Both extremes of redox balance are known to cause cardiac injury, with mounting evidence revealing that the injury induced by both oxidative and reductive stress is oxidative in nature. During reductive stress, when electron acceptors are expected to be mostly reduced, some redox proteins can donate electrons to O2 instead, which increases reactive oxygen species (ROS) production. However, the high level of reducing equivalents also concomitantly enhances ROS scavenging systems involving redox couples such as NADPH/NADP+ and GSH/GSSG. Here our objective was to explore how reductive stress paradoxically increases net mitochondrial ROS production despite the concomitant enhancement of ROS scavenging systems. Using recombinant enzymes and isolated permeabilized cardiac mitochondria, we show that two normally antioxidant matrix NADPH reductases, glutathione reductase and thioredoxin reductase, generate H2O2 by leaking electrons from their reduced flavoprotein to O2 when electron flow is impaired by inhibitors or because of limited availability of their natural electron acceptors, GSSG and oxidized thioredoxin. The spillover of H2O2 under these conditions depends on H2O2 reduction by peroxiredoxin activity, which may regulate redox signaling in response to endogenous or exogenous factors. These findings may explain how ROS production during reductive stress overwhelms ROS scavenging capability, generating the net mitochondrial ROS spillover causing oxidative injury. These enzymes could potentially be targeted to increase cancer cell death or modulate H2O2-induced redox signaling to protect the heart against ischemia/reperfusion damage.

Reactive oxygen species (ROS) production by isolated complex I is steeply dependent on the NADH/NAD(+) ratio. We used alamethicin-permeabilized mitochondria to study the substrate-dependence of matrix NADH and ROS production when complex I is inhibited by piericidin or rotenone. When complex I was inhibited in the presence of malate/glutamate, membrane permeabilization accelerated O2 consumption and ROS production due to a rapid increase in NADH generation that was not limited by matrix NAD(H) efflux. In the presence of inhibitor, both malate and glutamate were required to generate a high enough NADH/NAD(+) ratio to support ROS production through the coordinated activity of malate dehydrogenase (MDH) and aspartate aminotransferase (AST). With malate and glutamate present, the rate of ROS production was closely related to local NADH generation, whereas in the absence of substrates, ROS production was accelerated by increase in added [NADH]. With malate alone, oxaloacetate accumulation limited NADH production by MDH unless glutamate was also added to promote oxaloacetate removal via AST. α-ketoglutarate (KG) as well as AST inhibition also reversed NADH generation and inhibited ROS production. If malate and glutamate were provided before rather than after piericidin or rotenone, ROS generation was markedly reduced due to time-dependent efflux of CoA. CoA depletion decreased KG oxidation by α-ketoglutarate dehydrogenase (KGDH), such that the resulting increase in [KG] inhibited oxaloacetate removal by AST and NADH generation by MDH. These findings were largely obscured in intact mitochondria due to robust H2O2 scavenging and limited ability to control substrate concentrations in the matrix. We conclude that in mitochondria with inhibited complex I, malate/glutamate-stimulated ROS generation depends strongly on oxaloacetate removal and on the ability of KGDH to oxidize KG generated by AST.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site II_f); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site II_f The decrease in matrix [dicarboxylate] allows O₂ access to reduced site II_f, thereby making electron donation to O₂ possible, explaining the rapid increase in ROS production provided that site II_f is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg²⁺ and NAD⁺ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca²⁺-induced PTP opening. We show that H₂O₂ production under these conditions is related to Mg²⁺-dependent NADH generation by malic enzyme. H₂O₂ production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg²⁺], [NAD⁺], and [ADP].

Particulate pollutants cause adverse health effects through the generation of oxidative stress. A key question is whether these effects are mediated by the particles or their chemical compounds. In this article we show that aliphatic, aromatic, and polar organic compounds, fractionated from diesel exhaust particles (DEPs), exert differential toxic effects in RAW 264.7 cells. Cellular analyses showed that the quinone-enriched polar fraction was more potent than the polycyclic aromatic hydrocarbon (PAH)-enriched aromatic fraction in O2 .- generation, decrease of membrane potential (Delta-Psi m), loss of mitochondrial membrane mass, and induction of apoptosis. A major effect of the polar fraction was to promote cyclosporin A (CsA)-sensitive permeability transition pore (PTP) opening in isolated liver mitochondria. This opening effect is dependent on a direct effect on the PTP at low doses as well as on an effect on Delta-Psi m at high doses in calcium (Ca2+)-loaded mitochondria. The direct PTP effect was mimicked by redox-cycling DEP quinones. Although the aliphatic fraction failed to perturb mitochondrial function, the aromatic fraction increased the Ca2+ retention capacity at low doses and induced mitochondrial swelling and a decrease in Delta-Psi m at high doses. This swelling effect was mostly CsA insensitive and could be reproduced by a mixture of PAHs present in DEPs. These chemical effects on isolated mitochondria could be reproduced by intact DEPs as well as ambient ultrafine particles (UFPs). In contrast, commercial polystyrene nanoparticles failed to exert mitochondrial effects. These results suggest that DEP and UFP effects on the PTP and Delta-Psi m are mediated by adsorbed chemicals rather than the particles themselves.

As mediators of the first enzymatic step in glucose metabolism, hexokinases (HKs) orchestrate a variety of catabolic and anabolic uses of glucose, regulate antioxidant power by generating NADPH for glutathione reduction, and modulate cell death processes by directly interacting with the voltage-dependent anion channel (VDAC), a regulatory component of the mitochondrial permeability transition pore (mPTP). Here we summarize the current state-of-knowledge about HKs and their role in protecting the heart from ischemia/reperfusion (I/R) injury, reviewing: 1) the properties of different HK isoforms and how their function is regulated by their subcellular localization; 2) how HKs modulate glucose metabolism and energy production during I/R; 3) the molecular mechanisms by which HKs influence mPTP opening and cellular injury during I/R; and 4) how different metabolic and HK profiles correlate with susceptibility to I/R injury and cardioprotective efficacy in cancer cells, neonatal hearts, and normal, hypertrophied and failing adult hearts, and how these difference may guide novel therapeutic strategies to limit I/R injury in the heart. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

In the adult heart, regulation of fatty acid oxidation and mitochondrial genes is controlled by the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators. However, in response to pathological stressors such as hemodynamic load or ischemia, cardiac myocytes downregulate PGC-1 activity and fatty acid oxidation genes in preference for glucose metabolism pathways. Interestingly, despite the reduced PGC-1 activity, these pathological stressors are associated with mitochondrial biogenesis, at least initially. The transcription factors that regulate these changes in the setting of reduced PGC-1 are unknown, but Myc can regulate glucose metabolism and mitochondrial biogenesis during cell proliferation and tumorigenesis in cancer cells. Here we have demonstrated that Myc activation in the myocardium of adult mice increases glucose uptake and utilization, downregulates fatty acid oxidation by reducing PGC-1alpha levels, and induces mitochondrial biogenesis. Inactivation of Myc in the adult myocardium attenuated hypertrophic growth and decreased the expression of glycolytic and mitochondrial biogenesis genes in response to hemodynamic load. Surprisingly, the Myc-orchestrated metabolic alterations were associated with preserved cardiac function and improved recovery from ischemia. Our data suggest that Myc directly regulates glucose metabolism and mitochondrial biogenesis in cardiac myocytes and is an important regulator of energy metabolism in the heart in response to pathologic stress.

Mitochondria are a common energy source for organs and organisms; their diverse functions are specialized according to the unique phenotypes of their hosting environment. Perturbation of mitochondrial homeostasis accompanies significant pathological phenotypes. However, the connections between mitochondrial proteome properties and function remain to be experimentally established on a systematic level. This uncertainty impedes the contextualization and translation of proteomic data to the molecular derivations of mitochondrial diseases. We present a collection of mitochondrial features and functions from four model systems, including two cardiac mitochondrial proteomes from distinct genomes (human and mouse), two unique organ mitochondrial proteomes from identical genetic codons (mouse heart and mouse liver), as well as a relevant metazoan out-group (drosophila). The data, composed of mitochondrial protein abundance and their biochemical activities, capture the core functionalities of these mitochondria. This investigation allowed us to redefine the core mitochondrial proteome from organs and organisms, as well as the relevant contributions from genetic information and hosting milieu. Our study has identified significant enrichment of disease-associated genes and their products. Furthermore, correlational analyses suggest that mitochondrial proteome design is primarily driven by cellular environment. Taken together, these results connect proteome feature with mitochondrial function, providing a prospective resource for mitochondrial pathophysiology and developing novel therapeutic targets in medicine.