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

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.

Dystrophia myotonica (myotonic dystrophy) type 1 (DM1), the most common muscular dystrophy affecting adults, is caused by expansion of a CTG repeat in the 3′ untranslated region of the gene encoding the DM protein kinase (DMPK) (1). Evidence suggests that DM1 is not caused by abnormal expression of DMPK protein, but rather that it involves a toxic gain of function by mutant DMPK transcripts that contain an expanded CUG repeat (CUG^exp) (reviewed in ref. 2). The transcripts containing a CUG^exp tract elicit abnormal regulation of alternative splicing, or spliceopathy (3). The splicing defect, which selectively affects a specific group of pre-mRNAs, is thought to result from reduced activity of splicing factors in the muscleblind (MBNL) family (4), increased levels of CUG-binding protein 1 (3, 5), or both. Decreased activity of MBNL proteins can be attributed to sequestration of these proteins in nuclear foci of CUG^exp RNA (6, 7).

Previously we showed that transgenic mice expressing CUG^exp RNA (human skeletal actin long repeat [HSA^LR] mice) displayed myotonia and chloride channel 1 (ClC-1) splicing defects similar to those observed in DM1 (8). We postulate that myotonia in the HSA^LR model results from abnormal inclusion of exon 7a in the ClC-1 mRNA, owing to sequestration of MBNL1, a factor required for repression of exon 7a splicing in muscle fibers (4). This mechanism is supported by several lines of evidence: (a) inclusion of exon 7a causes frame shift and introduction of a premature termination codon in the ClC-1 mRNA (5, 8); (b) truncated ClC-1 protein encoded by the exon 7a+ isoform is devoid of channel activity (9); and (c) disruption of Mbnl1 in mice leads to increased inclusion of ClC-1 exon 7a and myotonia (4). Our postulate that myotonia in DM1 results from deficiency of ClC-1 is based on observations that mouse models of DM1 display a 70%–80% reduction in muscle chloride conductance (8, 10), coupled with previous estimates that a 75% reduction of ClC-1 conductance is sufficient to cause myotonic discharges in muscle fibers (11). However, the mechanism of ClC-1 downregulation and its requirement for myotonia in DM1 are controversial. Effects on sodium or potassium channels have also been implicated in DM1-associated myotonia (12–14). In addition, evidence that chloride channelopathy in DM1 results from downregulation of ClC-1 transcription, rather than abnormal splicing, has been reported (15). To provide a causal link between ClC-1 alternative splicing, chloride channelopathy, and myotonia in DM1, we used a morpholino antisense oligonucleotide (AON) to selectively repress the inclusion of exon 7a.

The strategy for suppressing the inclusion of exon 7a is diagrammed in Figure 1A. The morpholino AONs were complementary to the 3′ or 5′ splice sites of exon 7a in the ClC-1 pre-mRNA (Figure 1B). To examine tissue uptake, we injected a carboxyfluorescein-labeled morpholino into tibialis anterior (TA) muscle of HSA^LR mice. Examination of tissue sections indicated that uptake of antisense morpholino was limited to the needle track (data not shown). To improve uptake and distribution of AON, we used voltage pulses to electroporate muscle fibers after the AON injection. This led to uptake of antisense morpholino throughout the TA muscle (Figure 2, A–C). Of note, the AON was present in both nucleus and cytoplasm but appeared to accumulate preferentially in the nucleus (Figure 2, A and E).

Figure 1

Design of antisense morpholinos. (A) Inclusion of ClC-1 exon 7a induces a frame shift and premature termination codon in exon 7. Annealing of antisense morpholino to the 3′ splice site of exon 7a in the ClC-1 pre-mRNA is intended to prevent spliceosomal recognition of this exon. (B) Alignment of ClC-1 pre-mRNA (top strand) with antisense morpholinos targeting the 3′ or 5′ splice sites of exon 7a is shown. The control morpholino is the 5′-3′ invert of the 3′ splice site blocker. Exonic sequences are in upper case, intronic sequences are in lower case.

Figure 2

Antisense morpholino localizes preferentially to muscle nuclei and restores ClC-1 expression at the sarcolemma. (A–C) Cross section of HSA^LR TA muscle showing distribution of 3′-carboxyfluorescein–labeled antisense morpholino (green) 3 weeks after injection. The morpholino was complementary to the 3′ splice site of ClC-1 pre-mRNA. Muscle fibers are outlined by wheat germ agglutinin (WGA; red), and nuclei are highlighted by DAPI (blue). Bright field (D) and fluorescence (E) images of a single FDB fiber showing preferential nuclear localization of the antisense morpholino (day 5 after injection). As compared with invert-treated control (F), immunofluorescence shows an increase of sarcolemmal ClC-1 protein (red) in HSA^LR TA muscle 3 weeks after treatment with antisense morpholino (G). Scale bars: 20 μm.

To determine the effect of morpholino on splicing in HSA^LR mice, total RNA was extracted from TA muscle after AON injection. Analysis of ClC-1 splicing by RT-PCR showed that antisense morpholino had the intended effect of suppressing the inclusion of exon 7a, whereas control morpholino with inverted sequence had no effect on ClC-1 splicing in the contralateral TA (Figure 3, A and D). AON targeting the 3′ splice site, or coinjection of AONs targeting the 3′ and 5′ splice sites, was more effective than targeting the 5′ splice site alone (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI33355DS1). Effective and sustained skipping of exon 7a was achieved after a single injection of morpholino AON. Inclusion of exon 7a was suppressed to WT levels for at least 3 weeks after a single injection (Figure 3, A and D), and a partial exclusion of exon 7a was still evident after 8 weeks (Figure 3, B and E). Notably, the antisense morpholino did not affect the formation of nuclear foci containing CUG^exp RNA and MBNL1 protein (Supplemental Figure 2), nor did it correct the alternative splicing of other genes that are misregulated in DM1, such as titin (Figure 3C), ZASP, or Serca1 (data not shown) (7). These data indicate that morpholino AON specifically corrects the ClC-1 splicing defect rather than producing a general reversal of DM-associated spliceopathy or Mbnl1 sequestration.

Figure 3

Antisense morpholino represses splicing of ClC-1 exon 7a. (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 HSA^LR 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 HSA^LR muscle. HSA^LR 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.

Morpholino AONs influence splicing outcomes without inducing degradation of their target RNAs (16). Therefore, the predicted effect of repressing exon 7a inclusion was to eliminate the premature termination codon in ClC-1 mRNA and thereby reduce its degradation through the nonsense-mediated decay pathway (17). Consistent with this prediction, treatment with antisense morpholino, but not control morpholino with inverted sequence, led to increased levels of ClC-1 mRNA, as determined by quantitative real-time RT-PCR (Figure 3F). These results indicate that effects of CUG^exp RNA on ClC-1 expression are mainly at the posttranscriptional level. Furthermore, treatment with morpholino AON increased the level of ClC-1 protein in the sarcolemma, as indicated by immunofluorescence using antibodies directed against the C terminus (Figure 2, F and G).

We previously used whole-cell patch-clamp analysis of single flexor digitorum brevis (FDB) muscle fibers to show that ClC-1 current density is reduced and channel deactivation accelerated in FDB fibers of untreated HSA^LR mice (10). Therefore, we next set out to determine the effect of AON treatment on ClC-1 channel function. Hind limb foot pads of 10- to 12-day-old WT and HSA^LR mice were injected/electroporated with carboxyfluorescein-labeled antisense or invert morpholino. Patch-clamp analysis was performed 3–5 days after injection, at a time when fibers were still small enough to maintain an effective voltage clamp (10). Individual FDB fibers were isolated, and macroscopic ClC-1 channel activity was measured in fibers exhibiting green fluorescence (Figure 2, D and E). ClC-1 current density (Figure 4, A and B) and deactivation kinetics (Figure 4D) were rescued to WT values as early as 3 days after morpholino AON injection, while current density and deactivation kinetics in fibers treated with invert morpholino were not different from those of untreated HSA^LR fibers. The slower rate of channel deactivation observed for WT and AON-treated fibers was most likely not due to a current-dependent effect on channel gating, as reducing WT ClC-1 current magnitude in half with a prepulse does not significantly alter the kinetics of channel deactivation (10). Rescue of ClC-1 activity was not due to a shift in channel activation, since the voltage dependence of relative channel open probability (Po) was not different between antisense- and invert-injected HSA^LR and WT fibers (Figure 4C). These results demonstrate that morpholino AON rescue of ClC-1 spliceopathy is sufficient to completely restore normal ClC-1 current density and channel deactivation kinetics.

Figure 4

Antisense morpholino rescues ClC-1 channel function and reverses myotonia in skeletal muscle of HSA^LR mice. (A) Representative ClC-1 currents obtained from FDB fibers isolated from HSA^LR 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) HSA^LR 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 HSA^LR 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), HSA^LR mice treated with invert morpholino (n = 12), and HSA^LR 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 (A_f/A_total), slow (A_s/A_total), and nondeactivating (C/A_total) 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 HSA^LR 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.

Next we determined the effects of repressing exon 7a inclusion on muscle physiology in vivo. Electromyography (EMG) analysis by an examiner blinded to the experimental protocol revealed that myotonia was markedly reduced or absent in TA muscles of HSA^LR mice after injection of antisense morpholino, whereas myotonia in the invert-injected contralateral TA was not different from that in uninjected muscle (Figure 4, E and F). Myotonia reduction correlated with the degree of exon 7a skipping at 3- and 8-week time points, indicating that a single injection of antisense morpholino provided a sustained reduction in myotonia.

Previously we showed that homozygous deletion of Mbnl1 exon 3 in mice (Mbnl1^ΔE3/ΔE3) resulted in loss of Mbnl1 protein from muscle, spliceopathy similar to that in DM1 patients and HSA^LR mice, reduction of ClC-1 expression and activity, and myotonia (4, 7, 10). To examine the effects of AON in this model, we injected antisense morpholino or invert control into TA muscle of Mbnl1^ΔE3/ΔE3 mice. As in the HSA^LR transgenic model, antisense morpholino repressed the inclusion of exon 7a (Figure 5, A and B), increased the expression of ClC-1 protein at the sarcolemma (Figure 5, C and D), and reduced the myotonia in Mbnl1^ΔE3/ΔE3 mice (Figure 5E). Thus, while the pathogenesis of spliceopathy in DM is a subject of debate, rescue of the myotonia by antisense morpholino does not depend on the exact manner in which the ClC-1 splicing defect is generated.

Figure 5

Antisense morpholino represses exon 7a inclusion, restores ClC-1 protein expression, and rescues myotonia in Mbnl1^ΔE3/ΔE3 mice. (A) RT-PCR shows reduced inclusion of exon 7a at 3 weeks after injection of antisense morpholino 1 (20 μg antisense or invert control). (B) Quantitation of splicing results shown in A as mean ± SD; n = 3 per group; **P < 0.001, antisense versus invert-treated control; t test. Immmunofluorescence for ClC-1 is increased 3 weeks after injection with antisense (D) as compared with invert-treated control (C). Scale bar: 20 μm. (E) Myotonia in Mbnl1^ΔE3/ΔE3 TA muscle was reduced 3 weeks after treatment with antisense morpholino but not in muscle treated with invert control. Mean ± SD; n = 3 per group; **P < 0.0001, antisense versus invert-treated control; ANOVA.

Current models of DM1 pathogenesis postulate that DMPK mRNA containing a CUG^exp alters the function of splicing factors, leading to misregulated alternative splicing for a specific group of pre-mRNAs (3). In operational terms, one difficulty with this model is that functional differences between alternative splice isoforms, or biological consequences of altering the ratio of 2 alternative splice products, are not easy to determine. Furthermore, in the context of dozens to hundreds of transcripts whose splicing is so affected, the phenotypic consequences of any particular splicing change may be difficult to ascertain. A key finding of the present study is that misregulated alternative splicing of ClC-1 is required for the development of myotonia, a cardinal symptom of DM1, in both HSA^LR and Mbnl1^ΔE3/ΔE3 mice. To our knowledge, these results provide the clearest indication to date that CUG^exp-induced spliceopathy is directly involved in producing a clinical feature of DM1. Furthermore, our results suggest an approach for dissecting the functional significance of any particular splicing change. AONs can be used to correct a specific splicing defect in DM1 cells or to induce a DM1-like effect in WT cells.

Myotonia is the symptom by which DM1 is most often recognized, and, due to preferential involvement of hand and forearm muscles, it compromises manual dexterity and contributes to disability. In light of the abnormal calcium homeostasis observed in DM1 cells and model systems (18, 19), and the effects of DM1 on alternative splicing of the SERCA1 calcium reuptake pump of the sarcoplasmic reticulum (7, 20), it also seems possible that excessive calcium release due to myotonic discharges may aggravate the degeneration of DM1 muscle fibers. Although previous studies have implicated sodium channels or calcium-activated potassium channels in DM1 (12–14), a second finding of the present study is that DM1-associated myotonia results primarily from a chloride channelopathy.

We previously found that the number of functional ClC-1 channels in the sarcolemma was markedly decreased, the rate of channel deactivation was increased, and the maximum ClC-1 channel Po was reduced in both HSA^LR and Mbnl1^ΔE3/ΔE3 mice (10). The observed acceleration in channel deactivation and reduction in maximal channel Po are consistent with previously reported dominant-negative effects imparted by exon 7a–encoded protein products (9). Our observations that electroporation of AON in HSA^LR muscle reduced levels of exon 7a–containing transcript (Figure 3, D and E), increased full-length ClC-1 transcript (Figure 3F), and completely normalized ClC-1 current density (Figure 4B) and deactivation gating (Figure 4D) support the assertion that chloride channelopathy in DM1 involves a complex combination of transdominant RNA- and protein-based mechanisms.

AONs influence RNA processing by annealing to pre-mRNA and blocking the access of splicing factors to splice sites or cis-acting regulatory elements (21). AONs that induce skipping of constitutively spliced exons have been used to bypass stop codons or restore the proper reading frame in the dystrophin mRNA (22–24). We postulated that exon 7a may show heightened susceptibility to AONs because splicing signals in alternative exons tend to be intrinsically weak and this exon is normally skipped in a fraction of ClC-1 transcripts (17). However, ClC-1 mRNAs that include exon 7a contain premature termination codons and undergo rapid degradation (17). Therefore, these splice products are underrepresented at steady state, and we cannot determine the exact efficiency of AON-induced exon skipping. Despite this limitation, the decrease of exon 7a+ isoforms to WT levels and the normalized activity of ClC-1 channels in treated muscle fibers suggest that this intervention is highly effective and surprisingly prolonged.

To our knowledge, these results are the first to show that symptoms of DM1 are reversible using a targeted approach to restore normal alternative splicing that does not rely on gene therapy. While several drugs with antimyotonia properties are currently available, they provide only partial relief of symptoms, and their use in DM1 is limited by the lack of controlled trials supporting their efficacy and safety (25). Results here indicate that targeting the ClC-1 splicing defect could be highly effective for treating the myotonia in DM1.

View Supplemental data

The authors thank Matt Krym, Linda Richardson, and Kirti Bhatt for technical assistance. This work was supported by the University of Rochester Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center (NIH/NS48843), with additional support from the NIH (AR46806, AR/NS48143), the Run America Foundation, the Saunders Family Neuromuscular Research Fund, the Schwab Research Fund, and a National Institute of Dental and Craniofacial Research training grant (T32DE07202).

Address correspondence to: Charles Thornton, Box 673, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14618, USA. Phone: (585) 275-2542; Fax: (585) 273-1255; E-mail: charles_thornton@urmc.rochester.edu.

Nonstandard abbreviations used: AON, antisense oligonucleotide; ClC-1, chloride channel 1; CUG^exp, expanded CUG repeat; DM, dystrophia myotonica (myotonic dystrophy); DMPK, DM protein kinase; EMG, electromyography; FDB, flexor digitorum brevis; HSA^LR, human skeletal actin long repeat; MBNL, muscleblind; Po, open probability; TA, tibialis anterior.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:3952–3957 (2007). doi:10.1172/JCI33355

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