'In situ' high pressure confocal Ca2+ -fluorescence microscopy in skeletal muscle: A new method to study pressure limits in mammalian cells

Friedrich, O., Wegner, F. V., Hartmann, M., Frey, B., Sommer, K., Ludwig, H. and Fink, R. H. A. (2006) 'In situ' high pressure confocal Ca2+ -fluorescence microscopy in skeletal muscle: A new method to study pressure limits in mammalian cells. Undersea & Hyperbaric Medicine, 33 3: 181-195.


Author Friedrich, O.
Wegner, F. V.
Hartmann, M.
Frey, B.
Sommer, K.
Ludwig, H.
Fink, R. H. A.
Title 'In situ' high pressure confocal Ca2+ -fluorescence microscopy in skeletal muscle: A new method to study pressure limits in mammalian cells
Formatted title
'In situ' high pressure confocal Ca2+ -fluorescence microscopy in skeletal muscle: Aa new method to study pressure limits in mammalian cells
Journal name Undersea & Hyperbaric Medicine   Check publisher's open access policy
ISSN 1066-2936
Publication date 2006-05
Sub-type Article (original research)
Volume 33
Issue 3
Start page 181
End page 195
Total pages 15
Editor Irving Jacoby
Claude A. Piantadosi
Place of publication Bethesda, MD, U.S.A.
Publisher Undersea and Hyperbaric Medical Society
Language eng
Subject 1103 Clinical Sciences
Formatted abstract
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 ∼5 MPa. In the intact fibre, Fluo-4 fluorescence exponentially decreased during pressurization to 35 MPa with a pressure constant of π∼5 MPa whereas mitochondrial Rhod-2 fluorescence exponentially increased with a four-fold larger π. 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 π. 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.
Copyright © 2006 Undersea and Hyperbaric Medical Society, Inc.
Keyword High pressure microscopy
in situ
Confocal laser scanning microscopy
Pressure
Ca2+ homeostasis
Mammalian skeletal muscle
Fluorescence
Mitochondria
Q-Index Code C1
Additional Notes Issue: May-Jun 2006

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Biomedical Sciences Publications
 
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