Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e., the sarcoplasmic reticulum; SR) and from extracellular space: (1) excitation–contraction (EC) coupling and (2) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of calcium entry units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR stacks and transverse tubules (TTs). Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in the presence of extracellular Ca2+. The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated. Here, we first verified whether CEUs can assemble ex vivo (in the absence of blood supply and innervation), subjecting excised EDL muscles from mice to an ex vivo incremental fatigue protocol (80 Hz tetanus stimulation lasting 45 min): the data collected demonstrate that CEUs can assemble ex vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C versus 25°C) and lower pH (7.2 versus 7.4) promotes formation of CEUs increasing the percentage of fibers containing SR stacks, the number of SR stacks/area, and the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36°C) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+, suggesting that CEUs assembled ex vivo are functional.

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