Volume 10, Issue 1, Pages 35-44 (July 2002) Expanded CUG Repeats Trigger Aberrant Splicing of ClC-1 Chloride Channel Pre- mRNA and Hyperexcitability of Skeletal Muscle in Myotonic Dystrophy  Ami Mankodi, Masanori P. Takahashi, Hong Jiang, Carol L. Beck, William J. Bowers, Richard T. Moxley, Stephen C. Cannon, Charles A. Thornton  Molecular Cell  Volume 10, Issue 1, Pages 35-44 (July 2002) DOI: 10.1016/S1097-2765(02)00563-4

Figure 1 Electrical Excitability Is Increased in HSALR Fibers (A) Threshold current intensity is reduced and action potential latency is prolonged in HSALR fibers. On average, the threshold stimulus intensity was 42% lower for HSALR than wild-type (WT) fibers (70.5 ± 14.0 nA, n=10 and 120 ± 10 nA, n=10; p < 0.02), and the latency was prolonged nearly 3-fold (58.7 ± 1.7 ms, n=10 and 20.5 ± 3.1 ms, n = 10; p < 0.02). The membrane potential at threshold was not statistically different for HSALR (−52.7 ± 1.7 mV, n = 10) compared to WT fibers (−50.1 ± 0.8 mV, n = 10; p = 0.2). (B) Repetitive discharges are elicited in HSALR fibers by a current intensity of 2× threshold, whereas WT fibers discharge only once (data not shown). Molecular Cell 2002 10, 35-44DOI: (10.1016/S1097-2765(02)00563-4)

Figure 2 The Input Resistance Is Increased and the Chloride Conductance Is Markedly Reduced in HSALR Fibers (A) The subthreshold voltage transients for depolarizing current pulses (10 nA increments) applied from −80 mV are slower and larger for HSALR fibers. A locally reduced slope in the steady-state current-voltage relation (B) and increase in the membrane time constant, τm (C), reveal an abnormally high membrane resistance in HSALR fibers. In Cl−-free saline (open symbols), these same defects were observed in wild-type (WT) fibers, whereas the behavior of HSALR fibers was relatively unaltered, demonstrating a severe loss of GCl in HSALR fibers. The τm in HSALR fibers shows a larger relative change than the slope of the current-voltage relation (B) because τm is proportional to the specific membrane resistance, Rm, whereas the apparent input resistance (Rin = ΔV/ΔI) is proportional to the square root of Rm. The specific membrane resistance peaked at −55 mV where Rm was 4-fold higher for HSALR (4080 ± 590 Ω cm2, n = 11) than WT (1040 ± 100 Ω cm2, n = 8) fibers. Molecular Cell 2002 10, 35-44DOI: (10.1016/S1097-2765(02)00563-4)

Figure 3 Splicing of ClC-1 mRNA in HSALR Transgenic Mice (A) Northern blot of polyadenylated muscle RNA (1 ug) probed with a full-length murine ClC-1 cDNA reveals a single band (4.5 kb) in HSALR20b mice, of similar size but slightly reduced amount compared to wild-type (WT). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA serves as a loading control. (B) Novel ClC-1 exon 7a was generated from intron 6. The cross-hatch indicates regions that show high (>85%) mouse-human sequence conservation. (C) RT-PCR assay of ClC-1 exons 5 to 8 reveals four different products. Each band was sequenced to derive the splicing diagram shown in (D). Some exon 7a-inclusion isoforms have also included additional exon “8a” (band 3, generated out of intron 7) or part of intron 6 (band 4). The level of expanded CUG repeats in the RNA sample used for the splicing assay is shown by slot blot in the strip designated “(CUG)n”. tg/− indicates mice hemizygous for the indicated transgene, tg/tg indicates homozygotes. The fraction of exon 7a-inclusion isoforms (sum of bands 2, 3, and 4; expressed as a percentage of the sum for bands 1–4) in different transgenic lines was (mean ± SD of triplicates): WT, 6% ± 0.7%; HSASR, 5 ± 0.1%; neonatal WT, 29 ± 0.3%; hemizygous HSALR41, 7 ± 0.5%; homozygous HSALR41, 32% ± 2%; hemizygous HSALR20b, 21% ± 0.5%; homozygous HSALR20b, 44% ± 3%; and HSALR32a, 40% ± 0.6%. Molecular Cell 2002 10, 35-44DOI: (10.1016/S1097-2765(02)00563-4)

Figure 4 Alternative Splicing in Human and Murine ClC-1 cDNA Clones (B) Normal ClC-1 splice junctions are shown by the cDNA diagram in the center of each panel. Variant introns/exons are represented by rectangles above or below each cDNA diagram. The frequency of each variant exon/intron (i.e., the percentage of cDNAs that show a particular splice variant) is indicated by the height of the rectangle, relative to standards shown in the bar graph in (A). The position of added exons or intron inclusions (whole or partial) is shown by the rectangles above each cDNA diagram. The position of skipped exons (partial, single, or multiple) is shown by the rectangles below each cDNA diagram. The length of added or deleted cDNA sequence is indicated by the width of the rectangle. The shaded rectangle indicates exon 7a. The asterisk indicates an alternative splice acceptor site at exon 7, which adds a UAG triplet in frame to create a stop codon immediately following exon 6. Molecular Cell 2002 10, 35-44DOI: (10.1016/S1097-2765(02)00563-4)

Figure 5 ClC-1 Protein Is Reduced in HSALR Mice that Show Myotonia and in Human DM1 and DM2 A normal circumferential rim of ClC-1 is seen at the surface membrane in HSASR controls (A) and in hemizygous HSALR41 (non-myotonic) mice (C) that have low expression of expanded CUG repeats. (B) ClC-1 is absent in muscle fibers from ADR mice that have myotonia due to inactivating mutations in Clcn1. ClC-1 is absent or reduced in most fibers in homozygous HSALR41 (D) or HSALR20b (E) mice. (F) Immunofluorescence using a monoclonal antibody to the carboxyl terminus of dystrophin reveals a normal distribution of dystrophin in HSALR20b mice. In human muscle, the distribution of ClC-1 is normal in a patient with mitochondrial myopathy (G). Representative sections of quadriceps muscle from patients with DM1 (H) or DM2 (I) show that ClC-1 is markedly reduced. Molecular Cell 2002 10, 35-44DOI: (10.1016/S1097-2765(02)00563-4)