Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Abstract

Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG)n tract in the 3' UTR of the gene encoding myotonic dystrophy protein kinase (DMPK), which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNA-binding proteins (such as MBNL1) in ribonuclear inclusions. It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3' UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3' UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.

Figure 1

Transgene expression. (a) Transgenes used to create double transgenic tetracycline-inducible mice. (b) RNA-FISH and fluorescence microscopy of skeletal muscle in mice in which transgene expression was induced demonstrates GFP expression in all mice and RNA foci only in mice expressing the GFP-DMPK 3′ UTR (CTG)₂₀₀. (c) RNA blot of skeletal muscle RNA showing significant transgene induction in (CTG)₅ mice relative to endogenous Dmpk expression. Gapdh was used as loading control. (d) RNA-FISH and immunofluorescence for MBNL1 demonstrating colocalization in (CTG)₂₀₀ mice. (e) Antibodies to MBNL1 show that wild-type mice (FVB) and (CTG)₅ mice have diffuse nuclear staining, whereas (CTG)₂₀₀ mice have distinct MBNL foci.

Figure 2

Myotonic dystrophy phenotypes in transgenic mice. (a) Electromyography in skeletal muscles of mice in which transgene expression was not induced (‘No doxy’) shows a quiet baseline and classic myotonia in mice in which transgene expression was induced (‘Doxy’). ClC-1 immunohistochemistry showing normal sarcolemmal pattern of ClC-1 and loss of ClC-1 in myotonic mice. (b) Progressive cardiac conduction disturbances detected by serial ECGs on transgenic mice in which transgene expression was induced PR interval (PR int.) is prolonged by day 3. Note irregular and dropped beats by day 5 and lack of P waves by day 8. (c) Clcn1 and Tnnt3 RNA splicing abnormalities in skeletal muscle of mice in which transgene expression was induced. Lane 1: DNA marker; lane 2: wild-type; lane 3: uninduced 5-313; lane 4: induced 5-313. Note increased amounts of larger splice products. (d) H&E staining of skeletal muscle demonstrating increased numbers of central nuclei, fiber size variation and nuclear clumping in mice in which transgene expression was induced.

Figure 3

Reversal of RNA toxicity. (a) ClC-1 immunohistochemistry shows loss of ClC-1 in mice in which transgene expression was induced and reversion to normal patterns with transgene silencing. (b) ECGs demonstrating examples of first-degree heart block (prolonged PR interval) in mice in which transgene expression was induced. Mouse #3074 reverted to normal from first-degree heart block. Mouse #2090 had second-degree heart block (Wenkebach-type) refractory to reversal (* indicates dropped beat). (c) Clcn1 and Tnnt3 RNA splicing abnormalities in skeletal muscle were reversed. Lanes 1,2: uninduced; lanes 3,4: induced; lanes 5,6: mice that reverted. (d) H&E staining of skeletal muscle showing reversal of histopathology. Mice reverted for over two months. Note obvious reductions in small fibers, central nuclei and nuclear clumping in mice that have reverted.

Figure 4

CUG-BP1 levels elevated by toxic RNA. Protein blots showing expression of CUG-BP1, MBNL1 and GFP in mouse skeletal muscle and heart. GAPDH was used as a loading control. Note induction of CUB-BP1 in skeletal muscle and reversion with transgene silencing. Also, there was no change in MBNL1 expression in mouse tissues in which transgene expression was induced. The 5-313 mice used in this figure had been induced continuously for over 10 months, and tissues from mice that reverted were collected within 20 d after stopping doxycycline.