Pre-assembled Ca2+ entry units and constitutively active Ca2+ entry in skeletal muscle of calsequestrin-1 knockout mice

Abstract

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of Ca2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca2+ entry, as well as sustained Ca2+ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca2+-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca2+ store content and marked SR Ca2+ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm2 versus 2.0 ± 0.3/100 µm2) and FDB (16.7 ± 1.0/100 µm2 versus 2.7 ± 0.5/100 µm2) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca2+ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca2+ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca2+ transient amplitude, increased dependence of luminal SR Ca2+ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca2+ content by assembling CEUs to promote constitutive and store-operated Ca2+ entry.

Figure 1.

Incidence and size of SR stacks. (A and B) Representative EM images of cross sections of EDL muscle fibers from WT (A) and CASQ1-null (B) mice (empty arrows point to stacks of SR membranes). (C and D) Representative EM images of longitudinal sections of EDL muscle fibers showing stacks of SR membranes in proximity to triads (SR-TT-SR). (E–H) Quantitative analyses of the percentage of muscle fibers exhibiting SR stacks (E), the number of SR stacks per 100 µm² of cross-sectional area (F), the average SR stack length (G; labeled by dots in C and D), and the frequency of the number of SR stack elements (H; numbered in C and D) in EDL muscles WT and CASQ1-null mice. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of fibers analyzed. Number of mice: n = 4 WT; n = 4 CASQ1-null. Data for EDL muscles from WT mice shown in E–G are the same as those reported in Boncompagni et al. (2017) and Michelucci et al. (2019). Scale bars: 0.2 µm, A and B; 0.1 µm, C and D.

Figure S1.

SR stack incidence, number/area, and TT extension in FDB muscle fibers. (A–C) Quantitative analyses of the percentage of muscle fibers exhibiting SR stacks (A), number of SR stacks per 100 µm² of cross-sectional area (B), and TT length in the I band (μm/100 µm² of cross-sectional area; C) determined from longitudinal sections of FDB muscle fibers from WT and CASQ1-null mice. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of FDB fibers analyzed. Number of mice: n = 3 WT; n = 3 CASQ1-null.

Figure 2.

TT extension and TT/SR stack contact lengths. (A–D) Representative EM images of longitudinal sections of EDL muscle fibers from WT (A and B) and CASQ1-null (C and D) mice showing TTs stained in black with ferrocyanide precipitate. (E and F) Quantitative analyses of total TT length (E) and TT-SR contact length (F) per 100 µm² of cross-sectional area in EDL muscle from WT and CASQ1-null mice. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of EDL fibers analyzed. Number of mice: n = 4 WT; n = 4 CASQ1-null. Data for EDL muscles from WT mice shown in E and F are the same as those reported by Boncompagni et al. (2017) and Michelucci et al. (2019). Scale bars: 1 µm, A and C; 0.5 µm, B and D.

Figure 3.

Maximum rate of Mn²⁺ quench in the presence or absence of store depletion. (A and B) Representative traces of fura-2 fluorescence during application of 0.5 mM Mn²⁺ in FDB fibers isolated from WT and CASQ1-null mice following prior store depletion induced with 1 µM TG + 15 µM CPA (A; +depletion) and in the absence of store depletion (B; −depletion). (C and D) Quantitative analyses of the maximum rate of Mn²⁺ quench in the presence (C) and absence (D) of prior store depletion. (E and F) Frequency histograms of the percentage of FDB fibers exhibiting different levels of maximum rate of Mn²⁺ quench in the presence (E) and absence (F) of prior store depletion. Histogram data were fit with a single Gaussian distribution. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of FDB fibers analyzed. Number of mice: n = 6 WT; n = 5 CASQ1-null.

Figure S2.

BTP-2 sensitivity of store-operated and constitutive Mn²⁺ quench in FDB fibers. (A–D) Representative superimposed traces of fura-2 fluorescence (A and B) and quantitative analyses of the maximum rate of Mn²⁺ quench (C and D) during application of 0.5 mM Mn²⁺ followed by 0.5 mM Mn²⁺ supplemented with either 10 µM BTP-2 (blue) or DMSO (red) vehicle in FDB fibers from CASQ1-null mice in the presence (+depletion; A and C) or absence (−depletion; B and D) of prior store depletion. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of FDB fibers analyzed. Number of mice: n = 2 CASQ1-null.

Figure 4.

Western blot analyses of proteins that coordinate SOCE and SR Ca²⁺ uptake. (A–C) Representative immunoblots showing expression levels of the long (STIM1L) and short (STIM1S) STIM1 isoforms (A), Orai1 (B), and SERCA (C) in EDL muscle homogenates from WT and CASQ1-null mice. For Orai1 expression, EDL muscles from constitutive, muscle-specific Orai1 KO (cOrai1 KO) mice were used as a control for validation of the Orai1 antibody employed. (D–G) Relative band intensities (normalized to glyceraldehyde 3-phosphate dehydrogenase antibody [GAPDH]) for each of the proteins shown in A. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of mice analyzed.

Figure 5.

Ca²⁺ transients during repetitive stimulation. (A) Representative mag-fluo-4 (ΔF/F₀) traces during the 1st, 2nd, 20th, and 40th stimulus trains in FDB fibers from WT and CASQ1-null mice. (B) Time course of relative change in peak mag-fluo-4 fluorescence during 40 consecutive stimulus trains. (C and D) Quantitative analyses of the relative decay during the second stimulus train (ST_2nd/ST_1st; C) and the peak of the bump phase (ST_peak/ST_2nd; D) from the data shown in B. Data are shown as mean ± SEM; *, P < 0.05. Numbers in bars (n) indicate the number of FDB fibers analyzed. Number of mice: n = 4 WT; n = 5 CASQ1-null.

Figure S3.

Tail integral and end/peak ratio during repetitive stimulation. (A) Representative mag-fluo-4 fluorescence trace elicited during a single stimulus train (500 ms at 50 Hz) highlighting analysis of tail integral (left, gray shaded area) and end/peak ratio (right). (B) Representative superimposed mag-fluo-4 (ΔF/F₀) traces during the 1st (left) and 40th (right) stimulus train in FDB fibers isolated from WT and CASQ1-null mice. (C and D) Ca²⁺ transient tail integral (C) and end/peak ratio (D) analyses in FDB fibers from WT and CASQ1-null mice. Data are shown as mean ± SEM; *, P < 0.05. Numbers in parentheses (n) indicate the number of FDB fibers analyzed. Number of mice: n = 4 WT; n = 5 CASQ1-null. AUC, area under the curve.

Figure 6.

Measurements of total releasable Ca²⁺ store content and luminal free SR Ca²⁺. (A and B) Representative superimposed fura-FF fluorescence traces (A) and quantitative analyses of total releasable Ca²⁺ store content (B) elicited during application of Ca²⁺ release cocktail (ICE). (C) Representative immunofluorescence confocal image for D1ER (green) and α-actinin (red) in an FDB fiber from a WT mouse. (D) Representative D1ER emission (Em₄₈₀ and Em₅₃₅) and D1ER_535/480 ratio traces during the 1st (upper) and 40th (lower) stimulus train recorded from a representative FDB fiber of a CASQ1-null mouse. (E and F) Quantitative analyses of the average resting D1ER ratio (D1ER_535/480; E) and time course change of D1ER_535/480 during 40 consecutive stimulus trains (F) in FDB fibers from WT and CASQ1-null mice in the presence or absence of 10 µM BTP-2. Data are shown as mean ± SEM; *, P < 0.05, WT compared with both CASQ1-null and CASQ1-null + BTP-2. #, P < 0.05, CASQ1-null compared with CASQ1-null + BTP-2. Numbers in bars (n) indicate the number of FDB fibers analyzed. Number of mice: n = 6 WT; n = 4 WT + BTP-2; n = 3 CASQ1-null; n = 3 CASQ1-null + BTP-2.

Figure 7.

Contractile force during repetitive stimulation in the presence or absence of extracellular Ca²⁺ influx. (A–C) Time course of peak specific force recorded in EDL muscles from WT and CASQ1-null mice during 40 consecutive stimulus trains in the presence of either standard Ringer’s solution containing 2.5 mM Ca²⁺ (A), nominally Ca²⁺-free Ringer’s solution (B), or standard Ringer’s solution supplemented with 10 µM BTP-2 (C). (D–F) Quantitative analyses of the relative decay during the second stimulus train (ST_2nd/ST_1st) and the peak of the bump phase (ST_peak/ST_2nd) from the corresponding data shown in A–C. Data are shown as mean ± SEM; *, P < 0.05. Numbers in parentheses (n) indicate the number of EDL muscles analyzed. Number of mice: n = 8 WT, n = 7 CASQ1-null, A and D; n = 7 WT, n = 6 CASQ1-null, B and E; n = 7 WT, n = 6 CASQ1-null, C and F.

Figure S4.

Contractile force and decay during repetitive stimulation in the presence or absence of Ca²⁺ influx. (A) Representative superimposed specific force traces elicited during the 1st (left), 20th (middle), and 40th (right) stimulus train in EDL muscles from CASQ1-null mice conducted in either standard Ringer’s solution containing 2.5 mM Ca²⁺ (black), nominally Ca²⁺-free Ringer’s (blue), or standard Ringer’s solution supplemented with 10 µM BTP-2 (green). (B and C) Time course analyses of specific force (B) and end/peak ratio (C) under the three different experimental conditions. Data are shown as mean ± SEM; *, P < 0.05. Numbers in parentheses (n) indicate the number of EDL muscles analyzed. Number of mice: n = 6 CASQ1-null (2.5 mM Ca²⁺); n = 6 CASQ1-null (Ca²⁺-free); n = 7 CASQ1-null (2.5 mM Ca²⁺ + BTP-2).

Figure S5.

Immunostaining for RyR1 and Orai1 in EDL fibers. (A and B) Representative immunofluorescence images obtained in EDL fibers from WT (A) and CASQ1-null (B) mice that were double-labeled for RyR1 (red) and Orai1 (green). (C and D) Fluorescence intensity profiles along four sarcomeres for the EDL fibers from WT (C) and CASQ1-null (D) mice shown by the dashed line in A. Scale bar, 5 µm (inset, 1 µm).