'In situ' high pressure confocal Ca(2+)-fluorescence microscopy in skeletal muscle: a new method to study pressure limits in mammalian cells.

摘要: We combined 'in situ' high pressure microscopy with confocal laser scanning to directly study Ca2+ homeostasis in intact mammalian (murine) skeletal muscle fibres during exposure up 35 MPa. Cytosolic Fluo-4 and mitochondrial Rhod-2 fluorescence were simultaneously monitored. To separate changes direct/indirect effects of on the dye, experiments permeabilized ('skinned') performed at a fixed concentration. Normalized sharply declined 10 MPa but showed plateau between -35 In fibre, exponentially decreased pressurization constant pi-5 whereas increased four-fold larger pi. Holding almost did not change fluorescence. However, started decrease after -40 min. Upon decompression, similar initial values restored. Our results are agreement induced leakage from sarcoplasmic reticulum. might then be taken large amounts by mitochondria preventing cytosolic increase Ca2+. Prolonged applications (-40 min MPa) seem destabilize function release back into cytosol eventually mechanical activation resulting irreversible contractures. The disturbance have important implications for limits and/or dive profiles deep sea mammals.