Dynamics of the Mitochondrial Permeability Transition Pore: Transient and Permanent Opening Events
| Autor: | Christopher W. Ward, Andrew K. Coleman, Andrew P. Wescott, Guiling Zhao, B. Maura Greiser, W. J. Lederer, Mariusz Karbowski, Liron Boyman, Humberto C. Joca |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Vascular smooth muscle Heart Ventricles Biophysics Context (language use) Mitochondrion Biochemistry Mitochondrial Membrane Transport Proteins Article Mitochondria Heart Rats Sprague-Dawley 03 medical and health sciences chemistry.chemical_compound Inner membrane Animals Molecular Biology Cells Cultured chemistry.chemical_classification Membrane Potential Mitochondrial Reactive oxygen species 030102 biochemistry & molecular biology Mitochondrial Permeability Transition Pore MPTP Depolarization Rats 030104 developmental biology chemistry Mitochondrial permeability transition pore Reactive Oxygen Species |
| Zdroj: | Arch Biochem Biophys |
| Popis: | A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨ(M)) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 “super-resolution” or “high-resolution” imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 seconds (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 minutes. The time to first depolarization events was 8.4 min (± 1.1 SEM n=21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 minutes) and virtually no permanent depolarization events occurred for over 1 hour of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon “stress” in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1–5], we show that cyclosporine-A (CsA, 10 mM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80 % and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ±6 s (n=478) at 37°C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca(2+). In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨ(M) depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6, 7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨ(M) depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool (“Optical Stress-Probe”), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks. |
| Databáze: | OpenAIRE |
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