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. 2007 Dec 17;179(6):1163-77.
doi: 10.1083/jcb.200704059.

The mitochondrial respiratory chain is a modulator of apoptosis

Jennifer Q Kwong  1 , Matthew S Henning, Anatoly A Starkov, Giovanni Manfredi
Affiliations

The mitochondrial respiratory chain is a modulator of apoptosis

Jennifer Q Kwong et al. J Cell Biol. .

Abstract

Mitochondrial dysfunction and dysregulation of apoptosis are implicated in many diseases such as cancer and neurodegeneration. We investigate here the role of respiratory chain (RC) dysfunction in apoptosis, using mitochondrial DNA mutations as genetic models. Although some mutations eliminate the entire RC, others target specific complexes, resulting in either decreased or complete loss of electron flux, which leads to impaired respiration and adenosine triphosphate (ATP) synthesis. Despite these similarities, significant differences in responses to apoptotic stimuli emerge. Cells lacking RC are protected against both mitochondrial- and endoplasmic reticulum (ER) stress-induced apoptosis. Cells with RC, but unable to generate electron flux, are protected against mitochondrial apoptosis, although they have increased sensitivity to ER stress. Finally, cells with a partial reduction in electron flux have increased apoptosis under both conditions. Our results show that the RC modulates apoptosis in a context-dependent manner independent of ATP production and that apoptotic responses are the result of the interplay between mitochondrial functional state and environmental cues.

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Figures

Figure 1.
Figure 1.
Characterization of the mtDNA mutant cell lines. (A) Schematic representation of the human mtDNA. The mutants used in this study are indicated. (B) Coupled and uncoupled KCN-sensitive oxygen consumption was measured in whole cells with 1 mM pyruvate as a substrate. Cells were uncoupled with the addition of 1 μM FCCP. Cells harboring mtDNA mutations have severely reduced coupled and uncoupled respiration (n = 6; error bars represent SD).
Figure 2.
Figure 2.
Apoptosis in the mtDNA mutant cybrid cells. (A) Caspase 3 activity in response to 1 μM STS for 6 h, expressed as a percentage of the response observed in WT cells (n = 4; error bars represent SEM). ρ0, COX, CYTB, and MERRF cells are protected, whereas NARP cells are hypersensitive to STS-induced caspase 3 activation. (B) Caspase 3 activity in response to 10 μM ET for 24 h, expressed as a percentage of the response observed in WT cells (n = 4; error bars represent SEM). ρ0, MERRF, COX, and CYTB show a trend for protection, whereas NARP cells are hypersensitive to ET-induced caspase 3 activation. (C) Caspase 3 activity in response to 1 μM TG for 24 h, expressed as a percentage of the response observed in WT cells (n = 8; error bars represent SEM). ρ0 and MERRF cells were protected against TG, whereas COX, CYTB, and NARP cells were hypersensitive to TG. (D) Caspase 3 activity in response to 1 μM TN for 24 h, expressed as a percentage of the response observed in WT cells (n = 4; error bars represent SEM). The pattern of TN-induced caspase 3 activity mirrored the pattern induced by TG (C).
Figure 3.
Figure 3.
ΔΨm generated by the RC is necessary to maintain normal mitochondrial morphology. (A) Representative traces of the ΔΨm generated in isolated mitochondria from WT and ρ0 cells. Incubation medium (see Materials and methods) was supplemented with 5 mM succinate (S). 1 μM antimycin A was added to deenergize mitochondria (AA). (B) Quantification of ΔΨm generated in mtDNA mutant cells (n = 3). Only WT and NARP cells were able to generate RC-supported ΔΨm. (C) Confocal images of cells stained with 100 nM MitoTracker Red CMXRos for 30 min at 37°C. WT and NARP cells have intact mitochondrial networks, whereas ρ0, COX, CYTB, and MERRF cells display fragmented and punctate mitochondrial morphology. (D) Mean mitochondrial length in the mtDNA mutant cybrids. ρ0, COX, CYTB, and MERRF mitochondria had significantly decreased mean lengths as compared with WT and NARP mitochondria. 100 mitochondria were scored for each cell line (error bars represent SEM). (E) Distribution of mitochondrial lengths in the various cell lines. ρ0, COX, CYTB, and MERRF cells had a significant decrease in the proportion of mitochondria >2 μm.
Figure 4.
Figure 4.
ER stress is induced equally in mtDNA mutant cells. Western blot of the ER stress marker Grp78. TG treatment (1 μM for 24 h) strongly up-regulated Grp78 protein expression in all cell lines. Tim23, a protein unaffected by ER stress, was used as a loading control.
Figure 5.
Figure 5.
Mitochondrial ATP synthesis and cellular ATP content are not implicated in ER stress–induced apoptosis. (A) Caspase 3 activity in cells treated with either 1 μM TG alone or 1 μg/ml TG in combination with the mitochondrial ATP synthase inhibitor oligomycin for 24 h (n = 6; error bars represent SEM). Inhibition of ATP synthase did not modify the response in WT cells, whereas mutant cells exhibited increased caspase activity. (B) WT, ρ0, CYTB, and NARP cells were treated with 1 μM TG for various times and total cellular ATP content was measured (n = 3; error bars represent SEM). ATP content at each time point is expressed as a percentage of the untreated values, suggesting that acute ER stress does not result in ATP depletion.
Figure 6.
Figure 6.
Acute mitochondrial Ca2+ uptake upon ER stress does not correlate with the pattern of ER stress–induced apoptosis. (A and B) Representative traces of fluorometric measurement of cytosolic Ca2+ levels are shown using the Ca2+-sensitive dye Fura 2AM. Mitochondrial Ca2+ stores were released with FCCP and ER calcium stores were released by the addition of TG. The amount of Ca2+ in mitochondria was measured before (A, mCa before) and after ER calcium release with TG (B, mCa after). (C) Quantification of mitochondrial Ca2+ uptake after TG treatment. The amount of Ca2+ taken up by mitochondria in the mtDNA mutant cell lines after Ca2+ release by the ER was quantified by measuring the difference between mCa after and mCa before.
Figure 7.
Figure 7.
Mitochondrial Ca2+ uptake correlates with the ability to maintain ΔΨm. (A) Representative trace of Ca2+ uptake in isolated WT mitochondria. 100 μg mitochondria were incubated in buffer containing Calcium Green-5N, 7 mM pyruvate, and 1 mM of the RC substrate malate. Mitochondria were challenged with sequential additions of 100 nmol CaCl2 (arrows). After each addition there is a spike in fluorescence caused by increase Ca2+ concentration in the buffer. The downward slope reflects mitochondrial uptake of Ca2+. Note that under these conditions, WT mitochondria undergo permeability transition as indicated by release of Ca2+ in the buffer. (B) Representative trace of lack of Ca2+ uptake in isolated mitochondria from ρ0 cells. In comparison to A, it is clear that there is no progressive mitochondrial Ca2+ uptake, but the fluorescence increases stepwise because of increased concentration of Ca2+ in the buffer. (C) Representative traces of the ATP-supported ΔΨm generation in WT and ρ0 mitochondria. ΔΨm supported by ATP hydrolysis was taken as the difference between the maximal signal generated with the addition of ATP and the minimal signal generated with the addition of carboxyatractylate (C). (D) Quantification of ΔΨm generated by ATP hydrolysis in cybrid cell lines (n = 3). WT, COX, CYTB, MERRF, and NARP mitochondria were able to generate ΔΨm using ATP as a substrate, whereas ρ0 mitochondria were not. (E) ROS production before and after stimulation with 1 μM TG. Hydroperoxides measured with H2DCFDA are expressed as fluorescence units per micrograms of cellular proteins. NARP and CYTB cells have significantly increased ROS at 18 and 24 h after TG. Error bars represent SEM.
Figure 8.
Figure 8.
BN gels of RC complexes and mitochondrial levels of antiapoptotic Bcl-2 proteins. (A) WT cells have a full complement of RC complexes, whereas ρ0 and MERRF cells lack assembled complexes containing mtDNA-specified subunits (complexes I, III, IV, and V) and have partially assembled complex V intermediates. COX cells lack complex IV and have reduced complex I. CYTB cells lack complex III and also have severely reduced complex I. NARP cells contain all five RC complexes. (B) Western blot of the antiapoptotic proteins Bcl-2 and Bcl-XL in enriched mitochondrial fractions. Hsp60 was used as a marker of the mitochondrial matrix, Tim23 was used as a marker of the mitochondrial inner membrane, and VDAC was used as a marker of the mitochondrial outer membrane.
Figure 9.
Figure 9.
Electron flux through mitochondrial RC modulates ER stress–induced apoptosis. (A) Caspase 3 activation in response to TG and FCCP. Cells were pretreated with 5 μM FCCP for 30 min and then cotreated with 1 μM TG for 24 h. The effect of FCCP on TG-induced caspase 3 activation is expressed as a percentage of protection as compared with the TG treatment alone (n = 4; error bars represent SEM). (B) Caspase 3 activation in response to TG and TMPD. Cells were pretreated with 140 μM TMPD for 30 min and then cotreated with 1 μM TG for 24 h. Caspase 3 activity in WT cells treated with both TG and TMPD is defined as 100% (n = 4; error bars represent SEM). (C) Caspase 3 activity in untreated or 1-μM TG-treated WT cells with or without the addition of 100 nM of RC inhibitor rotenone, 4 μM of RC inhibitor piericidin A, and 1 μM of RC inhibitor antimycin A for 24 h. Caspase 3 activity is expressed as a percentage of WT cells treated with TG alone (set at 100%; n = 3; error bars represent SEM). All inhibitors of the RC enhance the apoptotic response in WT cells.
Figure 10.
Figure 10.
Correlation between RC composition and apoptotic responses in mtDNA mutant cybrids. The RC components of the cybrids are depicted. WT cells have a full complement of RC complexes. ρ0 cells are missing complexes I, III, IV, and V. COX cells are missing complex IV and have reduced complex I (depicted as a smaller circle). CYTB cells are missing complex III and have reduced complex I. MERRF cells are missing complexes I, III, IV, and V, detectable by BN gel, but have some residual ATP hydrolytic complex V activity. NARP cells have all of the complexes in place, but have reduced amounts and activities. Normal response to apoptosis induction is represented by +, high response by +++, and low response by −.
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