Purpose: Mitochondrial fission is a cellular response to stress that has an important role in neuronal cell death in neurodegenerative diseases. The purpose of this study was to determine whether elevated hydrostatic pressure induces mitochondrial fission and dysfunction in cultured retinal ganglion cells.
Methods: RGC-5 cells were differentiated with succinyl concanavalin A (50 microg/mL) and transferred to a pressurized incubator in which 30 mm Hg of pressure was applied for 1, 2, or 3 days. As a control, differentiated cells from an identical passage were incubated simultaneously in a conventional incubator at each of the time points. Live RGC-5 cells were then labeled with a red fluorescent mitochondrial dye and mitochondrial morphology was assessed by fluorescence microscopy and electron microscopy. After elevated hydrostatic pressure, the cellular adenosine triphosphate (ATP) levels were also measured by a luciferase-based assay.
Results: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered in >74.3% +/- 1.9% of mitochondria at 3 days after elevated hydrostatic pressure. Only 4.7% +/- 1.4% of nonpressurized control cells displayed mitochondrial fission after 3 days. Electron microscopy showed that elevated hydrostatic pressure for 3 days induced abnormal cristae depletion and decreased the length of the mitochondria. On elevation of hydrostatic pressure, the fission-linked protein, Drp-1 was translocated from the cytosol to the mitochondria. Elevated hydrostatic pressure also resulted in a significant, time-dependent reduction of cellular ATP.
Conclusions: Elevated hydrostatic pressure triggered mitochondrial fission, abnormal cristae depletion, Drp-1 translocation, and cellular ATP reduction in differentiated RGC-5 cells. Increased understanding of the molecular mechanisms that regulate the cellular response to elevated pressure including mitochondrial fission may provide new therapeutic targets for protecting RGCs from elevated hydrostatic pressure.