Contents

📒 Zorov 2014

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release1

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INTRODUCTION

mPTP activation by ROS => RIRR

ROS: GENERAL DEFINITIONS

  • superoxide anion radical (O2·−)
  • hydroxyl radical (·OH)
  • hydrogen peroxide (H2O2)
  • singlet oxygen (1O2)
  • Derived compounds: nitric oxide (NO), peroxynitrite, lipid hydroperoxides (LOOH), alkoxyl radical (RO·), peroxyl radical (·OOH), nitrogen-centered radical, sulfate radical (SO4·−) and metal-oxygen complexes

ROS: FROM SIGNALING TO PATHOLOGICAL

  • regulated oxidative stress could initiate diverse cellular responses ranging from triggering signaling pathways involved in cell protection e.g. mitochondrial fission and autophagy
  • Physiological ROS emmision : 2% of vO2

ROS: REDOX STRESS

  • apparent heterogeneity in ROS levels and types when comparing different cells and organs
  • ROS beyond the “normal” or “physiological” level => oxidative stress
  • But the term oxidative stress is often used in the literature in a very general term
  • when the reduced glutathione levels are too high, “reductive stress” occurs and demonstrates potentially detrimental consequences for the cell
  • both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are deleterious

ROS GENERATION IN MITOCHONDRIA

Complex II (SDH)

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  • Typically, complex II is excluded from the list of potential candidates for important physiological contributors of ROS
  • ROS generation when oxidizing high levels of succinate (10mM)
  • FAD-derived ROS
  • Dissociation of matrix subunits upon low pH

Complex I

  • FET (G/M substrate): rotenone-induced ROS. FMN site
  • at least 40% of all mitochondrial disorders are associated with mutations in subunits of complex I e.g. Parkinson’s disease (PD)
  • under pathological conditions, conformational rearrangements may be involved in the changes of the efficiency of ROS-producing machinery in complex I.
  • generating ROS in mitochondria in the cell remains constant or even increases when Po2 drops dramatically but not in isolated mitochondria

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  • hypoxia-induced superoxide production occurs through activation of NADPH oxidase located in the cell membrane. NO synthesis in mitochondria continues during hypoxia
  • NO can partially block cytochrome oxidase => favoring generation of superoxide at hypoxic conditions
  • In a tissue with high metabolic rate (such as heart muscle), capillary density during maximum or moderate exercise would not be sufficient to supply tissue with oxygen https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930003.jpeg
  • very steep regional redox transitions have been observed across the borderline of the ischemic area https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930004.jpeg

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930005.jpeg
the ROS production proton motive force sensitive

Can ROS production be decreased in mitochondria without jeopardizing ATP production? Mild uncoupling as a possible downregulator of ROS production

  • moderate lowering of Δψ could result in a lower ROS production in mitochondria without a significant effect on ATP production
  • UCPs, fatty acids, metformin, ANT, glutamate/aspartate transporter, thyroid hormones

Complex III (bcl1 complex)

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  • Under normal conditions, the probability of existence of unstable semiquinone (Q_.) is low due to its fast oxidation
  • conformational changes detected in bcl1 complex after antimycin A binding
  • ROS generation is pmf-dependent

Redox State of NAD(P)H/NAD(P)+ in Mitochondrial ROS Production

  • 50 μM NADH was used, while in the presence of 1 mM NADH, ROS production was remarkably suppressed; NAD+ revealed the same superoxide suppressive ability
  • ammonium-sensitive NADH oxidase ? => H2O2
  • dihydrolipoyl dehydrogenase: KGDH & PDH complexes => H2O2

Other Mitochondrial ROS-Producing Sites

  • NADPH-oxidase (Nox)
  • Monoaminoxidase (MAO)
  • p66shc
  • α-Glycerophosphate dehydrogenase
    • activity is relatively low in the liver, heart, and brain but high in brown adipose tissue
    • one of the most efficient ROS generators in mitochondria
  • Electron transfer flavoprotein (ETF) and ETF quinone oxidoreductase (ETF dehydrogenase)
    • fatty acid β-oxidation-related: acyl CoA dehydrogenase → ETF → ETF-QO → Ubiquinone → complex III
  • Aconitase
    • cubane-type [4Fe-4S] center highly susceptible to inactivation by superoxide
    • ROS sensor in the mitochondria
    • Producing H2O2 and OH radical
    • doxorubicin cardiotoxicity was explained mainly by an aconitase inactivation accompanied by hydroxyl radical release

ROS-INDUCED ROS RELEASE ASSOCIATED WITH THE mPTP

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  • Δψ loss in a significant fraction of mitochondria, caused by hypoxia/reoxygenation. Depolarized mitochondria (red-fluorescence ”holes“; bottom panels) are associated with increased ROS (green; bottom left panel). Hypoxic PC or pharmacological preconditioning (PC), represented by diazoxide (Dz), prevents mitochondrial depolarization, and 5-hydroxydecanoate (5HD) accentuates the loss.
  • mPTP and Ischemia/Reperfusion Injury
  • RIRR https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930008.jpeg https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930009.jpeg
  • Transient Δψ hyperpolarization and depolarization (flickering) preceding MPT induction can be observed

RIRR AND Ca2+

  • mitochondria have quite a high Ca2+-buffering capacity

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  • spontaneous ROS-induced Ca2+ release by the SR ryanodine receptor
  • the role of Ca2+ in ROS production in mitochondria remains controversial
  • experiments with intact cardiac myocytes and neurons demonstrated that the mPTP is largely insensitive to increased cytosolic Ca2+, opposed to isolated mitochondria

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930011.jpeg
Ca2+ dependence of the mPTP in rat adult cardiac myocytes

  • isolated mitochondria are stripped from cytoskeletal proteins that maintain mitochondrial integrity within the intracellular energetic unit, which might be involved in fine regulation of enzymatic parameters in the live cell and be responsible for observed differences

MITOCHONDRIAL COMPARTMENTATION

  • close proximity of mitochondria to ER/SR found in the cell
  • may dramatically change kinetics and, thus, enzymatic parameters of interactive systems
  • intramitochondrial compartmentation???
  • intermitochondrial compartmentation: mitochondrial matrix lumen continuity

MITOCHONDRIAL RIRR IN OSCILLATING MODE

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930012.jpeg
ROS-induced fluctuations of the mitochondrial Δψ

IMAC-ASSOCIATED RIRR

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  • By B. O’Rourke’s lab
  • Δψ oscillations and RIRR induced by local oxidative stress and by perfusion with hydrogen peroxide have been detected in live, intact myocardium
  • propagation of superoxide signal within the cardiac tissue with a velocity ∼20 μm/s.
  • With in silicon model

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930014.jpeg https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930015.jpeg

  • mPTP threshold is highly dependent on ambient molecular oxygen, since ROS production in the system is a reaction of the first order with respect to molecular oxygen

  • The term mitochondrial criticality was coined (16, 17) for the situation when the mitochondrial network in the cardiac myocyte becomes highly sensitive to the changes of ambient/intramitochondrial ROS

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930016.jpeg

  • in vivo mitochondrial states that may contribute to the fate of the cell

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930017.jpeg

PRACTICAL ASPECTS OF USING MITOCHONDRIAL Δψ AS A DRIVER AND REPORTER

  • delivery of drugs to the mitochondrial interior: mitochondrial targeting signal sequences and mitochondriaphillic (positively charged lipophilic) compounds

CONCLUDING COMMENTS

mPTP

  1. release valve for ROS and Ca2+ (flickering)
  2. trigger for mitophagy (permanant opening)
  3. trigger for apoptosis (permanant opening)

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/physrev/2014/physrev.2014.94.issue-3/physrev.00026.2013/production/images/large/z9j0031426930018.jpeg

IMAC

  • Faster wave of RIRR in the mitochondrial network
  • IMAC-associated redox instability during pathological stress is quite different from a safe, physiological flickering mode of the mPTP

Reference


  1. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 2014;94(3):909-50. PMC4101632↩︎