Orai1 enhances muscle endurance by promoting fatigue-resistant type I fiber content but not through acute store-operated Ca2+ entry

Abstract

Orai1 is a transmembrane protein that forms homomeric, calcium-selective channels activated by stromal interaction molecule 1 (STIM1) after depletion of intracellular calcium stores. In adult skeletal muscle, depletion of sarcoplasmic reticulum calcium activates STIM1/Orai1-dependent store-operated calcium entry. Here, we used constitutive and inducible muscle-specific Orai1-knockout (KO) mice to determine the acute and long-term developmental effects of Orai1 ablation on muscle structure and function. Skeletal muscles from constitutive, muscle-specific Orai-KO mice exhibited normal postnatal growth and fiber type differentiation. However, a significant reduction in fiber cross-sectional area occurred by 3 mo of age, with the most profound reduction observed in oxidative, fatigue-resistant fiber types. Soleus muscles of constitutive Orai-KO mice exhibited a reduction in unique type I fibers, concomitant with an increase in hybrid fibers expressing both type I and type IIA myosins. Additionally, ex vivo force measurements showed reduced maximal specific force and in vivo exercise assays revealed reduced endurance in constitutive muscle-specific Orai-KO mice. Using tamoxifen-inducible, muscle-specific Orai-KO mice, these functional deficits were found to be the result of the delayed fiber changes resulting from an early developmental loss of Orai1 and not the result of an acute loss of Orai1-dependent store-operated calcium entry.-Carrell, E. M., Coppola, A. R., McBride, H. J., Dirksen, R. T. Orai1 enhances muscle endurance by promoting fatigue-resistant type I fiber content but not through acute store-operated Ca²⁺ entry.

Keywords: development; exercise; myosin; skeletal muscle.

Figure 1.

Specificity and efficiency of muscle-specific cOrai-KO in mice. A) Diagram of muscle-specific Orai-KO breeding strategy. Mice containing 2 floxed Orai1 alleles (WT) were crossed with mice that contained 2 floxed alleles and Cre recombinase cassette driven by MCK promoter (cOrai-KO mice). B) Six-month weekly growth curves for male and female WT and Orai-KO mice (n = 5–10 animals/group). C, D) Orai1 transcript levels normalized to Cacna1s transcript levels in EDL, FDB, TA, and Sol muscles from young (C) and adult (D) WT and cOrai-KO mice, measured using semiquantitative PCR with 6-FAM-labeled primers (n = 3–4 animals). E) Orai1 transcript levels normalized to Gapdh transcript levels in nonskeletal muscle tissues measured as in C (n = 3–4 animals). F) Representative Mn²⁺ quench traces. Scale bars: x = 100 s, y = 4 × 10⁵ counts. G) Maximal rate of Mn²⁺ quench in store-depleted FDB fibers isolated from young and adult WT and cOrai-KO mice (n = 5–7 animals). H) Total releasable Ca²⁺ content measured using fura-FF in single isolated FDB fibers. ΔRatio = 340/380_max –340/380 _baseline (n = 4–5 animals).

Figure 2.

Sol muscles from Orai-KO mice develop normally and subsequently undergo fiber type-specific atrophy. A) Average weights of EDL and Sol muscles from WT and cOrai-KO mice (n = 25–36 muscles). B) Average fiber CSA in TA, EDL, and Sol muscle sections from WT and cOrai-KO mice (n = 3–6 animals). C, F) Top: sections of Sol muscles taken from young (C) and adult (F) WT animals. Bottom: sections of Sol muscles taken from young (C) and adult (F) cOrai-KO animals. Sections were stained for type I (magenta), type IIB (blue), and type IIA (red) myosin isotypes. Dystrophin (green) was used as marker of sarcolemma. Scale bar, 100 μm (white line) shown in applies to all images. D, G) Fractional contribution of fibers containing only type I, only type IIA, or both type I and type IIA myosin in Sol muscles from young (D) or adult (G) WT and cOrai-KO mice (n = 4–7 animals). E, H) Average CSA of different fiber types in Sol muscles from young (E) or adult (H) WT and cOrai-KO mice (n = 4–7 animals).

Figure 3.

Maximal and sustained contractile force during high-frequency stimulation is reduced in muscles from cOrai-KO mice. A–C) Representative ex vivo specific force traces in Sol muscles from adult WT and cOrai-KO mice stimulated at 1 (A), 20 (B), or 100 Hz (C). Timescale shown in B also applies to panels C, F, and G. Total duration for twitch traces in panels A and E is 250 ms. D) Force-frequency summary for Sol muscles from adult WT and Orai-KO mice (n = 12–13 muscles). E–G) Representative ex vivo specific force traces in EDL muscles from adult WT and cOrai-KO mice stimulated at 1 (E), 20 (F), or 100 Hz (G). H) Force-frequency summary of EDL muscles from adult WT and cOrai-KO mice (n = 14–15 muscles). I) Normalized specific force during repetitive, high-frequency stimulation (1 s tetani, 50 Hz, 0.4 duty cycle) for Sol muscles from adult WT and cOrai-KO mice (n = 6–9 muscles). J) Normalized specific force decay during repetitive, high-frequency stimulation (500 ms, 50 Hz, 0.2 duty cycle) in EDL muscles from WT and cOrai-KO mice (n = 6–10 muscles).

Figure 4.

Loss of Orai1 in skeletal muscle does not alter motor coordination but results in reduction in sustained grip strength and treadmill endurance. A) Motor coordination quantified from average latency to fall from rotarod at indicated speeds in WT and cOrai-KO mice (n = 11–12 animals). B) Sustained grip strength, measured from average latency to fall from inverted grid. Bars represent average latency to fall per trial averaged over 3 d of experiments (n = 8–10 animals). C) Cumulative number of falls during rotarod endurance task in WT and cOrai-KO mice (n = 10–12 animals). D) Summary of average total distance run by WT and Orai-KO mice during 1 h treadmill endurance protocol (n = 7–8 animals).

Figure 5.

Despite significant reductions in Orai1 transcript, force generation and whole-animal fatigue do not differ at 1 mo after Tam administration. A) Four-month-old animals were given 5 injections on 5 consecutive days of Tam (40 mg/kg/d) or equivalent volume of CO (vehicle), then collected and measured 1 mo later. B) Orai1 transcript normalized to Cacna1s transcript in Sol, EDL, and TA muscles isolated from mice 1 mo after 5 injections of CO or Tam (n = 3 animals). C) Representative Mn²⁺ quench traces (top; scale bars: x = 100 s, y = 4 × 10⁵ counts) and maximal rate of Mn²⁺ quench in isolated FDB fibers (n = 6–9 cells from 2 animals). D, F) Representative traces of ex vivo force generation by Sol (D) and EDL (F) stimulated at 150 Hz for 500 ms. E, G) Maximal specific force-frequency summary generated by excised Sol (E) and EDL (G) muscles (n = 3–7 muscles). H, I) Total treadmill distance run (H) and cumulative number of rests taken (I) by CO- and Tam-injected mice during 1 h endurance treadmill protocol (n = 3–4 animals).