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

Abstract

Skeletal muscle excitation-contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca²⁺ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and L-type Ca²⁺ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca²⁺ channel is still enigmatic. Here we show that Ca²⁺ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca²⁺ influx is eliminated. Homozygous knock-in mice display SR Ca²⁺ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca²⁺ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca²⁺ influx in mammalian skeletal muscle is an evolutionary remnant.In mammalian skeletal muscle, the DHPR functions as a voltage sensor to trigger muscle contraction and as a Ca²⁺ channel. Here the authors show that mice where Ca²⁺ influx through the DHPR is eliminated display no difference in skeletal muscle function, suggesting that the Ca²⁺ influx through this channel is vestigial.

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 depletion. 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 fatigue was observed in SOL and EDL muscles isolated from either young (left graph) ncDHPR (SOL: T80% = 50.12 ± 14.65 n; n = 12; EDL: T50% = 25.95 ± 1.04 n; n = 19) and wt (SOL: T80% = 48.0 ± 16.66 n; n = 9; EDL: T50% = 25.33 ± 1.05 n; n = 18) mice or aged (right graph) ncDHPR (SOL: T80% = 27.22 ± 4.89 n; n = 18; EDL: T50% = 18.12 ± 1.14 n; n = 17) and wt (SOL: T80% = 25.5 ± 3.31 n; n = 18; EDL: T50% = 21.42 ± 2.61 n; n = 19) mice. All recordings were performed at room temperature (~26 °C). Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

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 muscle fatigue and endurance. The cumulative number of rests at the end of the task was also indistinguishable (P > 0.05) between both genotypes (139.94 ± 37.97 for ncDHPR and 232.18 ± 52.89 for wt mice). d Mean cumulative resting time during 1-h treadmill run (0.55 km) in aged ncDHPR mice (3.54 ± 0.34 s; n = 23) was analogous to the wt (5.04 ± 0.85 s; n = 27) siblings. Cumulative number of rests at the end of the task was again comparable (P > 0.05) between aged ncDHPR and wt mice (19.65 ± 2.27 s and 24.52 ± 3.82 s, respectively). Data are represented as mean ± s.e.m.; P determined by unpaired Student’s t-test

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). Comparable results were obtained from SOL and EDL muscles of adult mice with EEF1A2 as the reference gene (Supplementary Fig. 10). Abbreviations: CSQ, calsequestrin; DHPRα_1S, dihydropyridine receptor α_1S 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 molecule; TRPC, transient receptor potential cation channel. b Quantitative western blot analysis revealed no compensatory translational regulation (P > 0.05) of crucial proteins involved in EC coupling and Ca²⁺ handling in the ncDHPR mouse (n = 3) compared with wt controls (n = 3). Band densities, quantified by densitometry, were standardized to GAPDH and normalized to the wt control sample on each gel. The corresponding western blots are shown in Supplementary Fig. 11 and in toto in Supplementary Fig. 12. Bars represent mean ± s.e.m. fold-change relative to wt; P determined by unpaired Student’s t-test