Role of STIM1 And Orai1 in the Formation of Tubular Aggregates in Ageing Skeletal Muscle Fibers

Abstract

Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) tubes, form in ageing fast-twitch fibers of mice, preferentially in males. TAs are also found in biopsies from patients affected by TA Myopathy (TAM), a muscle disorder linked to mutations in STIM1 and Orai1 (proteins involved in store-operated Ca2+ entry, SOCE) We have previously shown using Electron Microscopy (EM) that tubes of TAs appear linked by small bridges, resembling aggregated STIM1 molecules. Here, we compared extensor digitorum longus (EDL) muscles from 2 groups of mice (∼4 months, adults and ≥24 months, aged) to determine: a) presence of STIM1 (and Orai1) in TAs; b) the relative contribution of Ca2+ entry to muscle function during repetitive stimulation in ageing muscle. Immunofluorescence indicates that ageing causes STIM1 (a SR protein), but not Orai (that resides in TTs) to accumulate in TAs. This finding is consistent with: a) EM data showing that TTs are rarely seen in TAs; and b) western-bot analysis showing increased expression levels of both STIM1 splicing variants (arbitrary units: STIM1-short=0.87±0.09 vs. 1.18±0.04; STIM1-long=0.81±0.1 vs. 1.11±0.05, respectively adult vs. aged). During a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds): i) in 2.5 mM Ca2+ external solution, EDL muscles from aged mice exhibit a decreased capability to maintain contractile (relative force after 10 tetani in adult vs. aged: 61.0±3.2%, and 49.9±2.4%); ii) in Ca2+-free external solution, aged EDL muscles display a lower decay in contractile force (relative force after 10 tetani in adult vs. aged: 41.0±3.1%, and 53.4±1.5%). Our results indicate that TAs a) accumulate STIM1, which is likely visible as electron-dense strands between tubes; b) while contain STIM1, do not functionally contribute to Ca2+ entry during repetitive stimulation.