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1.  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
2.  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
3.  Cellular and molecular pathways to myocardial necrosis and replacement fibrosis 
Heart failure reviews  2011;16(1):23-34.
Fibrosis is a fundamental component of the adverse structural remodeling of myocardium present in the failing heart. Replacement fibrosis appears at sites of previous cardiomyocyte necrosis to preserve the structural integrity of the myocardium, but not without adverse functional consequences. The extensive nature of this microscopic scarring suggests cardiomyocyte necrosis is widespread and the loss of these contractile elements, combined with fibrous tissue deposition in the form of a stiff in-series and in-parallel elastic elements, contributes to the progressive failure of this normally efficient muscular pump. Cellular and molecular studies into the signal-transducer-effector pathway involved in cardiomyocyte necrosis have identified the crucial pathogenic role of intracellular Ca2+ overloading and subsequent induction of oxidative stress, predominantly confined within its mitochondria, to be followed by the opening of the mitochondrial permeability transition pore that leads to the destruction of these organelles and cells. It is now further recognized that Ca2+ overloading of cardiac myocytes and mitochondria serves as a prooxidant and which is counterbalanced by an intrinsically coupled Zn2+ entry serving as antioxidant. The prospect of raising antioxidant defenses by increasing intracellular Zn2+ with adjuvant nutriceuticals can, therefore, be preferentially exploited to uncouple this intrinsically coupled Ca2+–Zn2+ dyshomeostasis. Hence, novel yet simple cardioprotective strategies may be at hand that deserve to be further explored.
PMCID: PMC2920342  PMID: 20405318
Hypocalcemia; Hypomagnesemia; Hypozincemia; Secondary hyperparathyroidism; Intracellular calcium overloading; Mitochondria; Oxidative stress; Aldosteronism
4.  Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria 
British Journal of Cancer  2012;106(6):1224-1233.
Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues.
MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells.
MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than that of plasma membrane MRP-1 (11–22% P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 n, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria.
Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.
PMCID: PMC3304412  PMID: 22353810
MRP-1; mitochondria; human; multi-drug resistance; Ewing's sarcoma family of tumours
5.  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
6.  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
7.  Akt mediated mitochondrial protection in the heart 
Cardiomyocyte death is now recognized as a critical factor in the development of heart disease. Mitochondria are not only responsible for energy production to ensure that cardiac output meets the body’s energy demands, but they serve as critical integrators of cell survival signals. Numerous stressors are known to induce cell death by necrosis and/or apoptosis mediated through mitochondrial dysregulation. Anti- and pro-apoptotic Bcl-2 family proteins regulate apoptosis by controlling mitochondrial outer membrane permeability, whereas opening of the mitochondrial permeability transition pore (PT-pore) induces large amplitude permeability of the inner membrane and consequent rupture of the outer membrane. Akt is one of the best described survival kinases activated by receptor ligands and its activation preserves mitochondrial integrity and protects cardiomyocytes against necrotic and apoptotic death. The mechanisms responsible for Akt-mediated mitochondrial protection have not been fully elucidated. There is, however, accumulating evidence that multiple Akt target molecules, recruited through both transcriptional and post-transcriptional mechanisms, directly impinge upon and protect mitochondria. In this review we discuss mechanisms by which Akt activation can effect changes at the mitochondria that protect cardiomyocytes and attenuate pathophysiological responses of the heart.
PMCID: PMC2732429  PMID: 19377835
Akt; Mitochondria; Heart; Hexokinase-II
8.  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
9.  Mitochondrial Ca2+ Influx and Efflux rates in Guinea Pig Cardiac Mitochondria: Low and High Affinity Effects of Cyclosporine A 
Biochimica et biophysica acta  2011;1813(7):1373-1381.
Ca2+ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca2+ is controlled primarily by the mitochondrial Ca2+ uniporter (mCU) and the mitochondrial Na+/Ca2+ exchanger (mNCE), influencing NADH production through Ca2+-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca2+-triggered mitochondrial permeability transition pore (PTP) in cell death, it has been proposed that the PTP might also contribute to physiological mitochondrial Ca2+ release. Here we selectively measure Ca2+ influx rate through the mCU and Ca2+ efflux rates through Na+-dependent and Na+-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mNCE (CGP 37157) or the PTP (CsA). CsA suppressed the negative bioenergetic consequences (ΔΨm loss, Ca2+ release, NADH oxidation, swelling) of high extramitochondrial Ca2+ additions, allowing mitochondria to tolerate total mitochondrial Ca2+ loads of >400 nmol/mg protein. For Ca2+ pulses up to 15µM, Na+-independent Ca2+ efflux through the PTP accounted for ~5% of the total Ca2+ efflux rate compared to that mediated by the mNCE (in 5mM Na+). Unexpectedly, we also observed that CsA inhibited mNCE-mediated Ca2+ efflux at higher concentrations (IC50=2µM) than those required to inhibit the PTP, with a maximal inhibition of ~40% at 10µM CsA, while having no effect on the mCU. The results suggest a possible alternative mechanism by which CsA could affect mitochondrial Ca2+ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of CsA at high concentrations.
PMCID: PMC3109245  PMID: 21362444
mitochondrial Na+/Ca2+ exchanger; permeability transition pore; mitochondrial calcium uniporter; oxidative phosphorylation; bioenergetics; calcium transport
10.  Nix Nought Nothing: Fairy Tale or Real Deal 
Nix was first described in the heart as the protein product of a differentially expressed mRNA detected by hybridiztion to a partial cDNA sequence tag on an RNA expression array. Over the subsequent 8 years Nix has become the prototypical transcriptionally-regulated cardiac myocyte “suicide” gene and has been used as a model to interrogate mechanisms of programmed cardiomyocyte death in hypertrophy and heart failure. Nix stimulates conventional apoptosis mediated via the intrinsic mitochondrial pathway, but emerging evidence indicates that Nix also controls programmed necrosis dependent upon sarcoplasmic reticular-mitochondrial tethering, calcium cross-talk, and the mitochondrial permeability transition. Recent studies have also described Nix labeling of senescent cardiomyocyte mitochondria for autophagic elimination, elucidated a physiological mitochondrial quality control Nix function; so-called “mitochondrial pruning.
PMCID: PMC3036779  PMID: 20858501
apoptosis; programmed necrosis; autophagy; heart failure; BNip3 proteins; necrosis; mitochondrial autophagy
11.  Down-regulation of OPA1 alters mouse mitochondrial morphology, PTP function, and cardiac adaptation to pressure overload 
Cardiovascular Research  2012;94(3):408-417.
The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1+/− mice, having a 50% reduction in OPA1 protein expression in cardiac tissue.
Methods and Results
In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1+/+. Electron and fluorescence microscopy revealed altered morphology of the Opa1+/− mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibers, mitochondrial functional properties were maintained, but direct energy channeling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction (TAC), Opa1+/− hearts demonstrated hypertrophy almost two-fold higher (p<0.01) than in wild type mice with altered ejection fraction (decrease of 43% versus 22% in Opa1+/+ mice, p<0.05).
These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.
PMCID: PMC3863708  PMID: 22406748
Adaptation, Biological; Animals; Down-Regulation; GTP Phosphohydrolases; metabolism; Mice; Mice, Knockout; Mitochondria; genetics; metabolism; ultrastructure; Mitochondrial Membrane Transport Proteins; genetics; metabolism; Mitochondrial Membranes; metabolism; Mitochondrial Proteins; genetics; physiology; Myocytes, Cardiac; cytology; metabolism; Optic Atrophy, Autosomal Dominant; genetics; metabolism; physiopathology; Permeability; Pressure; cardiac energy metabolism; mitochondria; mitochondria dynamics; permeability transition pore; hypertrophy
12.  Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release 
Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.
Hearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively.
30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.
The results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.
PMCID: PMC3224577  PMID: 19671187
13.  The Permeability Transition Pore Controls Cardiac Mitochondrial Maturation and Myocyte Differentiation 
Developmental cell  2011;21(3):469-478.
Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z-bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.
PMCID: PMC3175092  PMID: 21920313
14.  NIM811, a Mitochondrial Permeability Transition Inhibitor, Attenuates Cholestatic Liver Injury But Not Fibrosis in Mice 
Cholestasis causes hepatocyte death, possibly due to mitochondrial injury. This study investigated whether NIM811, an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterwards. Mitochondrial depolarization, cell death and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123), propidium iodide (PI) and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis and apoptosis all increased. NIM811 decreased ALT, necrosis and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high power field (hpf), and non-viable cells were ∼1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria and blocked release of cytochrome c. Hepatic TNFα, TGF-β1 and procollagen α1(I) mRNA, α-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.
PMCID: PMC2582973  PMID: 18801946
15.  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
16.  Pyrroloquinoline Quinone Preserves Mitochondrial Function and Prevents Oxidative Injury in Adult Rat Cardiac Myocytes 
We investigated the ability of pyrroloquinoline quinone (PQQ) to confer resistance to acute oxidative stress in freshly isolated adult male rat cardiomyocytes. Fluorescence microscopy was used to detect generation of reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) depolarization induced by hydrogen peroxide. H2O2 caused substantial cell death, which was significantly reduced by preincubation with PQQ. H2O2 also caused an increase in cellular ROS levels as detected by the fluorescent indicators CM-H2XRos and dihydroethidium. ROS levels were significantly reduced by a superoxide dismutase mimetic Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or by PQQ treatment. Cyclosporine-A, which inhibits mitochondrial permeability transition, prevented H2O2-induced Δψm depolarization, as did PQQ and MnTBAP. Our results provide direct evidence that PQQ reduces oxidative stress, mitochondrial dysfunction, and cell death in isolated adult rat cardiomyocytes. These findings provide new insight into the mechanisms of PQQ action in the heart.
PMCID: PMC2844438  PMID: 17880922
mitochondria; rat; ischemia; oxidative stress
17.  ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle 
Biophysical Journal  2010;98(12):2785-2793.
Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.
PMCID: PMC2884246  PMID: 20550890
18.  ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle 
Biophysical Journal  2010;98(12):2785-2793.
Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.
PMCID: PMC2884246  PMID: 20550890
19.  Post-Transcriptional Alterations in the Expression of Cardiac Sodium Channel Subunits in Chronic Heart Failure 
Clinical and experimental evidence has recently accumulated about the importance of alterations of Na+ channel (NaCh) function and slow myocardial conduction for arrhythmias in infarcted and failing hearts (HF). The present study evaluated the molecular mechanisms of local alterations in the expression of NaCh subunits which underlie Na+ current (INa) density decrease in HF. HF was induced in 5 dogs by sequential coronary microembolization and developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 27±7%). 5 normal dogs served as a control group. Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium and INa was measured by whole-cell patch-clamp. The mRNA encoding the cardiac-specific sodium channel (NaCh) α-subunit Nav1.5, and one of its auxiliary subunits β1 (NaChβ1), was analyzed by competitive RT-PCR. Protein levels of Nav1.5, NaChβ1 and NaChβ2 were evaluated by Western blotting. The maximum density of INa/Cm was decreased in HF (n=5) compared to control hearts (32.3±2.6 vs. 50.8±6.5 pA/pF, mean±SEM, n=5, P<0.05). The steady-state inactivation and activation of INa remained unchanged in HF compared to control hearts. The levels of mRNA encoding Nav1.5, and NaChβ1 were unaltered in failing hearts. However, Nav1.5 protein expression was reduced about 30% in HF, while NaChβ1 and NaChβ2 protein were unchanged. We conclude that experimental HF in dogs results in post-transcriptional changes in cardiac NaCh α-subunit expression.
PMCID: PMC2408747  PMID: 15242739
heart failure; sodium channel; β-subunits; patch clamp; RT-PCR; Western blot
20.  Mitochondrial cyclophilin-D as a critical mediator of ischaemic preconditioning 
Cardiovascular Research  2010;88(1):67-74.
It has been suggested that mitochondrial reactive oxygen species (ROS), Akt and Erk1/2 and more recently the mitochondrial permeability transition pore (mPTP) may act as mediators of ischaemic preconditioning (IPC), although the actual interplay between these mediators is unclear. The aim of the present study is to determine whether the cyclophilin-D (CYPD) component of the mPTP is required by IPC to generate mitochondrial ROS and subsequently activate Akt and Erk1/2.
Methods and results
Mice lacking CYPD (CYPD−/−) and B6Sv129 wild-type (WT) mice were used throughout. We have demonstrated that under basal conditions, non-pathological mPTP opening occurs (indicated by the percent reduction in mitochondrial calcein fluorescence). This effect was greater in WT cardiomyocytes compared with CYPD−/− ones (53 ± 2% WT vs. 17 ± 3% CYPD−/−; P < 0.01) and was augmented by hypoxic preconditioning (HPC) (70 ± 9% WT vs. 56 ± 1% CYPD−/−; P < 0.01). HPC reduced cell death following simulated ischaemia–reperfusion injury in WT (23.2 ± 3.5% HPC vs. 43.7 ± 3.2% WT; P < 0.05) but not CYPD−/− cardiomyocytes (19.6 ± 1.4% HPC vs. 24.4 ± 2.6% control; P > 0.05). HPC generated mitochondrial ROS in WT (four-fold increase; P < 0.05) but not CYPD−/− cardiomyocytes. HPC induced significant Akt phosphorylation in WT cardiomyocytes (two-fold increase; P < 0.05), an effect which was abrogated by ciclosporin-A (a CYPD inhibitor) and N-2-mercaptopropionyl glycine (a ROS scavenger). Finally, in vivo IPC of adult murine hearts resulted in significant phosphorylation of Akt and Erk1/2 in WT but not CYPD−/− hearts.
The CYPD component of the mPTP is required by IPC to generate mitochondrial ROS and phosphorylate Akt and Erk1/2, major steps in the IPC signalling pathway.
PMCID: PMC2936122  PMID: 20400621
Cyclophilin-D; Mitochondrial permeability transition pore; Ischaemic preconditioning; Akt; Erk1/2; Reactive oxygen species
21.  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
22.  Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial KATP Channel Activation 
PLoS ONE  2010;5(5):e10666.
The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function.
Methodology/Principal Findings
Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca2+ induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K+ transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K+ channels (mitoKATP). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoKATP activation.
We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoKATP activation.
PMCID: PMC2871796  PMID: 20498724
23.  Blockade of Electron Transport During Ischemia Preserves bcl-2 and Inhibits Opening of the Mitochondrial Permeability Transition Pore 
FEBS letters  2011;585(6):921-926.
Myocardial ischemia damages the electron transport chain and augments cardiomyocyte death during reperfusion. To understand the relationship between ischemic mitochondrial damage and mitochondrial-driven cell death, the isolated perfused heart underwent global stop-flow ischemia with and without mitochondrial protection by reversible blockade of electron transport. Ischemic damage to electron transport depleted bcl-2 content and favored mitochondrial permeability transition (MPT). Reversible blockade of electron transport preserved bcl-2 content and attenuated calcium-stimulated mitochondrial swelling. Thus, the damaged electron transport chain leads to bcl-2 depletion and MPT opening. Chemical inhibition of bcl-2 with HA14-1 also dramatically increased mitochondrial swelling, augmented by exogenous H2O2 stress, indicating that bcl-2 depleted mitochondria are poised to undergo MPT during the enhanced oxidative stress of reperfusion.
PMCID: PMC3076511  PMID: 21354418
24.  The Role of Sulfur Dioxide in the Regulation of Mitochondrion-Related Cardiomyocyte Apoptosis in Rats with Isopropylarterenol-Induced Myocardial Injury 
The authors investigated the regulatory effects of sulfur dioxide (SO2) on myocardial injury induced by isopropylarterenol (ISO) hydrochloride and its mechanisms. Wistar rats were divided into four groups: control group, ISO group, ISO plus SO2 group, and SO2 only group. Cardiac function was measured and cardiomyocyte apoptosis was detected. Bcl-2, bax and cytochrome c (cytc) expressions, and caspase-9 and caspase-3 activities in the left ventricular tissues were examined in the rats. The opening status of myocardial mitochondrial permeability transition pore (MPTP) and membrane potential were analyzed. The results showed that ISO-treated rats developed heart dysfunction and cardiac injury. Furthermore, cardiomyocyte apoptosis in the left ventricular tissues was augmented, left ventricular tissue bcl-2 expression was down-regulated, bax expression was up-regulated, mitochondrial membrane potential was significantly reduced, MPTP opened, cytc release from mitochondrion into cytoplasm was significantly increased, and both caspase-9 and caspase-3 activities were increased. Administration of an SO2 donor, however, markedly improved heart function and relieved myocardial injury of the ISO-treated rats; it lessened cardiomyocyte apoptosis, up-regulated myocardial bcl-2, down-regulated bax expression, stimulated mitochondrial membrane potential, closed MPTP, and reduced cytc release as well as caspase-9 and caspase-3 activities in the left ventricular tissue. Hence, SO2 attenuated myocardial injury in association with the inhibition of apoptosis in myocardial tissues, and the bcl-2/cytc/caspase-9/caspase-3 pathway was possibly involved in this process.
PMCID: PMC3676849  PMID: 23698774
isopropylarterenol; sulfur dioxide; myocardium; apoptosis; mitochondrial membrane potential
25.  Minocycline and N-Methyl-4-Isoleucine Cyclosporin (NIM811) Mitigate Storage/Reperfusion Injury After Rat Liver Transplantation Through Suppression of the Mitochondrial Permeability Transition 
Hepatology (Baltimore, Md.)  2008;47(1):236-246.
Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca2+ uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05).
Minocycline and NIM811 attenuated graft injury after rat liver transplantation and improved graft survival. Minocycline and/or NIM811 might be useful clinically in hepatic surgery and transplantation.
PMCID: PMC2656601  PMID: 18023036

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