Exercise-dependent formation of new SR-TT junctions containing STIM1 and Orai1

S. Boncompagni,¹ A. Michelucci,¹ L. Pietrangelo,¹ R.T. Dirksen² and F. Protasi,¹ ¹CeSI-Met - Center for Research on Ageing and Translational Medicine & DNICS - Department of Neuroscience, Imaging and Clinical Sciences, University G. d'Annunzio of Chieti, I-66100 Italy and ²Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.

Depletion of calcium (Ca²⁺) from intracellular stores (endoplasmic reticulum, ER), triggers Ca²⁺ entry across the plasma membrane, a process known as capacitative Ca²⁺ entry or store-operated Ca²⁺ entry (SOCE) (Parekh & Penner, 1997). SOCE is important for intracellular Ca²⁺ regulation and is mediated by the interaction between STIM1 (stromal interaction molecule-1), which functions as the Ca²⁺ sensor in the ER (Liou et al., 2005), and Ca²⁺ permeable Orai1 channels in the external membrane (Vig et al., 2006). SOCE in skeletal muscle fibres (Kurebayashi & Ogawa, 2001) is also mediated by interaction between STIM1 in the sarcoplasmic reticulum (SR) and Orai1 channels in transverse tubules (TT) (Lyfenko & Dirksen, 2008).

The question. As this mechanism is likely the primary mechanism of Ca²⁺ entry in muscle during repetitive activity, it has been proposed that SOCE is important to prevents/delays fatigue (Wei-Lapierre et al., 2013). Despite its importance for proper muscle function, the subcellular sites for SOCE in skeletal fibres have not been unequivocally identified.

Main findings. Using electron microscopy we show that prolonged muscle activity (treadmill running in mice) drives the formation of previously unidentified intracellular junctions between SR and extensions of the TT membrane at the I band, between Ca²⁺-release units (CRUs, the sites of excitation-contraction coupling) and Z-line. Using immunohistochemistry and immunogold labeling we also demonstrate that these newly formed junctions contain the molecular machinery known to mediate SOCE in muscle: STIM1 Ca²⁺ sensor proteins in the SR, already present in the I band in control conditions, and Ca²⁺-permeable Orai1 channels, which move into the I band with TTs during prolonged muscle activity.

The Figure Panels A-B: show that following exercise, internal membranes at the I band rearrange into stacks of flat cisternae (empty arrow in B). C: Incidence of membrane stacks (as the one shown in B) is much higher in exercised muscles. D-F: Following exercise, TTs (stained black with ferrocyanide-precipitate in D and E) grows into the I band and become part of stacks of cisternae. G and H) Colocalization between STIM1 and Orai1 is low in control samples, but increases significantly following exercise. SR-TT-SR labels a triad (the site of excitation-contraction coupling in muscle). Data in C are shown as mean ± SEM: **p<0.01. Scale bars: A-F, 0.1 μm; G and H, 2.5 μm (insets: 1 μm).

Conclusions. The activity-dependent formation of these unique SR-TT junctions reflects a striking and unexpected remodeling of the existing sarcotubular system at the I band of the sarcomere. We here name these junctions Ca²⁺ Entry Units (CEUs), and propose that these structural entities are the preferential site for SOCE, at least during repetitive activity, representing the ideal pathway to rapidly recover Ca²⁺ ions from the extracellular space and, hence, important to prevent/delay fatigue.

Additional implications. As a) reduced SOCE activity also contributes to muscle dysfunction in ageing (Zhao et al., 2008), and b) mutations in STIM1 and Orai1 are linked to Tubular Aggregate Myopathy (Nesin et al., 2014), our findings could also have important implications for the understanding of muscular dysfunction in different physio-pathological conditions.

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