Correlation of Ca_V1.1 E29 skipping and muscle strength in DM. (A) Schematic representation of Ca_V1.1 topology and exon organization. Ca_V1.1 is composed of four membrane-spanning repeat domains (I–IV). Each repeat consists of six transmembrane segments (S1–S6). The protein segment encoded by the alternatively spliced E29 (57 nts, highlighted in red) is located in the IVS3–IVS4 extracellular loop. Positive charges in the S4 segments of each repeat are indicated by ‘+’ symbols. Locations of the α interacting domain in the I–II loop, which binds the β_1α subunit, and the EC coupling domain in the II–III intracellular loop, are also indicated. (B) RT–PCR assay for alternative splicing of Ca_V1.1 E29 (Ca_V1.1-E29, left) and SERCA1 exon 22 (SERCA1-E22, right) in muscle from normal (lanes 1 and 2), DM1 (lanes 3 and 4), DM2 (lanes 5 and 6) and FSHD (lanes 7 and 8) individuals. Inc% denotes the fractional inclusion rate, calculated as the signal intensity of inclusion (upper) band divided by the summed intensities of the corresponding inclusion and exclusion (lower) bands. Negative control (-ve) refers to the absence of template. (C) Upper panel shows representative RT–PCR E29 inclusion results for several normal (lanes 1–5) and DM1-affected (lanes 6–28) individuals. Negative control (-ve) refers to the absence of template. Lower panel shows correlation of ADF strength with fractional E29 inclusion in TA muscles from healthy individuals (n = 5, ‘NL’), DM1 protomutation (n = 5, ‘Proto’) and classical DM1 (n = 41). Strength was determined by standardized manual muscle testing using Medical Research Council scales (46). An MRC scale value of 5 indicates normal strength, 4 indicates moderate weakness, 3 indicates full active range of movement against gravity with no added resistance and 2 indicates inability to dorsiflex fully against gravity. The Spearman rank correlation for E29 inclusion versus strength in classical DM1 was r = 0.6560 (P < 0.0001, excludes normal and protomutation subjects).

Analysis of Ca_V1.1 E29 inclusion in mouse hindlimb muscle during late fetal and early postnatal development. (A) The inclusion level of Ca_V1.1 E29 and (B) Serca1 exon 22 (Serca1-E22) is shown from E16 to P30. Hindlimb muscles from four mice were pooled for RNA extraction at embryonic stages and from two mice for postnatal stages.

Mbnl1 knockdown enhances Ca_V1.1 E29 skipping in C2C12 mouse myoblasts. (A) Immunoblot for Mbnl1 in C2C12 myoblasts treated with mock transfection (lane 1), si-RNA targeting Mbnl1 (lanes 2 and 3) and scrambled si-RNA control (si-ctrl). Efficient knockdown was achieved with si-Mbnl1-No.5 but not si-Mbnl1-No.8. RT–PCR assay for alternative splicing of Ca_V1.1-E29 (B), Cypher exon 11 (C) and Serca1-E22 (D) (same cells/lanes as labeled in A and C). Quantification of Ca_V1.1-E29 inclusion for each condition is shown in the lower panel of (B). Mbnl1-knockdown significantly reduced Ca_V1.1-E29 inclusion (**P < 0.01).

Ca_V1.1 E29 is only weakly skipped in skeletal muscle of DM1 mouse models, but more strongly repressed in hearts. (A) RT–PCR assay of Ca_V1.1-E29 (upper) and Serca1-E22 (lower) splicing in quadriceps muscle of FVBn WT, HSA^LR transgenic and Mbnl1-knockout (Mbnl1^ΔE3/ΔE3) mice. (B) RT–PCR assays of Ca_V1.1-E29 (upper), Serca1-E22 (middle) and Ca_V1.2 exon 33 (lower) splicing in cardiac ventricular muscle of adult (6 months) male WT, Mbnl1-heterozygote (Mbnl1^+/ΔE3) and Mbnl1-knockout (Mbnl1^ΔE3/ΔE3) littermates. Note that strong amplification products were observed for Ca_V1.2-E33 after 25 cycles of RT–PCR, when compared with weaker products for Ca_V1.1-E29 after 30 cycles, as expected because Ca_V1.2 is the predominant L-type Ca²⁺ channel expressed in cardiomyocytes.

Overexpression of CUGBP1 represses Ca_V1.1-E29 in mouse TA muscles. (A) Immunoblot for the CUGBP1 protein in mouse TA 9 days after intramuscular injection and electroporation of CUGBP1 expression construct (lanes 1 and 3) or empty vector control (lanes 2 and 4). GAPDH was used as a loading control. (B) RT–PCR assay of Ca_V1.1 E29 (upper), Serca1-E22 (middle) and Capzb—exon 8 (lower) splicing in mouse TA muscle 9 days after injection/electroporation of CUGBP1 expression construct (lanes 1 and 3) or empty vector control (lanes 2 and 4).

Ca_V1.1-E29 skipping enhances L-type Ca²⁺ channel conductance and voltage sensitivity in mouse FDB muscle fibers. (A) Diagram of splice shifting antisense morpholino oligonucleotides used to induce Ca_V1.1 E29 skipping. The ▵E29 morpholinos target sequences near the 3′ and 5′ splice sites of Ca_V1.1 E29. (B) RT–PCR assay for Ca_V1.1-E29 (upper) and Serca1-E22 (lower) splicing 16 and 18 days following a single injection and electroporation of ▵E29 morpholinos into FDB muscle. ‘C’ denotes control FDB muscle that was injected and electroporated with vehicle (saline) alone. RNA for the assay was recovered from FDB fibers remaining after completion of patch-clamp recordings. (C) Representative calcium current (I_Ca) recordings in control (Ctrl, black) and Ca_V1.1-E29 skipping (ΔE29, grey) fibers obtained during 200 ms depolarizations to −30, −10, +10 and +30 mV. (D) Average (±SE) I_Ca–V relationships for control (black circles) and ΔE29-deleted fibers (grey circles) fitted by equation (1) with the following parameters for control: G_max = 108 nS/nF, V_0.5 = 1.1 mV, k_g = 6.9 mV, V_rev = 62.2 mV and for ▵E29: G_max = 135 nS/nF, V_0.5 = −21.3 mV, k_g = 3.5 mV, V_rev = 59.7 mV. Data were obtained from n = 14 fibers for each group. *P < 0.05, **P < 0.01 and ***P < 0.001. (E) The voltage dependence of fractional inactivation, represented as I_end/I_peak, plotted against test depolarization voltage. All symbols have the same meaning as in (D). Data are presented as mean ± SEM, 14 fibers were recorded for both control and ▵E29 morpholino-treated fibers.

Ca_V1.1-E29 skipping increases electrically evoked Ca²⁺ transients in FDB fibers. (A) Representative normalized mag-fluo-4 fluorescence transients (ΔF/F₀) elicited during a 1 Hz train of electrical stimulation in mouse FDB fibers treated with either control morpholino (left, control-Mo) or ▵E29 morpholinos (right, ▵E29-Mo). Following measurement of Ca²⁺ transients, remaining fibers were collected for mRNA extraction to quantify the amount of Ca_V1.1-E29 skipping by RT–PCR (Supplementary Material, Fig. S2, mice 7–10, and data not shown). (B) Bar graph summarizing peak electrically evoked mag-fluo-4 transients (ΔF/F₀) in mouse FDB fibers treated with either control (n = 16 fibers) or ▵E29 morpholinos (n = 18 fibers). Mag-fluo-4 transients were recorded both before (left) and after (right) addition of 0.5 mm Cd²⁺ and 0.2 mm La³⁺ for 2 min. *P < 0.05, t-test. No significant difference was found between the two groups in the presence of 0.5 mm Cd²⁺ and 0.2 mm La³⁺ (P > 0.3, t-test). FDB fibers were obtained from five mice each treated with either ▵E29 or control morpholinos. (C) Cd²⁺/La³⁺-sensitive component of electrically evoked mag-fluo-4 transients (ΔF/F₀) in mouse FDB fibers treated with either control (n = 16 fibers) or ▵E29 morpholinos (n = 18 fibers). Addition of 0.5 mm Cd²⁺ and 0.2 mm La³⁺ produced a significantly greater reduction in the peak electrically evoked mag-fluo-4 transient in FDB fibers treated with ▵E29 morpholinos (*P< 0.05, t-test).

Ca_V1.1-E29 skipping in TA of HSA^LR mice increases the percentage of fibers with central nuclei. (A) RT–PCR analysis of Ca_V1.1-E29 (upper) and Serca1-E22 (lower) splicing in TA muscles of HSA^LR mice, 3 months following a single injection and electroporation of ΔE29 (T) or control (C) morpholinos. The left TA (LTA) or right TA (RTA) muscles of the same mouse were randomly selected for the treatment with either control or ΔE29 morpholinos. (B) Immunoblot of the Ca_V1.1 protein in TA muscles treated with ΔE29 or control morpholinos for the same 10 samples shown in (A). The Ca_V1.1-specific antibody detects a band around 200 kDa (arrow); * indicates non-specific cross-reactive bands. M, molecular weight marker. (C) Representative images of hematoxylin- and eosin-stained sections showing an increased fraction of fibers with central nuclei in TA muscles treated with ΔE29-morpholino (lower) compared with control morpholino (upper). (D) Quantification of the percentage of fibers with central nuclei in sections of contralateral TA muscles treated with control- (left) or ΔE29-skipping morpholinos (right) (P < 0.05, paired t-test).