Respiration Of Heart Mitochondria Research Articles

Reduction in activity and abundance of mitochondrial supercomplexes in right ventricle of pulmonary hypertension rats

Pulmonary hypertension results in RV hypertrophy, fibrosis and impaired function resulting in RV failure. PH is associated with impaired heart metabolism and mitochondrial respiration. Mitochondrial supercomplexes (mSC) are assemblies of multiple electron transport chain (ETC) complexes that consist of physically associated complex I, III and IV to enhance respiration. We induced PH in rats by Sugen-Hypoxia (3 weeks) followed by normoxia (4 weeks). Control (n=11) and PH (n=10) were subjected to echocardiography, CN-PAGE to assess SC abundance and activity.

Abstract 14366: Pulmonary Hypertension-Induced RV Hypertrophy and Dysfunction is Associated With Reduced Activity and Abundance of Complex IV in Respiratory Supercomplexes

Introduction: PH results in RV hypertrophy, fibrosis and impaired function resulting in RV failure. PH is associated with impaired heart metabolism and mitochondrial respiration. Mitochondrial supercomplexes (mSC) are assemblies of multiple electron transport chain (ETC) complexes that consist of physically associated complex I, III and IV to enhance respiration. We determined if activity and/or abundance of mSC’s is altered in a rat model of PH. Methods: PH was induced in SD-rats by Sugen-Hypoxia (3 weeks) followed by normoxia (4 weeks) . Control (n=11) and PH (n=10) were subjected to echocardiography and isolation of LV and RV mitochondria. Clear native PAGE with equal amounts mitochondrial protein was used for SC abundance and activity with immunoblot or in gel activity with specific substrates. Results: PH animals had significantly reduced PAT and TAPSE, increased RV wall thickness and hypertrophy (RV/LVS). Western analysis of total protein fractions demonstrated reductions in representative CI, II, III, and IV proteins in RV indicating reduced mitochondrial content in PH. Assays with normalized mitochondrial protein showed activity (100+/- 3.6 vs 64.0+/-3.8 %, p<.01) and abundance (100+/-1.8 vs 49.3 +/-1.1, p=.02) of complex IV in mitochondrial SCs was severely reduced in PH animals compared to control. There were no differences in total CIV activity or abundance in lower order ETC assemblies (100 vs 97.5%, p=.78). TAPSE, PAT and RV Wall thickness significantly correlated with CIV SC activity (r=.71, .66, and -.80 respectively P<.01). CIII abundance was lower in SCs (P=.01) but total CIII unchanged. CI activity in SC assemblies was reduced in PH but this was not significant (100+/-12 vs 77.4 +/-8.2, p=.14). Overall levels of CI were unchanged. There were no changes in CII activity or abundance in RV or LV. In the LV, activity of CIV in SC was reduced in PH (100+/-6 vs 78+/-6, p=.02) but to a lesser extent than RV. There were also decreases in overall abundance of CIII and CIV assemblies in the LV in PH. Conclusion: The data support the concept that although mitochondrial abundance may be altered, there are also significant alterations associated with assembly and activity of mitochondrial SCs in the RV with pronounced differences in complex IV assembled into SCs.

Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3−/−, Grx2−/−), and double-knockout mice (Grx2−/−/Sirt3−/−) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2−/−/Sirt3−/− mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2−/− and the Sirt3−/− we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3−/−. Similar changes were observed but not worsened in hearts from Grx2−/−/Sirt3−/− mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

Protamine Sulfate Induces Mitochondrial Hyperpolarization and a Subsequent Increase in Reactive Oxygen Species Production.

Protamine sulfate (PS) is widely used in heart surgery as an antidote for heparin, albeit its pharmacological effects are not fully understood and applications are often accompanied by unwanted side effects. Here we show the effect of PS on mitochondrial bioenergetics profile resulting in mitochondrial reactive oxygen species (ROS) production. Polarographic measurements were performed in parallel to membrane potential and ROS measurements by FACS analyzer using tetramethylrhodamine ethyl ester and MitoSOX fluorescent dyes, respectively. PS inhibited intact rat heart mitochondrial respiration (stimulated by ADP) to 76% (P < 0.001) from the baseline of 51.6 ± 6.9 to 12.4 ± 2.3 nmol O2⋅min-1⋅ml-1 The same effect was found when respiration was inhibited by antimycin A (101.0 ± 8.9 vs. 38.0 ± 9.9 nmol O2 ⋅min-1⋅ml-1, P < 0.001) and later stimulated by substrates of cytochrome oxidase (CytOx) i.e., ascorbate and tetramethyl phenylene diamine, suggesting that PS exerted its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial respiration by PS was concentration dependent and accompanied by hyperpolarization of the mitochondrial membrane potential (Δψ m), i.e., 18% increase at 50 µg/ml and an additional 3.3% increase at 250 µg/ml PS compared with control. This effect was associated with a strong consequent increase in the production of ROS, i.e., 85% and 88.6% compared with control respectively. We propose that this excessive increase in ROS concentrations results in mitochondrial dysfunction and thus might relate to the "protamine reaction," contributing to the development of various cardiovascular adverse effects.

Hemodynamic-Directed Cardiopulmonary Resuscitation Improves Neurologic Outcomes and Mitochondrial Function in the Heart and Brain.

Less than half of the thousands of children who suffer in-hospital cardiac arrests annually survive, and neurologic injury is common among survivors. Hemodynamic-directed cardiopulmonary resuscitation improves short-term survival, but its impact on longer term survival and mitochondrial respiration-a potential neurotherapeutic target-remains unknown. The primary objectives of this study were to compare rates of 24-hour survival with favorable neurologic outcome after cardiac arrest treated with hemodynamic-directed cardiopulmonary resuscitation versus standard depth-guided cardiopulmonary resuscitation and to compare brain and heart mitochondrial respiration between groups 24 hours after resuscitation. Randomized preclinical large animal trial. A large animal resuscitation laboratory at a large academic children's hospital. Twenty-eight 4-week-old female piglets (8-11 kg). Twenty-two swine underwent 7 minutes of asphyxia followed by ventricular fibrillation and randomized treatment with either hemodynamic-directed cardiopulmonary resuscitation (n = 10; compression depth titrated to aortic systolic pressure of 90 mm Hg, vasopressors titrated to coronary perfusion pressure ≥ 20 mm Hg) or depth-guided cardiopulmonary resuscitation (n = 12; depth 1/3 chest diameter, epinephrine every 4 min). Six animals (sham group) underwent anesthesia and instrumentation without cardiac arrest. The primary outcomes were favorable neurologic outcome (swine Cerebral Performance Category ≤ 2) and mitochondrial maximal oxidative phosphorylation utilizing substrate for complex I and complex II (OXPHOSCI+CII) in the cerebral cortex and hippocampus. Favorable neurologic outcome was more likely with hemodynamic-directed cardiopulmonary resuscitation (7/10) than depth-guided cardiopulmonary resuscitation (1/12; p = 0.006). Hemodynamic-directed cardiopulmonary resuscitation resulted in higher intra-arrest coronary perfusion pressure, aortic pressures, and brain tissue oxygenation. Hemodynamic-directed cardiopulmonary resuscitation resulted in higher OXPHOSCI+CII (pmol oxygen/s × mg/citrate synthase) in the cortex (6.00 ± 0.28 vs 3.88 ± 0.43; p < 0.05) and hippocampus (6.26 ± 0.67 vs 3.55 ± 0.65; p < 0.05) and higher complex I respiration (pmol oxygen/s × mg) in the right (20.62 ± 1.06 vs 15.88 ± 0.81; p < 0.05) and left ventricles (20.14 ± 1.40 vs 14.17 ± 1.53; p < 0.05). In a model of asphyxia-associated pediatric cardiac arrest, hemodynamic-directed cardiopulmonary resuscitation increases rates of 24-hour survival with favorable neurologic outcome, intra-arrest hemodynamics, and cerebral and myocardial mitochondrial respiration.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Mercury and protein thiols: Stimulation of mitochondrial F1FO-ATPase and inhibition of respiration

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg2+ effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F1FO-ATPase (F-ATPase) activity promoted by micromolar Hg2+ concentrations were investigated. Hg2+ was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg2+ doses which inhibited the NADH–O2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg2+ dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg2+ interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria.

Insulin treatment increases myocardial ceramide accumulation and disrupts cardiometabolic function.

BackgroundStates of hyperinsulinemia, particularly insulin resistance and type 2 diabetes mellitus, are becoming remarkably common, with roughly half a billion people likely to suffer from the disorder within the next 15 years. Along with this rise has been an associated increased burden of cardiovascular disease. Considering type 2 diabetics treated with insulin are more likely to suffer from heart complications, we sought to determine the specific effect of insulin on ceramide-dependent cardiometabolic risk factors, including insulin resistance and altered heart mitochondrial physiology.MethodsH9c2 cardiomyocytes and adult mice were treated with insulin with or without myriocin to inhibit ceramide biosynthesis. Insulin and glucose changes were tracked throughout the study and mitochondrial bioenergetics was determined in permeabilized cardiomyocytes and myocardium.ResultsHerein, we demonstrate that insulin is sufficient to disrupt heart mitochondrial respiration in both isolated cardiomyocytes and whole myocardium, possibly by increasing mitochondrial fission. Further, insulin increases ceramide accrual in a time-dependent manner, which is necessary for insulin-induced alterations in heart mitochondrial respiration and insulin resistance.ConclusionsCollectively, these observations have two implications. First, they indicate a pathological role of insulin in heart complications stemming from mitochondrial disruption. Second, they identify ceramide as a possible mediator of insulin-related heart disorders.

Macrophage‐secreted Ceramides are Necessary for Myocardial Mitochondrial Disruption with LPS Treatments

Lipopolysaccharide (LPS) is a prevalent circulating inflammatory agent in obesity, potentially mediating some of the pathologies associated with weight gain, including cardiomyopathy. Considering the reliance of the myocardium on healthy mitochondrial function, we sought to determine whether LPS disrupts myocardial mitochondrial respiration. Given our previous findings of both LPS-induced ceramide accrual and ceramide-mediated mitochondrial fission and diminished mitochondrial respiration, we questioned whether ceramide is necessary for LPS-induced mitochondrial disruption. We found that heart tissue from mice receiving intraperitoneal LPS injections for four wk failed to respire in response to various substrates as robustly as mice receiving PBS injections. However, heart respiration from mice receiving both LPS and myriocin injections was similar to PBS-injected animals. In order to determine the direct effect of LPS on cardiomyocytes, we treated murine H9C2 cardiomyocytes with similar conditions as animal injections. However, in contrast to the whole heart response to LPS, respiration from cardiomyocytes was increased with LPS alone, and even higher with both myriocin alone and myriocin with LPS. This prompted us to utilize a two-culture system, wherein macrophages receive initial treatments, followed by transfer of conditioned medium to cardiomyocytes. Following this scheme, cardiomyocytes incubated with medium from LPS-treated macrophages experienced a reduction in respiration, but not with myriocin co-treatment. Altogether, these data suggest that the cellular heterogeneity of myocardium, particularly the presence of macrophages, is necessary for the deleterious effects of LPS on heart mitochondrial respiration.

Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c

Anthocynanins, found in fruits and vegetables, have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, antioxidants are generally strong reductants, and some reductants have been found to block apoptosis by reducing cytosolic cytochrome c, which prevents caspase activation. We tested the ability of various anthocyanins: to reduce cytochrome c, to support cytochrome c-induced mitochondrial respiration and to inhibit apoptosis induced by heart ischemia. Anthocyanins such as delphinidin-3-glucoside (Dp3G) and cyanidin-3-glucoside (Cy3G) were able to reduce cytochrome c directly and rapidly, whereas pelargonidin-3-glucoside (Pg3G), malvinidin-3-glucoside (Mv3G) and peonidin-3-glucoside (Pn3G) had relatively low cytochrome c reducing activities. Dp3G and Cy3G but not Pg3G supported mitochondrial state 4 respiration in the presence of exogenous cytochrome c. Pre-perfusion of hearts with 20μM Cy3G but not Pg3G prevented ischemia-induced caspase activation. This suggests that the ability of anthocyanins to block caspase activation may be due to their ability to reduce cytosolic cytochrome c. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Mitochondrial Transporter ATP Binding Cassette Mitochondrial Erythroid Is a Novel Gene Required for Cardiac Recovery After Ischemia/Reperfusion

Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Effects of Ginkgo biloba extract on heart and liver mitochondrial functions: mechanism(s) of action

Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H(2)O(2) generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.

Mitochondrial Preservation in Celsior Versus Histidine Buffer Solution During Cardiac Ischemia and Reperfusion

Various stressful conditions such as ischemia in cold cardioplegic solutions and reperfusion occur during heart transplantation. Since ATP production is essential for the maintenance of contractile activity, mitochondrial function may be a mediator of ischemia and ischemia/reperfusion (I/R) injury. We aimed at testing the ability of two distinct cardioplegic solutions, Celsior (Cs) and Histidine Buffer (HBS), to protect rat heart mitochondria (HM) function during ischemia alone or ischemia followed by reperfusion. A standard Krebs-Henseleit solution (KH) was used as "negative" control. Male and Female Wistar rats were divided into control (Ctrl), reperfusion control (Ctrl_R), ischemia and I/R groups. Ischemia and I/R were divided into three subgroups depending on the cardioplegic solution used (Cs, HBS or KH) and subjected to 4-or 6-h ischemia alone or followed by reperfusion. HM respiration and transmembrane electric potential (Deltapsi) were measured with oxygen and TPP(+)-selective electrodes, respectively. Mitochondrial electron microscopy and detection of protein carbonyl groups content were also performed. After ischemic heart preservation, mitochondrial respiration and Deltapsi were not significantly affected except for the respiratory control ratio (RCR). After I/R, state 3 respiration, RCR and Deltapsi were decreased, especially in HM from male and for complex I substrates (CxI). HM preserved in HBS had less membrane disruption, segregation or disintegration. We conclude that (a) female HM were less sensitive to I/R, (b) CxI was particularly affected by I/R, (c) two cardioplegic solutions tested act in different mitochondrial targets preventing mitochondrial collapse.

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.

Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy.

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated alpha-oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.

Quantitative analysis of the ‘phosphocreatine shuttle’: I. A probability approach to the description of phosophocreatine production in the coupled creatine kinase-ATP/ADP translocase-oxidative phosphorylation reactions in heart mitochondria

For the first time, a probability approach was used to describe heart mitochondrial respiration in the medium with ATP, Cr and PCr but without ADP. Respiring mitochondria were considered as a three-component system, including (1) oxidative phosphorylation reactions which provide stable ATP concentration in the mitochondrial matrix; (2) adenine nucleotide translocase, which provides exchange transfer of matrix ATP for outside creatine kinase-supplied ADP when both substrates are simultaneously bound to translocase and (3) creatine kinase, starting these reactions when activated by the substrates from medium. The specific feature of this system is a close proximity of creatine kinase and translocase molecules. This results in high probability of direct activation of translocase by creatine kinase-derived ADP without its leak into the medium. In turn, the activated translocase with the same high probability directly provides creatine kinase with matrix-derived ATP. The catalytic complexes of creatine kinase with ATP from matrix together with those formed from substrates from medium provide high activation of creatine kinase coupled to translocase activation. The considered probabilities were arranged into a mathematical model. The model satisfactorily simulates the experimental data by Jacobus, W.E. and Saks, V.A. ((1982) Arch. Biochem. Biophys. 219, 167–178), who investigated this system in all regimens of functioning. The results suggest the observed kinetic and thermodynamic irregularities in the behavior of structurally-bound creatine kinase as a direct consequence of its tight coupling to translocase.

Lipid composition and mitochondrial respiration in warm‐ and cold‐adapted sea bass

The response to cold of liver and heart membrane lipid composition and mitochondrial respiration in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was followed during the natural seasonal cycle from August to March. The data on the fatty acid composition of liver and heart polar lipids and on total lipids of liver mitochondria and microsomes did not indicate any increase in unsaturation in response to cold. The enzyme complexes of the liver and heart mitochondrial respiratory chain showed a repeated negative compensation for cold acclimation. The constancy of the break in the Arrhenius plot of liver cytochrome oxidase (EC 1.9.3.1) was consistent with the lack of homeoviscous adaptation of membrane lipids. A thermoadaptive strategy based on the reduction of sea bass metabolic activity is suggested.

Inhibition of rat heart mitochondrial respiration by cadmium chloride

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0–120 sec) and concentration-dependent (0–10 μ m CdCl 2) effects of cadmium on pyruvate-malatesupported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37°C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 μ m. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 μ m, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 μ m carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 μ m CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvatemalate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.

The relationship between the cardiac contractile function, adenine nucleotides and amino acids of cardiac tissue and mitochondria at acute respiratory hypoxia.

The effect of asphyxia and subsequent resumption of respiration on the content of adenine nucleotides and some amino acids in heart tissue and mitochondria, as well as respiration of heart mitochondria was studied in rats. The depression of cardiac contractile function during asphyxia showed a better correlation with losses in mitochondrial adenine nucleotides (ATP + ADP + AMP) than those in cardiac tissue. The decrease in the heart work index was accompanied by a decrease in state 3 respiration with glutamate and malate as well as uncoupled respiration with these substrates. This did not occur with succinate. Nonphosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by asphyxia, when respiratory substrates of both types were used. The decreased level of glutamic acid in the tissue and mitochondria of asphyxic hearts was simultaneously observed with a significant increase of alanine in cardiac tissue and of aspartic acid in the mitochondria. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during asphyxia were associated with a reduction of glutamic acid level in mitochondria. The recovery of cardiac function during resumption of respiration was related to the restoration of mitochondrial respiration supported by glutamate and malate, as well as to the restoration of mitochondrial adenine nucleotides and glutamic acid. The results suggest that the depression of cardiac function caused by acute respiratory hypoxia may be attributed to impairment of electron transport, particularly in complex I of the respiratory chain and changes in metabolism of glutamic acid.

Effect of superoxide generation on rat heart mitochondrial pyruvate utilization

Previous research has shown that heart mitochondria are able to produce reactive species of oxygen such as superoxide radicals, hydrogen peroxide and hydroxyl radicals [ 10, 11]. When these compounds are formed beyond a certain level they are not completely removed by the enzymatic and metabolic processes which neutralize their toxicity, and as a result they are able to produce structural and functional damages that impair mitochondrial function [ 5, 10]. In order to study the molecular mechanism/s by which the oxygen radicals may function as mediators of cellular injury a flow of these radicals by chemical, enzymatic or photochemical methods has been generated in vitro in the presence of cellular preparations. For example, the exposure of isolated subcellular particles to the enzymatic flow of oxygen radicals produced by the reaction of xanthine oxidase upon xanthine reduced both calcium uptake velocity and Ca 2+-ATPase activity in sarcoplasmic reticulum [ 7], while it reduced Ca 2+-stimulated ATPase activity in myofibrillar preparations [ 4]. In addition, incubation with the xanthine oxidase reaction produced an impairment of the respiratory functions associated with an increased lipid peroxidation in the isolated mitochondria [ 5, 10]. These negative effects were augmented in α-tocopherol-deficient mitochondria [ 3], but were opposed by the exogenous addition of superoxide dismutase [ 10]. This report shows that the superoxide radicals generated by the xanthine oxidase reaction reduced rat heart mitochondrial respiration induced by pyruvate. This negative effect was partially prevented by superoxide dismutase and catalase and by thiol protecting agents. Moreover, the generation of free radicals caused a significant reduction in the rate of (1- 14C)-pyruvate decarboxylation, while it did not change the transport of pyruvate into mitochondria.