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

Ju WK, Kim KY, Lindsey JD, Angert M, Patel A, Scott RT, Liu Q, Crowston JG, Ellisman MH, Perkins GA, Weinreb RN - Mol. Vis. (2009)

Bottom Line: Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture.Elevated pressure also activated caspase-3 and induced apoptotic cell death.These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

Purpose: 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.

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Cytochrome C release following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and immunostained with cytochrome c antibody. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). However, pressure treatment resulted in dispersed cytochrome C immunoreactivity in the cytoplasm (B). Size bar represents 10 µm (A and B). To quantify the observation of cytochrome C release from mitochondria exposed to elevated hydrostatic pressure, the percentage of cells with completely released cytochrome C in the cytoplasm was determined (C). Mitochondria were separated from cytosol by differential centrifugation and cytochrome C content was analyzed. Three protein samples from three independent experiments were loaded for each group. The cytochrome C protein bands show the positions, based on comparison with size standards, of the 15 kDa form of cytochrome C in cytosolic and mitochondrial fraction. The blot was stripped and reprobed with anti-actin antibody (~42 kDa) for cytosol fraction and anti-VDAC antibody (~31 kDa) for mitochondria fraction to confirm similar protein loading in each lane (D). Relative intensity of chemiluminescence for each protein band was normalized using actin as cytosolic fraction calibrator and VDAC as mitochondrial fraction calibrator (E). Data represent the means±SD of three independent experiments.
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f4: Cytochrome C release following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and immunostained with cytochrome c antibody. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). However, pressure treatment resulted in dispersed cytochrome C immunoreactivity in the cytoplasm (B). Size bar represents 10 µm (A and B). To quantify the observation of cytochrome C release from mitochondria exposed to elevated hydrostatic pressure, the percentage of cells with completely released cytochrome C in the cytoplasm was determined (C). Mitochondria were separated from cytosol by differential centrifugation and cytochrome C content was analyzed. Three protein samples from three independent experiments were loaded for each group. The cytochrome C protein bands show the positions, based on comparison with size standards, of the 15 kDa form of cytochrome C in cytosolic and mitochondrial fraction. The blot was stripped and reprobed with anti-actin antibody (~42 kDa) for cytosol fraction and anti-VDAC antibody (~31 kDa) for mitochondria fraction to confirm similar protein loading in each lane (D). Relative intensity of chemiluminescence for each protein band was normalized using actin as cytosolic fraction calibrator and VDAC as mitochondrial fraction calibrator (E). Data represent the means±SD of three independent experiments.

Mentions: Cytochome C immunoreactivity was prominent within the mitochondria of non-pressurized control RGC-5 cells (Figure 4A). Pressure treatment for 3 days resulted in dispersed cytochrome C immunoreactivity throughout the cytoplasm (Figure 4B). To assess cytochrome C release after elevated hydrostatic pressure, we counted cells with completely released cytochrome C in the cytoplasm. The percentage of control cells with completely released cytochrome C in the cytoplasm was 4.2±2.9% and the percentage of pressure-treated cells with completely released cytochrome C in the cytoplasm was 19.6±3.8% at 3 days (n=3, Figure 4C). Parallel experiments analyzed using western blotting found that pressure treatment increased cytochrome C release by 1.48±0.28 fold, compared to the non-pressurized control cells. In contrast, cytochrome C content in the mitochondrial fraction was decreased by 0.74±0.10 fold after 3 day of elevated hydrostatic pressure (n=3, Figure 4D,E).


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

Ju WK, Kim KY, Lindsey JD, Angert M, Patel A, Scott RT, Liu Q, Crowston JG, Ellisman MH, Perkins GA, Weinreb RN - Mol. Vis. (2009)

Cytochrome C release following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and immunostained with cytochrome c antibody. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). However, pressure treatment resulted in dispersed cytochrome C immunoreactivity in the cytoplasm (B). Size bar represents 10 µm (A and B). To quantify the observation of cytochrome C release from mitochondria exposed to elevated hydrostatic pressure, the percentage of cells with completely released cytochrome C in the cytoplasm was determined (C). Mitochondria were separated from cytosol by differential centrifugation and cytochrome C content was analyzed. Three protein samples from three independent experiments were loaded for each group. The cytochrome C protein bands show the positions, based on comparison with size standards, of the 15 kDa form of cytochrome C in cytosolic and mitochondrial fraction. The blot was stripped and reprobed with anti-actin antibody (~42 kDa) for cytosol fraction and anti-VDAC antibody (~31 kDa) for mitochondria fraction to confirm similar protein loading in each lane (D). Relative intensity of chemiluminescence for each protein band was normalized using actin as cytosolic fraction calibrator and VDAC as mitochondrial fraction calibrator (E). Data represent the means±SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629709&req=5

f4: Cytochrome C release following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and immunostained with cytochrome c antibody. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). However, pressure treatment resulted in dispersed cytochrome C immunoreactivity in the cytoplasm (B). Size bar represents 10 µm (A and B). To quantify the observation of cytochrome C release from mitochondria exposed to elevated hydrostatic pressure, the percentage of cells with completely released cytochrome C in the cytoplasm was determined (C). Mitochondria were separated from cytosol by differential centrifugation and cytochrome C content was analyzed. Three protein samples from three independent experiments were loaded for each group. The cytochrome C protein bands show the positions, based on comparison with size standards, of the 15 kDa form of cytochrome C in cytosolic and mitochondrial fraction. The blot was stripped and reprobed with anti-actin antibody (~42 kDa) for cytosol fraction and anti-VDAC antibody (~31 kDa) for mitochondria fraction to confirm similar protein loading in each lane (D). Relative intensity of chemiluminescence for each protein band was normalized using actin as cytosolic fraction calibrator and VDAC as mitochondrial fraction calibrator (E). Data represent the means±SD of three independent experiments.
Mentions: Cytochome C immunoreactivity was prominent within the mitochondria of non-pressurized control RGC-5 cells (Figure 4A). Pressure treatment for 3 days resulted in dispersed cytochrome C immunoreactivity throughout the cytoplasm (Figure 4B). To assess cytochrome C release after elevated hydrostatic pressure, we counted cells with completely released cytochrome C in the cytoplasm. The percentage of control cells with completely released cytochrome C in the cytoplasm was 4.2±2.9% and the percentage of pressure-treated cells with completely released cytochrome C in the cytoplasm was 19.6±3.8% at 3 days (n=3, Figure 4C). Parallel experiments analyzed using western blotting found that pressure treatment increased cytochrome C release by 1.48±0.28 fold, compared to the non-pressurized control cells. In contrast, cytochrome C content in the mitochondrial fraction was decreased by 0.74±0.10 fold after 3 day of elevated hydrostatic pressure (n=3, Figure 4D,E).

Bottom Line: Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture.Elevated pressure also activated caspase-3 and induced apoptotic cell death.These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus