Skeletal muscle delimited myopathy and verapamil toxicity in SUR2 mutant mouse models of AIMS

Figures

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  Figure 1. Exercise intolerance in SUR2‐STOP mice ATwo SUR2‐STOP lines were generated with CRISPR‐Cas9‐mediated indel mutations in ABCC9. SUR2‐STOP1149 mice (as previously described (Smeland et al, 2019)) and SUR2‐STOP475 in which a 59‐base deletion in exon 9 causes a frameshift and premature stop codon at amino acid position 475 (see Materials and Methods).B, CPerformance in the multitrail inverted screen test is impaired in (B) SUR2‐STOP1149 (WT n = 9 [3 male; 6 female]; STOP n = 9 [3 male; 6 female]; P = 0.011 according to Mann–Whitney U test) and (C) SUR2‐STOP475 mice (WT n = 9 [4 male; 5 female]; STOP n = 15 [5 male; 10 female]; P = 2.9 × 10−4 according to Mann–Whitney U test).DTreadmill exercise tolerance is also reduced in SUR2‐STOP475 mice (WT n = 4 [2 male; 2 female]; STOP n = 8 [4 male; 4 female]; P = 0.008 according to Mann–Whitney U test). Treadmill workload tolerated (see Materials and Methods) was also reduced (WT 18.1 ± 2.2 vs. STOP475 9.2 ± 0.9 J; P = 0.008 from Man Whitney U test), mean mouse body weight was WT 22.1 ± 0.9 vs. STOP475 21.9 ± 0.6 g. Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes P‐value < α =…

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  Figure 2. Effect of KATP LoF in skeletal muscle AExample traces of inside‐out patch clamp recordings of acutely isolated FDB myofibers of WT (black, top left), SUR2‐STOP475 (purple, top right), and skeletal muscle dominant‐negative (SkM‐DN; magenta, bottom left) mice. Patches were voltage clamped at −50 mV and excised at time points indicated by arrows. ATP (5 mM) was administered to the cytoplasmic face of membrane patches as indicated by horizontal bars. Dashed lines denote the zero KATP current level.BMaximal ATP‐sensitive current levels from excised patches. Bars show mean (± SEM) with individual recordings from each myofiber shown as dots/squares (cells from ≥ 3 mice; WT n = 10 recordings; STOP n = 8; SkM‐DN n = 11; Dunn's test following Kruskal–Wallis omnibus test P = 1.0 × 10−4 for WT vs. STOP and 0.014 for WT vs. SkM‐DN; * denotes P‐value < adjusted α = 0.0167).C Left—Performance of WT and SkM‐DN mice in the multi‐trial inverted screen test. Right—Cumulative performance across six trials for WT (black circles), single‐transgenic mice carrying either the Kir6.1‐AAA transgene alone (AAA+; light gray diamonds) or the Myf5‐Cre transgene alone (Myf5‐cre; dark gray triangles), and…

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  Figure 3. Histopathology due to skeletal muscle KATP LoF A Left—Example of HE‐stained tibialis anterior (TA) muscles from WT and SUR2‐STOP475 mice. Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers. Right—Quantification of centrally nucleated fibers. Bars show mean (± SEM) with individual biological replicates shown as dots/squares (WT n = 5; STOP n = 4; P = 0.016 according to Mann–Whitney U test).B Left—Example of HE‐stained TA muscles from control and SkM‐DN mice. Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers. Right—Quantification of centrally nucleated fibers. Bars show mean (± SEM) with individual biological replicates shown as dots/squares (Control n = 7 [comprising 2 × WT, 3 × Myf5+‐cre, and 2 × AAA+ littermate controls]; SkM‐DN n = 5; P = 0.002 according to Mann–Whitney U test).CExample of HE‐stained WT TA muscle (left) and two examples of HE‐stained SkM‐DN TA muscle alongside immunolabeling showing the presence of CD68+/CD206+ (macrophage marker) cells indicated by green arrows.D Left—Picrosirius red stained TA muscle from WT and SkM‐DN mice. Right—Quantification of red stained area (WT n =…

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  Figure 4. Effect of SUR2 LoF on isolated skeletal muscle contractility AExample traces showing force generation in isometric contractility studies of isolated EDL muscle from WT (left, black) and SUR2‐STOP475 (right, purple) mice. Muscle was stimulated every 2 s. Scale bars show 20 mN (y‐axis) and 2 s (x‐axis).BEnlarged representations of the 1st (at 0 s), 10th (20 s) and 30th (60 s) contractions, identified in concatenated traces in (A) by arrows. Scale bars show 20 mN (y‐axis) and 0.4 s (x‐axis).CAverage unstimulated force (taken prior to each stimulation) for WT and SUR2‐STOP475 myofibers across 60 stimulations. Unstimulated force was normalized to the peak tetanic force generated by the first stimulation. Error bars show SEM (n = 6 for both groups).DSummary of unstimulated force (normalized by physiological cross‐sectional area) at 60 s for each experiment. Bars show mean ± SEM. (WT n = 6 [3 male; 3 female]; STOP n = 6 [3 male; 3 female]; P = 2.5 × 10−4 according to Mann–Whitney U test). * denotes P‐value < α = 0.05.EAverage peak stimulated tetanic force for WT and SUR2‐STOP475 myofibers (n = 6 for both groups). Data shown as mean (± SEM). Source data are available online for this…

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  Figure 5. Effects of verapamil administration in KATP‐deficient mice ASurvival of WT and SUR2‐STOP475 mice administered with verapamil in drinking water (n = 7 [4 male, 3 female] for WT administered 0.9 g/l verapamil; n = 14 [8 male, 6 female] for SUR2‐STOP475 administered 0.9 g/l verapamil; n = 6 [3 male, 3 female] for SUR2‐STOP475 administered 0.3 g/l verapamil). Verapamil‐induced deaths were observed in both male and female mice (7 of 8 male and 4 of 6 female SUR2‐STOP475 mice died within 28 days of 0.9 g/l verapamil).BSurvival of WT and SUR2‐STOP1149 mice administered with verapamil in drinking water (n = 10 [3 male, 7 female] for SUR2‐STOP1149 mice administered 0.9 g/l verapamil).CFootpad ECG recordings from WT and SUR2‐STOP475 mice either without or with 0.9 g/l verapamil administration.DHeart rates calculated from ECG recordings from WT and SUR2‐STOP mice administered with no verapamil (0 Verap) or 0.9 g/l verapamil in drinking water.EVariance in interval between R spikes in QRS complex from footpad ECG recordings.FCumulative durations of inversion in multitrial inverted screen test for WT mice (black circles) and SkM‐DN mice without (magenta squares) or with 0.9 g/l verapamil (pink…

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  Figure 6. Ca2+ influx through CaV1.1 does not affect AIMS‐related pathology A Left—Average performance in the inverted screen test of mice homozygous for the ncDHPR allele alone (ncDHPR) or homozygous for both the ncDHPR and SUR2‐STOP475 alleles (STOP475/ncDHPR). Right—Summary of inverted screen test data. WT and SUR‐STOP475 data from experiments shown in Fig 1C. Data shown as mean ± SEM (WT n = 9 [4 male; 5 female]; ncDHPR n = 15 [male 6; female 9]; STOP475 n = 15 [5 male; 10 female]; STOP475/ncDHPR n = 12 [4 male; 12 female]; Dunn's test P = 4.0 × 10−4 for WT vs. STOP475, 6.9 × 10−4 for WT vs. STOP475/ncDHPR, 0.001 for ncDHPR vs. STOP475/ncDHPR verapamil, following Kruskal–Wallis omnibus test). * denotes P‐value < adjusted α = 0.0083.B Left—Example of HE‐stained tibialis anterior muscle from STOP475/ncDHPR mouse, black arrow shows peripheral nuclei and yellow arrows show central nuclei. Right—Quantification of centrally nucleated fibers, data from WT and SUR2‐STOP475 mice same as shown in Fig 3A (WT n = 5; STOP475 n = 4; STOP475/ncDHPR n = 8; Dunn's test P = 0.007 for WT vs. STOP475 and 0.011 for WT vs. STOP475/ncDHPR, following Kruskal–Wallis test, * denotes P‐value < adjusted α =…