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Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K + channel (mitoK ATP ) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C ε (PKCε) has been identified as a component of the mitoK ATP signaling cascade. We hypothesized that PKCε and mitoK ATP interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K + flux in liposomes containing partially purified mitoK ATP . The reconstituted proteins were obtained after detergent extraction of isolated mitochondria, 200-fold purification by ion exchange chromatography, and reconstitution into lipid vesicles. Immunoblot analysis revealed the presence of PKCε in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKCε peptide agonist, ψεRACK, each activated mitoK ATP -dependent K + flux in the reconstituted system. This effect of PKCε was prevented by chelerythrine, by the specific PKCε peptide antagonist, εV 1-2 , and by the specific mitoK ATP inhibitor 5-hydroxydecanoate. In addition, the activating effect of PKC agonists was reversed by exogenous protein phosphatase 2A. These results demonstrate persistent, functional association of mitochondrial PKCε and mitoK ATP .

The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The Ki values for ATP inhibition were 50 μm (UCP1), 70 μm (UCP2), and 120 μm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 μm at pH 7.2). The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 μmol·min–1·mg–1, depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATPopening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATPand leaves it in a persistent open state (Costa AD, Garlid KD. Am J Physiol Heart Circ Physiol 295, H874–H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O2·−), hydrogen peroxide (H2O2), and hydroxyl radical (HO˙), each of which has been proposed as the signaling ROS. Feedback activation of mitoKATPprovides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO˙ excludes H2O2as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO˙ and that it is probably a product of phospholipid oxidation.

Calcium-independent phospholipase A2γ (iPLA2γ) catalyses the hydrolysis of fatty acids from phospholipids with the consequent generation of lysophospholipids. iPLA2γ is targeted to peroxisomes and mitochondria and results from our laboratory have shown that the activity of mitochondrial iPLA2γ is increased by a redox-sensitive process [1]. Here we aimed to characterize the properties of isolated recombinant iPLA2γ and test the hypothesis that iPLA2γ is activated by thiol oxidation directly. We expressed His-tagged human iPLA2γ using HepG2 or HEK293 cell lines and reconstituted recombinant purified iPLA2γ into liposomes composed of L-α-Phosphatitylcholine and cardiolipin. Using selected specific fluorescent probes and GC/MS analysis, we observed H2O2-induced release of fatty acids in liposomes containing purified recombinant iPLA2γ but not in liposomes lacking the enzyme. The H2O2-activated enzyme released free fatty acids with a specific activity of about 20 nmol.min-1.mg-1. The activity increased several fold in the presence of bovine serum albumin or in mixed micelles containing non-ionic detergent, indicating a possible inhibition of iPLA2γ by the products of its enzymatic activities. H2O2 activated iPLA2γ with a half-maximal constant corresponding to about 1.5 mol H2O2 per mol of enzyme and the active enzyme was fully inhibited by thiol reducing compounds. These results indicate that the regulation of iPLA2γ is dependent on reversible oxidation of accessible cysteine residues and also on a feedback regulation by either fatty acids or lysophospholipids, which is consistent with the physiological participation of iPLA2γ in cellular redox signaling.Supported by the grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.1. Jaburek M, Ježek J, Zelenka J, Ježek P. Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen. Int. J. Biochem & Cell Biol 45, 816-825, 2013

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (DeltaPsim) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high DeltaPsim (similar to DeltaPsim in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with DeltaPsim. The application of DeltaPsim up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high DeltaPsim and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA- remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease DeltaPsim more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by DeltaPsim may be key in mitochondrial respiration and metabolism.

Functional activation of mitochondrial uncoupling protein-2 (UCP2) is proposed to decrease reactive oxygen species production. Skulachev and Goglia (Skulachev, V. P., and Goglia, F. (2003) FASEB J. 17, 1585-1591) hypothesized that hydroperoxy fatty acid anions are translocated by UCPs but cannot flip-flop across the membrane. We found that the second aspect is otherwise; the addition of synthesized linoleic acid hydroperoxides (LAOOH, a mix of four isomers) caused a fast flip-flop-dependent acidification of liposomes, comparable with the linoleic acid (LA)-dependent acidification. Using Escherichia coli-expressed UCP2 reconstituted into liposomes we found that LAOOH induced purine nucleotide-sensitive H(+) uniport in UCP2-proteoliposomes with higher affinity than LA (K(m) values 97 microM for LAOOH and 275 microM for LA). In UCP2-proteoliposomes LAOOH also induced purine nucleotide-sensitive K(+) influx balanced by anionic charge transfer, indicating that LAOOH was also transported as an anion with higher affinity than linoleate anion, the K(m) values being 90 and 350 microM, respectively. These data suggest that hydroperoxy fatty acids are transported via UCP2 by a fatty acid cycling mechanism. This may alternatively explain the observed activation of UCP2 by the externally generated superoxide. The ability of LAOOH to induce UCP2-mediated H(+) uniport points to the essential role of superoxide reaction products, such as hydroperoxyl radical, hydroxyl radical, or peroxynitrite, initiating lipoperoxidation, the released products of which support the UCP2-mediated uncoupling and promote the feedback down-regulation of mitochondrial reactive oxygen species production.

The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The Ki values for ATP inhibition were 50 microm (UCP1), 70 microm (UCP2), and 120 microm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 microm at pH 7.2). The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 micromol.min(-1).mg(-1), depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.

Thyroid hormones (TH) govern cardiac phenotype including myocardial bioenergetics, a finely tuned process, possibly by affecting expression of a number of proteins. Chronic hyperthyroidism is associated with cardiac hypertrophy, which may lead to serious heart problems perhaps through higher expression of uncoupling protein 2 (UcP2), which is present in the failing heart. We were investigating effects of silybin (SB) and dehydrosilybin (DHSB) on TH-regulated cardiomyocyte bioenergetics, including UcP2 expression levels. Both substances displayed concentration dependent down-regulation of UcP2 expression induced by THs in neonatal rat cardiomyocytes. Because DHSB uncoupled the respiration of isolated rat heart mitochondria while limiting reactive oxygen species (ROS) formation, it may be affecting UcP2 expression in a feedback control fashion. However, SB does neither. Therefore we explored the possibility of both substances limiting TH uptake into cardiomyocytes. TH presence in cardiomyocytes was evaluated by mass-spectrometry and we did not observe any limitation to the uptake of hormones. Because TH actions are primarily mediated by nuclear thyroid receptors and their transcriptional activation, we used reporter plasmid system to assess the SB and DHSB effect on thyroid hormone receptor transcriptional activity in cardiomyocytes. Our data suggest that SB and DHSB modulation of TH-mediated UcP2 expression is not related to their antioxidant ability (DHSB) or lack thereof (SB). Rather both substances influence TH-related processes by affecting the TH-dependent signaling pathway with possible beneficial effects in hyperthyroid patients.(Supported by GACR 303/08/0658 and MSM 6198959216)

Quercetin is a flavonoid, proposed to be capable of modulating mitochondrial activity and thereby exerting biological effects, including signaling by reactive oxygen species production. These effects are instrumental to ischemia-reperfusion induced changes in the heart. We tested the effect of quercetin on respiration of whole H9c2 cells, a model cell line derived from cardiomyoblasts. Brief treatment with quercetin up to 25μM had no effect, while 24 hour treatment with quercetin caused a decrease in the routine respiration relative to vehicle (