Search

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The customdesigned intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 x 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dualwavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state. [ABSTRACT FROM AUTHOR]

Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans. [ABSTRACT FROM AUTHOR]

Mitochondrial control of cellular redox states is a fundamental component of cell signaling in the coordination of core energy metabolism and homeostasis during normoxia and hypoxia. We investigated the relationship between cytochrome redox states and mitochondrial oxygen consumption at steady-state levels of hypoxia in mitochondria isolated from beef and mouse heart (BHImt, MHImt), comparing two species with different cardiac dynamics and local oxygen demands. A low-noise, rapid spectrophotometric system using visible light for the measurement of cytochrome redox states was combined with high-resolution respirometry. Monophasic hyperbolic relationships were observed between oxygen consumption, JO2, and oxygen partial pressure, PO2, within the range <1.1 kPa (8.3 mmHg; 13 μM). P50j (PO2 at 0.5.Jmax) was 0.015 ± 0.0004 and 0.021 ± 0.003 kPa (0.11 and 0.16 mmHg) for BHImt and MHImt, respectively. Maximum oxygen consumption, Jmax, was measured at saturating ADP levels (OXPHOS capacity) with Complex I-linked substrate supply. Redox states of cytochromes aa3 and c were biphasic hyperbolic functions of PO2. The relationship between cytochrome oxidation state and oxygen consumption revealed a separation of distinct phases from mild to severe and deep hypoxia. When cytochrome c oxidation increased from fully reduced to 45% oxidized at 0.1 Jmax, PO2 was as low as 0.002 kPa (0.02 μM), and trace amounts of oxygen are sufficient to partially oxidize the cytochromes. At higher PO2 under severe hypoxia, respiration increases steeply, whereas redox changes are small. Under mild hypoxia, the steep slope of oxidation of cytochrome c when flux remains more stable represents a cushioning mechanism that helps to maintain respiration high at the onset of hypoxia. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower-to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] atpH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi]. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase is the enzyme responsible for oxygen consumption by mitochondrial oxidative phosphorylation and coupling site 3 of oxidative phosphorylation. In this role it determines the cellular rate of ATP synthesis by oxidative phosphorylation and is the key to understanding how energy metabolism is regulated. Four electrons are required for the reduction of oxygen to water, and these are provided by the one-electron donor, cytochrome c. The rate of oxygen consumption (ATP synthesis) is dependent on the fraction of cytochrome c reduced (fred), oxygen pressure (pO2), energy state ([ATP]/[ADP][Pi]), and pH. In coupled mitochondria (high energy state) and pO2 >60 torr, the rate increases in an exponential-like fashion with increasing fred. When the dependence on fred is fitted to the equation rate = a(fred)b, a decreased from 100 to near 20, and b increased from 1.3 to 4 as the pH of the medium increased from 6.5 to 8.3. During oxygen depletion from the medium fred progressively increases and the rate of respiration decreases. The respiratory rate falls to ½ (P50) by about 1.5 torr, at which point fred is substantially increased. The metabolically relevant dependence on pO2 is obtained by correcting for the increase in fred, in which case the P50 is 12 torr. Adding an uncoupler of oxidative phosphorylation eliminates the dependence of the cytochrome c oxidase activity on pH and energy state. The respiratory rate becomes proportional to fred and the P50 decreases to less than 1 torr. [ABSTRACT FROM AUTHOR]

It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. The outcome of this signaling on mitochondrial enzyme complexes is yet to be understood. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with the specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 mm or subjected to four cycles of 5-mm ischemiareperfusion (PC) before 20-mm global ischemia and 120-mm reperfusion. PC and OSK Inhib VIII both improved recovery of postischemic left ventricular developed pressure, decreased infarct size, and reduced lactate production during ischemia compared with their time-matched controls. We used proteomics to examine mitochondrial protein levels/posttranslational modifications that were common between PC and OSK Inhib VIII. Levels of cytochrome-c oxidase subunits Va and Vib, ATP synthase-coupling factor 6, and cytochrome b-cl complex subunit 6 were increased while cytochrome c was decreased with PC and GSK Inhib VIII. Furthermore, the amount of cytochrome-c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function. [ABSTRACT FROM AUTHOR]

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

During low O2 (hypoxia), hypoxia-inducible factor (HIF)-α is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2α expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O2) caused a concentration- and time-dependent increase in HIF-2α induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (p0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 p~M), myxothiazol (1 μM), antimycin A (1 μg/ml), and cyanide (1 mM), blocked HIF-2α induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O2), HIF-2α induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2α induction. These data suggest that in this O2-sensitive cell line, mitochondrial 02 consumption, rather than changes in ROS, regulates HIF-2α during hypoxia. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion (l/R) injury is a commonly encountered clinical problem in liver surgery and transplantation. The pathogenesis of I/R injury is multifactorial, but mitochondrial Ca2+ overload plays a central role. We have previously defined a novel pathway for mitochondrial Ca2+ handling and now further characterize this pathway and investigate a novel Ca2+-channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB), for preventing hepatic I/R injury. The effect of 2-APB on cellular and mitochondrial Ca2+ uptake was evaluated in vitro by using 45Ca2+. Subsequently, 2-APB (2 mg/kg) or vehicle was injected into the portal vein of anesthetized rats either before or following 1 h of inflow occlusion to 70% of the liver. After 3 h of reperfusion, liver injury was assessed enzymatically and histologically. Hep O2 cells transfected with green fluorescent protein-tagged cytochrome c were used to evaluate mitochondrial permeability. 2-APB dose-dependently blocked Ca2+ uptake in isolated liver mitochondria and reduced cellular Ca2+ accumulation in Hep G2 cells. In vivo I/R increased liver enzymes 10-fold, and 2-APB prevented this when administered pre- or postischemia. 2-APB significantly reduced cellular damage determined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of liver tissue. In vitro I/R caused a dissociation between cytochrome c and mitochondria in Hep G2 cells that was prevented by administration of 2-APB. These data further establish the role of cellular Ca2+ uptake and subsequent mitochondrial Ca2+ overload in I/R injury and identify 2-APB as a novel pharmacological inhibitor of liver I/R injury even when administered following a prolonged ischemic insult. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with anti-oxidants has been shown to ameliorate these processes. The c-Jun NH2-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H2O2-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 μM H2O2, and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.4550 1, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients. [ABSTRACT FROM AUTHOR]

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H2O2 (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H2O2 and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, Vmax) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H2O2-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn2+-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy. [ABSTRACT FROM AUTHOR]

Cd2+ induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd2+-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 µM Cd2+ induced ∼5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosisinducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd2+ concentrations at 24 h, suggesting that elevated Cd 2+ may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd2+ caused swelling of energized isolated kidney cortex mitochondria (EC50 ∼9 µM) and cytochrome c release, which were independent: of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd2+ inhibited swelling and cytochrome c release induced by PTP openers (PO43- or H2O2 + Ca2+). The mitochondrial Ca2+ uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La3+, ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd2+ inhibition of PO43- -induced swelling. Using the Cd2+-sensitive fluorescent indicator FluoZin-1, Cd2+ was also taken up by mitoplasts. The aquaporin inhibitor AgNO3 abolished Cd2+-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd2+ concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to ’plugging’ of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9–5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis. [ABSTRACT FROM AUTHOR]

Examines the influence of polyamine depletion in preventing capotothecin-induced apoptosis. Inhibition of the release of cythocrome; Regulation of apoptosis in mammalian cells; Role of the mitochondria.