A mitochondrial megachannel resides in monomeric F1FO ATP synthase.

Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.

The proton pumping activity of H(+)-ATPases: an improved fluorescence assay.

A new method for the estimation of steady-state delta pH, and the rate of acidification, by H(+)-ATPases (and other proton transporters) in inverted membrane vesicles is described. The method is based on a combination of two widely used fluorescent delta pH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine. It is demonstrated that 9-amino-6-chloro-2-methoxyacridine fluorescence quenching, which is very sensitive to small pH gradients, is not sensitive to the magnitude of large pH gradient, while 9-aminoacridine, which does not sense small gradients, is very sensitive to large pH gradients. A proper mixture of the two probes provides a method which is equally sensitive to pH gradients from very small values up to 3.5 pH units. The probe response was evaluated by titrations of the fluorescence signal with nigericin and adjusted by changing the concentration ratio and the emission wavelength. In liposomes, submitochondrial particles and bacterial vesicles an almost linear dependence of quenching on delta pH over the entire range can be obtained with this method. It is demonstrated that the new method can be used to obtain more reliable estimates of the rate of acidification as well as the magnitude of delta pH, whereas each of these and similar probes, by themselves are not as reliable. A determination of the ratio delta Gp/delta muH over a wide range of values reveal that this ratio is not constant but decreases with delta Gp. This finding should be taken into consideration when attempting to estimate the H+/ATP ratio form the measurement of delta Gp/delta muH.

Functional studies on in situ-like mitochondria isolated in the presence of polyvinyl pyrrolidone.

Mitochondria isolated and maintained in sucrose mannitol medium show a large intermembrane space and a condensed matrix unlike the appearance of in situ mitochondria. Mitochondria resembling in situ organelles are obtained when the isolation medium is supplemented with certain macromolecules such as polyvinyl pyrrolidone. We found that the in situ appearance was acquired also by the conventionally isolated mitochondria when they were exposed to 2% polyvinyl pyrrolidone supplemented medium. Paradoxically, however, these in situ looking mitochondria proved functionally inferior in that their brief incubation without substrates led to a marked loss of their ability to respire with subsequently added substrates such as pyruvate, acylcarnitines or glutamate. The oxidation of succinate was, however, not so affected. This phenomenon was shared by heart and skeletal muscle mitochondria of different animal species but not by rat liver mitochondria. The inhibition of respiration could not be related to the failure to oxidize NADH, to the tieing up of mitochondrial free CoASH, or to the increased matrix space of mitochondria that was observed in the presence of polyvinyl pyrrolidone. The polyvinyl pyrrolidone-exposed mitochondria regained their respiratory ability on being freed from polyvinyl pyrrolidone. The same phenomenon was seen also when the medium contained 2% albumin or 20% Ficoll.

[Estimation of intact outer and inner membranes of heart mitochondria]. / Opredelenie intaktnosti naruzhnoi i vnutrennei membran mitokhondriiserdtsa.

A simple procedure is developed for estimation of the damage rate of inner membrane of heart mitochondria. In the assay the rate of succinate oxidation was measured using bromthymol blue as an inhibitor of succinate transport. Bromthymol blue at low concentration (12 microM) functioned as a mixed type inhibitor of succinate oxidation, whereas at high concentrations--as uncompetitive inhibitor. Polarographic registration of cytochrome c content and of the rate of ascorbate oxidation in the samples containing Triton X-100 and free of the detergent was more sensitive procedure as compared with spectrophotometric measurement of reduced cytochrome c oxidation in estimation of the damage rate of outer mitochondrial membranes. The damage rates of outer and inner membranes of heart mitochondria isolated by a procedure which included the treatment with trypsin were equal to 8.43 +/- 0.74% and 8.04 +/- 1.9%, respectively, while in those isolated without the trypsin treatment--12.8 +/- 1.5% and 13.3 +/- 1.8%, respectively.

A slow-releasing form of prostacyclin agonist (ONO1301SR) enhances endogenous secretion of multiple cardiotherapeutic cytokines and improves cardiac function in a rapid-pacing-induced model of canine heart failure.

OBJECTIVES: Cardiac functional deterioration in dilated cardiomyopathy (DCM) is known to be reversed by intramyocardial up-regulation of multiple cardioprotective factors, whereas a prostacyclin analog, ONO1301, has been shown to paracrinally activate interstitial cells to release a variety of protective factors. We here hypothesized that intramyocardial delivery of a slow-releasing form of ONO1301 (ONO1301SR) might activate regional myocardium to up-regulate cardiotherapeutic factors, leading to regional and global functional recovery in DCM. METHODS AND RESULTS: ONO1301 elevated messenger RNA and protein level of hepatocyte growth factor, vascular endothelial growth factor, and stromal-derived factor-1 of normal human dermal fibroblasts in a dose-dependent manner in vitro. Intramyocardial delivery of ONO1301SR, which is ONO1301 mixed with polylactic and glycolic acid polymer (PLGA), but not that of PLGA only, yielded significant global functional recovery in a canine rapid pacing-induced DCM model, assessed by echocardiography and cardiac catheterization (n = 5 each). Importantly, speckle-tracking echocardiography unveiled significant regional functional recovery in the ONO1301-delivered territory, consistent to significantly increased vascular density, reduced interstitial collagen accumulation, attenuated myocyte hypertrophy, and reversed mitochondrial structure in the corresponding area. CONCLUSIONS: Intramyocardial delivery of ONO1301SR, which is a PLGA-coated slow-releasing form of ONO1301, up-regulated multiple cardiotherapeutic factors in the injected territory, leading to region-specific reverse left ventricular remodeling and consequently a global functional recovery in a rapid-pacing-induced canine DCM model, warranting a further preclinical study to optimize this novel drug-delivery system to treat DCM.

An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum.

A comparative study of various cryofracturing techniques has been conducted on the mammalian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube-like structures in the scanning electron microscope (SEM). Regular cross-bridging often seen at the Z-band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably. Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometers under the surface. The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol-impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles. Separation of the cell organelles caused by freezing or by critical point drying revealed thread-like structures extending from the mitochondrial surface. Re-examination of SEM-processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron-dense material, here named 'mito-reticular junctional fibres', bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specific anchoring function.

Post-embedding immuno-electron microscopy with rapid freezing and freeze-substitution techniques for the localization of manganese superoxide dismutase.

Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen-antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase.

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.

Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane.

Rotenone-sensitive NADH dehydrogenase activity and Lubrol stimulation of cytochrome oxidase activity were measured to assess the opposite membrane polarity of beef heart mitoplast and inside-out particle preparations. The ATP-Pi exchange activity of mitoplasts was not affected by their incubation at pH 8.9 in the presence of 5 mM EDTA (a treatment known to extract coupling factor B (F beta) from submitochondrial particles), nor was it stimulated by the addition of F beta to intact and alkaline treated mitoplast preparations. In contrast, the exchange activity of inside-out particles was decreased 18 fold by the alkaline/EDTA treatment and was almost completely restored by the addition of F beta to F beta-depleted particles. From these results it is concluded that in beef heart mitochondria, the coupling factor F beta is bound to the matrix-side of the inner mitochondrial membrane.

Cryo-ultramicrotomy as a tool for the assessment of doxorubicine cardiotoxicity in rats.

Cryo-ultramicrotomy was tested as a tool for the assessment of cardiac damage induced by doxorubicine in rats. Slightly fixed specimens were cut at -80 or -90 degrees C (knife temperature -60 or -70 degrees C), and the ultrastructural changes observed were compared with those found in conventional epoxy sections. It is concluded that the cryo method, although using more elaborate equipment and being more painstaking, might yield more sensitive parameters for cardiac damage than epoxy sections.

Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations.

Differences in oxidative metabolism between subsarcolemmal and interfibrillar heart mitochondria were investigated. Interfibrillar mitochondria oxidized substrates donating reducing equivalents at Complex I (NADH-CoQ reductase), Complex II (succinate-CoQ reductase), and Complex III (CoQH2-cytochrome c reductase) more rapidly than did subsarcolemmal mitochondria. There was no difference in oxidation of substrates entering the electron transport chain at Complex IV (cytochrome c oxidase). Differences expressed in normal-ionic-strength medium at Complexes II and III but not I were eliminated in low-ionic-strength medium. The concentrations of cytochromes and activities of NADH and cytochrome c oxidase were virtually the same in the two populations. In permeabilized mitochondria, activities of succinate-duroquinone and TMPD plus ascorbate oxidase were significantly lower in the subsarcolemmal mitochondria. Differences in membrane permeability between the populations were suggested by the greater permeability of subsarcolemmal mitochondria to exogenous NADH. The influence of isolation buffers and preparative procedures on the two classes of mitochondria were also examined. Characteristic biochemical and morphological properties of the two populations were unchanged by exposing each to the preparative procedure used to isolate the alternate population; the oxidative performance of the two populations cannot be equalized by experimental manipulation.

Physiological state of submitochondrial particles and their susceptibility to Triton X-100.

The solubilizing effect of Triton X-100 on beef heart submitochondrial particles (ETPH) has been studied under various physiological conditions. Coupled, uncoupled and azide-inhibited ETPH particles have been studied. Quantitative and qualitative differences are found in the proteins solubilized by the detergent from ETPH particles under the various conditions tested.

Comparative morphometry of the mitochondria and activity of some enzymes in the myocardium of small mammals.

Heart rate in mammals is inversely related to body weight. In the etruscan shrew it exceeds 960 beats/min. Since the heart depends primarily on energy from aerobic sources, it was interesting to examine some morphological parameters of the mitochondria in some mammals with a high heart frequency. Volume fraction and surface to volume ratio of the mitochondria in the myocardium were determined in the white rat (221 g), white mouse (36 g), white-toothed shrew (8 g) and the etruscan shrew (2 g) using morphometric methods. The volume fraction and surface to volume ratio of the mitochondria increased progressively and significantly as body weight of the animal decreased. The increase was higher in the surface to volume ratio than in the volume fraction. It reached a value which was 62% higher in the etruscan shrew than that of the white rat, while in the volume fraction of the mitochondria the maximal increase was only 34%. In accordance with the morphometric data, enzymatic activity of succinic dehydrogenase in the myocardium was inversely related to body weight. Creatine phosphokinase activity revealed a similar but not statistically significant trend. Lactic dehydrogenase activity was about three-fold higher in the white rat than in the white toothed shrew. It is concluded that one of the adaptive responses of the heart in small sized mammals to increase ATP production is not only elevation of the volume fraction of the mitochondria, but also an increase of their surface to volume ratio to provide a higher rate of oxygen diffusion to them.

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium.

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.

Recovery of monkeys after myocardial infarction with ventricular fibrillation. Effects of PGB.

PGBx, a polymeric, stable, free radical derivative of 15-keto-prostaglandin B1, that conserves oxidative phosphorylation in mitochondria under degenerative conditions in vitro, affected survival of male Rhesus monkeys (5-9 kg) anesthetized with pentobarbital and subjected to coronary ligation and induced ventricular fibrillation (VF). In tests performed in sequence with intervening periods for recover, intracardiac injections of norepinephrine (NE), cardiac massage (CM), and electrical defibrillation (EDF) were used to restore cardiac function both in controls and experimental animals, but the latter were injected also with 1 mg/kg PGBx. Recovery was established by maintenance of effective blood pressure without exogenous support. In the control group the cumulative survival for fibrillation episodes of 4, 6, 8, and 12 min was 60, 40, 31, and 25% respectively. In the PGBx-treated group survival for equivalent periods was 100, 93, 93, and 88% respectively. In separate studies, African Green monkeys were subjected to single episodes of VF of either 8 or 12 min. Combined survival was 36% for the controls, 93% for the PGBx-treated animals. Clearly PGBx radically improved cardiac recovery after circulatory arrest due to VF in the presence of acute myocardial infarction. The results also suggest a synergistic action between norepinephrine and PGBx in achieving such recovery.

Transmembrane topography of the mitochondrial oxoglutarate carrier assessed by peptide-specific antibodies and enzymatic cleavage.

The folding of the peptide chain of the bovine heart oxoglutarate carrier in the inner mitochondrial membrane and in the membrane of reconstituted proteoliposomes has been investigated by enzymatic and immunochemical approaches using proteinase K and polyclonal site-directed antibodies, respectively. Two peptides corresponding to the amino acid sequences 2-12 (N-terminal peptide) and 303-314 (C-terminal peptide) have been synthesized and coupled to ovalbumin before being used to immunize rabbits. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and by Western blot analysis. Both anti-N-terminal and anti-C-terminal antibodies reacted specifically with the corresponding peptides and with the isolated oxoglutarate carrier, whereas only anti-C-terminal antibodies immunodetected the carrier in mitochondrial lysates and reacted with the membrane-bound carrier in mitoplasts and in freeze-thawed mitochondria. This result indicated that the last 12 C-terminal amino acid residues of the oxoglutarate carrier protein are accessible from the cytosolic side of the inner mitochondrial membrane. Anti-C-terminal antibodies did not recognize the oxoglutarate carrier in reconstituted proteoliposomes unless the membrane was inverted, indicating that the carrier was inserted unidirectionally in proteoliposomes, with an orientation opposite that found in mitochondria. The immunological data were complemented by data from a limited proteolysis study performed on the membrane-bound oxoglutarate carrier in proteoliposomes, using proteinase K. Cleavage of the carrier caused a time-dependent inhibition of the oxoglutarate-oxoglutarate exchange activity of the reconstituted system. Four cleavage sites were identified, between Val-39 and Gln-40, between Tyr-61 and Lys-62, between Phe-169 and Arg-170, and between Arg-182 and Gly-183.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on a possible molecular basis for the structure of mitochondrial cristae.

We have investigated a possible molecular basis for mitochondrial cristae formation. Proteoliposomes containing electron transport proteins, cytochrome oxidase, or complex III in their proper orientation bind to pig heart mitoplasts but not pig heart mitochondria. Using Leydig tumor cells, we have confirmed earlier reports that chloramphenicol causes a diminution in cristae content and a change in its characteristic lamellar form. We show that the proteoliposomes containing cytochrome oxidase or complex III in the proper orientation bind to mitoplasts from Leydig tumor cells but do not bind as well to mitoplasts from chloramphenicol-treated Leydig tumor cells. These experiments provide a possible mechanism to explain cristae formation.

Interactions among mitochondrial aspartate aminotransferase, malate dehydrogenase, and the inner mitochondrial membrane from heart, hepatoma, and liver.

The inner mitochondrial membranes from bovine heart, rat liver, and Morris hepatoma 7777 all bound the mitochondrial isozymes of aspartate aminotransferase and malate dehydrogenase with comparable affinities and binding ratios (mg of enzyme bound per mg of membrane protein). A low molecular weight fraction separated from a detergent extract of the heart membrane by chromatography on Sephacryl S-300 contained most of the binding activity of the extract for the aminotransferase and had a dissociation constant for the aminotransferase of 0.2 microM. The protein component of the membrane binding sites for the aminotransferase was apparently present in this fraction because binding activity was largely eliminated by proteolysis with trypsin. When this fraction was chromatographed on an aminotransferase affinity column, only the portion that was bound and eluted by 0.25 M KCl associated with added aminotransferase. Unlike the membrane, which was markedly inhibited by the non-ionic detergent Genapol but was inhibited only 20% by trypsin, the binding activity of this subfraction was completely inhibited by trypsin but not by Genapol. This suggests, on the membrane, that the aminotransferase binds to the binding protein and is then transferred to lipids specifically associated with the binding protein. These putative lipids are presumably removed on the affinity column. Although the yield of the binding protein was low, there is probably ample binding protein in mitochondria to accommodate the aminotransferase. In every case, binding of the aminotransferase to the membrane inactivated the malate dehydrogenase binding site whereas malate dehydrogenase had little effect on the binding of the aminotransferase and only associated with the higher molecular weight fractions from the Sephacryl column that contained Complex I activity. Inactivation of the malate dehydrogenase site by the aminotransferase, but not vice versa, could result from aminotransferase associating with the binding protein and malate dehydrogenase with Complex I followed by association of the enzymes with lipids located in the same region of the membrane. However, since aminotransferase is more cationic, it is not displaced readily from the lipids by malate dehydrogenase. The relevance of these interactions to the organization of the enzymes is discussed.