TAM-associated CASQ1 mutants diminish intracellular Ca2+ content and interfere with regulation of SOCE

Abstract

Tubular aggregate myopathy (TAM) is a rare myopathy characterized by muscle weakness and myalgia. Muscle fibers from TAM patients show characteristic accumulation of membrane tubules that contain proteins from the sarcoplasmic reticulum (SR). Gain-of-function mutations in STIM1 and ORAI1, the key proteins participating in the Store-Operated Ca²⁺ Entry (SOCE) mechanism, were identified in patients with TAM. Recently, the CASQ1 gene was also found to be mutated in patients with TAM. CASQ1 is the main Ca²⁺ buffer of the SR and a negative regulator of SOCE. Previous characterization of CASQ1 mutants in non-muscle cells revealed that they display altered Ca²⁺dependent polymerization, reduced Ca²⁺storage capacity and alteration in SOCE inhibition. We thus aimed to assess how mutations in CASQ1 affect calcium regulation in skeletal muscles, where CASQ1 is naturally expressed. We thus expressed CASQ1 mutants in muscle fibers from Casq1 knockout mice, which provide a valuable model for studying the Ca²⁺ storage capacity of TAM-associated mutants. Moreover, since Casq1 knockout mice display a constitutively active SOCE, the effect of CASQ1 mutants on SOCE inhibition can be also properly examined in fibers from these mice. Analysis of intracellular Ca²⁺ confirmed that CASQ1 mutants have impaired ability to store Ca²⁺and lose their ability to inhibit skeletal muscle SOCE; this is in agreement with the evidence that alterations in Ca²⁺entry due to mutations in either STIM1, ORAI1 or CASQ1 represents a hallmark of TAM.

Keywords: Calcium; Myopathy; Sarcoplasmic reticulum; Skeletal muscle; Store operated calcium entry.

Fig. 1

Evaluation of Ca²⁺ store content in isolated FDB muscle fibers. Ca²⁺ store content was analyzed in FDB muscles from Casq1 knockout mice and in fibers expressing CASQ1WT-GFP and CASQ1-GFP mutants following treatment with ICE store depletion medium. (a) Representative traces of Fura-2 fluorescence ratio of 340/380 nm, starting from ICE addition, and quantitative analyses of total releasable Ca²⁺ store content (b). Numbers on bars indicate the number of fibers tested for each sample. Experiments were conducted using 3 to 5 male mice. Bars indicate mean values of AUC ± SEM. Statistically significant differences compared to fibers expressing CASQ1^WT are indicated by * (p values ≤ 0.05), ** (p values ≤ 0.01), *** (p values ≤ 0.001). Statistically significant differences compared to FDB fibers from Casq1 knockout mice are indicated by §§§ (p values ≤ 0.001)

Fig. 2

Evaluation of SOCE in isolated FDB muscle fibers. SOCE was analyzed in FDB muscle fibers from Casq1 knockout mice and in fibers expressing CASQ1WT-GFP and CASQ1 mutants. (a) Representative traces of maximal rate of Fura-2 fluorescence quench by Mn²⁺, normalized for the initial baseline rate of Fura-2 decay, starting with the application of 0.5 mM Mn²⁺ in FDB fibers. (b) Bars indicate mean dF/dt values ± SEM. Numbers on bars indicate the number of fibers tested for each sample. Experiments were conducted using 3 to 5 male mice. Statistically significant differences compared to fibers expressing CASQ1^WT are indicated by * (p values ≤ 0.05), ** (p values ≤ 0.01), *** (p values ≤ 0.001). Statistically significant differences compared to FDB fibers from Casq1 knockout mice are indicated by § (p values ≤ 0.05), §§§ (p values ≤ 0.001)

Fig. 3

Expression of CASQ1WT and CASQ1 mutants in FDB from Casq1 knockout mice. Representative FDB muscle fibers isolated from Casq1 knockout mice transfected with CASQ1^WT (B), CASQ1^Asp44Asn (E), CASQ1^Gly103Asp (H), CASQ1^Ile385Thr (K). FDB muscle fibers were stained with rabbit primary antibodies against RyR1 and Alexa Fluor 555-conjugated anti rabbit secondary antibodies to label triads (A, D, G and J). To evaluate colocalization, the fluorescence intensity of RyR1 (red) and CASQ1 (green) was plotted as a function of distance for six to seven successive triads (C, F, I and L). Pearson coefficients of co-localization range from 0.712 to 0.741. Scale bar = 3 μm