Control Mitochondria Research Articles

Metabolic Reprogramming of Human Macrophages by Sickle Cell Hemoglobin

BACKGROUND: Sickle cell disease is caused by a single nucleotide mutation in the β-globin gene, resulting in the substitution of valine for glutamic acid, polymerization of hemoglobin and sickling of red blood cells (RBCs) under low oxygen conditions causing chronic hemolysis, and organs damage. SCA is characterized by the presence of cell-free plasma hemoglobin. Organ damage is associated with chronic inflammation and pro-inflammatory macrophages phenotype. Typically, anti-inflammatory response is elicited by the phagocytosis of senescent RBCs and cell-free hemoglobin. We have demonstrated that treatment of human macrophages with sickle cell hemoglobin (HbS) leads to lysosome accumulation, autophagy activation, and pro-inflammatory phenotype. The propensity of mutant HbS to polymerize under low pH may promote lysosomal HbS polymer accumulation, hindering degradation, and inducing macrophage differentiation towards a pro-inflammatory state. The differentiation of macrophages into specific phenotypes is associated with significant metabolic reprogramming. HYPOTHESIS: We hypothesize that the uptake of HbS by human macrophages induces their metabolic reprogramming via mitochondrial remodeling. METHODS: Human THP-1 cells were differentiated into macrophages with PMA and treated with either purified HbS or normal hemoglobin (HbA). Mitochondria were detected with MitoTrackerTM Green FM dye, and mitochondrial ROS (mtROS) level was detected by Mito SOX Red Kit. Transmission electron microscopy imaging was performed at Talos 200X transmission electron microscope. EPView (Olympus) software was used for the structure analysis. Total RNA was isolated by Trizol and sequenced on an Illumina® NextSeq 500. Comparisons between treatments were conducted using Gene Set Enrichment Analysis (GSEA). RESULTS: Immunofluorescent staining demonstrated accumulation of mitochondria after treatment with HbS compared to untreated (ctrl) or HbA-treated macrophages. Electron microscopy revealed an increase in the number and size of mitochondria. In 25 random microphotographs of cytoplasm, we detected 51 mitochondria in control, 79 in HbA treated and, 96 in HbS treated macrophages. The mitochondria volume and cristae size were significantly increased in macrophages treated with HbS. There was no increase in mtROS production in HbS treatment compared to ctrl. As expected, HbA treatment reduced mtROS production. The mitochondrial gene set (242 genes) was the second most enriched GSEA set in HbS-treated compared to HbA-treated macrophages. The larger mitochondrial size may be associated with a reduction of fission or an increase in fusion. We did not find differences in the expression of mitochondrial fission and fusion genes between HbS and HbA-treated macrophages. The increase in the mitochondrial size and cristae size may also be associated with increased oxidative phosphorylation. We found significant enrichment of genes responsible for oxidative phosphorylation in the HbS-treated macrophages. We did not find significant differences in the expression of glycolysis genes. CONCLUSION: Treatment of human macrophages with HbS significantly increases the number and volume of mitochondria, size of the cristae, and oxidative phosphorylation without upregulation of mtROS production and reduction of glycolysis. This work was supported by NIH Research Grants SC1HL150685, P50HL118006, and R01HL125005. We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3024: Mitochondrial Volume Abnormalities In Gastrocnemius Muscle Of Patients With Peripheral Artery Disease

Background: The mechanisms of claudication in patients with peripheral artery disease (PAD) are poorly understood. Mitochondria are responsible for ATP production during aerobic conditions but may become structurally impaired in patients with chronic ischemia and PAD. Research Question: Are mitochondrial volumes different in patients with PAD compared with age-matched controls? Aims: To better understand mitochondrial abnormalities and ultrastructure in patients with PAD. Methods/Approach: Patients with PAD (defined as ABI <0.85) and age-matched controls (ABI 1.00-1.40) were recruited and underwent gastrocnemius muscle biopsy. Muscle samples were collected in optimal cutting temperature compound. Mitochondria were labeled with cytochrome oxidase IV antibody and Cy3 fluorescence secondary antibody and images were rendered in three dimensions using structured illumination microscopy to quantify mitochondrial size, content, and morphology. Comparisons were done with a Mann-Whitney U test. Results/Data: Forty-eight subjects (median [IQR] age 66.5 [59.0, 74.0] years, 32 (66.7%) male) were recruited and underwent gastrocnemius muscle biopsy, including 16 controls and 32 with PAD. The largest singular mitochondrial volume was greater in those with PAD (22.6 vs. 4.2μm 3 , p=0.01, Figure 1A) but the median mitochondrial volume was smaller (0.07 vs. 0.01μm 3 , p=0.03, Figure 1B). While no control mitochondria were larger than 7.2μm 3 , 1% of mitochondria in patients with PAD were larger than 10μm 3 (Figure 1C). There was no difference in the total mitochondrial volume or content between PAD and controls. Conclusion: Patients with PAD have larger maximum but smaller median mitochondria sizes in the gastrocnemius muscle, suggesting greater heterogeneity of mitochondrial size in those with PAD. The changes in mitochondrial morphology may result in mitochondrial inefficiency and an impaired compensatory response in the setting of chronic ischemia from PAD.

Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has beendescribed as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regardingreactive oxygen species(ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrialrespiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

Diabetic mitochondria are resistant to palmitoyl CoA inhibition of respiration, which is detrimental during ischemia.

The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP‐stimulated state 3 respiration and increased the apparent K m for ADP twofold. The half maximal inhibitory concentration (IC50) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the K m for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose‐dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31P‐magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.

Selective anticancer activity of superparamagnetic iron oxide nanoparticles (SPIONs) against oral tongue cancer using in vitro methods: The key role of oxidative stress on cancerous mitochondria

Today, it has been proven that the nanoparticles such as superparamagnetic iron oxide nanoparticles (SPIONs) have widespread use in biomedical applications, for instance, in magnetic resonance imaging and targeted delivery of drugs. Despite many studies on SPIONs in diagnosing some diseases like cancer, it has not been investigated on the oral tongue squamous cell carcinoma (OTSCC) detection by the NPs. Hence, the present study has been designed to assess the in vitro cytotoxicity of SPIONs on the isolated mitochondria of OTSCC by mitochondrial tests. Isolated mitochondria were removed from the separated cancer and control tissues from the squamous cells of tango in male Wistar rats (6 or 8 weeks) and exposed to the different concentrations of SPIONs (30, 60, and 120 nM). A rise in the production of reactive oxygen species is one of the significant mechanisms of this study, followed by a collapse of mitochondrial membrane potential, the escape of mitochondrial cytochrome c, and mitochondrial swelling in the exposed isolated mitochondria of OTSCC with SPIONs. Furthermore, our results indicated that the exposure to the SPIONs reduced the activity of succinate dehydrogenase in complex II of the mitochondria obtained from cancerous oral tongue squamous. So the SPIONs can induce selective cytotoxicity on the OTSCC mitochondria without significant effects on the control mitochondria. Based on the results and further studies about in vivo experiments in this regard, it is concluded the SPIONs may be a hopeful therapeutic candidate for the treatment of OTSCC.

Coenzyme Q 10 supplementation: A potential therapeutic option for the treatment of intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease characterized by pruritus, elevated serum bile acids and abnormal liver function that may be associated with severe adverse pregnancy outcomes. We previously reported that plasma coenzyme Q10 (CoQ10) is decreased in women with ICP as it is its analogue coenzyme Q9 (CoQ9) in rats with ethinyl estradiol (EE)-induced cholestasis. The aim of the present study was to evaluate the possible therapeutic role of CoQ10 in experimental hepatocellular cholestasis and to compare it with ursodeoxycholic acid (UDCA) supplementation. Bile acids, CoQ9, CoQ10, transaminases, alkaline phosphatase, retinol, α-tocopherol, ascorbic acid, thiobarbituric acid reactive substances, carbonyls, glutathione, superoxide dismutase and catalase were assessed in plasma, liver and/or hepatic mitochondria in control and cholestatic rats supplemented with CoQ10 (250 mg/kg) administered alone or combined with UDCA (25 mg/kg). CoQ10 supplementation prevented bile flow decline (P < 0.05) and the increase in serum alkaline phosphatase and bile acids, particularly lithocholic acid (P < 0.05) in cholestatic rats. Furthermore, it also improved oxidative stress parameters in the liver, increased both CoQ10 and CoQ9 plasma levels and partially prevented the fall in α-tocopherol (P < 0.05). UDCA also prevented cholestasis, but it was less efficient than CoQ10 to improve the liver redox environment. Combined administration of CoQ10 and UDCA resulted in additive effects. In conclusion, present findings show that CoQ10 supplementation attenuated EE-induced cholestasis by promoting a favorable redox environment in the liver, and further suggest that it may represent an alternative therapeutic option for ICP.

Kidney dysfunction induced by a sucrose-rich diet in rat involves mitochondria ROS generation, cardiolipin changes, and the decline of autophagy protein markers.

The mechanistic link between obesity and renal failure has been proposed to involve mitochondria reactive oxygen species generation and lipotoxicity. These pathological conditions make mitochondria of particular interest in the regulation of cell function and death by both apoptosis and autophagy. Therefore, this work was undertaken to investigate mitochondria function, autophagy, and apoptosis protein markers in the kidney from a rat model of intra-abdominal obesity and renal damage induced by a high-sucrose diet. Mitochondria from sucrose-fed (SF) kidneys in the presence of pyruvate-malate generated H2O2 at a higher rate than from control (79.81 ± 4.98 vs. 65.84 ± 1.95 pmol·min-1·mg protein-1). With succinate, the release of H2O2 was significantly higher compared with pyruvate-malate, and it remained higher in SF than in control mitochondria (146.4 ± 8.8 vs. 106.1 ± 5.9 pmol·min-1·mg protein-1). However, cytochrome c release from SF kidney mitochondria was lower than from control. In addition, cardiolipin, a mitochondria-specific phospholipid, was found increased in SF mitochondria due to the enhanced amount of both cardiolipin synthase and tafazzin. Cardiolipin was also found enriched with saturated and monounsaturated fatty acids, which are less susceptible to peroxidative stress involved in cytochrome c release. Furthermore, beclin-1 and light chain 3-B, as autophagy protein markers, and caspase-9, as apoptosis protein marker, were found decreased in SF kidneys. These results suggest that the decline of autophagy protein markers and the lack of apoptosis process could be a pathological mechanism of cell dysfunction leading to the progression of renal disease in SF rats.

COmplexome Profiling ALignment (COPAL) reveals remodeling of mitochondrial protein complexes in Barth syndrome.

MotivationComplexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.ResultsWe have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multi-dimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodeling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (mitochondrial contact site and cristae organizing system, prohibitins), or are attached to it (the large ribosomal subunit).Availability and implementationCOPAL is written in python and is available from http://github.com/cmbi/copal.Supplementary information Supplementary data are available at Bioinformatics online.

Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography

SummaryMitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease.

Punica Granatum Rind Extract Promotes Mouse Liver Mitochondrial Function and Attenuates Paraquat Toxicity in a Caenorhabditis Elegans Model

Punica granatum (pomegranate) has drawn the interest of the scientific community due to its rich array of phenolic compounds and antioxidant capacity. Evidence supports the fruit's role in preventing free radical damage in humans. Although various studies have concluded the beneficial effects of pomegranate juice, limited studies have characterized the antioxidant effects of its rind (outer covering/peel; PRE). The goal of our study was to characterize the effects of PRE on oxidative stress. We utilized mouse liver mitochondria and paraquat (PQ) toxicity in a Caenorhabditis elegans (C. elegans) model to determine the beneficial effects of PRE. Mitochondria were isolated from C57 male mice liver tissue (6 weeks of age). Phosphatidylcholine/cholesterol liposomes (Lp) were used to induce oxidative stress. Samples were then incubated at 37□ for 2 hours with or without the addition of PRE at two levels (PRE1 and PRE2 represent 60 and 120 μM Trolox equivalent/mL, respectively). Oxygen consumption rate (OCR; nmol O2 consumed/min per mg mitochondria) was used to express the activity of NADH‐CoQ oxidoreductase, succinate dehydrogenase, and cytochrome c oxidase (complex I, II, and IV of electron transport chain, respectively). Antioxidant level in PRE was determined by DPPH‐, ABTS‐ and Folin‐Ciocalteu assays. The experiment was repeated four times. Pre‐incubation of mitochondria with Lp decreased (p &lt; 0.05) OCR by 50% compared with control mitochondria without Lp. However, incubation of Lp with PRE2 increased (p &lt; 0.05) OCR for complex II and IV. No significant changes (p &gt; 0.05) in OCR were observed for complex I with incubation of Lp and PRE 1 or PRE2. Using C. elegans, an oxidative stress resistance assay was performed to explore PREs effects on paraquat toxicity. Wild‐type adult/L4 hermaphrodites were placed in 96‐well plates. PQ was dissolved to a final concentration of 50 μM and half concentration of PRE1 was added for experimental groups. Resistance to oxidative stress was measured through percentage survival at hour time intervals. After 5 hours, mean survival for PQ alone was 61.91±6.56%; PQ groups supplemented with PRE had a mean survival of 88.29±6.18%. The current study suggests that PRE may play a role in preserving mitochondrial function during oxidative stress and attenuates PQ toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptomic analysis reveals inflammatory and metabolic pathways that are regulated by renal perfusion pressure in the outer medulla of Dahl-S rats.

Studies exploring the development of hypertension have traditionally been unable to distinguish which of the observed changes are underlying causes from those that are a consequence of elevated blood pressure. In this study, a custom-designed servo-control system was utilized to precisely control renal perfusion pressure to the left kidney continuously during the development of hypertension in Dahl salt-sensitive rats. In this way, we maintained the left kidney at control blood pressure while the right kidney was exposed to hypertensive pressures. As each kidney was exposed to the same circulating factors, differences between them represent changes induced by pressure alone. RNA sequencing analysis identified 1,613 differently expressed genes affected by renal perfusion pressure. Three pathway analysis methods were applied, one a novel approach incorporating arterial pressure as an input variable allowing a more direct connection between the expression of genes and pressure. The statistical analysis proposed several novel pathways by which pressure affects renal physiology. We confirmed the effects of pressure on p-Jnk regulation, in which the hypertensive medullas show increased p-Jnk/Jnk ratios relative to the left (0.79 ± 0.11 vs. 0.53 ± 0.10, P < 0.01, n = 8). We also confirmed pathway predictions of mitochondrial function, in which the respiratory control ratio of hypertensive vs. control mitochondria are significantly reduced (7.9 ± 1.2 vs. 10.4 ± 1.8, P < 0.01, n = 6) and metabolomic profile, in which 14 metabolites differed significantly between hypertensive and control medullas ( P < 0.05, n = 5). These findings demonstrate that subtle differences in the transcriptome can be used to predict functional changes of the kidney as a consequence of pressure elevation.

340. Cell Penetrating Peptide Can Improve the Efficacy of Mitochondrial Transfer

Cell-based therapies for organ regeneration are currently emerging as a new promising approach to treat various diseases, including cardiovascular and neurodegenerative diseases. However, the mechanism supporting these therapeutic effects remains poorly understood. Several recent studies suggest that the intercellular transfer of organelles, including mitochondria, might contribute to these regenerative responses.Mitochondria are considered cellular “power plants” because they largely synthesize the universal energy “currency” of the cells, i.e., adenosine triphosphate (ATP). Mitochondrial dysfunction is associated with many diseases, including metabolic and neurodegenerative disorders. In vitro experiments revealed that mitochondrial DNA (mtDNA)-depleted mammalian cells recovered aerobic respiration after intercellular mitochondrial transfer from intact cells. The therapeutic potential of mitochondrial transfer was supported by an in vivo study conducted on rabbit model of myocardial infarction. Direct injection of autologous mitochondria into the ischemic heart considerably improved post-infarct cardiac functions. Mitochondria dysfunction also plays central role in the pathogenesis of ischemia reperfusion injury and subsequent cardiomyocytes death. The beneficial effect of mitochondria replenishment for damaged cells and tissues has been reported recently.Our previous studies demonstrated that the mitochondrial transfer could occur by simple co-culture of mammalian cells with isolated mitochondria. We assessed the impact of the transfer on the mitochondrial function in the recipient cells. The mechanism of mitochondrial internalization was investigated using endocytosis inhibitors. We showed that mammalian cells engulfed isolated mitochondria by macropinocytosis, which survived in heteroplasmy state and functioned to generate ATP in recipient cells. However, the internalization for isolated mitochondria remains inefficient to apply as a clinical therapeutic strategy.Here, we report the use of TAT, which is cell penetrating peptide derived of human immunodeficiency virus, dextran complexes (TAT-DEX) to enhance the cellular uptake of exogenous mitochondria and improve the protective effect of mitochondria replenishment on rat neonatal cardiomyocytes (NRCM) from IR injury in vitro. TAT-DEX modification significantly neutralized surface negative charge in mitochondria isolated from the human endometrium-derived mesenchymal cells and H9c2 rat cardiomyoblast. TAT-DEX modified mitochondria isolated from H9c2 showed a significantly higher level of cellular uptake. Compared with control mitochondria, co-incubation of TAT-DEX modified mitochondria with NRCM during IR significantly reduced IR-induced reactive oxygen species production and apoptosis in NRCM. These results indicate that surface modification of isolated mitochondria at the molecular level can improve the efficiency of cellular uptake of exogenous mitochondria, and enhanced mitochondrial internalization into cardiomyocytes may improve the potential effect of mitochondria replenishment therapy in myocardial IR injury.

The Protective Roles of Selenium on Hepatic Tissue Ultrastructure and Mitochondrial Antioxidant Capacity in Copper-Overloaded Rats.

The aim of this study was to explore the effects of selenium addition on hepatic tissue ultrastructure and mitochondrial antioxidant capacity in copper-overloaded rats. Eighteen adult male Sprague-Dawley (SD) rats were randomly divided into three groups (n = 6 per group). Each group received 1mL (intragastrically) of water (control, group I) or water containing copper chloride (CuCl2, 1mol/L) (group II) or a mixture of CuCl2 (1mol/L) with sodium selenite (Na2SeO3, 0.05mol/L) (group III) once daily for 30days. Histological examination revealed normal hepatocyte structure and no ultrastructural changes in mitochondria in controls. In contrast, group II exhibited severe ultrastructural alterations, fuzzy mitochondrial membranes, irregularly shaped and fragmented rough endoplasmic reticulum (RER), and the high melanin content; group III also exhibited larger amounts of engulfing vesicles (EV) in the cytoplasm. Compared to controls, the CuCl2 treatment lowered (P < 0.05) hepatocyte enzymatic activities and increased lipid peroxidation as indicated by an increase in malondialdehyde (MDA) levels. The effects of CuCl2 were attenuated by simultaneous administration with Na2SeO3. These results indicated that the adverse effects of copper toxicity can be partially attenuated by providing a source of selenium.

Activation of the Mitochondrial Permeability Transition Pore Leads to the Increase in Amount of C-Subunit of ATP Synthase Associated with Channel-Forming Complex of Polyhydroxybutyrate and Inorganic Polyphosphate

Low permeability of the mitochondrial inner membrane is critical for maintaining the mitochondrial electrochemical potential - the driving force for ATP production. Acute stress conditions, lead to the increase in the mitochondrial inner membrane permeability due to the opening of the permeability transition pore (PTP). PTP allows free movement of ions and small molecules leading to mitochondrial depolarization, ATP depletion and cell death. Recent studies suggest that C-subunit of the mitochondrial ATP synthase plays a central role in PTP. Previous work in our laboratory showed that mitochondria contain non-protein complex composed of polyhydroxybutyrate, inorganic polyphosphate and calcium that forms an ion channel with properties resembling PTP. Here we explore the possibility of interactions between these non-protein components and C-subunit during the induction of PTP.To induce PTP,isolated energized mitochondria were treated with calcium. Control mitochondria were treated with calcium either in the presence of ruthenium red, inhibitor of calcium uptake or Cyclosporin A, inhibitor of PTP. This was followed by a water-free chloroform extraction of channel forming fraction of PTP. Components of the extract were analyzed using immunoblot analysis. We found significantly increased amount of C-subunit associated with channel forming fraction extracted from mitochondria with activated PTP. In contrast, C-subunit was not detectable in the extract when Ruthenium Red was present and significantly decreased in the presence of Cyclosporin A or in the absence of calcium.These results show that C-subunit is likely an interacting partner of the pore-forming complex of polyphosphate, calcium and polyhydroxybutyrate. We hypothesize that fully functional PTP requires calcium-induced formation of the pore made of both the complexed polymers and the c-subunit of ATP synthase

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) — sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Activation of mitochondrial‐u‐calpain sensitizes opening of the mitochondrial permeability transition pore during ischemia‐reperfusion (648.11)

BACKGROUND: Opening of the mitochondrial permeability transition pore (MPTP) increases cardiac injury during ischemia‐reperfusion (IR). IR activates both cytosolic and mitochondrial calpains. Administration of MDL‐28170 (MDL, a calpain inhibitor) decreases injury in isolated hearts during IR. We asked if inhibition of calpains during IR decreases MPTP opening and if activation of mitochondrial u‐calpain (m‐u‐calpain) predisposes to MPTP.METHODS: Buffer‐perfused mouse hearts (C57BL/6) were subjected to 25 min ischemia and 30 min reperfusion with or without MDL (10 uM) treatment. Opening of MPTP was evaluated by calcium retention capacity (CRC, nmol calcium/mg protein) in mitochondria isolated from IR hearts. In control mitochondria (protease‐purified to remove cytosolic contamination), exogenous calcium was used to activate mito‐u‐calpain. The ADP‐stimulated change in inner membrane potential (ΔΨ) during the transition from state 4 to state 3 respiration (reflected by alteration in TMRM fluorescence arbitrary units, A.U.) was used to assess inner membrane integrity.RESULTS: Inhibition of calpains improved CRC in mitochondria following IR, supporting that calpain activation enhanced MPTP opening. In control mitochondria, exogenous calcium progressively depolarized ΔΨ, whereas inhibition of m‐u‐calpain attenuated the extent of depolarization.CONCLUSION: Inhibition of calpain in purified mitochondria reduces the calcium‐induced permeation of the inner membrane, indicating that activation of m‐u‐calpain likely contributes to MPTP opening and cardiac injury during IR. Isolated hearts LDH release LVDP CRC IR 615±47 13±2 424±50 MDL + IR 383±55* 29±5* 560±16* Isolated mito ΔΨ, TMRM, A.U. Calcium uM 0 50 100 DMSO + mito 0.088±0.009 0.050±0.003†0.013±0.003†MDL+ mito 0.087±0.006 0.066±0.004‡ 0.036±0.013‡ Mean ± SEM; LVDP‐developed pressure mmHg; LDH‐mU/g/min; *p&lt;0.05 vs. IR; †p&lt;0.05 vs. calcium (0 uM); ‡ p&lt;0.05 vs. untreated mitochondria (mito).Grant Funding Source: Supported by Scientist Development Grant from AHA

ADP Protects Cardiac Mitochondria under Severe Oxidative Stress

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca2+ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca2+-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.

The STAT3 Inhibitor Stattic Impairs Cardiomyocyte Mitochondrial Function Through Increased Reactive Oxygen Species Formation

The signal transducer and activator of transcription 3 (STAT3) transduces stress signals from the plasma membrane to the nucleus but has recently also been identified in mitochondria. Inhibition of cardiomyocyte mitochondrial STAT3 with the STAT3-specific inhibitor Stattic decreases ADP-stimulated respiration and enhances calcium-induced mitochondrial permeability transition pore (MPTP) opening. The aim of the present study was to analyze whether or not these effects of STAT3 inhibition by Stattic are mediated by the formation of reactive oxygen species (ROS). The H2O2 formation from isolated rat left ventricular mitochondria was measured continuously in the presence of the complex 1 substrates glutamate and malate using the H2O2 indicator Amplex UltraRed. Stattic dose-dependently increased mitochondrial ROS formation (slope of Amplex UltraRed fluorescence/time; DMSO: 0.39±0.01; 1 µM Stattic: 0.40±0.03; 10 µM Stattic: 0.71±0.04; 25 µM Stattic: 1.43±0.05; 50 µM Stattic: 3.53±0.23; 100 µM Stattic: 9.23±0.69, n=5 mitochondrial preparations, p<0.05 for 10-100 µM Stattic). The increase in the ROS signal by 100 µM Stattic was abolished in the presence of the ROS scavenger N-acetylcysteine (Nac, 0.46±0.02, n=7, p<0.05). Mitochondria treated with 100 µM Stattic produced less ATP than control mitochondria (86±3 arbitrary units (a.u.) vs. 128±7 a.u., n=9, p<0.05). Again, in the presence of Nac ATP production was similar between Stattic-treated and control mitochondria (142±4 a.u. vs. 147±12 a.u., n=5, p=ns). MPTP opening was induced by lower amounts of calcium in Stattic-treated than in control mitochondria (in nmol CaCl2/mg protein, Stattic: 507±57, n=7; control: 857±70, n=7, p<0.05). There was no difference in calcium-induced MPTP opening between Stattic-treated (833±57, n=6) and control mitochondria (921±75, n=7, p=ns) in the presence of Nac. Taken together, our data show that inhibition of mitochondrial STAT3 by Stattic impacts on mitochondrial ATP production and MPTP opening through enhanced ROS formation.