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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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Mitochondrial fission and loss of cristae following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and stained with MitoTracker Red. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). Pressurized cells show mitochondrial fission, which is characterized by the conversion of tubular fused mitochondria into isolated small organelles (B). Electron micrographs of thin sections of RGC-5 cells show normal, elongated forms of mitochondria in non-pressurized control cells (C), whereas elevated hydrostatic pressure produces smaller mitochondria (arrow) and mitochondria with abnormal, severe cristae depletion (arrowheads; D). Closely apposed smaller mitochondria were also observed, such as the six shown in panel E, suggesting that fission had occurred. To quantify the observation of severe cristae depletion in the mitochondria exposed to elevated hydrostatic pressure, the cristae density in tomographic volumes was determined after segmentation. This parameter was calculated by dividing the sum of the cristae volumes by the mitochondrial volume for each mitochondrion (F). There were 13 control (from two experiments) and 14 pressurized mitochondria (from two experiments) fully segmented into constituent compartments and analyzed (The asterisk indicates significance at p<0.001 compared to non-pressurized cells). Data represent the means±SD. Size bar represents 20 µm (A and B) and 500 nm (C-E).
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f1: Mitochondrial fission and loss of cristae following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and stained with MitoTracker Red. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). Pressurized cells show mitochondrial fission, which is characterized by the conversion of tubular fused mitochondria into isolated small organelles (B). Electron micrographs of thin sections of RGC-5 cells show normal, elongated forms of mitochondria in non-pressurized control cells (C), whereas elevated hydrostatic pressure produces smaller mitochondria (arrow) and mitochondria with abnormal, severe cristae depletion (arrowheads; D). Closely apposed smaller mitochondria were also observed, such as the six shown in panel E, suggesting that fission had occurred. To quantify the observation of severe cristae depletion in the mitochondria exposed to elevated hydrostatic pressure, the cristae density in tomographic volumes was determined after segmentation. This parameter was calculated by dividing the sum of the cristae volumes by the mitochondrial volume for each mitochondrion (F). There were 13 control (from two experiments) and 14 pressurized mitochondria (from two experiments) fully segmented into constituent compartments and analyzed (The asterisk indicates significance at p<0.001 compared to non-pressurized cells). Data represent the means±SD. Size bar represents 20 µm (A and B) and 500 nm (C-E).

Mentions: In agreement with our previous study [27], mitochondria in non-pressurized control cells at 3 days showed a typical filamentous and fused mitochondrial network (Figure 1A,C). In contrast, mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small round organelles, was induced at 3 days after elevated hydrostatic pressure (Figure 1B,D,E). Transmission EM analysis of images collectively showing several hundred mitochondria provided evidence that non-pressurized control cells contained classical long tubular mitochondria with abundant cristae (Figure 1C). In contrast, cells exposed to elevated hydrostatic pressure contained elongated mitochondria that had few cristae (Figure 1D) and a mix of small round mitochondria (Figure 2E). Again, EM images showing hundreds of mitochondria were analyzed. To gain insight into the mitochondrial ultrastructure of non-pressurized versus pressurized cells, cultures were fixed to preserve mitochondrial morphology, and processed by electron microscope (EM) tomography, to obtain 3D reconstruction showing detailed mitochondrial ultrastructure. Quantative analysis of tomographic volumes showed that pressure treatment induced a fourfold reduction of cristae density (Figure 1F). Tomographic reconstructions of control samples showed an intact outer mitochondrial membrane (OMM; blue) and lamellar cristae (various colors), occupying the mitochondrial matrix space (Figure 2B-D). The tomographic reconstruction of pressurized cells was dramatically different in two ways (Figure 2E-L). First, cristae depletion was often observed. Figure 2F-H shows a typical example of a mitochondrion that is devoid of cristae in much of its volume. Second, mitochondrial fission was apparent. Mitochondria of pressurized cells, were sometimes observed to be much smaller and more globular and in close proximity (Figure 2J-L), suggesting recent fission before fixation. The movies (movie 1 and movie 2) have been made to illustrate the above reconstructions. These can be viewed in Figure 2.


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)

Mitochondrial fission and loss of cristae following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and stained with MitoTracker Red. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). Pressurized cells show mitochondrial fission, which is characterized by the conversion of tubular fused mitochondria into isolated small organelles (B). Electron micrographs of thin sections of RGC-5 cells show normal, elongated forms of mitochondria in non-pressurized control cells (C), whereas elevated hydrostatic pressure produces smaller mitochondria (arrow) and mitochondria with abnormal, severe cristae depletion (arrowheads; D). Closely apposed smaller mitochondria were also observed, such as the six shown in panel E, suggesting that fission had occurred. To quantify the observation of severe cristae depletion in the mitochondria exposed to elevated hydrostatic pressure, the cristae density in tomographic volumes was determined after segmentation. This parameter was calculated by dividing the sum of the cristae volumes by the mitochondrial volume for each mitochondrion (F). There were 13 control (from two experiments) and 14 pressurized mitochondria (from two experiments) fully segmented into constituent compartments and analyzed (The asterisk indicates significance at p<0.001 compared to non-pressurized cells). Data represent the means±SD. Size bar represents 20 µm (A and B) and 500 nm (C-E).
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Related In: Results  -  Collection

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f1: Mitochondrial fission and loss of cristae following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and stained with MitoTracker Red. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). Pressurized cells show mitochondrial fission, which is characterized by the conversion of tubular fused mitochondria into isolated small organelles (B). Electron micrographs of thin sections of RGC-5 cells show normal, elongated forms of mitochondria in non-pressurized control cells (C), whereas elevated hydrostatic pressure produces smaller mitochondria (arrow) and mitochondria with abnormal, severe cristae depletion (arrowheads; D). Closely apposed smaller mitochondria were also observed, such as the six shown in panel E, suggesting that fission had occurred. To quantify the observation of severe cristae depletion in the mitochondria exposed to elevated hydrostatic pressure, the cristae density in tomographic volumes was determined after segmentation. This parameter was calculated by dividing the sum of the cristae volumes by the mitochondrial volume for each mitochondrion (F). There were 13 control (from two experiments) and 14 pressurized mitochondria (from two experiments) fully segmented into constituent compartments and analyzed (The asterisk indicates significance at p<0.001 compared to non-pressurized cells). Data represent the means±SD. Size bar represents 20 µm (A and B) and 500 nm (C-E).
Mentions: In agreement with our previous study [27], mitochondria in non-pressurized control cells at 3 days showed a typical filamentous and fused mitochondrial network (Figure 1A,C). In contrast, mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small round organelles, was induced at 3 days after elevated hydrostatic pressure (Figure 1B,D,E). Transmission EM analysis of images collectively showing several hundred mitochondria provided evidence that non-pressurized control cells contained classical long tubular mitochondria with abundant cristae (Figure 1C). In contrast, cells exposed to elevated hydrostatic pressure contained elongated mitochondria that had few cristae (Figure 1D) and a mix of small round mitochondria (Figure 2E). Again, EM images showing hundreds of mitochondria were analyzed. To gain insight into the mitochondrial ultrastructure of non-pressurized versus pressurized cells, cultures were fixed to preserve mitochondrial morphology, and processed by electron microscope (EM) tomography, to obtain 3D reconstruction showing detailed mitochondrial ultrastructure. Quantative analysis of tomographic volumes showed that pressure treatment induced a fourfold reduction of cristae density (Figure 1F). Tomographic reconstructions of control samples showed an intact outer mitochondrial membrane (OMM; blue) and lamellar cristae (various colors), occupying the mitochondrial matrix space (Figure 2B-D). The tomographic reconstruction of pressurized cells was dramatically different in two ways (Figure 2E-L). First, cristae depletion was often observed. Figure 2F-H shows a typical example of a mitochondrion that is devoid of cristae in much of its volume. Second, mitochondrial fission was apparent. Mitochondria of pressurized cells, were sometimes observed to be much smaller and more globular and in close proximity (Figure 2J-L), suggesting recent fission before fixation. The movies (movie 1 and movie 2) have been made to illustrate the above reconstructions. These can be viewed in Figure 2.

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