scholarly journals Defective kinetics of cytochrome c oxidase and alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres (‘MERRF’) at position 8344 nt

1999 ◽  
Vol 342 (3) ◽  
pp. 537-544 ◽  
Author(s):  
Hana ANTONICKÁ ◽  
Daniel FLORYK ◽  
Petr KLEMENT ◽  
Leona STRATILOVÁ ◽  
Jana HEŘMANSKÁ ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Control Analysis ◽  
Hybrid Cells ◽  
Myoclonus Epilepsy ◽  
Increased Sensitivity ◽  
Hybrid Clones

We have investigated pathogenic effects of the tRNALys A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased Vmax for both the low-affinity and high-affinity phases; however, the Km values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, δΨ, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in δΨ at state 4 and an increased sensitivity of δΨ to an uncoupler in fibroblasts from the patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship between heteroplasmy of the MERRF mutation, COX activity and δΨ. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and δΨ was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and δΨ in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of δΨ inhibition.

Tight control of mitochondrial membrane potential by cytochrome c oxidase

Mitochondrion ◽  
2011 ◽  
Vol 11 (2) ◽  
pp. 334-341 ◽  
Author(s):  
Consiglia Pacelli ◽  
Dominga Latorre ◽  
Tiziana Cocco ◽  
Ferdinando Capuano ◽  
Christian Kukat ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Tight Control

Abstract 371: Mitochondrial Antiviral Signaling (MAVS) Protects Cardiomyocytes Under Oxidative Stress by Interfering with Bax-Mediated Cell Death

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Toshitaka Yajima ◽  
Stanley Park ◽  
Hanbing Zhou ◽  
Michinari Nakamura ◽  
Mitsuyo Machida ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Antiviral Signaling ◽  
Cell Death Pathway ◽  
The Impact

MAVS is a mitochondrial outer membrane protein that activates innate antiviral signaling by recognizing cytosolic viral RNAs and DNAs. While the discovery of MAVS is the first molecular evidence that links mitochondria to innate immune mechanisms, it is still unclear whether MAVS affects mitochondrial cell death as a member of caspase activation and recruitment domain (CARD)-containing proteins. We found that MAVS interacts with Bax through CARD by Yeast two-hybrid and a series of immunoprecipitation (IP) assay, which led us to hypothesize that MAVS functions not only in the innate antiviral mechanisms but also in the mitochondrial cell death pathway. Methods: 1) We examined molecular interaction between MAVS and Bax under oxidative stress by IP using isolated myocytes with H2O2 stimulation and the heart post ischemia-reperfusion (I/R). 2) We evaluated the effect of MAVS on mitochondrial membrane potential and apoptosis under H2O2 stimulation using isolated myocytes with adenoviral MAVS knockdown. 3) We investigated the impact of MAVS on %myocardial infarction (%MI) post I/R using cardiac-specific MAVS knockout (cKO) and transgenic (cTg) mice which we have originally generated. 4) We examined the effect of MAVS on recombinant Bax (rBax)-mediated cytochrome c release using isolated mitochondria from wild type (WT) and MAVS KO mice. Results: 1) The amount of Bax pulled down with MAVS was significantly increased in isolated myocytes with 0.2 mM H2O2 compared to those without stimulation (mean±SD; 1.808±0.14, n=5, p<0.001) and in the heart post I/R compared to sham (2.2±1.19, n=3, p=0.0081). 2) Myocytes with MAVS knockdown showed clear abnormalities in mitochondrial membrane potential and caspace-3 cleavage with 0.2 mM H2O2 compared to control cardiomyocytes. 3) MAVS cKO had significantly larger %MI than WT (81.9 ± 5.8% vs. 42.6 ± 13.6%, n=8, p=0.0008). In contrast, MAVS cTg had significantly smaller %MI that WT (30.0 ± 4.8% vs. 49.2 ± 4.8%, n=10, p=0.0113). 4) Mitochondria from MAVS KO exhibited cytochrome c release after incubation with 2.5 μ g of rBax while those from WT required 10 μ g of rBax. Conclusion: These results demonstrate that MAVS protects cardiomyocyte under oxidative stress by interfering with Bax-mediated cytochrome c release from mitochondria.

Volatile Anesthetics Induce Caspase-dependent, Mitochondria-mediated Apoptosis in Human T Lymphocytes In Vitro 

2005 ◽  
Vol 102 (6) ◽  
pp. 1147-1157 ◽  
Author(s):  
Torsten Loop ◽  
David Dovi-Akue ◽  
Michael Frick ◽  
Martin Roesslein ◽  
Lotti Egger ◽  
...  
Keyword(s):  
Membrane Potential ◽  
T Lymphocytes ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Volatile Anesthetics ◽  
Human T Lymphocytes ◽  
Induced Apoptosis

Background Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

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