Rat Heart Mitochondria Research Articles

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.

Phosphorylation and Kinetics of Mammalian Cytochrome c Oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.

Sirolimus-Induced Vascular Dysfunction: Increased Mitochondrial and Nicotinamide Adenosine Dinucleotide Phosphate Oxidase-Dependent Superoxide Production and Decreased Vascular Nitric Oxide Formation

This study sought to analyze mechanisms that mediate vascular dysfunction induced by sirolimus. Despite excellent antirestenotic capacity, sirolimus-eluting stents have been found to trigger coronary endothelial dysfunction and impaired re-endothelialization. To mimic the continuous sirolimus exposure of a stented vessel, Wistar rats underwent drug infusion with an osmotic pump for 7 days. Sirolimus treatment caused a marked degree of endothelial dysfunction as well as a desensitization of the vasculature to the endothelium-independent vasodilator nitroglycerin. Also, sirolimus stimulated intense transmural superoxide formation as detected by dihydroethidine fluorescence in aortae. Increased superoxide production was mediated in part by the vascular nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase as indicated by a marked stimulation of p67(phox)/rac1 NADPH oxidase subunit expression and by increased rac1 membrane association. In addition, superoxide production in rat heart mitochondria was up-regulated by sirolimus, as measured by L012-enhanced chemiluminescence. As a consequence, electron spin resonance measurements showed a 40% reduction in vascular nitric oxide bioavailability, which was further supported by decreased serum nitrite levels. Sirolimus causes marked vascular dysfunction and nitrate resistance after continuous treatment for 7 days. This impaired vasorelaxation may, in part, be induced by up-regulated mitochondrial superoxide release as well as by an up-regulation of NADPH oxidase-driven superoxide production. Both processes could contribute to endothelial dysfunction observed after coronary vascular interventions with sirolimus-coated stents.

Seasonal variations in properties of rat heart mitochondria: Effect on membrane fluidity and ATPase activity in diabetes

Seasonal variations in properties of rat heart mitochondria: Effect on membrane fluidity and ATPase activity in diabetes

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O 2 − ) from rat heart mitochondria (RHM), while O 2 − generated in intact RHM do not escape from mitochondria. This was shown by a set of O 2 − -sensitive spin probes with varying hydrophobicity. The levels of O 2 − detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O 2 − levels. Elevated O 2 − levels became sensitive to SOD but in a different manner. The determination of O 2 − with water-soluble PPH was fully sensitive to SOD, while the determination of O 2 − with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O 2 − into the surrounding medium.

Effect of Adriamycin on superoxide radical generation in isolated heart mitochondria

The influence of Adriamycin (doxorubicin) on the rate of superoxide radical formation in isolated rat heart mitochondria was studied by EPR with the Tiron spin trap not penetrating the mitochondrial inner membrane. Adriamycin at 10–150 μM considerably enhanced superoxide generation in the presence of succinate (substrate of the respiratory chain complex II) and glutamate/malate (complex I substrate) when electron transfer was blocked in complex III with antimycin A. Such effects may partly account for the known cardiotoxicity of this antitumor drug.

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K+ channels (mitoKATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca2+-induced mitochondrial permeability transition, and hydrogen peroxide (H2O2) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 muM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (DeltaPsi), the phosphorylation rate, and the membrane permeability to H+, K+ and Ca2+ were not affected either. However, sildenafil citrate decreased H2O2 generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O2 (*-) ) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 microM do not affect either rat heart mitochondrial bioenergetics or Ca2+-induced mitochondrial permeability transition, but it depresses H2O2 generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.

71 Ischemic damage to the electron transport chain increases the production of reactive oxygen species from isolated rat heart mitochondria

71 Ischemic damage to the electron transport chain increases the production of reactive oxygen species from isolated rat heart mitochondria

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca 2+ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe–S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca 2+. GE also inhibits Ca 2+ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca 2+ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe–S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.

Cannabinoid receptor agonists are mitochondrial inhibitors: A unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Δ-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10–50 μM) and significant decreases at 100 μM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II–III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.

Inhibition of anion channels derived from mitochondrial membranes of the rat heart by stilbene disulfonate—DIDS

The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2',2-disulfonic acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM KCl, cis/trans) and symmetric (150 mM KCl) conditions was approximately 100 pS. DIDS caused a dramatic decrease in the channel activity (IC(50) = 11.7 +/- 3.1 microM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade was complete and irreversible when concentration of DIDS was increased up to 200 microM. Our data indicate that DIDS is an allosteric blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion channels on the functional level.

Nitration of Tryptophan 372 in Succinyl-CoA:3-Ketoacid CoA Transferase during Aging in Rat Heart Mitochondria

The main objective of this study was to test the hypothesis that in vivo post-translational modifications in proteins, induced by the endogenously generated reactive oxygen and nitrogen molecules, can alter protein function and thereby have an effect on metabolic pathways during the aging process. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in rat heart to undergo age-associated increase in a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity ( approximately 10 A) of the enzyme active site. Between 4 and 24 months of age, the molar content of nitration was more than doubled while specific enzyme activity increased significantly. The amount of SCOT protein, however, remained unchanged. In vitro treatment of heart mitochondrial soluble proteins with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of SCOT. Results of this study identify tryptophan to be a specific target of nitration in vivo, for the first time. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption as an alternative source of energy supply in the heart.

Involvement of SH-groups during interaction of diazoxide with the inner membrane of rat heart mitochondria

It is known that adenine nucleotide translocase plays an important role in the formation of calcium-dependent mitochondrial permeability transition pore in the inner mitochondrial membrane [1] and activation of basal mitochondrial respiration by diazoxide, an agonist of mitochondrial K ATP channels [2]. Recent findings suggest that mitochondrial permeability transition pore and mitochondrial K ATP channel, which are regulated by [Ca 2+ ] m and ∆Ψ , respectively, and play a key role in the initiation of apoptosis [3], have close structural interconnections. It was hypothesized that some molecules in the inner mitochondrial membrane (in particular, adenine nucleotide translocase) are involved in formation of both the calcium-dependent mitochondrial permeability transition pore and mitochondrial K ATP channel [4]. It was previously shown that diazoxide, which mimicked the effect of pharmacological preconditioning during ischemia/reperfusion of an isolated heart [5], induced the opening of calcium-dependent mitochondrial permeability transition pore in the low-conductance state in the presence of

Effect of diazoxide and Ca2+ on rat heart mitochondria loaded with Na+

Effect of diazoxide and Ca2+ on rat heart mitochondria loaded with Na+

Mitochondrial membrane fluidity, potential, and calcium transients in the myocardium from acute diabetic ratsThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

In this study, we report for the first time concurrent measurements of membrane potential and dynamics and respiratory chain activities in rat heart mitochondria, as well as calcium transients in the hearts of rats in an early phase of streptozotocin diabetes, not yet accompanied with diabetes-induced complications. Quantitative relationships among these variables were assessed. The mitochondria from diabetic rats exhibited decreased fluorescence anisotropy values of diphenylhexatriene. This indicates that hydrophobic core of the membranes was more fluid compared with controls (p<0.05). We discuss the changes in fluidity as having been associated with augmented energy transduction through the diabetic membranes. Reduced ratio of JC-1 fluorescence (aggregates to monomers) in the mitochondria from diabetic hearts reflected descendent transmembrane potential. A significant negative association between membrane fluidity and potential in the diabetic group was found (p<0.05; r=0.67). Further, we observed an increase in calcium transient amplitude (CTA) in the diabetic cardiomyocytes (p=0.048). We conclude that some of the calcium-induced regulatory events that dictate fuel selection and capacity for ATP production in diabetic heart occur at the membrane level. Our findings offer new insight into acute diabetes-induced changes in cardiac mitochondria.

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H2O2), hydroxyl radical (HO•) or superoxide anion () on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO• slightly decreased F1F0-ATPase activity and HO• and partially inhibited the activity of adenine nucleotide translocase; H2O2 failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for and alpha-ketoglutarate dehydrogenase for H2O2 and HO•. Our results show that , H2O2 and HO• act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.

Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: Induction of permeability transition and cytochrome c release

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, Δ Ψ collapse, release of preaccumulated Ca 2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca 2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca 2+ for MPT induction and cytochrome c release. This synergistic effect of Ca 2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca 2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.

Regucalcin increases Ca2+-ATPase activity in the heart mitochondria of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of Ca2+-ATPase activity in rat heart mitochondria was investigated. Mitochondrial Ca2+-ATPase activity was significantly increased by increasing concentrations of CaCl2 (2.5-50 microM). An increase in the enzyme activity was saturated at 50 microM CaCl2. The addition of regucalcin (10(-11)-10(-8) M) in the enzyme reaction mixture caused a significant increase in Ca2+-ATPase activity in heart mitochondria in the presence of 50 microM CaCl2. Regucalcin did not have a significant effect on mitochondrial Mg2+-ATPase activity. Regucalcin (10(-9) M) did not have a significant effect on Ca2+-ATPase activity in the presence of digitonin (10(-3) or 10(-2) %), which is a solubilization effect on membranous lipids. The effect of regucalcin in increasing mitochondrial Ca2+-ATPase activity was not observed in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), which is an inhibitor of Ca2+ uniporter. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca2+-ATPase activity was not significantly enhanced in the presence of calmodulin (5 microg/ml) or dibutyryl cyclic AMP (10(-4) M), which is an intracellular signaling factor that can cause a significant increase in the enzyme activity. Mitochondrial regucalcin localization was significantly increased in the heart of regucalcin transgenic rats as compared with that of normal rats using Western blot analysis. Ca2+-ATPase activity was significantly increased in the heart mitochondria of regucalcin transgenic rats. This study demonstrates that regucalcin has an activating effect on Ca2+-ATPase in rat heart mitochondria, suggesting its role in the regulation of heart mitochondrial function.

Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion☆

Reenergization of ischemic cardiomyocytes may be associated with acute necrotic cell death due in part to cytosolic Ca2+ overload and opening of a permeability transition pore (PTP) in mitochondria. It has been suggested that Ca2+ overload during ischemia primes mitochondria for PTP opening during reperfusion. We investigated the ability of mitochondria to uptake Ca2+ during simulated ischemia (SI) and whether this uptake determines PTP opening and cell death upon simulated reperfusion (SR). Rat heart mitochondria were submitted to either hypoxia (anoxic chamber) or to SI (respiratory inhibition, substrate depletion and acidosis) and subsequent SR. Mitochondrial Ca2+ uptake was monitored using Ca2+ microelectrodes after exposure to different [Ca2+] up to 25 microM during SI, and PTP opening was assessed by quantification of mitochondrial swelling (changes in absorbance rate at 540 nm) and calcein release. Mitochondrial Ca2+ uptake (Rhod-2 fluorescence) and cytosolic Ca2+ rise (Fura-2 ratio fluorescence) were further investigated in HL-1 cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 microM ruthenium red) or blocking PTP opening (with 0.5 microM cyclosporin A) on the rate of cell death was investigated in adult cardiomyocytes exposed to SI/SR. SI induced a progressive dissipation of mitochondrial membrane potential (TMRE fluorescence); however, prior to the completion of depolarization, high levels of Ca2+ uptake were observed in mitochondria. SR induced PTP opening but this phenomenon was not influenced by the magnitude of mitochondrial Ca2+ uptake during previous SI. Blockade of the mitochondrial Ca2+ uniporter during SI in cardiomyocytes attenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR. Mitochondrial Ca2+ uptake during SI buffers cytosolic Ca2+ overload but its magnitude appears not to be an important determinant of PTP opening upon subsequent SR.