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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 (CUGexp) (reviewed in ref. 2). The transcripts containing a CUGexp 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 CUGexp RNA (6, 7).
Previously we showed that transgenic mice expressing CUGexp RNA (human skeletal actin long repeat [HSALR] mice) displayed myotonia and chloride channel 1 (ClC-1) splicing defects similar to those observed in DM1 (8). We postulate that myotonia in the HSALR 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 Figure1A.1A. The morpholino AONs were complementary to the 3′ or 5′ splice sites of exon 7a in the ClC-1 pre-mRNA (Figure (Figure1B).1B). To examine tissue uptake, we injected a carboxyfluorescein-labeled morpholino into tibialis anterior (TA) muscle of HSALR 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 (Figure2,2, A–C). Of note, the AON was present in both nucleus and cytoplasm but appeared to accumulate preferentially in the nucleus (Figure (Figure2,2, A and E).
To determine the effect of morpholino on splicing in HSALR 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 (Figure3,3, 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 (Figure3,3, A and D), and a partial exclusion of exon 7a was still evident after 8 weeks (Figure (Figure3,3, B and E). Notably, the antisense morpholino did not affect the formation of nuclear foci containing CUGexp 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 (Figure3C),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.
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 (Figure3F).3F). These results indicate that effects of CUGexp 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 (Figure2,2, 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 HSALR 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 HSALR 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 (Figure2,2, D and E). ClC-1 current density (Figure (Figure4,4, A and B) and deactivation kinetics (Figure (Figure4D)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 HSALR 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 HSALR and WT fibers (Figure (Figure4C).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.
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 HSALR mice after injection of antisense morpholino, whereas myotonia in the invert-injected contralateral TA was not different from that in uninjected muscle (Figure (Figure4,4, 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 (Mbnl1ΔE3/ΔE3) resulted in loss of Mbnl1 protein from muscle, spliceopathy similar to that in DM1 patients and HSALR 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 Mbnl1ΔE3/ΔE3 mice. As in the HSALR transgenic model, antisense morpholino repressed the inclusion of exon 7a (Figure (Figure5,5, A and B), increased the expression of ClC-1 protein at the sarcolemma (Figure (Figure5,5, C and D), and reduced the myotonia in Mbnl1ΔE3/ΔE3 mice (Figure (Figure5E).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.
Current models of DM1 pathogenesis postulate that DMPK mRNA containing a CUGexp 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 HSALR and Mbnl1ΔE3/ΔE3 mice. To our knowledge, these results provide the clearest indication to date that CUGexp-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 HSALR and Mbnl1ΔE3/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 HSALR muscle reduced levels of exon 7a–containing transcript (Figure (Figure3,3, D and E), increased full-length ClC-1 transcript (Figure (Figure3F),3F), and completely normalized ClC-1 current density (Figure (Figure4B)4B) and deactivation gating (Figure (Figure4D)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.
Morpholino oligonucleotides (Gene Tools LLC) were 5′-CCAGGCACGGTctgcaacagagaag-3′, targeting the ClC-1 3′ splice site; 5′-gaagagacaacgtctggcacggacc-3′ inverted control; and 5′-ggaagtgaaacttgcCTCCATCAGG-3′, targeting the ClC-1 5′ splice site. (Upper case indicates exonic sequences, lower case indicates intronic sequences.)
Animal experiments were approved by the University Committee on Animal Resources at the University of Rochester Medical Center. HSALR (26) or Mbnl1ΔE3/ΔE3 mice (4) were anesthetized by intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine. TA muscle was pretreated by intramuscular injection of bovine hyaluronidase (15 μl, 0.4 U/μl) (Sigma-Aldrich) (27). Two hours later, 10 or 20 μg of morpholino in a total volume of 20 μl PBS was injected using a 30-gauge needle. TA muscle was then electroporated using electrodes placed parallel to the long axis of the muscle. Electroporation parameters were 100 V/cm, 10 pulses at 1 Hz, and 20-ms duration per pulse. Antisense or control morpholino with inverted sequence was injected into TA muscles of opposite limbs. The determination of which TA received antisense morpholino was randomized, and investigators remained blinded to this assignment until EMG analyses were completed. Other analyses were performed without blinding. For experiments to determine the distribution of injected oligonucleotides, the antisense morpholino was labeled with carboxyfluorescein, and cryosections of muscle (10 μM) were examined by fluorescence microscopy, with or without fixation in 4% paraformaldehyde. Some sections were colabeled with TRITC–wheat germ agglutinin (28) (50 μg/ml in PBS; Sigma-Aldrich) and DAPI to highlight the surface membranes and nuclei of muscle fibers.
Mice were sacrificed 3 or 8 weeks after morpholino injection. TA muscles were removed and frozen in liquid nitrogen. Total RNA was isolated with TriReagent (Molecular Research Center). cDNA synthesis was primed with oligo-dT as described previously (8). Assays for alternative splicing of ClC-1 and titin were described previously (7, 8). Primer sequences were: ClC-1 forward, ClCm-7 5′-TGAAGGAATACCTCACACTCAAGG-3′, and reverse, ClCm-30 5′-CACGGAACACAAAGGCACTG-3′; mtitin forward, mTTN1 5′-GTGTGAGTCGCTCCAGAAACG-3′, and reverse, mTTN2 5′-CCACCACAGGACCATGTTATTTC-3′.
RT-PCR products (22 cycles) were separated on agarose gels, stained with SYBR green II, and scanned on a laser fluorimager (Molecular Dynamics). Band intensity was quantified using ImageQuant software (GE Healthcare). Total levels of ClC-1 mRNA were determined by quantitative real-time RT-PCR (Taqman; Applied Biosystems) relative to housekeeping gene RNA polymerase II transcription factor IIB.
Frozen transverse sections of TA muscle (10 μM) were stained with affinity-purified rabbit polyclonal anti–ClC-1 antibody (1:50; Alpha Diagnostic International) as previously described (29). Muscle sections from ClC-1–null mice and WT FVB mice served as negative and positive controls on each slide (data not shown). z-Plane stacks consisting of 8 images separated by 0.25 μM were captured and deconvolved using Autoquant version 9.3 software (Autoquant Imaging). Maximum projection images were obtained using Metavue software (Universal Imaging Corp.). Exposure time and thresholding were identical for all comparisons of antisense and invert controls.
Delivery of antisense and invert morpholinos into FDB fibers was achieved by injection and electroporation of hind limb footpads. Briefly, 12- to 14-day-old HSALR mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine. Hind limb foot pads then were injected with bovine hyaluronidase followed 1 hour later with 20 mg (10 μl, 2 mg/ml in PBS) of antisense or invert carboxyfluorescein-labeled morpholino. Uptake of morpholinos was enhanced by electroporation (100 V/cm, 20 pulses at 1 Hz, and 20 ms per pulse) of the foot pad immediately after injection (30). Three to 5 days after injection/electroporation, individual FDB muscle fibers were isolated as previously described (10). Bright field and fluorescence (488 nm excitation) images of single FDB fibers were acquired using a 40× (1.4 NA) objective and a TILL IMAGO-QE cooled-CCD camera. Only fibers exhibiting clear striations, clean surfaces, and green fluorescence were chosen for electrophysiological recordings. ClC-1 currents were measured and analyzed in whole-cell patch clamp experiments (31) using an approach identical to that described in detail elsewhere (10). ClC-1 current density (pA/pF) was calculated in order to compare data across fibers of different sizes.
EMG was performed under general anesthesia as described previously (4). Images and video recordings of electromyographic myotonia in HSALR and Mbnl1ΔE3/ΔE3 mice are shown in previous reports (4, 26). A minimum of 15 needle insertions were performed for each muscle examined. Myotonic discharges were graded on a 4-point scale: 0, no myotonia; 1, occasional myotonic discharge in less than 50% of needle insertions; 2, myotonic discharge in greater than 50% of needle insertions; 3: myotonic discharge with nearly every insertion.
Group data are expressed as mean ± SD, except for patch clamp data in Figure Figure4,4, which are expressed as mean ± SEM. Between-group comparison was performed by 2-tailed Student’s t test or 1-way ANOVA as indicated. A P value < 0.05 was considered significant.
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).
Nonstandard abbreviations used: AON, antisense oligonucleotide; ClC-1, chloride channel 1; CUGexp, expanded CUG repeat; DM, dystrophia myotonica (myotonic dystrophy); DMPK, DM protein kinase; EMG, electromyography; FDB, flexor digitorum brevis; HSALR, 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.
Citation for this article: J. Clin. Invest. 117:3952–3957 (2007). doi:10.1172/JCI33355