Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells.
OBJECTIVE
This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells.


METHODS
Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimensional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method.


RESULTS
Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35+/-14% on day 2, but reduced expression by 36+/-4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death.


CONCLUSIONS
Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.
Ju
Won-Kyu
WK
The Sophie and Arthur Brody Optic Nerve Laboratory, Hamilton Glaucoma Center, University of California San Diego, La Jolla, CA 92037-0946, USA. danielju@glaucoma.ucsd.edu
Kim
Keun-Young
KY
Lindsey
James D
JD
Angert
Mila
M
Patel
Ankur
A
Scott
Ray T
RT
Liu
Quan
Q
Crowston
Jonathan G
JG
Ellisman
Mark H
MH
Perkins
Guy A
GA
Weinreb
Robert N
RN
D000818:Animals; D017209:Apoptosis; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006874:Hydrostatic Pressure; D007150:Immunohistochemistry; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000201:enzymology* / Q000648:ultrastructure; D018800:Thy-1 Antigens / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D012333:RNA, Messenger; D018800:Thy-1 Antigens; D045304:Cytochromes c; D020558:GTP Phosphohydrolases; C498811:Opa1 protein, rat