Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells

[摘要] Purpose: This study was conducted todetermine whether elevated hydrostatic pressure alters mitochondrialstructure, triggers release of the dynamin-related guanosinetriphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome Cfrom mitochondria, alters OPA1 gene expression, and candirectly induce apoptotic cell death in cultured retinal ganglion cell(RGC)-5 cells. Methods: Differentiated RGC-5 cells wereexposed to 30 mmHg for three days in a pressurized incubator. As acontrol, differentiated RGC-5 cell cultures were incubatedsimultaneously in a conventional incubator. Live RGC-5 cells were thenlabeled with MitoTracker Red and mitochondrial morphology was assessedby fluorescence microscopy. Mitochondrial structural changes were alsoassessed by electron microscopy and three-dimenstional (3D) electronmicroscope tomography. OPA1 mRNA was measured by Taqmanquantitative PCR. The cellular distribution of OPA1 protein andcytochrome C was assessed by immunocytochemistry and western blot.Caspase-3 activation was examined by western blot. Apoptotic cell deathwas evaluated by the terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) method. Results: Mitochondrial fission,characterized by the conversion of tubular fused mitochondria intoisolated small organelles, was triggered after three days exposure toelevated hydrostatic pressure. Electron microscopy confirmed thefission and noted no changes to mitochondrial architecture, nor outermembrane rupture. Electron microscope tomography showed that elevatedpressure depleted mitochondrial cristae content by fourfold. Elevatedhydrostatic pressure increased OPA1 gene expression by 35±14%on day 2, but reduced expression by 36±4% on day 3. Total OPA1 proteincontent was not changed on day 2 or 3. However, pressure treatmentinduced release of OPA1 and cytochrome C from mitochondria to thecytoplasm. Elevated pressure also activated caspase-3 and inducedapoptotic cell death. Conclusions: Elevated hydrostaticpressure triggered mitochondrial changes including mitochondrialfission and abnormal cristae depletion, alteration of OPA1 geneexpression, and release of OPA1 and cytochrome C into the cytoplasmbefore the onset of apoptotic cell death in differentiated RGC-5 cells.These results suggest that sustained moderate pressure elevation maydirectly damage RGC integrity by injuringmitochondria.

[学科分类] 生物化学/生物物理