Abstract
Store-operated Ca2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca2+ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca2+ sensor, and Orai1, the Ca2+-permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca2+, compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca2+ with Mg2+ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca2+ Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca2+ ions from the extracellular space during repetitive muscle activity.
Highlights
We used 4 month old male skeletal muscle-specific, tamoxifen-inducible Orai[1] KO mice (Orai1fl/fl:HSA-MCM) that were fed either normal or tamoxifen-infused chow to induce knock-out of the Orai[1] gene for 4 weeks prior to isolating muscles and quantifying: (1) Orai[1] transcript levels in tibialis anterior (TA) muscles (Fig. 1A); (2) maximum rate of Mn2+ quench following thapsigargin-induced store depletion in single, acutely dissociated flexor digitorum brevis (FDB) fibers (Fig. 1B); and (3) IF localization of Orai[1] in isolated EDLmuscle bundles (Fig. 1C–F)
Identical gains for confocal excitation, emission and magnification were used, permitting a direct comparison between signals observed in the presence or absence of Orai[1]
An intense double-row of Orai[1] fluorescence that co-localizes with the RyR1 signal was only observed in fibers from control mice
Summary
We used 4 month old male skeletal muscle-specific, tamoxifen-inducible Orai[1] KO mice (Orai1fl/fl:HSA-MCM) that were fed either normal (control) or tamoxifen-infused chow to induce knock-out of the Orai[1] gene (iOrai[1] KO) for 4 weeks prior to isolating muscles and quantifying: (1) Orai[1] transcript levels in tibialis anterior (TA) muscles (Fig. 1A); (2) maximum rate of Mn2+ quench following thapsigargin-induced store depletion in single, acutely dissociated flexor digitorum brevis (FDB) fibers (Fig. 1B); and (3) IF localization of Orai[1] in isolated EDLmuscle bundles (Fig. 1C–F). The IF experiments in EDL muscle bundles from control and iOrai[1] KO mice shown in Fig. 1C–F were conducted using the same Orai[1] (PA5-26378) and RyR1 (34 C) antibodies, dilutions and conditions as described in the Article. Identical gains for confocal excitation, emission and magnification were used, permitting a direct comparison between signals observed in the presence (control) or absence (iOrai[1] KO) of Orai[1].
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