.

Total RNA isolated from adult α-MHC-LacZ-(CTG)₄₀₀ and adult α-MHC-LacZ hearts and wild-type postnatal day1 and day 2 mouse hearts was subjected to RT-PCR analysis using the Tnnt2, Alp, Zasp and m-Ttn primers as described in . Gapdh RNA was amplified in parallel as an internal control. The experiments were carried out in triplicate and the results are tabulated in .

Panels A–B: Protein extracts were prepared from α-MHC-LacZ, α-MHC-LacZ-(CTG)₄₀₀TG^high, and α-MHC-LacZ-(CTG)₄₀₀TG^low mouse hearts and 6 or 10 µg of the total proteins from the tissue extracts were resolved on SDS-PAGE followed by Western blot analyses and immunostaining with CUG-BP1 and MBNL1 monoclonal antibodies (mAb), respectively. The blots were re-probed for GAPDH using anti-GAPDH polyclonal antibodies as an internal control. The experiments were carried out in triplicate and mean values of steady-state Cug-bp1 and Mbnl1 levels are shown. Panel C: Cytoplasmic and nuclear proteins extracts (10 µg) from α-MHC-LacZ, α-MHC-LacZ-(CTG)₄₀₀TG^high, and α-MHC-LacZ-(CTG)₄₀₀TG^low mouse hearts were resolved on SDS-PAGE followed by Western blot analyses and immunostaining with CUG-BP1 mAb. The blots were re-probed for TATA binding protein (TBP), and for GAPDH, which were used as nuclear and cytoplasmic markers respectively. The experiments were carried out in triplicate and mean values of steady-state Cug-bp1 levels are shown.

Panel A: Nuclear DAPI staining of cardiomyocytes derived from α-MHC-LacZ, α-MHC-LacZ-(CTG)₄₀₀ mice and normal human and DM1 myoblasts is shown. Transcripts encoding the expanded CUG tracts were detected by hybridization with a (CAG)₁₀-Cy3 probe (red signal). CUG foci are observed in the cytoplasm in α-MHC-LacZ-(CTG)₄₀₀ cardiomyocytes (b, c) and in both the cytoplasm and nucleus of DM1 myoblasts (e) (120× magnification). Panel B: Nuclear DAPI staining of heart sections of α-MHC-LacZ, α-MHC-LacZ-(CTG)₄₀₀TG^high, and α-MHC-LacZ-(CTG)₄₀₀TG^low are shown. CUG foci are observed in the cytoplasm of both α-MHC-LacZ-(CTG)₄₀₀TG^high, and α-MHC-LacZ-(CTG)₄₀₀TG^low heart tissue sections [red signals; e, f (40× magnification) and h, i (120× magnification)]. Transcripts containing expanded repeats are not observed in cardiomyocytes and heart sections of α-MHC-LacZ mice and in normal human myoblasts [Panel A; a, d and Panel B; d, g]. >400 cells were examined in both α-MHC-LacZ-(CTG)₄₀₀ cardiomyocyte preparations and in α-MHC-LacZ-(CTG)₄₀₀ cardiac sections.

Panel A: Nuclear DAPI staining of normal and DM1 fibroblasts and myoblasts, either untreated or immortalized with SV40, are shown in Panels a, d, g, j, m, p, s & v. DMPK transcripts encoding the expanded CUG tracts were detected by hybridization with a (CAG)₁₀-Cy3 probe (red signal; Panels h, k, n, q, t & w). Transcripts containing expanded CUG repeats are not observed in the normal fibroblasts and normal myoblasts (b & e), respectively. Merged images of DAPI and (CAG)₁₀-Cy3 stains show CUG RNA foci as red signals (i, l, o, r, u & x) within the nucleus and in the cytoplasm in DM1 fibroblasts and myoblasts. The percent of DM1 fibroblasts and myoblasts containing both nuclear and cytoplasmic foci are tabulated in . Images of CUG RNA foci in additional DM1 fibroblasts and myoblasts are shown in supplementary (Panels A–E).

Panel A: Distribution of endogenous Mbnl1 is visualized as a green signal (a, d, g & j) using anti-MBNL1 (MB1a) monoclonal antibody and secondary antibodies conjugated with FITC. The mutant transcripts encoding the expanded CUG tracts were detected by hybridization with a (CAG)₁₀-Cy3 probe (red signals; e & h). Transcripts containing expanded CUG repeats are not observed in the α-MHC-LacZ cardiomyocytes (b) and normal myoblasts (k). Merged images (f & i), where super-imposition of green and red signals are observed as a yellow signals, show that Mbnl1 co-localizes with the expanded CUG tracts in the α-MHC-LacZ-(CUG)₄₀₀ cardiomyocytes (f) and in DM1 myoblasts (i). Panel B: Graphical representation of Mbnl1 distribution in each compartment (nucleus, cytoplasm and foci) of α-MHC-LacZ, α-MHC-LacZ-(CUG)₄₀₀ cardiomyocytes and DM1 and normal myoblasts is shown and the results are tabulated in . No significant difference is observed in the fraction of Mbnl1 which colocalizes with the foci in α-MHC-LacZ-(CTG)₄₀₀ cardiomyocytes and in DM1 myoblasts (p = 0.37). The specificity of MBNL1 (MB1a) monoclonal antibody was assessed by immunofluorescence using cardiomyocytes derived from Mbnl1^−/− mice (Supplementary ).

Panel A: The α-MHC-LacZ-(CTG)₄₀₀ transgene encoding the α-myosin heavy chain promoter (α-MHC) used to drive cardiac specific expression of the β-galactosidase (LacZ) gene followed by a CTG tract of ∼400 repeats and the bovine growth hormone polyA (BGH-PolyA) sequence is shown. Panel B: Restriction digestion of plasmids encoding the α-MHC-LacZ-(CTG)₄₀₀ sequences with SfiI, which allows excision of the CTG repeat tract, demonstrates the instability of the CTG repeats when propagated in E. coli at 37°C. Panel C: Southern blot analysis of mouse tail-clip DNA digested with PvuII. The 280 bp probe used for hybridization (Panel A) is shown. The majority of the detected bands contained 350∼380 CTG repeats in α-MHC-LacZ-(CTG)₄₀₀TG^high (band intensities ∼76%) or 300∼350 CTG repeats in α-MHC-LacZ-(CTG)₄₀₀TG^low (band intensities ∼80%) in tail clip DNAs. Panel D: Northern blot analysis of RNA derived from α-MHC-LacZ-(CTG)₄₀₀ and α-MHC-LacZ mouse hearts probed with β-galactosidase and Gapdh sequences is shown.

Panels A–B: RT-PCR analyses of the steady-state expression levels of LacZ-(CTG)₀, LacZ-(CTG)₄₀₀, and DMPK11-15(CTG)₅, DMPK 11-15(CTG)₃₀₀ cassettes are shown. Synthesized cDNA (100 ng) from normal myoblsts expressing LacZ-(CTG)_{0 or 400} and DMPK 11-15(CTG)_{5 or 300} were subjected to RT-PCR analysis. GAPDH RNA was amplified in parallel as an internal control. The experiments were carried out in triplicate and the results are tabulated in . Relative steady-state expression levels of the LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ cassettes were not significantly different (p = 0.479). Panels C–F: Real-time PCR analysis of serial dilutions of plasmid DNAs encoding LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ sequences and of LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ cDNAs is shown. PCR reactions were carried using 10⁻² to 10⁻⁶ fmoles of plasmid DNAs encoding LacZ-(CTG)₄₀₀ or DMPK 11-15(CTG)₃₀₀ sequences. To quantitate the expression levels of expanded CUG repeat encoding transcripts, cDNAs (5 ng) from human myoblasts expressing LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ were subjected to Real-time PCR analysis in parallel. LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ cDNAs are present at approximately similar levels (Panels C, E & ). Melting curves of LacZ-(CTG)₄₀₀ or DMPK 11-15(CTG)₃₀₀ PCR reactions are shown (Panels D & F). GAPDH was used as an internal control for RNA quality and the reverse transcriptase reaction (Ct values for GAPDH in LacZ-(CTG)₄₀₀ and DMPK 11-15(CTG)₃₀₀ samples was 19.8 in each case).

Panel A: DMPK 11-15(CTG)_{5 or 300} (a), GFP-DMPK 3′UTR (CTG)_{5 or 400} (b) and LacZ-(CTG)_{0 or 400} (c) cassettes under the transcriptional control of the cytomegalovirus (CMV) promoter are shown. Panel B: Nuclear DAPI staining of human normal myoblasts expressing DMPK11-15(CTG)₅ (a), DMPK 11-15(CTG)₃₀₀ (b), GFP-DMPK 3′UTR(CTG)₅ (c), GFP-DMPK 3′UTR(CTG)₄₀₀ (d), LacZ-(CTG)₀ (e), LacZ-(CTG)₄₀₀ (f) cassettes are shown. The mutant transcripts encoding the expanded CUG tracts were detected by hybridization with a (CAG)₁₀-Cy3 probe. CUG RNA foci are observed primarily within the nucleus in normal myoblasts expressing DMPK11-15(CTG)₃₀₀ (red signal; b) and GFP-DMPK 3′UTR (CTG)₄₀₀ (red signal; d). CUG RNA foci are observed in the cytoplasm (red signal; f) in normal myoblasts expressing the LacZ-(CTG)₄₀₀ cassette. Normal myoblasts expressing DMPK11-15(CTG)₅ (a), GFP-DMPK 3′UTR(CTG)₅ (c), and LacZ-(CTG)₀ (e) constructs did not show RNA foci. Panel C: IR and cTNT RNA splicing in myoblasts expressing the indicated cassettes are shown. Synthesized cDNAs (150 ng) were subjected to RT-PCR analysis using the IR and cTNT primers described in . GAPDH RNA was amplified in parallel as an internal control. The experiments were carried out in triplicate. Representative panels are shown in Panel C and the results are tabulated in .