Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy
Thurman M. Wheeler, … , Robert T. Dirksen, Charles A. Thornton
Thurman M. Wheeler, … , Robert T. Dirksen, Charles A. Thornton
Published November 15, 2007
Citation Information: J Clin Invest. 2007;117(12):3952-3957. https://doi.org/10.1172/JCI33355.
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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

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Figure 4

Antisense morpholino rescues ClC-1 channel function and reverses myotonia in skeletal muscle of HSALR mice.

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Antisense morpholino rescues ClC-1 channel function and reverses myotoni...
(A) Representative ClC-1 currents obtained from FDB fibers isolated from HSALR mice electroporated with either invert (left) or antisense (middle) morpholino and WT mice electroporated with antisense morpholino (right). The dashed lines represent the 0 current level. Capacitative currents recorded from each fiber are shown in the inset of each panel (scale bars: vertical, 3 nA; horizontal, 4 ms). Superimposed traces (solid lines) of normalized ClC-1 current deactivation at –100 mV in FDB fibers obtained from invert- (circles) and antisense-treated (squares) HSALR mice and antisense-treated WT mice (triangles) fit with a second-order exponential are shown in the inset to the left panel. Note that accelerated ClC-1 deactivation kinetics of FDB fibers obtained from HSALR mice are normalized only following treatment with antisense morpholino. (B) Membrane potential (Vm) dependence of average instantaneous ClC-1 current density recorded from FDB fibers of 16- to 18-day-old WT mice treated with invert morpholino (n = 11), WT mice treated with antisense morpholino (n = 10), HSALR mice treated with invert morpholino (n = 12), and HSALR mice treated with antisense morpholino (n = 16). (C) Average relative Po-Vm curves for the same experiments shown in B. Smooth curves through each data set were generated using a modified Boltzmann equation (10). (D) Average relative contribution of the fast (Af/Atotal), slow (As/Atotal), and nondeactivating (C/Atotal) components of ClC-1 current deactivation elicited from a voltage step to –100 mV for the same experiments shown in B. Mean ± SEM; *P < 0.05 invert-treated HSALR fibers compared with each of the other experimental conditions; t test. Myotonia was significantly reduced 3 (E) and 8 (F) weeks following injection of antisense morpholino. Mean ± SD; n = 3–7 per group. Antisense morpholino was injected into one TA; invert morpholino was injected into the contralateral TA; and gastrocnemius muscle served as an untreated control. **P < 0.0001 for antisense versus invert-treated control; ANOVA.