Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy
Thurman M. Wheeler, John D. Lueck, Maurice S. Swanson, Robert T. Dirksen, Charles A. Thornton
Thurman M. Wheeler, John D. Lueck, Maurice S. Swanson, Robert T. Dirksen, Charles A. Thornton
View: Text | PDF
Research Article Genetics

Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy

Abstract

In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3′ splice site of ClC-1 exon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this exon, the AON restored the full-length reading frame in ClC-1 mRNA, upregulated the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM.

Authors

Thurman M. Wheeler, John D. Lueck, Maurice S. Swanson, Robert T. Dirksen, Charles A. Thornton

×

Figure 3

Antisense morpholino represses splicing of ClC-1 exon 7a.

Options: View larger image (or click on image) Download as PowerPoint
Antisense morpholino represses splicing of ClC-1 exon 7a. (A) RT-PCR sho...
(A) RT-PCR showed reduction of exon 7a inclusion 3 weeks after injection of antisense (anti) morpholino (antisense 1 + antisense 2; 5 μg each) into TA muscle of HSALR mice. Pairs of injected TA muscles from each mouse are identified as 1, 2, and 3. Muscle injected with control morpholino (inv) (10 μg) was not different from untreated HSALR muscle. HSALR and WT mice have the same (FVB) inbred strain background. int, intron, ex, exon. (B) Inclusion of exon 7a remained partially suppressed 8 weeks after injection of antisense morpholino (20 μg antisense 1 vs. 20 μg invert control). (C) ClC-1 antisense morpholino did not correct the misregulated alternative splicing of titin M-line exon 5. The percentage of ClC-1 splice products that include exon 7a is shown at 3 (D) and 8 (E) weeks following morpholino injection. Mean ± SD; n = 3 per group; **P < 0.001; *P = 0.035 antisense versus invert-treated controls; t test. (F) The level of ClC-1 mRNA was increased 3 weeks after treatment with antisense moropholino. ClC-1 mRNA level is expressed in arbitrary units relative to housekeeping gene RNA polymerase II transcription factor IIB. Mean ± SD; n = 3 per group; *P = 0.06 for antisense versus invert-treated control; t test.