Exacerbated Skeletal Muscle Phenotype in Mice with ‘Homotypic’ Expression of the Tubular Aggregate Myopathy ORAI1 G100S Mutation

Figures

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  Figure 1. Phenotypic characterization of 4M homotypic cORAI1-KO/GS mice. (A) Average (±SEM) ORAI1 transcript levels normalized to GAPDH as a reference gene in tibialis anterior muscles from 4M cfl/+, cfl/GS, cORAI1-KO/+ and cORAI1-KO/GS mice using RT-qPCR. (B,C) Average (±SEM) body mass (B) and EDL muscle mass (C) of 4M cfl/+, cfl/GS, cORAI1-KO/+ and cORAI1-KO/GS mice. (D) Representative traces of constitutive entry (upper) and average (±SEM) peak maximum rate of constitutive entry (lower) in FDB fibers from 4M cfl/+, cfl/GS, cORAI1-KO/+ and cORAI1-KO/GS mice. (E) Representative traces of store-operated entry (upper) and average (±SEM) peak maximum rate of store-operated entry (lower) in FDB fibers from 4M cfl/+, cfl/GS, cORAI1-KO/+ and cORAI1-KO/GS mice. Significance was calculated using a 1-way ANOVA test followed by Tukey’s multiple-comparisons post hoc test. Exact p-values where p < 0.05 are provided in the figure.

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  Figure 2. Homotypic cORAI1-KO/GS mice exhibit muscle weakness and exercise intolerance. (A) Wire-hang test of 4M cfl/+, cfl/GS, cORAI1-KO/+, and cORAI1-KO/GS mice where the time mice remain hanging on the grip were plotted (upper). The fractional reduction in hanging time was calculated as the difference in latency between the first grip (T1) and the third grip (T3), divided by the latency of the first grip: (T1-T3)/T1 (lower). (B,C) Rotarod endurance (B) and treadmill endurance (C) tests in 4M cfl/+, cfl/GS, cORAI1-KO/+, and cORAI1-KO/GS mice where the fraction of mice remaining on the apparatus during experiment (upper) and the total running distance (lower) were plotted. Significance was assessed using a two-way ANOVA (A) or one-way ANOVA (B,C), followed by Tukey’s multiple-comparisons post hoc test. * indicates p < 0.05 for cORAI1-KO/GS vs. cfl/+, cfl/GS and cORAI1-KO/+ mice. For bar graphs, the exact p-values are shown where p < 0.05.

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  Figure 3. Homotypic cORAI1-KO/GS mice exhibit reduced ventilatory function. 4M cORAI1-KO/GS mice exhibited significantly reduced breathing frequency (A), mid-tidal expiratory flow (EF50) (B), peak expiratory flow (PeFb) (C), and peak inspiratory flow (PiFb) (D). Significance was determined using 1-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. Exact p-values where p < 0.05 are shown.

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  Figure 4. Homotypic cORAI1-KO/GS mice exhibit reduced ex vivo EDL peak force production. Average (±SEM) specific force-frequency curves (A) and peak specific force production during repetitive, high-frequency stimulation (60 trains of 50 H for 500 ms/train delivered every 2.5 s) (B) in EDL muscles from 4M cfl/+, cfl/GS, cORAI1-KO/+, and cORAI1-KO/GS mice. Significance was determined using 2-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. & indicates p < 0.05 for cfl/+ vs. cfl/GS mice; @ indicates p < 0.05 for cfl/+ vs. cORAI1-KO/GS mice; $ indicates p < 0.05 for cORAI1-KO/+ vs. cORAI1-KO/GS mice; # indicates p < 0.05 for cfl/+ vs. cORAI1-KO/+ mice. N = 4, 4, 6, 5 for cfl/+, cfl/GS, cORAI1-KO/+ and cORAI1-KO/GS respectively.

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  Figure 5. Fiber-type analysis of homotypic cORAI1-KO/GS mice. (A) Representative fiber type immunohistochemistry staining images of EDL muscles from 4M cfl/+, cfl/GS, cORAI1-KO/+, and cORAI1-KO/GS mice. Examples of each fiber type are indicated with the appropriate color-coded arrowhead (Type I, cyan; Type IIa, red; Type IIb, green; Type IIx, black) in the cfl/GS image (upper right). (B,C) Average (±SEM) cross-sectional area (CSA) (B), relative fiber type distribution (C) of different muscle fiber types and (D) number of fibers in EDL muscles of 4M cfl/+, cfl/GS, cORAI1-KO/+, and cORAI1-KO/GS mice. Significance was determined using 1-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. Exact p-values where p < 0.05 are shown.

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  Figure 6. Summary of the main findings and predictions. ORAI1 hexameric channels can be comprised of only WT subunits (normal channels), heteromers of WT and GS subunits (heterotypic channels), or only GS subunits (homotypic GS channels) that exhibit distinct effects of ORAI1 channel function (i.e., constitutive- and store-operated calcium entry) and muscle structure/function during development and adulthood.