'In situ' high pressure confocal Ca(2+)-fluorescence microscopy in skeletal muscle: a new method to study pressure limits in mammalian cells.
We combined 'in situ' high pressure microscopy with confocal laser scanning microscopy to directly study Ca2+ homeostasis in intact mammalian (murine) skeletal muscle fibres during high pressure exposure up to 35 MPa. Cytosolic Fluo-4 and mitochondrial Rhod-2 Ca2+ fluorescence were simultaneously monitored. To separate changes in Ca2+ and direct/indirect effects of pressure on the dye, experiments in permeabilized ('skinned') muscle fibres were performed at a fixed Ca2+ concentration. Normalized Fluo-4 fluorescence sharply declined up to 10 MPa but showed a plateau between 10 MPa and -35 MPa. In the intact fibre, Fluo-4 fluorescence exponentially decreased during pressurization to 35 MPa with a pressure constant of pi-5 MPa whereas mitochondrial Rhod-2 fluorescence exponentially increased with a four-fold larger pi. Holding the pressure at 35 MPa almost did not change Fluo-4 fluorescence. However, Rhod-2 fluorescence started to decrease after -40 min. Upon decompression, Rhod-2 and Fluo-4 fluorescence increased exponentially with similar pi. However, initial Fluo-4 fluorescence values were not restored. Our results are in agreement with pressure induced Ca2+ leakage from the sarcoplasmic reticulum. Ca2+ might then be taken up in large amounts by mitochondria preventing cytosolic increase in Ca2+. Prolonged pressure applications (-40 min at 35 MPa) seem to destabilize mitochondrial function with release of Ca2+ from mitochondria back into the cytosol and eventually mechanical activation resulting in irreversible contractures. The pressure induced disturbance of Ca2+ homeostasis might have important implications for the pressure exposure limits and/or dive profiles of deep sea mammals.
Friedrich
O
O
Medical Biophysics, Institute of Physiology and Pathophysiology, INF 326, Ruprecht-Karls-University, Heidelberg, Germany.
Wegner
F V
FV
Hartmann
M
M
Frey
B
B
Sommer
K
K
Ludwig
H
H
Fink
R H A
RH
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006575:Heterocyclic Compounds, 3-Ring / Q000378:metabolism; D006706:Homeostasis; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011312:Pressure; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D014966:Xanthenes; C068483:rhod-2; D002118:Calcium