Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca2+ Entry Units in Aged Muscle

Abstract

Tubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in STIM1 and ORAI1. STIM1 and ORAI1 are the two main proteins mediating store-operated Ca²⁺ entry (SOCE), a mechanism activated by depletion of intracellular Ca²⁺ stores (e.g., SR) that allows recovery of Ca²⁺ from the extracellular space during repetitive muscle activity. We have recently shown that exercise triggers the formation of unique intracellular junctions between SR and transverse tubules named Ca ²⁺ entry units (CEUs). CEUs promote colocalization of STIM1 with ORAI1 and improve muscle function in presence of external Ca²⁺. TAs virtually identical to those of TAM patients are also found in fast-twitch fibers of aging male mice. Here, we used a combination of electron and confocal microscopy, Western blotting, and ex vivo stimulation protocols (in presence or absence of external Ca²⁺) to evaluate the presence of TAs, STIM1-ORAI1 localization and expression and fatigue resistance of intact extensor digitorum longus (EDL) muscles in wild-type male adult (4-month-old) and aged (24-month-old) mice and in mice trained in wheel cages for 15 months (from 9 to 24 months of age). The results collected indicate that (i) aging causes STIM1 and ORAI1 to accumulate in TAs and (ii) long-term exercise significantly reduced formation of TAs. In addition, (iii) EDL muscles from aged mice exhibited a faster decay of contractile force than adult muscles, likely caused by their inability to refill intracellular Ca²⁺ stores, and (iv) exercise in wheel cages restored the capability of aged EDL muscles to use external Ca²⁺ by promoting maintenance of CEUs. In conclusion, exercise prevented improper accumulation of STIM1 and ORAI1 in TAs during aging, maintaining the capability of aged muscle to refill intracellular Ca²⁺ stores via SOCE.

Keywords: electron microscopy; excitation-contraction coupling; sarcoplasmic reticulum; store opereted calcium entry; transverse tubule.

FIGURE 1

Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for RYR1 (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B). Raw images for the individual fluorescence channel used to construct these overlays are shown in Supplementary Figure 1. (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B), 5 μm (insets 2 μm); (C,D), 1 μm (inset 0.1 μm).

FIGURE 2

Representative EM images and corresponding cartoons showing SR linkers. SR vesicles in adult control mice [(A), and relative cartoon in panel (B)], SR stacks in adult exercised mice [(C), and relative cartoon in panel (D)], and TA tubes in aged EDL muscle fibers [(E), and relative cartoon in panel (F)]. SR linkers are pointed by empty arrows in EM images and represented as red rods in the cartoons. Image and numeric data in panels (B,D) originates from adult mice acutely exercised in Boncompagni et al. (2017). Data are shown as mean ± SEM; *p < 0.01 (adult vs. adult exercised). Scale bar: (A,C,E), 0.1 μm; inset, 0.05 μm.

FIGURE 3

Quantitative histological and EM analysis of TAs incidence in EDL fibers. (A–D) Representative histological (A,B) and EM (C,D) images from transversal sections of EDL muscles from aged (A,C) and aged trained (B,D) mice. TAs are marked with an asterisk in panels (A,B), whereas TAs are outlined with a blue line in panels (C,D). Representative histological and EM images of adult EDL muscle (which do not contain TAs) are shown in Supplementary Figure 2. (E–G) Bar plots showing the quantitative analysis of the percentage of EDL fibers containing TAs (E), the number of TAs per fiber (F), and finally the average size of TAs (G). Data are shown as mean ± SEM; **p < 0.01 in panels (E,F); *p < 0.05 in G; n = number of fibers analyzed. Scale bars: (A,B), 10 μm; (C,D), 2 μm.

FIGURE 4

Ex vivo fatigue protocols in EDL muscles. (A,B) Time course of specific (A) and relative (B) force decay (normalized to the first stimulus train) during 30 consecutive stimulus trains (60 Hz, 1-s duration, every 5 s) in presence of a standard KH solution containing 2.5 mM Ca²⁺. The asterisks in panels (A,B) indicate the window in which there is a significant statistical difference between aged (control) and the other two groups of samples (adult and aged trained). (C) Bar plot showing the fold change of force, relative to adult mice, calculated at the 10th stimulus train [pointed by arrows in panels (A,B)]. Data are shown as mean ± SEM; *p < 0.05; n = number of EDL muscles.

FIGURE 5

Ex vivo fatigue protocols in EDL muscles in presence or absence of extracellular Ca²⁺. (A–C) Time courses of relative force decay (normalized to the first stimulus train) during 30 consecutive stimuli (60 Hz, 1-s duration, every 5 s) in presence of standard KH solution either containing 2.5 mM Ca²⁺ (green), nominally Ca²⁺-free solution (dark blue), or standard KH solution containing 2.5 mM Ca²⁺ and supplemented with 10 μM BTP-2 (orange). The asterisks in panels (A,C) indicate the window in which there is a significant statistical difference between presence of 2.5 mM Ca²⁺ and the other two conditions (0 Ca²⁺ and presence of BTP2). (D–F) Bar plots showing the fold change of the force, relative to the 2.5 mM Ca²⁺ condition, calculated at the 10th stimulus train [see arrows in panels (A–C)]. Data are shown as mean ± SEM; *p < 0.05; n = number of EDL muscles.

FIGURE 6

Quantitative analysis by EM of SR stacks and TT length at the I band in EDL fibers. (A–F) Representative EM images of transversal sections of EDL fibers from adult (A,D), aged (B,E) and aged trained (C,F) mice. In (D–F) panels, TTs are stained with ferrocyanide (dark precipitate). (G,H) Bar plots showing the incidence of SR stacks (G) and the TT length at the I band (H). Arrows point to SR stacks in panels (A–C); arrows point to TTs stained with ferrocyanide in panels (D–F). Data are shown as mean ± SEM; *p < 0.05; n = number of measurements. Scale bar: (A–F), 0.1 mm.