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Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (ΔΨm). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3′dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on ΔΨm. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure–activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on ΔΨm and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.Cell Death and Differentiation (2006) 13, 151–159. doi:10.1038/sj.cdd.4401732; published online 29 July 2005 [ABSTRACT FROM AUTHOR]

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the F O F 1 -ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial F O F 1 -ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of F O F 1 -ATPase supports mitochondrial polarization.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Background: Dithiocarbamates and derivatives (including disulfiram, DSF) are currently investigated as antineoplastic agents. We have revealed earlier the ability of hydroxocobalamin (vitamin В 12b ) combined with diethyldithiocarbamate (DDC) to catalyze the formation of highly cytotoxic oxidized derivatives of DSF (DSFoxy, sulfones and sulfoxides)., Methods: Electron and fluorescent confocal microscopy, molecular biology and conventional biochemical techniques were used to study the morphological and functional responses of MCF-7 human breast cancer cells to treatment with DDC and B 12b alone or in combination., Results: DDC induces unfolded protein response in MCF-7 cells. The combined use of DDC and B 12b causes MCF-7 cell death. Electron microscopy revealed the separation of ER and nuclear membranes, leading to the formation of both cytoplasmic and perinuclear vacuoles, with many fibers inside. The process of vacuolization coincided with the appearance of ER stress markers, a marked damage to mitochondria, a significant inhibition of 20S proteasome, and actin depolimerization at later stages. Specific inhibitors of apoptosis, necroptosis, autophagy, and ferroptosis did not prevent cell death. A short- time (6-h) exposure to DSFoxy caused a significant increase in the number of entotic cells., Conclusions: These observations indicate that MCF-7 cells treated with a mixture of DDC and B 12b die by the mechanism of paraptosis. A short- time exposure to DSFoxy caused, along with paraptosis, a significant activation of the entosis and its final stage, lysosomal cell death., General Significance: The results obtained open up opportunities for the development of new approaches to induce non-apoptotic death of cancer cells by dithiocarbamates., (Copyright © 2022. Published by Elsevier B.V.)

We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t 1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism., (© 2022 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Background: Postoperative atrial fibrillation (PoAF) is a common complication after cardiac surgery. A pre-existing atrial substrate appears to be important in postoperative development of dysrhythmia, but its preoperative estimation is challenging. We tested the hypothesis that a combination of clinical predictors, noninvasive surrogate markers for atrial fibrosis defining abnormal left atrial (LA) mechanics, and biomarkers of collagen turnover is superior to clinical predictors alone in identifying patients at-risk for PoAF., Methods: In patients without prior AF undergoing coronary artery bypass grafting, concentrations of biomarkers reflecting collagen synthesis and degradation, extracellular matrix, and regulatory microRNA-29s were determined in serum from preoperative blood samples and correlated to atrial fibrosis extent, alteration in atrial deformation properties determined by 3D speckle-tracking echocardiography, and AF development., Results: Of 90 patients without prior AF, 34 who developed PoAF were older than non-PoAF patients (72.04 ± 10.7 y; P = 0.043) with no significant difference in baseline comorbidities, LA size, or ventricular function. Global (P = 0.007) and regional longitudinal LA strain and ejection fraction (P = 0.01) were reduced in PoAF vs. non-PoAF patients. Preoperative amino-terminal-procollagen-III-peptide (PIIINP) (103.1 ± 39.7 vs. 35.1 ± 19.3; P = 0.041) and carboxy-terminal-procollagen-I-peptide levels were elevated in PoAF vs. non-PoAF patients with a reduction in miR-29 levels and correlated with atrial fibrosis extent. Combining age as the only significant clinical predictor with PIIINP and miR-29a provided a model that identified PoAF patients with higher predictive accuracy., Conclusions: In patients without a previous history of AF, using age and biomarkers of collagen synthesis and regulation, a noninvasive tool was developed to identify those at risk for new-onset PoAF., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells., Methods: Stable isotope metabolic labeling, media creatine depletion, and Na + /K + -ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na + /K + -ATPase and creatine uptake., Results: Inhibition of the Na + /K + -ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na + /creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na + /K + -ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na + /K + -ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na + /K + -ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells., Conclusions: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na + /K + -ATPase pump activity., General Significance: These studies identify kinase regulation of the Na + /K + -ATPase as pivotal in regulating creatine uptake and energy metabolism in cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.

We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

Background: Excessive cardiac fibrosis due to maladaptive remodeling leads to progression of cardiac dysfunction and is modulated by TGF-β1-activated intracellular phospho-SMAD signaling effectors and transcription regulators. SMAD2/3 phosphorylation, regulated by protein-phosphatases, has been studied in different cell types, but its role in human ventricular fibroblasts (hVFs) is not defined as a target to reduce cytokine-mediated excessive fibrotic response and adverse cardiac remodeling. Statins are a class of drugs reported to reduce cardiac fibrosis, although underlying mechanisms are not completely understood. We aimed to assess whether simvastatin-mediated reduction in TGF-β1-augmented profibrotic response involves reduction in phospho-SMAD2/3 owing to activation of protein-phosphatase in hVFs., Methods and Results: Cultures of hVFs were used. Effect of simvastatin on TGF-β1-treated hVF proliferation, cytotoxicity, myofibroblast differentiation/activation, profibrotic gene expression and protein-phosphatase activity was assessed. Simvastatin (1 μM) reduced effect of TGF-β1 (5 ng/mL) on hVF proliferation, myofibroblast differentiation (reduced α-smooth muscle actin [α-SMA-expression]) and activation (decreased procollagen-peptide release). Simvastatin also reduced TGF-β1-stimulated time-dependent increases in SMAD2/3 phosphorylation and nuclear translocation, mediated through catalytic activation of protein-phosphatases PPM1A and PP2A, which physically interact with SMAD2/3, thereby promoting their dephosphorylation. Effect of simvastatin on TGF-β1-induced fibroblast activation was annulled by okadaic acid, an inhibitor of protein-phosphatase., Conclusions: This proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-β1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Energy production in myocardial cells occurs mainly in the mitochondrion. Although alterations in mitochondrial functions in the senescent heart have been documented, the molecular bases for the aging-associated decline in energy metabolism in the human heart are not fully understood. In this study, we examined transcription profiles of genes coding for mitochondrial proteins in atrial tissue from aged (≥65 years old) and comorbidities-matched adult (<65 years old) patients with preserved left ventricular function. We also correlated changes in functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes with gene expression changes. There was significant alteration in the expression of 10% (101/1,008) of genes coding for mitochondrial proteins, with 86% downregulated (87/101). Forty-nine percent of the altered genes were confined to mitochondrial energetic pathways. These changes were associated with a significant decrease in respiratory capacity of mitochondria oxidizing glutamate and malate and functional activity of complex I activity that correlated with the downregulation of NDUFA6, NDUFA9, NDUFB5, NDUFB8, and NDUFS2 genes coding for NADH dehydrogenase subunits. Thus, aging is associated with a decline in activity of OXPHOS within the broader transcriptional downregulation of genes regulating mitochondrial energetics, providing a substrate for reduced energetic efficiency in the senescent human atria.

Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury., (Copyright © 2016 the American Physiological Society.)

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2), third (18O3), and fourth (18O4) positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium.

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20 min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca(2+) uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca(2+) uptake and suppressed the Ca(2+)-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca(2+) uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU., (© 2013. Published by Elsevier Inc. All rights reserved.)

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the oxidized nicotinamide adenine dinucleotide (NAD(+) )/reduced nicotinamide adenine dinucleotide (NADH) ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD(+) /NADH ratio using (13) C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2-(13) C-glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3-(13) C-serine and 2,3-(13) C-serine isotopomers while reducing 2-(13) C-serine by 55%, demonstrating that ca. 20% of glycine-derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide-3-chlorophenylhydrazone both increased serine isotopomers, whereas rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that (13) C magnetic resonance spectroscopy monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2-(13) C-glycine reflects the changes of mitochondrial redox status., (Copyright © 2011 Wiley Periodicals, Inc.)

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT., (Published by Elsevier Inc.)

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 microM digitonin. Blockade of VDAC with Koenig's polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 microM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.

The utility of nitric oxide (NO)-releasing silica nanoparticles as novel antibacterial agents is demonstrated against Pseudomonas aeruginosa. Nitric oxide-releasing nanoparticles were prepared via co-condensation of tetraalkoxysilane with aminoalkoxysilane modified with diazeniumdiolate NO donors, allowing for the storage of large NO payloads. Comparison of the bactericidal efficacy of the NO-releasing nanoparticles to 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a small molecule NO donor, demonstrated enhanced bactericidal efficacy of nanoparticle-derived NO and reduced cytotoxicity to healthy cells (mammalian fibroblasts). Confocal microscopy revealed that fluorescently labeled NO-releasing nanoparticles associated with the bacterial cells, providing rationale for the enhanced bactericidal efficacy of the nanoparticles. Intracellular NO concentrations were measurable when the NO was delivered from nanoparticles as opposed to PROLI/NO. Collectively, these results demonstrate the advantage of delivering NO via nanoparticles for antimicrobial applications.

Unlabelled: 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 Ca(2+) 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)., Conclusion: 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.

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.

Intracellular calcium signaling plays a central role in cell proliferation. In leukemic cells, the calcium release-activated calcium channels provide a major pathway for calcium entry (I(CRAC)) perpetuating progression through the cell cycle. Although I(CRAC) is under mitochondrial regulation, targeting mitochondrial function has not been exploited to control malignant cell growth. The benzothiadiazine diazoxide, which depolarized respiration-dependent mitochondrial membrane potential, reduced the rate of proliferation and arrested human acute leukemic T cells in the G0/G1 phase. Diazoxide did not alter cellular energetics, but rather inhibited the mitochondria-controlled I(CRAC) and reduced calcium influx into tumor cells. The antiproliferative action of diazoxide was mimicked by removal of extracellular calcium or by the tyrphostin A9, an I(CRAC) inhibitor. Deletion of the mitochondrial genome, which encodes essential respiratory chain enzyme subunits, attenuated the inhibitory effect of diazoxide on I(CRAC)-mediated calcium influx and cell proliferation. Thus, manipulation of mitochondrial function and associated calcium signaling provides a basis for a novel anticancer strategy.

Mitochondria, the major source of cellular ATP, display high vulnerability to metabolic stress, in particular to excessive Ca2+ loading. Here, we show that Ca2+-inhibited mitochondrial ATP generation could be restored through stimulated Ca2+ discharge from mitochondrial matrix. This was demonstrated using a Ca2+ ionophore or through Na+/Ca2+ exchange-mediated decrease of mitochondrial Ca2+ load. Furthermore, diazoxide, a mitochondrial potassium channel opener, which maintained mitochondrial Ca2+ homeostasis, also restored Ca2+-inhibited ATP synthesis and preserved the structural integrity of Ca2+-challenged mitochondria. Thus, under conditions of excessive mitochondrial Ca2+ overload targeting mitochondrial Ca2+ transport pathways restores oxidative phosphorylation required for vital cellular processes. This study, therefore, identifies an effective strategy capable to rescue Ca2+-disrupted mitochondrial energetics.

Background: Heart muscle primarily relies on adenosine triphosphate produced by oxidative phosphorylation and is highly vulnerable to anoxic insult. Although a number of strategies aimed at improving myopreservation are available, no effective means of preserving mitochondrial energetics under conditions of anoxic injury have been developed. Openers of mitochondrial adenosine triphosphate-sensitive potassium channels have emerged as powerful cardioprotective agents presumably capable of maintaining mitochondrial function under metabolic stress. Here, we evaluated the ability of a prototype mitochondrial adenosine triphosphate-sensitive potassium channel opener, diazoxide, to preserve oxidative phosphorylation in mitochondria subjected to anoxia and reoxygenation., Methods: Mitochondria were isolated from rat hearts and subjected to 20 minutes of anoxia, followed by reoxygenation. Mitochondrial respiration and oxidative phosphorylation, as well as mitochondrial integrity, were assessed by means of ion-selective minielectrodes, high-performance liquid chromatography, fluorometry, and electron microscopy., Results: Anoxia-reoxygenation decreased the rate of adenosine diphosphate-stimulated oxygen consumption, inhibited adenosine triphosphate production, and disrupted mitochondrial integrity. On average, anoxic stress reduced adenosine diphosphate-stimulated respiration from 291 +/- 14 to 141 +/- 15 ng-atoms O(2). min(-1). mg(-1) protein and decreased the rate of adenosine triphosphate production from 752 +/- 14 to 414 +/- 34 nmol adenosine triphosphate. min(-1). mg(-1) protein. After anoxia, the majority (88%) of mitochondria was damaged or swollen and released adenylate kinase, a marker of mitochondrial integrity. Diazoxide (100 micromol/L), present throughout anoxia, preserved adenosine diphosphate-stimulated respiration at 255 +/- 7 ng-atoms O(2). min(-1). mg(-1) protein and adenosine triphosphate production at 640 +/- 39 nmol adenosine triphosphate. min(-1). mg(-1) protein. Diazoxide also protected mitochondrial structure from anoxia-mediated damage, so that after anoxic stress, 67% of mitochondria remained intact and adenylate kinase was confined to the mitochondria., Conclusions: The present study demonstrates that diazoxide diminishes anoxia-induced functional and structural deterioration of cardiac mitochondria. By protecting mitochondria and preserving myocardial energetics, diazoxide may be useful under conditions of reduced oxygen availability, including global surgical ischemia or storage of donor heart.

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 microM, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 +/- 15 mV, K+ channel openers, pinacidil (100 microM), cromakalim (25 microM), and levcromakalim (20 microM), induced membrane depolarization by 10 +/- 7, 25 +/- 9, and 24 +/- 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.

In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Regenerative Sr(++)-induced Sr++ release from isolated rat liver mitochondria was studied using ion-selective electrode techniques. Mitochondria, when exposed to a pulse of Sr++, demonstrated a reversible and transient increase in inner membrane permeability to K+ and H+ ions. The increase in permeability was an all-or-none process with a threshold dependent on the amplitude of the Sr++ pulse. The threshold concentration of Sr++ was lowered from 120-150 microM to 20-30 microM when mitochondria were preloaded with 100 nmoles Sr++/mg protein. Release of matrix-stored divalent cations provided a mechanism for amplification of the extramitochondrial Sr++ concentration (regenerative Sr(++)-induced Sr++ release). The mitochondrial inner membrane became refractory, since subsequent cycles of excitation could not be immediately induced. These experiments demonstrate that the inner mitochondrial membrane is an excitable membrane, with threshold-dependent, regenerative and subsequently refractile Sr(++)-induced Sr++ release characteristics.