Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.     

Acutely administered melatonin restores hepatic mitochondrial physiology in old mice

Damage to mitochondria as a result of the intrinsic generation of free radicals is theoretically involved in the processes of cellular aging. Herein, we investigated whether acutely administered melatonin, due to its free radical scavenging activity, would influence mitochondrial metabolism. Mitochondrial respiratory activity and respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated-prone mice (SAMP8) and a strain of senescence-accelerated-resistant mice (SAMR1) were measured when the animals were 12 months of age. Respiratory control index (RCI), ADP/O ratio, State 3 respiration and dinitrophenol (DNP)-dependent uncoupled respiration were significantly lower in SAMP8 than in SAMR1. In contrast, State 4 respiration was significantly higher in SAMP8 than in SAMR1. Activities of complexes I and IV in SAMP8 were significantly lower than in SAMR1. Melatonin administration (10mg/kg body weight, intraperitoneally) 1h prior to sacrifice significantly increased RCI, ADP/O ratio, State 3 respiration and DNP-induced uncoupled respiration in SAMP8 while also significantly reducing State 4 respiration in SAMP8. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in the liver of SAMP8 mice. These results document an age-related decrease in hepatic mitochondrial function in SAM which can be modified by an acute pharmacological injection of melatonin; the indole stimulated mitochondrial respiratory chain activity which would likely reduce deteriorative oxidative changes in mitochondria that normally occur in advanced age.     

'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress

Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that 'mild uncoupling' could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Deltapsi(m)) were determined in parallel as a function of FCCP concentration. Deltapsi(m) dropped by less than 10 mV before respiratory control was lost. Conditions for the valid estimation of matrix superoxide levels were determined from the rate of oxidation of the matrix-targeted fluorescent probe MitoSOX. No significant change in the level of matrix superoxide could be detected on addition of FCCP while respiratory control was retained, although cytoplasmic superoxide levels measured by dihydroethidium oxidation increased. 'Mild uncoupling' by 30 nmol/L FCCP did not alleviate neuronal dysregulation induced by glutathione depletion and significantly enhanced that due to menadione-induced oxidative stress. Low protonophore concentrations enhanced N-methyl-d-aspartate receptor-induced delayed calcium deregulation consistent with a decrease in the spare respiratory capacity available to match the bioenergetic demand of chronic receptor activation. It is concluded that the 'mild uncoupling' hypothesis is not supported by this model.     

Rat diaphragm mitochondria have lower intrinsic respiratory rates than mitochondria in limb muscles

The mitochondrial content of skeletal muscles is proportional to activity level, with the assumption that intrinsic mitochondrial function is the same in all muscles. This may not hold true for all muscles. For example, the diaphragm is a constantly active muscle; it is possible that its mitochondria are intrinsically different compared with other muscles. This study tested the hypothesis that mitochondrial respiration rates are greater in the diaphragm compared with triceps surae (TS, a limb muscle). We isolated mitochondria from diaphragm and TS of adult male Sprague Dawley rats. Mitochondrial respiration was measured by polarography. The contents of respiratory complexes, uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3), and voltage-dependent anion channel 1 (VDAC1) were determined by immunoblotting. Complex IV activity was measured by spectrophotometry. Mitochondrial respiration states 3 (substrate and ADP driven) and 5 (uncoupled) were 27 ± 8% and 24 ± 10%, respectively, lower in diaphragm than in TS (P < 0.05 for both comparisons). However, the contents of respiratory complexes III, IV, and V, UCP1, and VDAC1 were higher in diaphragm mitochondria (23 ± 6, 30 ± 8, 25 ± 8, 36 ± 15, and 18 ± 8% respectively, P ≤ 0.04 for all comparisons). Complex IV activity was 64 ± 16% higher in diaphragm mitochondria (P ≤ 0.01). Mitochondrial UCP2 and UCP3 content and complex I activity were not different between TS and diaphragm. These data indicate that diaphragm mitochondria respire at lower rates, despite a higher content of respiratory complexes. The results invalidate our initial hypothesis and indicate that mitochondrial content is not the only determinant of aerobic capacity in the diaphragm. We propose that UCP1 and VDAC1 play a role in regulating diaphragm aerobic capacity.     

Inhibition of respiration in mitochondria and in digitonin-treated rat hepatocytes by podophyllotoxin

Summary The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain.     

Effects of α-pinene on the mitochondrial respiration of maize seedlings

The effects of α-pinene, which is one of the major components of essential oils of several aromatic species, on energy metabolism of mitochondria isolated from maize (Zea mays L.) coleoptiles and primary roots were investigated. α-Pinene exerted similar effects on oxygen consumption irrespective of the source of mitochondria or of the substrate (L-malate, succinate or NADH). At concentrations lower than 250 μM, α-pinene stimulated respiration in state IV and inhibited respiration in state III. At higher concentrations the effect of α-pinene on state IV respiration was shifted toward inhibition. Complete suppression of respiratory control ratio was evident at α-pinene concentrations higher than 100 μM. When mitochondria were uncoupled with carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone (FCCP), α-pinene caused only inhibition of respiration. In the presence of α-pinene, the transmembrane potential was decreased as indicated by changes in the safranine binding by energized mitochondria. α-Pinene did not affect the activities of succinate dehydrogenase (EC 1.3.5.1) and L-malate dehydrogenase (L-malate:NAD⁺ oxidoreductase; EC 1.1.1.37). The results indicate that α-pinene acts by at least two mechanisms: uncoupling of oxidative phosphorylation and inhibition of electron transfer. Confirming the impairment of mitochondrial energy metabolism, α-pinene strongly inhibited mitochondrial ATP production. It is apparent that the actions of α-pinene on isolated mitochondria are consequences of unspecific disturbances in the inner mitochondrial membrane.     

Influence of glyoxylic acid on properties of isolated mitochondria]

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.     

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation

Apoptosis induced by K+/serum deprivation (low K+) in cerebellar granule neurons has been extensively investigated. The mitochondria play a key role in apoptosis by releasing proapoptotic factors into the cytoplasm, and mitochondrial dysfunction has been proposed as an early or initiating event in this model. To directly test this hypothesis, cellular and mitochondrial bioenergetics were quantified by determining the respiratory parameters of coverslip-attached neurons. While oxidative phosphorylation rate decreased 39-49% in low K+, this was due to decreased cellular ATP demand rather than impaired ATP/ADP exchange or respiratory chain inhibition. From 3 to 5 h in low K+, apoptosis progressed from 13 to 40% despite no appreciable change in respiratory parameters. Changes in steady-state O2-, assessed with dihydroethidium, were seen in granule but not hippocampal neurons. The O2- change correlated with changes in [Ca2+]c, but not mitochondrial respiration. Thus, early mitochondrial dysfunction can be excluded in this common model of neuronal apoptosis.     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

The Effect of the Lipid Peroxidation Product 4-Hydroxynonenal and of its Metabolite 4-Hydroxynonenoic Acid on Respiration of Rat Kidney Cortex Mitochondria

In rat kidney cortex mitochondria, 4-hydroxynonenal inhibits state 3 respiration as well as uncoupled respiration at micromolar concentrations. The inhibition is more distinct for NAD-linked than for FAD-linked respiration. 4-Hydroxynonenal increases the state 4 respiration. It is assumed that 4-hydroxynonenal behaves like a decoupling agent. 4-Hydroxynonenal augments the inhibitory effect of 2, 4-dinitrophenol observed at superoptimal concentrations. 4-Hydroxynonenal is metabolised by renal mitochondria, and 4-hydroxynonenoic acid is one of the metabolites generated. This metabolite is without effect on respiration at concentrations up to 50 μM. Therefore, the effect of 4-hydroxynonenal on respiration is not mediated by this fatty acid derivative formed during respiratory measurements.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

Redox State of Endogenous Coenzyme Q Modulates the Inhibition of Linoleic Acid-Induced Uncoupling by Guanosine Triphosphate in Isolated Skeletal Muscle Mitochondria

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.     

A possible mechanism of mitochondrial dysfunction during cerebral ischemia: inhibition of mitochondrial respiration activity by arachidonic acid.

The dramatic increase in the arachidonic acid (AA) level in the brain is a well-known molecular event during cerebral ischemia. As mitochondria are known to be one possible site of the cell damage, the effects of AA on the respiratory activity of rat brain mitochondria were investigated in vitro using an oxygen electrode. In NAD-linked respiration, respiratory control ratio was decreased significantly by AA, with an IC50 of 6.0 microM. AA had the dual effect on mitochondrial respiration, a decrease in state 3 and uncoupled state and an increase in state 4 (i.e., uncoupling) as reported by Hillered and Chan (J. Neurosci. Res. 19, 94-100, 1988). Furthermore, we found that other unsaturated long-chain free fatty acids (C18:1-C18:3, C20:1-C20:5) also showed such a dual effect. Cyclooxygenase metabolites of AA such as prostaglandins (D2, E2, F2 alpha, E1) and thromboxane B2, and lipoxygenase metabolites such as leukotrienes (D4, B4) and 5- or 12-hydroperoxyeicosatetraenoic acid had no significant effect. The inhibition of the uncoupled state by AA was more marked in NAD-linked than that in FAD-linked respiration, while the degree of uncoupling by AA were the same in both respirations. In spectrophotometrical measurement, the reduction of cytochromes and flavo-protein was markedly inhibited by AA in NAD-linked respiration, but not in the FAD-linked one. In addition, the activity of cytochrome c oxidase was scarcely inhibited by AA. These data suggest that AA itself, not its metabolites, may inhibit mitochondrial ATP production during brain ischemia and that AA may act on the site(s) closely related to NAD-linked respiration, but not the FAD-linked one, in addition to its uncoupling effect.     

Effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> 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. H₂O₂ 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.     

Non-phosphorylating bypass of the plant mitochondrial respiratory chain by stress protein CSP 310

Recently, it has been reported that the cold-stress protein CSP 310, discovered in the cytoplasm of cold-resistant winter cereals, causes uncoupling of oxidative phosphorylation during cold stress. To understand how the uncoupling mechanism of CSP differs from that of cyanide-insensitive alternative oxidase and plant mitochondrial uncoupling protein, we determined the effect of respiratory-chain inhibition on winter wheat (Triticum aestivum L. cv. Zalarinka) mitochondria. Our data show a possible involvement of stress protein CSP 310 in mitochondrial electron transport in winter wheat. CSP 310 shunts electrons around the main cytochrome pathway of the mitochondrial respiratory chain, i.e. electron flow bypasses ubiquinone and complex III via CSP 310 to complex IV.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.