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(Circulation Research. 2003;93:192.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Uncoupling Protein-2 Overexpression Inhibits Mitochondrial Death Pathway in Cardiomyocytes

Yasushi Teshima, Masaharu Akao, Steven P. Jones, Eduardo Marbán

From the Institute of Molecular Cardiobiology, The Johns Hopkins University, Baltimore, Md.

Correspondence to Eduardo Marbán, MD, PhD, FACC, Institute of Molecular Cardiobiology, The Johns Hopkins University, 720 Rutland Ave, 844 Ross Building, Baltimore, MD 21205. E-mail marban{at}jhmi.edu

	Abstract

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Uncoupling proteins (UCPs) are located in the mitochondrial inner membrane and partially dissipate the transmembrane proton electrochemical gradient. UCP2 is expressed in various human and rodent tissues, including the heart, where its functional role is unknown. In the present study, we tested the hypothesis that UCP2 overexpression could protect cardiomyocytes from oxidative stress–induced cell death by reducing reactive oxygen species (ROS) production in mitochondria. Using an adenoviral vector containing human UCP2, we investigated the effects of UCP2 overexpression on the mitochondrial death pathway induced by oxidative stress (100 µmol/L H₂O₂) in cultured neonatal cardiomyocytes. UCP2 overexpression significantly suppressed markers of cell death, including TUNEL positivity, phosphatidylserine exposure, propidium iodide uptake, and caspase-3 cleavage. Furthermore, UCP2 remarkably prevented the catastrophic loss of mitochondrial inner membrane potential induced by H₂O₂, which is a critical early event in cell death. Ca²⁺ overload and the production of ROS in mitochondria, both of which contribute to mitochondrial inner membrane potential loss, were dramatically attenuated by UCP2 overexpression. Thus, overexpression of UCP2 attenuates ROS generation and prevents mitochondrial Ca²⁺ overload, revealing a novel mechanism of cardioprotection.

Key Words: heart • mitochondria • membrane potential • calcium • reactive oxygen species

	Introduction

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Uncoupling proteins (UCPs), which are located in the mitochondrial inner membrane, partially dissipate the proton electrochemical gradient.¹ The best characterized uncoupling protein, UCP1, is exclusively expressed in brown adipose tissue and is a key molecule in thermogenesis.¹ UCP2, another member of the UCP family,^2,3 is expressed in various tissues, including the brain, lung, spleen, kidney, liver, adipose tissues, and heart.^1–3 Recently, UCP2 was reported to regulate insulin secretion from pancreatic islets by regulating ATP concentration.^4–6 Furthermore, UCP2 reportedly contributed to immune response to infection by regulating reactive oxygen species (ROS) production.⁷ These findings suggest that the physiological role of UCP2 may be organ specific; however, its function in the heart remains unknown.

Mitochondrial ATP-sensitive potassium (mitoK_ATP) channels have cardioprotective effects against ischemia/reperfusion injury.^8,9 The mitoK_ATP channel opener diazoxide prevents apoptosis induced by oxidative stress in cultured cardiomyocytes.¹⁰ Partial depolarization of mitochondrial inner membrane potential (_m) achieved by the opening of mitoK_ATP channels and subsequent inhibition of mitochondrial Ca²⁺ overload have been proposed as potential mechanisms of this effect.^11,12 Similarly, low concentrations of mitochondrial uncouplers such as carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or 2,4-dinitrophenol were reported to have cytoprotective effects. FCCP prevented permeability transition pore (PTP) opening and ROS production induced by staurosporine or chelerythrine in isolated mitochondria from rat liver^13,14; 2,4-dinitrophenol reduced infarct size and preserved cardiac function in a rat model of myocardial ischemia/reperfusion injury.¹⁵ These results suggest that depolarization of _m is responsible for cytoprotection. Furthermore, UCP2 overexpression in murine macrophages reduced ROS generation induced by lipopolysaccharide.¹⁶

Based on these observations, we hypothesized that UCP2 may have protective effects against oxidative stress in the heart. Using an adenoviral vector, we investigated the effects of UCP2 overexpression on cell injury induced by hydrogen peroxide (H₂O₂) in cultured cardiomyocytes.

	Materials and Methods

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All procedures were performed in accordance with the Johns Hopkins University animal care guidelines, which conform to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Primary Culture of Neonatal Rat Cardiac Ventricular Myocytes
Cardiac ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats and cultured as previously described.¹⁰

In Vitro Transduction
Adenovirus (AdEGI-UCP2) that was generated previously⁴ was used in the present study. Briefly, adenovirus vectors were generated using Cre-lox recombination of purified 5 viral DNA and shuttle vector pAdEGI-UCP2. This vector coexpressed green fluorescent protein (GFP) and UCP2, driven by the inducible ecdysone promoter. The recombinant vectors were expanded and purified using cesium chloride gradients, yielding concentrations of 1x10¹⁰ plaque-forming units per milliliter. Functional expression was inferred from GFP visualization by fluorescence microscopy. The control vector (AdEGI) expressed only GFP, again driven by the ecdysone promoter. Transductions were carried out in culture medium (QBSF; Sigma) for 2 hours at 37°C on day 6 after the isolation of cardiomyocytes. Afterward, the myocytes were washed with virus-free medium and incubated with ponasterone A (Invitrogen) to induce expression from ecdysone-inducible promoter. The experiments were carried out at least 48 hours after transduction.

Immunoblot Analysis
Subcellular fractions of cells were prepared as described before.¹⁷ Immunoblots were performed as described.¹⁰ Primary antibodies for UCP2 and cytochrome c oxidase subunit IV were purchased from Alpha Diagnostic International and Molecular Probes, respectively.

Mitochondrial Oxygen Consumption Rate
For oxygen consumption measurements, cardiomyocytes were trypsinized for detachment from the dishes. After centrifugation, cells were resuspended in DMEM containing 25 mmol/L HEPES. Oxygen consumption was measured using an oxygen sensor (FOXY Fiber Optic Oxygen Sensors; Ocean Optics Inc) at 37°C in a stirred bath. The data for oxygen consumption rate were normalized by the protein concentration in each sample.

TUNEL Staining
TUNEL staining was performed according to the manufacturer’s protocol (Roche) at 16 hours after application of 100 µmol/L H₂O₂. Fluorescein labels incorporated in nucleotide polymers were detected by laser scanning confocal microscopy.

Caspase-3 Activity Assay
Caspase-3 activity was measured as described previously¹⁸ by detection of the cleavage of a colorimetric caspase-3 substrate, N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline, using an assay kit, ApoAlert CPP32 (Clontech), at 8 and 16 hours of 100 µmol/L H₂O₂ stimulation.

Loading of Cells With Fluorescent Indicators
For quantification of cellular viability, cells were double-stained with annexin V and propidium iodide (PI) according to manufacturer’s instructions (Roche). To monitor _m, cells were loaded with 100 nmol/L tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) at 37°C for 20 minutes. To monitor mitochondrial Ca²⁺ level, cells were loaded with 2 µmol/L rhod-2 AM (Molecular Probes) at 37°C for 30 minutes. We assayed ROS production, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, using chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, Molecular Probes). Cells were loaded with 2 µmol/L CM-H₂DCFDA at 37°C for 30 minutes, and the formation of the oxidized derivative, CM-DCF, was monitored.

Confocal Imaging
Cells plated on glass-bottom dishes were loaded with fluorescent probes as described above. After loading with each dye, cells were placed in phenol red–free DMEM supplemented with 25 mmol/L HEPES (pH 7.4). Cells were illuminated (488- and 568-nm lines of a krypton/argon laser), and images were taken by confocal microscopy (UltraVIEW; Perkin-Elmer). Time-lapse confocal imaging was carried out with various intervals between frames, using a x40 objective lens for DCF and a x20 objective lens for the other dyes.

Image Analysis
Quantitative image analysis was performed using image analysis software (ImageJ; http://rsb.info.nih.gov/ij/).

FACS Analysis
For FACS analysis of _m, TMRE-loaded cells were harvested by trypsinization at the end of experimental protocols and analyzed using FACScan (20 000 cells/sample) (Becton Dickinson). The fluorescence intensity of TMRE was monitored at 582 nm (FL-2 channel). FACS data were analyzed using WinMDI software (http://facs.scripps.edu).

Statistical Analysis
Data are presented as mean±SEM. Multiple comparisons among groups were carried out by one-way ANOVA with Fisher’s least-significant difference as the post hoc test. A level of P<0.05 was accepted as statistically significant.

	Results

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Adenovirus-Mediated Delivery of UCP2 to Neonatal Cardiomyocytes
To deliver UCP2 to cultured cardiomyocytes, we used a recombinant adenovirus containing the full-length human UCP2 cDNA. Figure 1A shows the results of immunoblot analysis. UCP2 protein level was increased in AdEGI-UCP2 (AdUCP2, 20 multiplicity of infection [moi])-transduced cells compared with AdEGI-transduced cells in the mitochondrial fraction but not in the cytosolic fraction. UCP2 protein level was increased virus volume dependently up to 20 moi, but there was no difference between 20 and 40 moi (n=3). In Figure 1B, cells were stained with TMRE to examine the effect of UCP2 expression on _m under basal conditions. AdUCP2-transduced cells showed partial depolarization of _m (gray area) compared with AdEGI-transduced cells (thick line), verifying functional overexpression of UCP2. This degree of _m dissipation is measurable but very modest compared with the catastrophic depolarization produced by the potent pharmacological uncoupler FCCP (thin line). We analyzed the intensity of fluorescence in the peak of the histogram in each group (n=5). Significant but moderate dissipation of _m was observed in AdUCP2 cells compared with AdEGI. There was no difference between 20 and 40 moi. In Figure 1C, UCP2-transduced cells showed modest but statistically significant increases of oxygen consumption compared with AdEGI cells (n=7, P<0.05). A chemical uncoupler, FCCP (0.5 µmol/L), increased oxygen consumption in both AdEGI and AdUCP2 cells to a similar (presumably maximal) level. However, the magnitude of the FCCP-induced increase was smaller in AdUCP2 compared with AdEGI. This is consistent with the idea that AdUCP2 cells have a higher uncoupled respiration under basal conditions. Conversely, sodium cyanide (5 mmol/L) suppressed the oxygen consumption rate in each group.

View larger version (44K): [in this window] [in a new window]   Figure 1. Overexpression of UCP2 in cardiomyocytes. A, Representative images and quantitative results of immunoblots from mitochondrial and cytosol fractions. Expression of cytochrome c oxidase subunit IV was examined to confirm successful fractionation and equal loading of protein in each lane. Data are mean±SEM (n=3). *P<0.05 vs AdEGI or AdUCP2 0 moi. B, Representative histograms and quantitative results show the effects of UCP2 overexpression on TMRE fluorescence. Partial depolarization was observed in AdUCP2-transduced cells. Thick line shows AdEGI-transduced cells. Gray area shows UCP2-transduced cells. FCCP was used to show the fluorescence intensity at completely depolarized cells (thin line). Data are mean±SEM (n=5). *P<0.05 vs AdUCP2 0 moi. C, Representative oxygen consumption curve and quantitative results show the effect of UCP2 overexpression on oxygen consumption. Data are mean±SEM (n=7). *P<0.05 vs AdEGI.

Effects of Overexpression of UCP2 on Cell Death
First, we examined the long-term effects of UCP2 overexpression on cell death. We performed TUNEL staining 16 hours after H₂O₂ application. Representative images and quantitative data are shown in Figures 2A and 2B, respectively (n=4 for each group from two independent experiments). In the control group (AdEGI), 15.9±0.6% of the cells showed TUNEL-positive nuclei, and exposure to H₂O₂ (AdEGI+H₂O₂) increased the number of TUNEL-positive nuclei to 33.7±1.7% (P<0.001). This increase of TUNEL positivity was significantly suppressed in UCP2-transduced cells treated with H₂O₂ (AdUCP2+H₂O₂) (19.2±0.5%) (P<0.001 versus AdEGI+H₂O₂). UCP2 overexpression without H₂O₂ did not affect TUNEL positivity. Activation of caspase-3 is a critical step in the process of cell death.¹⁹ Figure 2C shows relative caspase-3 activity in each group at 8 and 16 hours after H₂O₂ application. Caspase-3 activity was significantly increased in AdEGI+H₂O₂ compared with AdEGI at each time point (P<0.001), and UCP2 overexpression significantly suppressed these increases (P<0.05 and P<0.001 versus AdEGI+H₂O₂ at 8 hours and 16 hours, respectively) (n=3 for each data point).

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of UCP2 overexpression on cell death. A, Representative images of TUNEL staining in each group. B, Quantitative percentages of the cells with TUNEL-positive nuclei. UCP2 overexpression suppressed the increase of TUNEL positivity induced by H2O2. Data are mean±SEM (n=4 for each group). *P<0.001 vs AdEGI; #P<0.001 vs AdEGI+H2O2. C, Effects of UCP2 overexpression on caspase-3 activity at 8 and 16 hours after H2O2 application. Average of control value at each time point is defined as 100%. Data at each time point are relative percentage of caspase-3 activity to control at the same time point. There are no actual data at 0 hours. UCP2 overexpression mitigated the activation of caspase-3 induced by H2O2 at each time point. Data are mean±SEM (n=3 for each group). *P<0.001 vs AdEGI; #P<0.05 vs AdEGI+H2O2; **P<0.001 vs AdEGI+H2O2.

Effects of Overexpression of UCP2 on Early Phase of Cell Death
To assess the effects of UCP2 overexpression in the early phase of cell injury, we investigated the alterations of annexin V and PI fluorescence in each group. Time-lapse confocal microscopy at 5-minute intervals began immediately after application of 100 µmol/L H₂O₂. In AdEGI+H₂O₂, annexin V fluorescence started to increase 20 minutes after H₂O₂ application and gradually increased to a plateau at 100 minutes (Figures 3A and 3B). UCP2 remarkably suppressed this increase of annexin V fluorescence (Figures 3A and 3B). PI fluorescence was increased after 90 minutes of latency and reached the plateau at 150 minutes in AdEGI+H₂O₂ (Figures 3C and 3D). This increase of PI fluorescence was also abrogated by UCP2 overexpression (Figures 3C and 3D). After 150 minutes of scanning, cells were permeabilized with saponin (Sigma) to calculate the percentage of PI-positive cells in each group. In AdEGI+H₂O₂, 86.3±3.4% of cells were PI-positive at the end of scanning. In contrast, only 7.5±4.9% of cells were PI-positive in AdUCP2+H₂O₂ (n=3, P<0.001) (Figure 3D, inset). Because annexin V is an indicator of apoptosis and PI of necrosis, these results suggest that UCP2 overexpression reduces both types of cell death.

View larger version (38K): [in this window] [in a new window]   Figure 3. Effects of UCP2 overexpression on early phase of cell death. A, Representative images of sequential changes of annexin V fluorescence in response to H2O2 application in each group. B, Quantitative results of time-lapse confocal microscopy using annexin V. Each plot is the average of 10 areas from one scanning. Similar results were obtained from 3 independent experiments. C, Representative images of sequential changes of PI fluorescence in response to H2O2 application in each group. D, Quantitative results of time-lapse confocal microscopy using PI. Each plot is the average of 10 areas from one scanning. Similar results were obtained from 3 independent experiments. The bar graph inserted shows the percentages of PI-positive cells at the end of scanning (n=3, P<0.01). Increases of annexin V and PI fluorescence induced by H2O2 were abrogated by UCP2 overexpression.

Effects of UCP2 Overexpression on m
Dissipation of _m is a critical event early in the process of cell death.²⁰ To examine whether preservation of _m is associated with cardioprotective effects of UCP2, we assessed the change of TMRE fluorescence by H₂O₂ stimulation in each group using FACS analysis. Incubation with 100 µmol/L H₂O₂ for 1 hour decreased TMRE fluorescence and shifted the distribution curve leftward, indicating the depolarization of _m (Figure 4A, top). In AdUCP2+H₂O₂, the decrease of TMRE fluorescence was remarkably suppressed, indicating the preservation of _m (Figure 4A, bottom). Summarized data from FACS analysis are shown in Figures 4B and 4C. We evaluated the percentage of cells that exhibited a high level of TMRE fluorescence (defined as >5x10¹ in this analysis). UCP2 preserved _m virus volume dependently (n=3 for each group) (Figure 4B). Treatment with 1 µmol/L ponasterone A without AdUCP2 transfection had no effect (data not shown). Inhibition of mitoK_ATP channels by 5-hydroxydecanoate (5HD, 500 µmol/L) or glibenclamide (10 µmol/L) did not cancel _m preservation by UCP2 overexpression (n=3 for each group) (Figure 4C). Thus, whereas the effects of UCP2 mimic those of mitoK_ATP channel openers,²¹ they are not dependent on mitoK_ATP channel activation.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of UCP2 overexpression on m. A, Representative histograms obtained by FACS analysis. Cells were loaded with TMRE. Top, Gray area shows AdEGI group. Black line shows AdEGI+H2O2. Bottom, Representative histogram from AdUCP2+H2O2. B, Quantitative results of FACS analysis. UCP2 overexpression preserved m virus volume dependently. C, Effects of 5HD or glibenclamide on m preservation by UCP2 overexpression. Neither 5HD nor glibenclamide canceled the effects of UCP2. Data are expressed as percentage of cells with high TMRE fluorescence (defined as >5x101 of fluorescence intensity). Data are mean±SEM (n=3 per group). *P<0.05 vs AdEGI+H2O2. glib indicates glibenclamide.

Time-Lapse Analysis of m Loss
To examine time-dependent changes of _m on a single-cell basis, confocal microscopy was performed using cells loaded with TMRE. Time-lapse scanning began immediately after application of 100 µmol/L H₂O₂. At first, we confirmed that TMRE fluorescence did not change during 70 minutes of scanning in AdEGI (Figure 5A). In contrast, cells treated with H₂O₂ progressively lost red fluorescence intensity, indicating irreversible dissipation of _m (Figure 5B). TMRE fluorescence was remarkably preserved in AdUCP2+H₂O₂ (Figure 5C). Twenty cells were randomly selected in each group, and TMRE fluorescence intensity from each cell was plotted in Figure 5D. TMRE fluorescence intensity of individual cells rapidly decreased in AdEGI+H₂O₂, but UCP2 overexpression prevented the loss of _m induced by H₂O₂ in most cells. The bottom panel in Figure 5D shows the average of TMRE fluorescence intensity from 20 randomly selected cells in each group.

View larger version (89K): [in this window] [in a new window]   Figure 5. Time-lapse analysis of m loss. A through C, Representative sequential images of TMRE fluorescence in each group. A, AdEGI. B, AdEGI+H2O2. C, AdUCP2+H2O2. D, Time course of TMRE fluorescence of 20 cells randomly selected in each group. Bottom graph shows mean fluorescence intensity in each group. UCP2 overexpression prevented rapid decrease of TMRE fluorescence that was observed in AdEGI+H2O2. Similar results were obtained from 3 independent experiments.

Effects of UCP2 Overexpression on Mitochondrial Ca²⁺ Overload
Dissipation of _m is caused by the opening of PTP. Ca²⁺ overload in mitochondria is one of the critical triggers of cell death and is known to open the PTP. We examined the effects of UCP2 overexpression on mitochondrial Ca²⁺ level using the mitochondrial Ca²⁺-sensitive dye rhod-2. Time-lapse confocal microscopy began after the addition of 100 µmol/L H₂O₂. We first confirmed that rhod-2 fluorescence did not change during 70 minutes of scanning in AdEGI (Figure 6A). Rhod-2 fluorescence was rapidly and dramatically augmented in AdEGI+H₂O₂ at 30 minutes after H₂O₂ application (Figure 6B). UCP2 overexpression almost completely inhibited the Ca²⁺ surge observed in AdEGI+H₂O₂ (Figure 6C). Figure 6D shows the average of rhod-2 fluorescence intensity from 20 randomly selected cells in each group.

View larger version (48K): [in this window] [in a new window]   Figure 6. Time-lapse analysis of mitochondrial Ca2+ level. A through C, Representative sequential images of rhod-2 fluorescence in each group. A, AdEGI. B, AdEGI+H2O2. C, AdUCP2+H2O2. D, Quantitative data of rhod-2 fluorescence. Each line indicates mean fluorescence intensity of 20 cells randomly selected in each group. UCP2 overexpression abrogated rapid increase of rhod-2 fluorescence that was observed in AdEGI+H2O2. Similar results were obtained from 3 independent experiments.

Effects of UCP2 Overexpression on ROS Production
ROS is another trigger of PTP opening. We investigated the effects of UCP2 overexpression on ROS production using time-lapse confocal microscopy. No changes in fluorescence were observed in AdEGI (Figure 7B, ). In AdEGI+H₂O₂, DCF fluorescence increased immediately after H₂O₂ application and gradually reached a plateau at 40 minutes (Figures 7A and 7B, ). UCP2 overexpression abrogated the increase in DCF fluorescence (Figures 7A and 7B, . Furthermore, the effects of UCP2 overexpression on ROS production were confirmed using an entirely different form of oxidant stress, namely doxorubicin, which exerts cardiotoxicity by increasing ROS production in mitochondria.^22,23 Incubation with 50 µmol/L doxorubicin for 3 hours markedly increased DCF fluorescence compared with AdEGI (P<0.001) (Figure 7C), but UCP2 overexpression significantly prevented the increase of fluorescence (P<0.001 versus AdEGI+doxorubicin) (n=6 for each group from two independent experiments) (Figure 7C).

View larger version (40K): [in this window] [in a new window]   Figure 7. Effects of UCP2 overexpression on ROS production. A, Representative images of sequential changes of DCF fluorescence in response to H2O2 application. B, Quantitative changes of DCF fluorescence using time-lapse confocal microscopy. Each plot is average of 10 areas from one scanning. Similar results were obtained from 3 independent experiments. , AdEGI group; , AdEGI+H2O2; , AdUCP2+H2O2. C, Effects of UCP2 overexpression on doxorubicin (Dox)-induced ROS production. Representative images are shown in the groups AdEGI, AdEGI+Dox, and AdUCP2+Dox. Quantitative data are also shown as bar graph in each group. Data are mean±SEM (n=6 for each group from 2 independent experiments). *P<0.001 vs AdEGI; #P<0.001 vs AdEGI+Dox.

	Discussion

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The major findings in the present study are as follows. First, UCP2 overexpression suppressed markers of cell death such as increased TUNEL positivity, caspase-3 activity, phosphatidylserine exposure, and PI positivity induced by oxidative stress. Second, mitochondrial Ca²⁺ overload and increased ROS production in mitochondria, and subsequent _m loss induced by oxidative stress, were prevented by UCP2 overexpression in a dose-dependent manner. These results demonstrate that overexpression of UCP2 protects cardiomyocytes against oxidative stress by preserving the integrity of early processes that would normally be recruited as part of the mitochondrial death pathway.

Cardiomyocytes contain abundant mitochondria to maintain cardiac function, which requires a large amount of energy. The plethora of mitochondria can potentially produce ROS that contribute to ischemia/reperfusion injury in the heart.²⁴ The mitochondrial respiratory chain generates ROS, especially in state IV respiration, when the electrochemical gradient between the mitochondrial inner membrane is high and the rate of electron transport is limited. In this context, UCP2 should affect ROS production by dissipating the electrochemical gradient. Indeed, the association of UCP2 and ROS production has been described in several reports. GDP, an inhibitor of UCP2, raised mitochondrial membrane potential and increased H₂O₂ production in mitochondrial fractions of liver, spleen, and thymus.²⁵ Macrophages from mice lacking UCP2 generate more ROS than wild-type mice and had a remarkably increased resistance to Toxoplasma gondii infection.⁷ Transfection of UCP2 cDNA in murine macrophage cell line reduced lipopolysaccharide-induced intracellular ROS production,¹⁶ and the lack of UCP2 in blood cells accelerates atherosclerotic plaque development, probably by increasing ROS production.²⁶ This evidence strongly supports the possibility that the principal role of UCP2 is regulation of ROS production in mitochondria, although the precise mechanism of this effect has not been established. Furthermore, a recent report demonstrated that superoxide activated UCPs in various tissues, indicating that UCPs may be recruited endogenously as a compensatory mechanism to counteract oxidative stress.²⁷ Consistent with these observations, UCP2 overexpression reduced ROS production in the present study. Exogenous H₂O₂-induced ROS production may not originate only from mitochondrial respiration but also from other reactions, such as the Fenton reaction or interactions with mitochondrial respiratory components. Therefore, we cannot be certain that reduced ROS production was caused by increased respiration rate in mitochondria. The present result that UCP2 overexpression suppressed the increase in ROS production by doxorubicin, which is known to increase ROS production in mitochondria,^22,28,29 indirectly supports our hypothesis. As another possibility, interaction of UCP2 with coenzyme Q, may prevent coenzyme Q from H₂O₂-oxidizing action and thereby reduce ROS production.³⁰

This is the first study that shows UCP2 overexpression inhibited mitochondrial Ca²⁺ overload, an important trigger of the mitochondrial death pathway. One possible mechanism is that this effect may simply be attributable to the partial depolarization of _m, which decreases the driving force for Ca²⁺ uptake into the mitochondrial matrix. Otherwise, inhibition of Ca²⁺ overload may be a secondary effect of reduced ROS production. In fact, ROS was reported to trigger Ca²⁺ increase during hypoxia in pulmonary arterial myocytes.³¹ On the contrary, ROS levels under oxidative stress may depend on extracellular Ca²⁺ concentration.³² Therefore, ROS and Ca²⁺ overload may be tightly coupled and there could be a mutual requirement for each to reach its maximal levels.³³

PTP is a major player in the cell death pathway. Opening of PTP results in the loss of _m, massive swelling of mitochondria, rupture of the outer membrane, and release of intermembrane components that induce cell death.³⁴ Mitochondrial Ca²⁺ overload and ROS favor PTP opening.^35,36 Hence, inhibition of _m loss by UCP2 overexpression may be mediated by prevention of Ca²⁺ overload or ROS production in mitochondria, such that PTP never opens.

Activation of mitoK_ATP channels has cardioprotective effects, and inhibition of mitochondrial Ca²⁺ overload and subsequent preservation of _m are considered the mechanisms of these effects.^10–12 We examined the possibility that the cardioprotective effects by UCP2 overexpression observed in the present study may be mediated by activation of mitoK_ATP channels. Neither 5HD nor glibenclamide altered the effects of UCP2 overexpression, indicating that the cardioprotection of UCP2 is not dependent on mitoK_ATP channels. Nevertheless, the fact that UCP2 overexpression mimics the effects of mitoK_ATP channel openers supports the idea that mitoK_ATP channels may be cardioprotective because they are mild uncouplers.¹¹

Although the present findings convincingly demonstrate protective effects of UCP2 overexpression, other models provide discordant results. Namely, a recent report demonstrated that overexpression of UCP2 in HeLa cells prompted cellular oncosis by dramatically decreasing the mitochondrial membrane potential.³⁷ Although we cannot define the reason for the discrepancy, UCP2 overexpression may result in different downstream effects in various cell types. In fact, UCP2 overexpression did not alter the growth and viability of fibroblasts, while causing fatal response in HeLa cells.³⁷ Furthermore, UCP2 negatively regulates insulin secretion in pancreatic ß cells.^4–6 In macrophages, UCP2 contributes to immune function by regulating ROS production.⁷ A recent study, published while the present article was in revision, demonstrated that transfection of UCP1 conferred cell-protective effects against hypoxia/reoxygenation in H9c2 cells.³⁸ Another study showed that UCP2 expression levels were inversely correlated with caspase-3 activation in neurons.³⁹ Taking these results together, the physiological role of UCP2 may actually be similar in the brain and heart.

Since the discovery of UCP2, several reports have described the regulation of UCP2 mRNA expression in the heart. Thyroid hormone, ß-adrenergic stimulation,⁴⁰ and fatty acids⁴¹ are known as regulators of UCP2 expression. However, the regulation and the functional role of UCP2 in the pathological heart are largely unexplored. In a recent study, UCP2 expression was increased in the hearts of rats with cardiomyopathy induced by aortic regurgitation.⁴² In contrast, UCP2 expression was decreased in human hearts with idiopathic cardiomyopathy, ischemic cardiomyopathy, and peripartum cardiomyopathy.⁴³ These studies shed some light on UCP2 expression in the pathological heart, but the functional role is still obscure. Although the results of the present study are restricted to cultured neonatal cardiomyocytes, they suggest that UCP2 may mitigate ischemia-reperfusion injury by reducing cell death. Future studies are necessary to identify the effects of UCP2 overexpression or upregulation on cardiac function in adult heart.

	Acknowledgments

 
This study was supported by the NIH (R37 HL36957) and the Banyu Fellowship in Cardiovascular Medicine (to M.A.). E.M. holds the Michel Mirowski, M.D., Professorship of Cardiology. The authors appreciate the technical assistance of Drs O’Rourke and Cortassa in oxygen consumption measurements.

	Footnotes

 
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received February 5, 2003; revision received June 25, 2003; accepted June 27, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ricquier D, Bouillaud F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem J. 2000; 345: 161–179.[CrossRef][Medline] [Order article via Infotrieve]
2. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet. 1997; 15: 269–272.[CrossRef][Medline] [Order article via Infotrieve]
3. Gimeno RE, Dembski M, Weng X, Deng N, Shyjan AW, Gimeno CJ, Iris F, Ellis SJ, Woolf EA, Tartaglia LA. Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis. Diabetes. 1997; 46: 900–906.[Abstract]
4. Chan CB, MacDonald PE, Saleh MC, Johns DC, Marbán E, Wheeler MB. Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets. Diabetes. 1999; 48: 1482–1486.[Abstract]
5. Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, ß cell dysfunction, and type 2 diabetes. Cell. 2001; 105: 745–755.[CrossRef][Medline] [Order article via Infotrieve]
6. Joseph JW, Koshkin V, Zhang CY, Wang J, Lowell BB, Chan CB, Wheeler MB. Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet. Diabetes. 2002; 51: 3211–3219.[Abstract/Free Full Text]
7. Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet. 2000; 26: 435–439.[CrossRef][Medline] [Order article via Infotrieve]
8. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K⁺ channels: possible mechanism of cardioprotection. Circ Res. 1997; 81: 1072–1082.[Abstract/Free Full Text]
9. Liu Y, Sato T, O’Rourke B, Marbán E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998; 97: 2463–2469.[Abstract/Free Full Text]
10. Akao M, Ohler A, O’Rourke B, Marbán E. Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells. Circ Res. 2001; 88: 1267–1275.[Abstract/Free Full Text]
11. Murata M, Akao M, O’Rourke B, Marbán E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca²⁺ overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res. 2001; 89: 891–898.[Abstract/Free Full Text]
12. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K⁺ channel openers prevent Ca²⁺ overload in rat cardiac mitochondria. J Physiol. 1999; 519: 347–360.[Abstract/Free Full Text]
13. Aronis A, Komarnitsky R, Shilo S, Tirosh O. Membrane depolarization of isolated rat liver mitochondria attenuates permeability transition pore opening and oxidant production. Antioxid Redox Signal. 2002; 4: 647–654.[CrossRef][Medline] [Order article via Infotrieve]
14. Stoetzer OJ, Pogrebniak A, Pelka-Fleischer R, Hasmann M, Hiddemann W, Nuessler V. Modulation of apoptosis by mitochondrial uncouplers: apoptosis-delaying features despite intrinsic cytotoxicity. Biochem Pharmacol. 2002; 63: 471–483.[CrossRef][Medline] [Order article via Infotrieve]
15. Minners J, van den Bos EJ, Yellon DM, Schwalb H, Opie LH, Sack MN. Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for a mitochondrial role in cardioprotection. Cardiovasc Res. 2000; 47: 68–73.[Abstract/Free Full Text]
16. Kizaki T, Suzuki K, Hitomi Y, Taniguchi N, Saitoh D, Watanabe K, Onoe K, Day NK, Good RA, Ohno H. Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci U S A. 2002; 99: 9392–9397.[Abstract/Free Full Text]
17. Akao M, O’Rourke B, Teshima Y, Seharaseyon J, Marbán E. Mechanically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes. Circ Res. 2003; 92: 186–194.[Abstract/Free Full Text]
18. Akao M, Teshima Y, Marbán E. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol. 2002; 40: 803–810.[Abstract/Free Full Text]
19. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281: 1312–1316.[Abstract/Free Full Text]
20. Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 1998; 60: 619–642.[CrossRef][Medline] [Order article via Infotrieve]
21. Akao M, O’Rourke B, Kusuoka H, Teshima Y, Jones SP, Marbán E. Differential actions of cardioprotective agents on the mitochondrial death pathway. Circ Res. 2003; 92: 195–202.[Abstract/Free Full Text]
22. Davies KJ, Doroshow JH. Redox cycling of anthracyclines by cardiac mitochondria, I: anthracycline radical formation by NADH dehydrogenase. J Biol Chem. 1986; 261: 3060–3067.[Abstract/Free Full Text]
23. Doroshow JH, Davies KJ. Redox cycling of anthracyclines by cardiac mitochondria, II: formation of superoxide anion, hydrogen peroxide, and hydroxyl radical. J Biol Chem. 1986; 261: 3068–3074.[Abstract/Free Full Text]
24. Lefer DJ, Granger DN. Oxidative stress and cardiac disease. Am J Med. 2000; 109: 315–323.[CrossRef][Medline] [Order article via Infotrieve]
25. Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L, Casteilla L. A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 1997; 11: 809–815.[Abstract]
26. Blanc J, Alves-Guerra MC, Esposito B, Rousset S, Gourdy P, Ricquier D, Tedgui A, Miroux B, Mallat Z. Protective role of uncoupling protein 2 in atherosclerosis. Circulation. 2003; 107: 388–390.[Abstract/Free Full Text]
27. Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD. Superoxide activates mitochondrial uncoupling proteins. Nature. 2002; 415: 96–99.[CrossRef][Medline] [Order article via Infotrieve]
28. Ogura R, Sugiyama M, Haramaki N, Hidaka T. Electron spin resonance studies on the mechanism of adriamycin-induced heart mitochondrial damages. Cancer Res. 1991; 51: 3555–3558.[Abstract/Free Full Text]
29. Doroshow JH. Anthracycline antibiotic-stimulated superoxide, hydrogen peroxide, and hydroxyl radical production by NADH dehydrogenase. Cancer Res. 1983; 43: 4543–4551.[Abstract/Free Full Text]
30. Echtay KS, Winkler E, Klingenberg M. Coenzyme Q is an obligatory cofactor for uncoupling protein function. Nature. 2000; 408: 609–613.[CrossRef][Medline] [Order article via Infotrieve]
31. Waypa GB, Marks JD, Mack MM, Boriboun C, Mungai PT, Schumacker PT. Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes. Circ Res. 2002; 91: 719–726.[Abstract/Free Full Text]
32. Sharikabad MN, Hagelin EM, Hagberg IA, Lyberg T, Brors O. Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation. J Mol Cell Cardiol. 2000; 32: 441–452.[CrossRef][Medline] [Order article via Infotrieve]
33. Tan S, Sagara Y, Liu Y, Maher P, Schubert D. The regulation of reactive oxygen species production during programmed cell death. J Cell Biol. 1998; 141: 1423–1432.[Abstract/Free Full Text]
34. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999; 341: 233–249.[CrossRef][Medline] [Order article via Infotrieve]
35. Hunter DR, Haworth RA. The Ca²⁺-induced membrane transition in mitochondria, III: transitional Ca²⁺ release. Arch Biochem Biophys. 1979; 195: 468–477.[CrossRef][Medline] [Order article via Infotrieve]
36. Crompton M, Costi A, Hayat L. Evidence for the presence of a reversible Ca²⁺-dependent pore activated by oxidative stress in heart mitochondria. Biochem J. 1987; 245: 915–918.[Medline] [Order article via Infotrieve]
37. Mills EM, Xu D, Fergusson MM, Combs CA, Xu Y, Finkel T. Regulation of cellular oncosis by uncoupling protein 2. J Biol Chem. 2002; 277: 27385–27392.[Abstract/Free Full Text]
38. Bienengraeber M, Ozcan C, Terzic A. Stable transfection of UCP1 confers resistance to hypoxia/reoxygenation in a heart-derived cell line. J Mol Cell Cardiol. 2003; 35: 861–865.[CrossRef][Medline] [Order article via Infotrieve]
39. Bechmann I, Diano S, Warden CH, Bartfai T, Nitsch R, Horvath TL. Brain mitochondrial uncoupling protein 2 (UCP2): a protective stress signal in neuronal injury. Biochem Pharmacol. 2002; 64: 363–367.[CrossRef][Medline] [Order article via Infotrieve]
40. Teshima Y, Saikawa T, Yonemochi H, Hidaka S, Yoshimatsu H, Sakata T. Alteration of heart uncoupling protein-2 mRNA regulated by sympathetic nerve and triiodothyronine during postnatal period in rats. Biochim Biophys Acta. 1999; 1448: 409–415.[Medline] [Order article via Infotrieve]
41. Van Der Lee KA, Willemsen PH, Van Der Vusse GJ, Van Bilsen M. Effects of fatty acids on uncoupling protein-2 expression in the rat heart. FASEB J. 2000; 14: 495–502.[Abstract/Free Full Text]
42. Murakami K, Mizushige K, Noma T, Tsuji T, Kimura S, Kohno M. Perindopril effect on uncoupling protein and energy metabolism in failing rat hearts. Hypertension. 2002; 40: 251–255.[Abstract/Free Full Text]
43. Razeghi P, Young ME, Alcorn JL, Moravec CS, Frazier OH, Taegtmeyer H. Metabolic gene expression in fetal and failing human heart. Circulation. 2001; 104: 2923–2931.[Abstract/Free Full Text]

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