The Ca2+ influx through the mammalian skeletal muscle dihydropyridine receptor is irrelevant for muscle performance

Fig. 1. N617D mutation completely abolishes DHPR Ca2+ influx without altering EC coupling. a, b Representative whole cell Ca2+ current recordings on myotubes isolated from 3 to 4 days old (a) wt and (b) ncDHPR pups, indicating the complete loss of inward Ca2+ current in ncDHPR mice in contrast (P < 0.001) to wt control mice. Scale bars, 50 ms (horizontal), 2 pA pF−1 (vertical). c Current–voltage relationships for DHPR-mediated Ca2+ currents recorded from ncDHPR (n = 13) and wt (n = 7) myotubes. d, e Representative intracellular SR Ca2+ release recordings from (d) wt and (e) ncDHPR myotubes. Scale bars, 50 ms (horizontal), ∆F/F 0 = 2 (vertical). f In contrast to DHPR Ca2+ influx, voltage dependence of maximal Ca2+ release is indistinguishable (P > 0.05) between ncDHPR ((ΔF/F 0)max = 3.75 ± 0.39; n = 12) and wt ((∆F/F 0)max = 3.39 ± 0.26; n = 8) myotubes, implying unaltered EC coupling in the ncDHPR mouse. Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 2. Unaltered voltage dependence of SR Ca2+ release in ncDHPR adult muscle fibres. a Representative traces of Ca2+ release flux signals determined from isolated adult toe fibres (musculus interosseus) at 100-ms depolarizing voltage steps. The flux exhibits an early peak and a rapid decline to a lower level (plateau). Scale bars, 200 ms (horizontal), 50 mM s−1 (vertical). b Voltage dependence of normalized peak Ca2+ release flux is indistinguishable (P > 0.05) between ncDHPR (n = 12) and wt (n = 10) adult muscle fibres. c Ca2+ release flux traces were converted to permeability with the assumption that the slow decline during the plateau phase results from SR depletion60. The calculated peak Ca2+ release permeability as a function of voltage showed no significant difference (P > 0.05) between ncDHPR and wt fibres. Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 3. Absence of DHPR-mediated Ca2+ influx has no impact on SR Ca2+ store content. a Representative trace of SR Ca2+ release in intact interosseous muscle fibres triggered by application (green arrow) of 500 µM of RyR agonist 4-CmC in the presence of 30 µM of SERCA blocker CPA. The resulting Ca2+ signals were measured with the low affinity indicator Fura-FF-AM to avoid dye saturation during Ca2+ release stimulation. b Comparison of 4-CmC-induced Ca2+ signals, measured as peak fluorescence ratio of Fura-FF (340/380) showed no significant difference (P > 0.05) between ncDHPR (1.0 ± 0.02; n = 53) and wt muscle fibres (1.04 ± 0.02; n = 48), indicating identical SR Ca2+ store filling. Bars represent mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 4. Lack of DHPR Ca2+ influx has no impact on body weight development and fertility. Growth curve of (a) male and (b) female ncDHPR and wt mice was monitored weekly for 150 days. a Male ncDHPR (n = 30) and wt (n = 35) siblings showed identical (P > 0.05) body weight development. b Similarly, female ncDHPR (n = 29) showed no difference (P > 0.05) in body weight development compared with their wt control siblings (n = 31). At the end of the observation period, the maximum body weight of male ncDHPR and wt mice was 29.76 ± 0.55 and 30.74 ± 0.54 g, respectively, and of female ncDHPR and wt mice was 28.96 ± 0.73 and 28.98 ± 0.60 g, respectively. b (Inset) The mean litter size of ncDHPR mice (7.64 ± 0.21; n = 100) was similar (P > 0.05) to wt mice (7.31 ± 0.37; n = 49). Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 5. Voluntary locomotor activity is identical in ncDHPR and wt mice. a Home cage ambulatory activity was measured for 2 days and 3 nights with an infrared video monitoring system. Young (3–6 months old) ncDHPR mice (n = 10) displayed high nighttime activity (41,520 ± 3,934 a.u.) and low daytime activity (5,794 ± 790 a.u) similar (P > 0.05) to the control wt mice (37,620 ± 1,079 a.u. and 6,247 ± 1,152 a.u., respectively; n = 6). b Average cumulative distance covered during 21 days of voluntary activity wheel task (binned every 24 h) was indistinguishable (P > 0.05) between young ncDHPR (n = 18) and wt (n = 17) mice. Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 6. Absence of DHPR Ca2+ influx has no impact on ex vivo muscle force and fatigue. Soleus (SOL) and extensor digitorum longus (EDL) muscles isolated from young and aged ncDHPR and wt control mice were subjected to (a, b) force-frequency and c repetitive tetanic fatigue tests (see also Supplementary Fig. 7). a The average maximum twitch force (at supramaximal voltage, 25 V for 1 ms) is identical (P > 0.05) in SOL and EDL muscles between young (left graph) ncDHPR (SOL: 16.78 ± 1.80 mN; n = 12; EDL: 55.33 ± 3.31 mN; n = 19) and wt (SOL: 17.39 ± 1.49 mN: n = 9; EDL: 55.14 ± 3.39 mN: n = 18) mice, as well as between aged (right graph) ncDHPR (SOL: 11.66 ± 0.91 mN; n = 19 and EDL: 9.76 ± 1.04 mN; n = 20) and wt (SOL: 13.33 ± 1.35 mN; n = 18 and EDL: 10.09 ± 1.06 mN; n = 22) mice. b Frequency dependence of average tetanic force in SOL and EDL muscles is also unaltered (P > 0.05) between age-matched ncDHPR and wt mice (left and right graphs). c Under high-frequency repetitive stimulations, the number of tetani till the tetanus amplitude decreased to 80% (T80%) of the initial force in case of SOL and 50% (T50%) for EDL was taken as an index of fatigue. No difference in susceptibility to…

Fig. 7. DHPR Ca2+ influx has no impact on whole animal muscle coordination and strength. a ncDHPR mice performed equally well similar to control littermates in wire hang test set for 600 s. The mean latency to fall from the cross-wired grid was similar between young ncDHR (453.85 ± 39.97 s; n = 26) and wt (453.04 ± 38.06 s; n = 25) mice, as well as between aged ncDHPR (153.12 ± 24.82 s; n = 25) and wt (100.44 ± 15.06 s; n = 27) mice. b The mean grip strength, an index of maximal muscle force of forelimbs, was not different in young (185.68 ± 4.71 g; n = 15) and aged (136.39 ± 3.59 g; n = 23) ncDHPR mice compared with their age-matched wt littermates (young wt: 181.38 ± 3.88 g; n = 6; aged wt: 142.29 ± 4.34 g; n = 27 mice). Normal age-dependent deterioration in muscle strength is evident in both genotypes. Bars represent mean ± s.e.m.; P determined by unpaired Student’s t-test

Fig. 8. DHPR Ca2+ influx has no impact on whole animal muscle endurance and fatigue. a In Rotarod performance test, the mean latency to fall from a rotating rod under steady acceleration (4–40 rpm for 300 s) was indistinguishable (P > 0.05) when young (188.78 ± 13.95 s; n = 16) and aged ncDHPR (133.36 ± 6.73 s; n = 26) mice were compared with their wt (174.50 ± 15.02 s; n = 16 and 132.85 ± 10.19 s; n = 28, respectively) counter mates. b The mean latency to fall during 600-s Rotarod endurance task at constant speed, 20 rpm or 15 rpm in young or aged mice, respectively, was indifferent (P > 0.05) between age-matched ncDHPR (young: 351.54 ± 58.64 s; n = 16 and aged: 312.22 ± 50.76 s; n = 25) and wt (young: 263.43 ± 64.66 s; n = 16 and aged: 220.67 ± 47.32 s; n = 27) mice. The number of mice that remained on the rotating rod for the entire duration of the task was similar for both genotypes (5 mice each for the young group and 10 aged ncDHPR versus 6 aged wt mice). c Mean cumulative resting time during an intense treadmill run (0.9 km in 1 h) in young ncDHPR (30.45 ± 8.75 s; n = 17) was similar (P > 0.05) to wt control (50.15 ± 12.36 s; n = 17) mice, indicating unaltered susceptibility to…

Fig. 9. ncDHPR mice do not show compensatory regulation of key triadic EC coupling proteins. a Schematic representation of the skeletal muscle triad with T-tubular invagination (T-tubule) of the sarcolemma adjacent to the sarcoplasmic reticulum Ca2+ store (SR). TaqMan qPCR assay (comparative C T method) on skeletal muscle from neonatal mice revealed that none of the investigated key EC coupling and Ca2+ handling proteins is transcriptionally up- or down-regulated (P > 0.05) in ncDHPR mice (n = 18) compared to wt controls (n = 18). All PCRs were performed in triplicates on 18 first strand replicates from 9 pups. For each gene of interest, the expression level was normalized to the expression of the reference gene EEF1A2 (Eukaryotic translation elongation factor 1 alpha 2)66. Comparable results were obtained from SOL and EDL muscles of adult mice with EEF1A2 as the reference gene (Supplementary Fig. 10). Abbreviations: CSQ, calsequestrin; DHPRα1S, dihydropyridine receptor α1S subunit; NCX, Na+–Ca2+ exchanger; Orai, Ca2+ release-activated Ca2+ channel; PMCA, plasma membrane Ca2+-ATPase; RyR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; STIM, stromal interaction…