Verapamil mitigates chloride and calcium bi-channelopathy in a myotonic dystrophy mouse model

Abstract

Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function displayed markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl- bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl- bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.

Keywords: Calcium channels; Chloride channels; Muscle; Muscle Biology; Therapeutics.

Figure 1. Ca_V1.1^Δe29 and ClC-1^–/– alleles exhibit synthetic lethality and result in significantly reduced body weight and severe muscle weakness in mice.

(A) Kaplan-Meier survival analysis of WT (n = 10; female = 5, male = 5), Ca_V1.1^Δe29/+ (n = 13; female = 7, male = 6), Ca_V1.1^Δe29 (n = 15; female = 7, male = 8), RyR1^Δe70/Δe70 (n = 21; female = 10, male = 11), SERCA1^Δe22/Δe22 (n = 19; female = 10, male = 9), Ca_V1.1^Δe29 RyR1^Δe70/Δe70 (n = 11; female = 5, male = 6), Ca_V1.1^Δe29 SERCA1^Δe22/Δe22 (n = 10; female = 6, male = 4), RyR1^Δe70/Δe70 SERCA1^Δe22/Δe22 (n = 15; female = 4, male = 11), Ca_V1.1^Δe29 RyR1^Δe70/Δe70 SERCA1^Δe22/Δe22 (n = 19; female = 6, male = 13), ClC-1^–/– (n = 27; female = 12, male = 15), RyR1^Δe70/Δe70 ClC-1^–/– (n = 14; female = 7, male = 7), SERCA1^Δe22/Δe22 ClC-1^–/– (n = 27; female = 14, male = 13), Ca_V1.1^Δe29/+ ClC-1^–/– (n = 8; female = 3, male = 5), and Ca_V1.1^Δe29/Δe29 ClC-1^–/– (n = 11; female = 6, male = 5) mice. (B) Weekly body weight change and (C) percentage of body weight change from weaning at 10 weeks of age for WT (n = 20; female = 10, male = 10), Ca_V1.1^Δe29 (n = 21; female = 12, male = 9), RyR1^Δe70/Δe70 (n = 15; female = 5, male = 10), SERCA1^Δe22/Δe22 (n = 14; female = 7, male = 7), Ca_V1.1^Δe29 RyR1^Δe70/Δe70 (n = 11; female = 5, male = 6), Ca_V1.1^Δe29 SERCA1^Δe22/Δe22 (n = 10; female = 6, male = 4), RyR1^Δe70/Δe70 SERCA1^Δe22/Δe22 (n = 15; female = 4, male = 11), Ca_V1.1^Δe29 RyR1^Δe70/Δe70 SERCA1^Δe22/Δe22 (n = 19; female = 6, male = 13), ClC-1^–/– (n = 23; female = 13, male = 10), Ca_V1.1^Δe29 ClC-1^–/– (n = 17; female = 8, male = 9), RyR1^Δe70/Δe70 ClC-1^–/– (n = 11; female = 5, male = 6), and SERCA1^Δe22/Δe22 ClC-1^–/– (n = 12; female = 8, male = 4) mice. (D) Representative traces and (E) average peak specific force elicited by 150 Hz (500 ms) tetanic stimulation of isolated EDLs from mice at 10 weeks. (F) Average frequency dependence of specific force generation elicited from isolated EDLs at 10 weeks. (E and F) WT (n = 17; female = 8, male = 9), Ca_V1.1^Δe29 (n = 10; female = 5, male = 5), ClC-1^–/– (n = 9; female = 4, male = 5), Ca_V1.1^Δe29 ClC-1^–/– (n = 19; female = 8, male = 11) mice were used. (G) Weekly TRR analysis of Ca_V1.1^Δe29 ClC-1^–/– (red), ClC-1^–/– (blue), Ca_V1.1^Δe29 (orange), WT (black) mice. (H) Correlation of the TRR to death for Ca_V1.1^Δe29 ClC-1^–/– mice. Dots represent individual mice, and duplications indicate TRR trials. In B–H, results in red represent heterozygous and homozygous Ca_V1.1^Δe29 mice that are ClC-1^–/–, and results in orange indicate heterozygous and homozygous Ca_V1.1^Δe29 alleles. Symbols and open circles indicate individual mice; bars and closed circles indicate the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by log-rank analysis (A); 2-way ANOVA (B, F, and G) and 1-way ANOVA (C and E) with Tukey’s post hoc analysis; and linear regression analysis (H).

Figure 2. Heterozygous and homozygous Ca_V1.1^Δe29 mice exhibit similar Ca_V1.1 voltage dependence and peak current densities in FDB muscle.

(A) Representative current density traces from whole-cell patch clamp recordings of FDB fibers isolated from 4-week-old WT (black), Ca_V1.1^Δe29/+ (red, dashed), and Ca_V1.1^Δe29/Δe29 (red, solid) mice at 0 mV (top), +20 mV (middle), and +40 mV (bottom). (B) Plot of average current-voltage relationship of Ca_V1.1 activity measured in WT (black), Ca_V1.1^Δe29/+ (red and black circles, red dashed), and Ca_V1.1^Δe29/Δe29 (red circle, solid red line) FDB fibers isolated from 4-week-old mice. (C) RT-PCR products of Ca_V1.1 RNA isolated from tibialis anterior from 10-week-old mice. PCR amplifications are from exons 27–31 of Cacna1s cDNA. Ld, ladder. Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc analysis (B).

Figure 3. Ca_V1.1^Δe29 ClC-1^–/– and ClC-1^–/– muscle exhibits severe transient weakness that is significantly improved by the addition of verapamil.

(A and C) Normalized representative force traces of the first 15 tetani (100 Hz, 500 ms) separated by 4 seconds, recorded ex vivo from EDL muscles isolated from 6-week-old ClC-1^–/– (blue) and Ca_V1.1^Δe29 ClC-1^–/– (red) mice in the (A) absence and (C) presence of 20 μM verapamil added to the bath. (B and D) Average peak tetanic EDL forces normalized to the initial stimulus, elicited by 44 subsequent 100 Hz, 500 ms tetanic stimulations separated by 4 seconds. EDL muscles were from 6-week-old WT (black, n = 4), ClC-1^–/– (blue, n = 4), Ca_V1.1^Δe29 (orange, n = 4), and Ca_V1.1^Δe29 ClC-1^–/– red, n = 4) mice in the (B) absence and (D) presence of 20 μM verapamil added to the bath for ClC-1^–/– (blue, n = 4) and Ca_V1.1^Δe29 ClC-1^–/– (red, n = 4) EDLs. Dashed lines in D represent the average data presented in B as a reference for pretreatment. (B and D) WT (n = 4; female = 2, male = 2), Ca_V1.1^Δe29 (n = 4; female = 2, male = 2), ClC-1^–/– (n = 4; female = 2, male = 2), Ca_V1.1^Δe29 ClC-1^–/– (n = 4; female = 2, male = 2). Symbols and closed circles indicate the mean ± SEM. (B and D) Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc analysis.

Figure 4. Verapamil significantly reduces myotonia in both Ca_V1.1^Δe29 ClC-1^–/– and ClC-1^–/– mouse muscle.

(A and C) Representative normalized specific force traces of the first (left) and third (right) tetani (150 Hz, 500 ms) from EDL muscles isolated from 6-week-old WT (black) and Ca_V1.1^Δe29 (orange) mice in the (A) absence and (C) presence of 20 μM verapamil added to the bath. Dashed lines represent accumulated force. (B and D) Average integration of normalized specific force for WT (black) and Ca_V1.1^Δe29 (orange) EDL muscles across 3 tetanic stimulations (150 Hz, 500 ms) in the (B) absence and (D) presence of 20 μM verapamil added to the bath. (E and G) Representative normalized specific force traces of the first (left) and third (right) tetani (150 Hz, 500 ms) from EDL muscles isolated from 6-week-old ClC-1^–/– (blue) and Ca_V1.1^Δe29 ClC-1^–/– (red) mice in the absence (E) and presence (G) of 20 μM verapamil added to the bath. Dashed lines represent accumulated force. (F and H) Average integration of specific force for ClC-1^–/– (blue) and Ca_V1.1^Δe29 ClC-1^–/– (red) EDL muscles across 3 tetanic stimulations (150 Hz, 500 ms) in the (B) absence and (D) presence of 20 μM verapamil added to the bath. (B, D, F, and H) WT (n = 4; female = 2, male = 2), Ca_V1.1^Δe29 (n = 4; female = 2, male = 2), ClC-1^–/– (n = 4; female = 2, male = 2), Ca_V1.1^Δe29 ClC-1^–/– (n = 4; female = 2, male = 2). All specific force traces were normalized to the peak specific force. Symbols and open circles represent individual mice; bars indicate the mean ± SEM. Notes: Contralateral EDL muscles were used for each untreated and treated experiment. All traces were plotted with the same scale of time and normalized force for comparison. The same traces without force normalization are shown in Supplemental Figure 8. (B, D, F, and H) **P < 0.01 and ****P < 0.0001, by 1-way ANOVA with Tukey’s post hoc analysis.

Figure 5. Verapamil treatment improves survival, body weight, and motility of Ca_V1.1^Δe29 ClC-1^–/– mice.

(A) Kaplan-Meier survival analysis of WT mice (n = 10; female = 5, male = 5), WT mice treated with 200 mg/kg/d verapamil (verap) (n = 10; female = 5, male = 5), Ca_V1.1^Δe29 ClC-1^–/– mice (n = 19; female = 9, male = 10), and Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 9; female = 5, male = 4). Verapamil was dosed in mouse nutrition/hydration food cups. Note: log-rank analysis of Ca_V1.1^Δe29 ClC-1^–/– versus WT mice, WT mice treated with 200 mg/kg/d verapamil, and Ca_V1.1^Δe29 ClC-1^–/– mice treated with 200 mg/kg/d verapamil; P < 0.0001, P < 0.0001, and P < 0.0001, respectively. (B) Percentage of body weight change from weaning at 10 weeks in WT mice (n = 20; female = 10, male = 10), WT mice treated with 200 mg/kg/d verapamil (n = 10; female = 5, male = 5), Ca_V1.1^Δe29 ClC-1^–/– mice (n = 35; female = 14, male = 21), and Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 9; female = 5, male = 4). (C) Weekly time of righting reflex analysis of Ca_V1.1^Δe29 ClC-1^–/– mice (red), ClC-1^–/– mice (light blue), Ca_V1.1^Δe29 mice (orange), WT mice (black), Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (green), and WT mice treated with verapamil (gray). (D) Average time of righting reflex in vehicle- and verapamil-treated mice at 10 and 20 weeks of age. Note: Untreated Ca_V1.1^Δe29 ClC-1^–/– mice did not survive to 20 weeks of age, therefore the last recording before death was documented. Box indicates Q1, the median, and Q3; whiskers show the minimum and maximum. (C and D) Ten- and 20-week-old WT mice (n = 10; female = 5, male = 5), WT mice treated with 200 mg/kg/d verapamil (n = 10; female = 5, male = 5), Ca_V1.1^Δe29 mice (n = 8; female = 4, male = 4), ClC-1^–/– mice (n = 19; female = 9, male = 10), Ca_V1.1^Δe29 ClC-1^–/– mice (n = 15; female = 7, male = 8), Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (10-week-old mice, n = 16; female = 7, male = 9), Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (20-week-old mice, n = 7; female = 4, male = 3). (E) Paired before (light circles) and after (dark circles) 2 weeks of verapamil treatment of ClC-1^–/– mice at 100 mg/kg/d (left; n = 5; female = 3, male = 2) and 200 mg/kg/d (right; n = 6; female = 3, male = 3) dosing in nutrition/hydration food cups. Symbols and closed circles indicate individual mice; open circles indicate the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by log-rank analysis (A); 1-way ANOVA (B and D) and 2-way ANOVA (C) with Tukey’s post hoc analysis; and paired t test (E).

Figure 6. Verapamil treatment significantly improves respiratory function and diaphragm strength in Ca_V1.1^Δe29 ClC-1^–/– mice.

WBP for (A, C, and E) 10-week-old WT mice (n = 18; female = 9, male = 9), WT mice treated with 200 mg/kg/d verapamil (n = 10; female = 5, male = 5), Ca_V1.1^Δe29 mice (n = 6; female = 3, male = 3), ClC-1^–/– mice (n = 17; female = 8, male = 9), Ca_V1.1^Δe29 ClC-1^–/– mice (n = 14; female = 7, male = 7), and Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 14, female = 7, male = 7) and (B, D and F) 20-week-old WT mice (n = 10; female = 5, male = 5), WT mice treated with 200 mg/kg/d verapamil (n = 10; female = 5, male = 5), Ca_V1.1^Δe29 mice (n = 10; female = 4, male = 6), ClC-1^–/– mice (n = 8; female = 4, male = 4), Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 7; female = 4, male = 3). (A and B) PIFR (mL/s) and (C and D) PEFR (mL/s) of respiration. (E and F) Tidal volume (mL) of respiration. (G) Representative tetanic (150 Hz, 500 ms) specific force traces from diaphragm strips isolated from 10-week-old (left) and 20-week-old (right) mice of the indicated genotypes and treatment groups. (H and I) Plot of the average stimulation frequency dependence of specific force generated from diaphragm strips isolated from (H) 10-week-old (n values indicate individual EDL muscles) WT mice (n = 10; female = 5, male = 5), WT mice treated with 200 mg/kg/d verapamil (n = 6; female = 3, male = 3), Ca_V1.1^Δe29 mice (n = 5; female = 3, male = 2), ClC-1^–/– mice (n = 5; female = 2, male = 3), Ca_V1.1^Δe29 ClC-1^–/– mice (n = 7; female = 4, male = 3), and Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 7; female = 3, male = 4)) and (I) 20-week-old (n values indicate individual EDL muscles) WT mice (n = 8; female = 4, male = 4), WT mice treated with 200 mg/kg/d verapamil (n = 8; female = 4, male = 4), Ca_V1.1^Δe29 mice (n = 7; female = 4, male = 3), ClC-1^–/– mice (n = 7; female = 3, male = 4), and Ca_V1.1^Δe29 ClC-1^–/– mice treated with verapamil (n = 7; female = 4, male = 3). Symbols and open circles represent individual mice; bars and closed circles indicate the mean ± SEM. Note: Untreated Ca_V1.1^Δe29 ClC-1^–/– mice did not survive to 20 weeks of age. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA (A–F) and 2-way ANOVA (H and I) with Tukey’s post hoc analysis.