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1.  A new twist on an old idea part 2: cyclosporine preserves normal mitochondrial but not cardiomyocyte function in mini‐swine with compensated heart failure 
Physiological Reports  2014;2(6):e12050.
We recently developed a clinically relevant mini‐swine model of heart failure with preserved ejection fraction (HFpEF), in which diastolic dysfunction was associated with increased mitochondrial permeability transition (MPT). Early diastolic function is ATP and Ca2+‐dependent, thus, we hypothesized chronic low doses of cyclosporine (CsA) would preserve mitochondrial function via inhibition of MPT and subsequently maintain normal cardiomyocyte Ca2+ handling and contractile characteristics. Left ventricular cardiomyocytes were isolated from aortic‐banded Yucatan mini‐swine divided into three groups; control nonbanded (CON), HFpEF nontreated (HF), and HFpEF treated with CsA (HF‐CsA). CsA mitigated the deterioration of mitochondrial function observed in HF animals, including functional uncoupling of Complex I‐dependent mitochondrial respiration and increased susceptibility to MPT. Attenuation of mitochondrial dysfunction in the HF‐CsA group was not associated with commensurate improvement in cardiomyocyte Ca2+ handling or contractility. Ca2+ transient amplitude was reduced and transient time to peak and recovery (tau) prolonged in HF and HF‐CsA groups compared to CON. Alterations in Ca2+ transient parameters observed in the HF and HF‐CsA groups were associated with decreased cardiomyocyte shortening and shortening rate. Cellular function was consistent with impaired in vivo systolic and diastolic whole heart function. A significant systemic hypertensive response to CsA was observed in HF‐CsA animals, and may have played a role in the accelerated the development of heart failure at both the whole heart and cellular levels. Given the significant detriment to cardiac function observed in response to CsA, our findings suggest chronic CsA treatment is not a viable therapeutic option for HFpEF.
In a recently developed a translational mini‐swine model of heart failure with preserved ejection fraction (HFpEF), we hypothesized inhibiting mitochondrial permeability transition using cyclosporine (CsA) would improve cardiomyocyte function and calcium handling by supporting mitochondrial function. The purpose of this study was to examine the impact of inhibiting cyclophilin D on mitochondrial function and subsequent cardiomyocyte calcium handling using a reduced, nonimmunosuppressive dose of CsA chronically. We found improved mitochondrial function following chronic CsA treatment was not associated with a parallel improvement in cardiomyocyte calcium handling and contractile function, and demonstrate for the first time impaired cardiomyocyte calcium handling and contractile function are present early in the disease process in our HFpEF model.
PMCID: PMC4208639  PMID: 24963034
Calcium; cardiomyocyte; cyclosporine; HFpEF; mitochondria
2.  Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells 
Anesthesiology  2010;113(4):906-916.
Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients, relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning.
H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain 2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters.
Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening.
APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.
PMCID: PMC2945423  PMID: 20823757
Circulation  2008;118(19):1970-1978.
Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning (PC) but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia-reperfusion injury.
Methods and Results
Constitutive overexpression of iNOS in transgenic (TG) mice (α-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but profoundly decreased infarct size in mice subjected to 30 min of coronary occlusion and 24 h of reperfusion. In comparison with wild-type (WT) hearts, isolated iNOS-transgenic (TG) hearts subjected to ischemia for 30 min followed by 40 min of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[3H]-glucose (DOG). Reperfusion-induced loss of NAD+ and mitochondrial release of cytochrome c were attenuated in iNOS-TG hearts, indicating reduced mitochondrial permeability transition (MPT). The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and MPT-induced NAD+ loss was decreased in WT but not iNOS-null mice treated with the NO donor DETA/NO, 24 h before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-TG hearts and was increased in WT hearts treated with the MPT inhibitor cyclosporin A.
Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.
PMCID: PMC2763350  PMID: 18936326
ischemia; reperfusion; nitric oxide; EPR; and free radicals
4.  Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes 
Basic research in cardiology  2011;106(3):385-396.
Protection achieved by ischemic preconditioning is dependent on A2B adenosine receptors (A2BAR) in rabbit and mouse hearts and, predictably, an A2BAR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A2BAR. The present study tested whether A2BAR could be demonstrated on rat cardiomyocytes.
Methods and Results
Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A2BAR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4±2.0 % of the risk zone in control hearts to 19.9±2.8 % indicating that A2BAR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A2BAR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A2BAR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A2BAR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A2BAR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A2BAR band.
Thus the present study demonstrates that activation of A2BAR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A2BAR are expressed in individual cardiomyocytes. However, surprisingly, A2BAR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A2BAR signalling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.
PMCID: PMC3533442  PMID: 21246204
adenosine A2B receptors; cardioprotection; mitochondria
5.  Dietary ω-3 Fatty Acids Alter Cardiac Mitochondrial Phospholipid Composition and Delay Ca2+-Induced Permeability Transition 
Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA+DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA+DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation (n=10–14), and fed a standard diet, or supplemented with EPA+DHA (2.3% of energy intake) for 12 weeks. EPA+DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA+DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA+DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA+DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.
PMCID: PMC2783943  PMID: 19703463
eicosapentaenoic acid; docosahexaenoic acid; myocardial infarction; mitochondrial permeability transition pore
6.  Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout 
BMC Cell Biology  2009;10:90.
Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.
Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria.
The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts.
PMCID: PMC2806299  PMID: 20017912
7.  Abnormal Mitochondrial L-Arginine Transport Contributes to the Pathogenesis of Heart Failure and Rexoygenation Injury 
PLoS ONE  2014;9(8):e104643.
Impaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury.
Methods and Results
In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress.
These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.
PMCID: PMC4128716  PMID: 25111602
8.  Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte 
Cell Stress & Chaperones  2004;9(3):281-293.
Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+-adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress–induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress–induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.
PMCID: PMC1065287  PMID: 15544166
9.  BMAP-28, an Antibiotic Peptide of Innate Immunity, Induces Cell Death through Opening of the Mitochondrial Permeability Transition Pore 
Molecular and Cellular Biology  2002;22(6):1926-1935.
BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca2+ and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.
PMCID: PMC135593  PMID: 11865069
10.  Bnip3 Mediates Permeabilization of Mitochondria and Release of Cytochrome c via a Novel Mechanism 
Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with loss of cardiac myocytes after a myocardial infarction. Previous studies have demonstrated that Bnip3 induces mitochondrial dysfunction, but the mechanisms involved in this process remain unknown. In this study, we demonstrate that Bnip3 induces permeabilization of the mitochondria via a novel mechanism that is different from other BH3-only proteins. We found that Bnip3 induced mitochondrial swelling and cytochrome c release in isolated heart mitochondria in vitro. Another BH3-only protein, tBid, also caused release of cytochrome c but failed to induce swelling of mitochondria. Swelling of mitochondria is a characteristic of mitochondrial permeability transition pore (mPTP) opening, but Bnip3-mediated mitochondrial swelling was insensitive to cyclosporine A, an inhibitor of the mPTP and independent of cyclophilin D (cypD), an essential component of the mPTP. Bnip3 also induced permeabilization of the mitochondrial membranes as evident by calcein release from the matrix in both wild type (WT) and cypD deficient mouse embryonic fibroblasts (MEFs). Moreover, Bnip3 induced mitochondrial matrix remodeling and large amplitude swelling of the inner membrane, which led to disassembly of OPA1 complexes and release from the mitochondria. Thus, these studies suggest that Bnip3 mediates mitochondrial permeabilization by a novel mechanism that is different from other BH3-only proteins.
PMCID: PMC2866782  PMID: 20025887
Bnip3; Bcl-2; BH3-only proteins; OPA1; mitochondria; cardiac myocytes; apoptosis; necrosis; mitochondrial permeability transition pore; cyclophilin D
11.  A comparison of Zn2+- and Ca2+- triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition 
Cell calcium  2009;45(5):10.1016/j.ceca.2009.03.002.
Intracellular Zn2+ toxicity is associated with mitochondrial dysfunction. Zn2+ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn2+-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn2+-induced depolarization with the effects of Ca2+ in single isolated rat liver mitochondria monitored with the potentiometric probe Rhodamine123. Consistent with previous work, we found that relatively low levels of Ca2+ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg2+, ADP and cyclosporine A. Zn2+ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn2+-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca2+ and Zn2+ in a calcein-retention assay. Consistent with the well-documented ability of Ca2+ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn2+-treated mitochondria. Considered together, our results suggest that Ca2+ and Zn2+ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn2+-induced depolarization, and that Zn2+ is not a particularly potent mitochondrial inhibitor.
PMCID: PMC3815704  PMID: 19349076
Intracellular zinc; intracellular calcium; reactive oxygen species; rhodamine123; mitochondrial transmembrane potential; cyclosporine A
12.  Lysosomal Iron Mobilization and Induction of the Mitochondrial Permeability Transition in Acetaminophen-Induced Toxicity to Mouse Hepatocytes 
Toxicological Sciences  2010;117(1):101-108.
Acetaminophen induces the mitochondrial permeability transition (MPT) in hepatocytes. Reactive oxygen species (ROS) trigger the MPT and play an important role in AAP-induced hepatocellular injury. Because iron is a catalyst for ROS formation, our aim was to investigate the role of chelatable iron in MPT-dependent acetaminophen toxicity to mouse hepatocytes. Hepatocytes were isolated from fasted male C3Heb/FeJ mice. Necrotic cell killing was determined by propidium iodide fluorometry. Mitochondrial membrane potential was visualized by confocal microscopy of tetramethylrhodamine methylester. Chelatable ferrous ion was monitored by calcein quenching, and 70 kDa rhodamine-dextran was used to visualize lysosomes. Cell killing after acetaminophen (10mM) was delayed and decreased by more than half after 6 h by 1mM desferal or 1mM starch-desferal. In a cell-free system, ferrous but not ferric iron quenched calcein fluorescence, an effect reversed by dipyridyl, a membrane-permeable iron chelator. In hepatocytes loaded with calcein, intracellular calcein fluorescence decreased progressively beginning about 4 h after acetaminophen. Mitochondria then depolarized after about 6 h. Dipyridyl (20mM) dequenched calcein fluorescence. Desferal and starch-desferal conjugate prevented acetaminophen-induced calcein quenching and mitochondrial depolarization. As calcein fluorescence became quenched, lysosomes disappeared, consistent with release of iron from ruptured lysosomes. In conclusion, an increase of cytosolic chelatable ferrous iron occurs during acetaminophen hepatotoxicity, which triggers the MPT and cell killing. Disrupted lysosomes are the likely source of iron, and chelation of this iron decreases acetaminophen toxicity to hepatocytes.
PMCID: PMC2923283  PMID: 20584761
acetaminophen; cell death; glutathione; iron; mitochondria
Biochimica et biophysica acta  2010;1813(7):1382-1394.
To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-XL in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-XL, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R.
PMCID: PMC3089816  PMID: 21185334
14.  Sequential Opening of Mitochondrial Ion Channels as a Function of Glutathione Redox Thiol Status*s 
The Journal of biological chemistry  2007;282(30):21889-21900.
Mitochondrial membrane potential (ΔΨm) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with ΔΨm and the NADH/NAD+ redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible ΔΨm depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in ΔΨm to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4′-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, ΔΨm depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized ΔΨm and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.
PMCID: PMC2292488  PMID: 17540766
15.  Potential Therapeutic Benefits of Strategies Directed to Mitochondria 
Antioxidants & Redox Signaling  2010;13(3):279-347.
The mitochondrion is the most important organelle in determining continued cell survival and cell death. Mitochondrial dysfunction leads to many human maladies, including cardiovascular diseases, neurodegenerative disease, and cancer. These mitochondria-related pathologies range from early infancy to senescence. The central premise of this review is that if mitochondrial abnormalities contribute to the pathological state, alleviating the mitochondrial dysfunction would contribute to attenuating the severity or progression of the disease. Therefore, this review will examine the role of mitochondria in the etiology and progression of several diseases and explore potential therapeutic benefits of targeting mitochondria in mitigating the disease processes. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate and manipulate mitochondrial function and genomics for therapeutic benefit. These approaches to treat mitochondrial dysfunction rationally could lead to selective protection of cells in different tissues and various disease states. However, most of these approaches are in their infancy. Antioxid. Redox Signal. 13, 279–347.
Introduction and Topics Reviewed
Anatomy and Function of Mitochondrial Membranes
Outer mitochondrial membrane and its potential role as therapeutic target
Inner mitochondrial membrane and its potential role as therapeutic target
Mitochondrial permeability transition pore
Electron Transport Chain and Oxidative Phosphorylation: Modulation by Mitochondrial Ion Channels and Exchangers
Mitochondrial ROS and RNS
Mitochondria and reactive oxygen species
Mitochondria and reactive nitrogen species
Mitochondrial ROS Scavenging and Its Potential Therapeutic Value
Manganese superoxide dismutase
Glutathione thioredoxin, and peroxiredoxin systems
Catalase and glutathione peroxidase
Cytochrome c
Mitochondria as scavengers of cytosolic O2•−
Uncoupling Proteins in Modulation of Mitochondrial Function: Physiological and Pharmacologic Relevance
Mitochondrial DNA-Related Pathologies and a Potential Therapeutic Target
Mitochondrial Interaction with other Organelles: Therapeutic Implications
Mitochondrion—mitochondrion interaction
Mitochondrion—nucleus interaction
Mitochondria—endoplasmic/sarcoplasmic reticulum interaction
Mitochondria-Related Diseases and Cell Injury
Mitochondria and cardiac ischemia and reperfusion injury
Mitochondria and the failing heart
Mitochondria and diabetes
Mitochondria and hypertension
Mitochondria and neurodegenerative diseases
Alzheimer's disease
Parkinson's disease
Amyotrophic lateral sclerosis
Friedreich's ataxia
Neoplastic diseases
Other mitochondria-related diseases
Mitochondria and psychiatric disorders
Mitochondria and migraine headache
Mitochondrial Pharmacology and Therapeutic Potential
Strategies for drug delivery to mitochondria
Mitochondria-targeted drugs
Approaches to improve mitochondrial function during ischemia and reperfusion
Other Mitochondrial Therapeutic Approaches
Lipid replacement therapy
Transactivator of transcription proteins and mitochondrial therapy
Molecular genetics approaches
Mitochondria and caloric restriction
Mitochondria and dietary supplements
Mitochondria Age and Lifespan
Mitochondria and age-associated diseases
Mitochondrial p66shc and lifespan
Caveats and Potential Limitations in Mitochondrial Drug Targeting
Conclusion and Perspectives
PMCID: PMC2936955  PMID: 20001744
16.  Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells 
Autophagy  2010;6(4):462-472.
Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) then cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.
PMCID: PMC3768271  PMID: 20364102
autophagy; cardiac myocyte; cyclophilin D; mitochondrial permeability transition
17.  Effects of 4′-chlorodiazepam on cellular excitation-contraction coupling and ischaemia-reperfusion injury in rabbit heart 
Cardiovascular research  2008;79(1):141-149.
Recent evidence indicates that the activity of energy-dissipating ion channels in the mitochondria can influence the susceptibility of the heart to ischaemia-reperfusion injury. In this study, we describe the effects of 4′-chlorodiazepam (4-ClDzp), a well-known ligand of the mitochondrial benzodiazepine receptor, on the physiology of both isolated cardiomyocytes and intact hearts.
Methods and results
We used current- and voltage-clamp methods to determine the effects of 4-ClDzp on excitation-contraction coupling in isolated rabbit heart cells. At the level of the whole heart, we subjected rabbit hearts to ischaemia/reperfusion in order to determine how 4-ClDzp influenced the susceptibility to arrhythmias and contractile dysfunction. In isolated rabbit cardiomyocytes, 4-ClDzp evoked a significant reduction in the cardiac action potential that was associated with a decrease in calcium currents and peak intracellular calcium transients. In intact perfused normoxic rabbit hearts, 4-ClDzp mediated a dose-dependent negative inotropic response, consistent with the observation that 4-ClDzp was reducing calcium influx. Hearts that underwent 30 min of global ischaemia and 30 min of reperfusion were protected against reperfusion arrhythmias and post-ischaemic contractile impairment when 4-ClDzp (24 μM) was administered throughout the protocol or as a single bolus dose given at the onset of reperfusion. In contrast, hearts treated with cyclosporin-A, a classical blocker of the mitochondrial permeability transition pore, were not protected against reperfusion arrhythmias.
The findings indicate that the effects on 4-ClDzp on both mitochondrial and sarcolemmal ion channels contribute to protection against post-ischaemic cardiac dysfunction. Of clinical relevance, the compound is effective when given upon reperfusion, unlike other pre-conditioning agents.
PMCID: PMC2562874  PMID: 18304929
Arrhythmia; Mitochondrial ion channels; Ischaemia; Reperfusion
18.  Human Stanniocalcin-1 Suppresses Angiotensin II-Induced Superoxide Generation in Cardiomyocytes through UCP3-Mediated Anti-Oxidant Pathway 
PLoS ONE  2012;7(5):e36994.
We have previously shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial membrane and transgenic over-expression of STC1 is associated with increased energy utilization.
We examined the hypothesis that STC1 uncouples mitochondrial oxidative phosphorylation - to suppress superoxide generation and modulate neurohormonal effects on cardiomyocytes.
Methods and Results
Compared to WT mouse heart, STC1 Tg heart displays: 2-fold higher uncoupling protein 3 (UCP3) levels, but no effect on UCP2 protein; 40% lower ATP levels; but similar activities of respiratory chain complexes I-IV. In cultured adult rat and freshly-isolated mouse cardiomyocytes, rSTC1 induces UCP3, but not UCP2. Treatment of cardiomyocytes with STC1 decreases mitochondrial membrane potential and suppresses baseline and angiotensin II (Ang II)-induced superoxide generation. Furthermore, baseline superoxide generation is higher in freshly-isolated adult UCP3−/− mouse cardiomyocytes compared to WT, suggesting an important role for UCP3 in regulating cardiomyocyte ROS under physiologic conditions. Treatment of UCP3−/− cardiomyocytes with rSTC1 failed to suppress superoxide generation, suggesting that the effects of STC1 on superoxide generation in cardiomyocytes are UCP3-dependent.
STC1 activates a novel anti-oxidant pathway in cardiac myocytes through induction of UCP3, and may play an important role in suppressing ROS in the heart under normal physiologic conditions and ameliorate the deleterious effects of Ang II-mediated cardiac injury. Importantly, our data point to a critical role for the mitochondria in regulating ROS generation in response to Ang II.
PMCID: PMC3365029  PMID: 22693564
19.  Novel Insights Into Interactions Between Mitochondria and Xanthine Oxidase in Acute Cardiac Volume Overload 
Free radical biology & medicine  2011;51(11):1975-1984.
Xanthine oxidoreductase (XOR) is increased in the left ventricle (LV) of humans with volume overload (VO) and mitochondrial inhibition of the respiratory chain occurs in animal models of VO. Since mitochondria are both a source and target of reactive oxygen and nitrogen species, we hypothesized that activation of XOR and mitochondrial dysfunction are interdependent. To test this we used the aortocaval fistula (ACF) rat model of VO and a simulation of the stretch response in isolated adult cardiomyocytes with and without the inhibitor of XOR, allopurinol, or the mitochondrially targeted antioxidant MitoQ. XO activity was increased in cardiomyocytes from ACF vs. sham rats (24h) without an increase in XO protein. A two-fold increase in LV end-diastolic pressure/wall stress and a decrease in LV systolic elastance with ACF were improved with allopurinol (100 mg/kg) started at ACF induction. Subsarcolemmal state 3 mitochondrial respiration was significantly decreased in ACF and normalized by allopurinol. Cardiomyocytes subjected to 3 hour cyclical stretch resulted in an increase in XO activity and mitochondrial swelling, which was prevented by allopurinol or MitoQ pretreatment. These studies establish an early interplay between cardiomyocyte XO activation and bioenergetic dysfunction that may provide a new target that prevents progression to heart failure in VO.
PMCID: PMC3364106  PMID: 21925594
volume overload; oxidative stress; MitoQ; stretch; mitochondria; allopurinol
20.  Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes 
PLoS ONE  2012;7(11):e50778.
Hypoxia/reoxygenation(H/R)-induced apoptosis of cardiomyocytes plays an important role in myocardial injury. Lycopene is a potent antioxidant carotenoid that has been shown to have protective properties on cardiovascular system. The aim of the present study is to investigate the potential for lycopene to protect the cardiomyocytes exposed to H/R. Moreover, the effect on mitochondrial function upon lycopene exposure was assessed.
Methods and Findings
Primary cardiomyocytes were isolated from neonatal mouse and established an in vitro model of H/R which resembles ischemia/reperfusion in vivo. The pretreatment of cardiomyocytes with 5 µM lycopene significantly reduced the extent of apoptosis detected by TUNEL assays. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and the process of mitochondria-mediated apoptosis were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of the mitochondrial permeability transition pore (mPTP) by reducing the intracellular reactive oxygen species (ROS) levels and inhibiting the increase of malondialdehyde (MDA) levels caused by H/R. Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in lycopene-treated cultures.
The present results suggested that lycopene possesses great pharmacological potential in protecting against H/R-induced apoptosis. Importantly, the protective effects of lycopene may be attributed to its roles in improving mitochondrial function in H/R-treated cardiomyocytes.
PMCID: PMC3511264  PMID: 23226382
21.  Measuring mitochondrial function in intact cardiac myocytes 
Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment – the cytosol of intact cells – and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca2+ and Na+ signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts.
PMCID: PMC3246130  PMID: 21964191
Cardiomyocytes; Mitochondrial function; Methods
22.  Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity 
Autophagy  2013;9(3):328-344.
Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.
PMCID: PMC3590254  PMID: 23298947
oxidative stress; mitochondrial dysfunction; autophagy; cardiomyocytes; rapamycin; MTOR
23.  Mitochondrial Dysfunction in Diabetes: From Molecular Mechanisms to Functional Significance and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2010;12(4):537-577.
Given their essential function in aerobic metabolism, mitochondria are intuitively of interest in regard to the pathophysiology of diabetes. Qualitative, quantitative, and functional perturbations in mitochondria have been identified and affect the cause and complications of diabetes. Moreover, as a consequence of fuel oxidation, mitochondria generate considerable reactive oxygen species (ROS). Evidence is accumulating that these radicals per se are important in the pathophysiology of diabetes and its complications. In this review, we first present basic concepts underlying mitochondrial physiology. We then address mitochondrial function and ROS as related to diabetes. We consider different forms of diabetes and address both insulin secretion and insulin sensitivity. We also address the role of mitochondrial uncoupling and coenzyme Q. Finally, we address the potential for targeting mitochondria in the therapy of diabetes. Antioxid. Redox Signal. 12, 537–577.
Basic Physiology
Electron transport
Reactive oxygen species and mitochondria
Mitochondrial nitric oxide
Role of calcium and the mitochondrial permeability transition pore
Assessing Mitochondrial Function
Respiration and potential
ATP production and the proton leak
ROS production by isolated mitochondria
Site specificity of mitochondrial superoxide production
Mitochondrial ROS production in intact cells
Oxidative damage to mitochondria in intact cells
Mitochondrial Metabolism and Diabetes
General considerations
Mitochondrial diabetes
Type 1 and type 2 diabetes
Mitochondrial number and morphology
Mitochondrial biogenesis
Mitochondrial function in type 2 diabetes and insulin-resistant states
Is mitochondrial impairment a cause of insulin resistance?
Mitochondrial respiratory coupling and insulin release
Mitochondrial function in insulin-deficient diabetes
Diabetes and mitochondrial function in non–insulin-sensitive tissues
Mitochondria and cell-fuel selectivity
Diabetic cardiomyopathy and mitochondrial function
Mitochondrial ROS and Diabetes
ROS production and the cause of diabetes
Oxidative damage and pancreatic islet β cells
ROS and oxidative damage in insulin-sensitive target tissues
ROS and the complications of diabetes
Non–insulin-sensitive tissues (retina, renal, neural cells)
ROS and vascular cells
Mitochondrial Membrane Potential and Diabetes
Role of uncoupling proteins
Does membrane potential actually protect against superoxide production?
Coenzyme Q and Diabetes
Therapeutic Implications
Improving mitochondrial metabolism
Lifestyle modification
Pharmacologic intervention
Controlling ROS production and oxidative damage
Mitochondria-targeted antioxidants
Metabolic effects of mitochondria-targeted antioxidants
Mitochondria-targeted antioxidant peptides
Targeting superoxide
PMCID: PMC2824521  PMID: 19650713
24.  KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury 
PLoS ONE  2014;9(7):e103402.
Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK−/− cardiomyocytes. Transmission electron microscopy of BK−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.
PMCID: PMC4114839  PMID: 25072914
25.  Loss of DJ-1 Does Not Affect Mitochondrial Respiration but Increases ROS Production and Mitochondrial Permeability Transition Pore Opening 
PLoS ONE  2012;7(7):e40501.
Loss of function mutations in the DJ-1 gene have been linked to recessively inherited forms of Parkinsonism. Mitochondrial dysfunction and increased oxidative stress are thought to be key events in the pathogenesis of Parkinson’s disease. Although it has been reported that DJ-1 serves as scavenger for reactive oxidative species (ROS) by oxidation on its cysteine residues, how loss of DJ-1 affects mitochondrial function is less clear.
Methodology/Principal Findings
Using primary mouse embryonic fibroblasts (MEFs) or brains from DJ-1−/− mice, we found that loss of DJ-1 does not affect mitochondrial respiration. Specifically, endogenous respiratory activity as well as basal and maximal respiration are normal in intact DJ-1−/− MEFs, and substrate-specific state 3 and state 4 mitochondrial respiration are also unaffected in permeabilized DJ-1−/− MEFs and in isolated mitochondria from the cerebral cortex of DJ-1−/− mice at 3 months or 2 years of age. Expression levels and activities of all individual complexes composing the electron transport system are unchanged, but ATP production is reduced in DJ-1−/− MEFs. Mitochondrial transmembrane potential is decreased in the absence of DJ-1. Furthermore, mitochondrial permeability transition pore opening is increased, whereas mitochondrial calcium levels are unchanged in DJ-1−/− cells. Consistent with earlier reports, production of reactive oxygen species (ROS) is increased, though levels of antioxidative enzymes are unaltered. Interestingly, the decreased mitochondrial transmembrane potential and the increased mitochondrial permeability transition pore opening in DJ-1−/− MEFs can be restored by antioxidant treatment, whereas oxidative stress inducers have the opposite effects on mitochondrial transmembrane potential and mitochondrial permeability transition pore opening.
Our study shows that loss of DJ-1 does not affect mitochondrial respiration or mitochondrial calcium levels but increases ROS production, leading to elevated mitochondrial permeability transition pore opening and reduced mitochondrial transmembrane potential.
PMCID: PMC3392228  PMID: 22792356

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