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2018
Kamunde, C., M. Sharaf, and N. MacDonald, "H2O2 metabolism in liver and heart mitochondria: Low emitting-high scavenging and high emitting-low scavenging systems.", Free radical biology & medicine, vol. 124, pp. 135-148, 2018 May 24. Abstract

Although mitochondria are presumed to emit and consume reactive oxygen species (ROS), the quantitative interplay between the two processes in ROS regulation is not well understood. Here, we probed the role of mitochondrial bioenergetics in HO metabolism using rainbow trout liver and heart mitochondria. Both liver and heart mitochondria emitted HO at rates that depended on their metabolic state, with the emission rates (free radical leak) constituting 0.8-2.9% and 0.2-2.5% of the respiration rate in liver and heart mitochondria, respectively. When presented with exogenous HO, liver and heart mitochondria consumed it by first order reactions with half-lives (s) of 117 and 210, and rate constants of 5.96 and 3.37 (× 10 s), respectively. The mitochondrial bioenergetic status greatly affected the rate of HO consumption in heart but not liver mitochondria. Moreover, the activities and contribution of HO scavenging systems varied between liver and heart mitochondria. The significance of the scavenging systems ranked by the magnitude (%) of inhibition of HO removal after correcting for emission were, liver (un-energized and energized): catalase > glutathione (GSH) ≥ thioredoxin reductase (TrxR); un-energized heart mitochondria: catalase > TrxR > GSH and energized heart mitochondria: GSH > TrxR > catalase. Notably, depletion of GSH evoked a massive surge in HO emission that grossly masked the contribution of this pathway to HO scavenging in heart mitochondria. Irrespective of the organ of their origin, mitochondria behaved as HO regulators that emitted or consumed it depending on the ambient HO concentration, mitochondrial bioenergetic state and activity of the scavenging enzyme systems. Indeed, manipulation of mitochondrial bioenergetics and HO scavenging systems caused mitochondria to switch from being net consumers to net emitters of HO. Overall, our data suggest that the low levels of HO typically present in cells would favor emission of this metabolite but the scavenging systems would prevent its accumulation.

2017
Sharaf, M. S., D. Stevens, and C. Kamunde, "Zinc and calcium alter the relationship between mitochondrial respiration, ROS and membrane potential in rainbow trout (Oncorhynchus mykiss) liver mitochondria.", Aquatic toxicology (Amsterdam, Netherlands), vol. 189, pp. 170-183, 2017 Aug. Abstract

At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨ) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨ, and reactive oxygen species (ROS, as hydrogen peroxide (HO)) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨ that was associated with relatively slow enhancement of HO release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨ. In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and HO release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨ, and caused transient and sustained stimulation of respiration and HO release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with highly elevated but not unrealistic Ca loads was not the cause but a result of mPTP opening in the high conductance mode.

Sharaf, M. S., D. Stevens, and C. Kamunde, "Mitochondrial transition ROS spike (mTRS) results from coordinated activities of complex I and nicotinamide nucleotide transhydrogenase.", Biochimica et biophysica acta, vol. 1858, issue 12, pp. 955-965, 2017 12. Abstract

Mitochondria exhibit suppressed ATP production, membrane potential (∆Ψ) polarization and reactive oxygen species (ROS) bursts during some cellular metabolic transitions. Although mitochondrial ROS release is influenced by ∆Ψ and respiratory state, the relationship between these properties remains controversial primarily because they have not been measured simultaneously. We developed a multiparametric method for probing mitochondrial function that allowed precise characterization of the temporal relationship between ROS, ∆Ψ and respiration. We uncovered a previously unknown spontaneous ROS spike - termed mitochondrial transition ROS spike (mTRS) - associated with re-polarization of ∆Ψ that occurs at the transition between mitochondrial energy states. Pharmacological inhibition of complex CI (CI), nicotinamide nucleotide transhydrogenase (NNT) and antioxidant system significantly decreased the ability of mitochondria to exhibit mTRS. NADH levels followed a similar trend to that of ROS during the mTRS, providing a link between CI and NNT in mTRS regulation. We show that (i) mTRS is enhanced by simultaneous activation of CI and complex II (CII); (ii) CI is the principal origin of mTRS; (iii) NNT regulates mTRS via NADH- and ∆Ψ-dependent mechanisms; (iv) mTRS is not a pH spike; and (v), mTRS changes in amplitude under stress conditions and its occurrence can be a signature of mitochondrial health. Collectively, we uncovered and characterized the biophysical properties and mechanisms of mTRS, and propose it as a potential diagnostic tool for CI-related dysfunctions, and as a biomarker of mitochondrial functional integrity.

2015
Sharaf, M. S., M. R. van den Heuvel, D. Stevens, and C. Kamunde, "Zinc and calcium modulate mitochondrial redox state and morphofunctional integrity.", Free radical biology & medicine, vol. 84, pp. 142-153, 2015 Jul. Abstract

Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H2O2 production, membrane potential (ΔΨm), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H2O2 production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨm and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨm and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions.