Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients’ cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls’ fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.     

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.     

Fumarate permeation in rat liver mitochondria: fumarate/malate and fumarate/phosphate translocators

Fumarate permeation in isolated rat liver mitochondria was demonstrated by measuring malate and phosphate efflux caused by fumarate added externally to the mitochondrial suspension. The existence of two specific fumarate translocators, fumarate/malate and fumarate/phosphate, is shown here. These carriers are distinguished in the light of different kinetic parameters (Km values are 50 microM and 150 microM, and Vmax values are 17 and 40 nmoles/min X mg mitochondrial protein, respectively) and of differing sensitivity to non-penetrant compounds. Fumarate was found to cause oxaloacetate efflux from mitochondria by means of an indirect process which involves the cooperation of both fumarate/malate and malate/oxaloacetate translocators. Results are discussed in the light of the physiological role played by fumarate translocation in both ureogenesis and aminoacid metabolism.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Gas chromatographic–mass spectrometric screening for organic acidemias using dried urine filter paper: determination of α-ketoacids

There are several organic acid disorders that require information on α-ketoacids, such as maple syrup urine disease or α-ketoadipic acidemia. The recovery, stability and diagnostic availability of α-ketoacids in dried urine filter paper analyzed by GC–MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of α-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain α-ketoacids, α-ketoadipate and α-ketoglutarate was stable for at least 28 days, although some α-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.     

Proton transfer in catalysis by fumarase.

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.     

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.     

Alterations of lipid-mediated mitophagy result in aging-dependent sensorimotor defects

The metabolic consequences of mitophagy alterations due to age-related stress in healthy aging brains versus neurodegeneration remain unknown. Here, we demonstrate that ceramide synthase 1 (CerS1) is transported to the outer mitochondrial membrane by the p17/PERMIT transporter that recognizes mislocalized mitochondrial ribosomes (mitoribosomes) via 39-FLRN-42 residues, inducing ceramide-mediated mitophagy. P17/PERMIT-CerS1-mediated mitophagy attenuated the argininosuccinate/fumarate/malate axis and induced d -glucose and fructose accumulation in neurons in culture and brain tissues (primarily in the cerebellum) of wild-type mice in vivo. These metabolic changes in response to sodium-selenite were nullified in the cerebellum of CerS1to/to (catalytically inactive for C18-ceramide production CerS1 mutant), PARKIN−/− or p17/PERMIT−/− mice that have dysfunctional mitophagy. Whereas sodium selenite induced mitophagy in the cerebellum and improved motor-neuron deficits in aged wild-type mice, exogenous fumarate or malate prevented mitophagy. Attenuating ceramide-mediated mitophagy enhanced damaged mitochondria accumulation and age-dependent sensorimotor abnormalities in p17/PERMIT−/− mice. Reinstituting mitophagy using a ceramide analog drug with selenium conjugate, LCL768, restored mitophagy and reduced malate/fumarate metabolism, improving sensorimotor deficits in old p17/PERMIT−/− mice. Thus, these data describe the metabolic consequences of alterations to p17/PERMIT/ceramide-mediated mitophagy associated with the loss of mitochondrial quality control in neurons and provide therapeutic options to overcome age-dependent sensorimotor deficits and related disorders like amyotrophic lateral sclerosis (ALS).     

The effect of malate on propionate mitochondrial toxicity.

Propionic acidemia occasionally produces a toxic encephalopathy resembling Reye's syndrome, indicating disruption of mitochondrial metabolism. Liver mitochondria respiratory control ratios were reduced 46% by 5 mM propionate; inhibition correlated with matrix propionyl-CoA levels. L-Malate prevented the toxic effect of propionate and reduced the propionyl-CoA matrix concentration by 62%. The beneficial effect of L-malate is apparently due to stimulation of succinate efflux because the effect is blocked by benzylmalonate, an inhibitor of the dicarboxylate carrier. Matrix concentration of label from [1-14C]propionate was not affected by L-malate and/or benzylmalonate. L-Malate may be useful in the treatment of patients with propionic acidemia.     

Neurotoxicity of ammonia and fatty acids: Differential inhibition of mitochondrial dehydrogenases by ammonia and fatty acyl coenzyme a derivatives

In several metabolic encephalopathies, hyperammonemia and organic acidemia are consistently found. Ammonia and fatty acids (FAs) are neurotoxic: previous workers have shown that ammonia and FAs can act singly, in combination, or synergistically, in inducing coma in experimental animals. However, the biochemical mechanisms underlying the neurotoxicity of ammonia and FAs have not been fully elucidated. FAs are normally converted to their corresponding CoA derivatives (CoAs) once they enter cells and it is known that these fatty acyl CoAs can alter intermediary metabolism. The present study was initiated to determine the effects of ammonia and fatty acyl CoAs on brain mitochondrial dehydrogenases. At a pathophysiological level (2 mM), ammonia is a potent inhibitor of brain mitochondrial -ketoglutarate dehydrogenase complex (KGDHC). Only at toxicological levels (10–20 mM) does ammonia inhibit brain mitochondrial NAD⁺- and NADP⁺-linked isocitrate dehydrogenase (NAD-ICDH, NADP-ICDH), and NAD⁺-linked malate dehydrogenase (MDH) and liver mitochondrial NAD-ICDH. Butyryl- (BCoA), octanoyl- (OCoA), and palmitoyl (PCoA) CoA were potent inhibitors of brain mitochondrial KGDHC, with IC₅₀ values of 11, 20, and 25 M, respectively; moreover, the inhibitory effect of fatty acyl CoAs and ammonia were additive. At levels of 250 M or higher, both OCoA (IC₅₀=1.15 mM) and PCoA (IC₅₀=470 M) inhibit brain mitochondrial NADP-ICDH; only at higher levels (0.5–1 mM) does BCoA inhibit this enzyme (by 30–45%). Much less sensitive than KGDHC and NADP-ICDH, brain mitochondrial NAD-ICDH is only inhibited by 1 mM BCoA, OCoA, and PCoA by 22%, 35%, and 44%, respectively. Even at 1 mM, OCoA and PCoA (but not BCoA) only slightly inhibited brain mitochondrial MDH (by 23%). In the presence of toxicological levels of ammonia (20 mM) and fatty acyl CoAs (1 mM), the inhibitory effect of fatty acyl CoAs and ammonia on brain mitochondrial NAD-ICDH, NADP-ICDH, and MDH are only partially additive. These results provide some support for our hypothesis that selective inhibition of a rate-limiting and regulated enzymatic step (e.g., KGDHC) by ammonia and fatty acyl CoAs may be one of the major mechanisms underlying the neurotoxicity of ammonia and FAs. The data also suggest that the same mechanism may acocunt for the synergistic effect of ammonia and FAs in inducing coma. Since the inhibition of KGDHC by ammonia and fatty acyl CoAs occurs at pathophysiological levels, the results may assume some pathophysiological and/or pathogenetic importance in metabolic encephalopathies in which hyperammonemia and organic acidemia are persistent features.We dedicate this paper to Dr. Santiago Grisolia. Dr. Grisolia has carried out many pioneering studies in urea metabolism and ammonia toxicity. His interesting ideas have been influential in these and related fields of research. He continues to contribute significantly in unravelling the mechanisms of ammonia toxicity.     

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.     

Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Anaerobic rat heart. Effects of glucose and tricarboxylic acid-cycle metabolites on metabolism and physiological performance

1. The ability of tricarboxylic acid-cycle metabolites to influence the physiological performance of the perfused anaerobic rat heart was investigated. Energy expenditure/h [(beats/min)×60×systolic pressure/g of protein] for various anoxic conditions compared with oxygenated control hearts were: 5mm-glucose, 4.5%; 20mm- or 40mm-glucose, 10%; 20mm-glucose plus fumerate+malate+glutamate, 29%; 20mm-glucose plus oxaloacetate and α-oxoglutarate, 31%. 2. The energy expenditure/lactate production ratio was increased by the tricarboxylic acid-cycle metabolites, indicating that alterations in anaerobic physiological performance did not result from changes in glycolysis. 3. Analysis of tissue constituents provided further indication of an enhanced energy status for fumarate+malate+glutamate- and oxaloacetate+α-oxoglutarate-perfused hearts; tissue concentrations of both glycogen and ATP were higher than in the 20mm-glucose-perfused groups. 4. A marked increase in the accumulation of succinate in tissues perfused with oxaloacetate+α-oxoglutarate or fumarate+malate+glutamate provided further evidence that these metabolites were stimulating mitochondrial energy production under anoxia. 5. These studies indicate that mitochondrial ATP production can be stimulated in an isolated mammalian tissue perfused under anaerobiosis with a resulting enhancement of cell function.     

Fumarate: Multiple functions of a simple metabolite

Although much is now known about fumarate metabolism, our knowledge of some aspects of its biological function remain far from comprehensive. In this short review we begin with an introductory overview of the role of fumarate in both plant and non-plant systems. We next highlight the relative importance of fumarate in relation to cell type and circumstance in contrast to other chemically similar organic acids. Considerable cumulative evidence is suggestive of a role for fumarate in pH regulation during nitrate assimilation and that fumarate has similar effects as malate during stomatal movement. Indeed it is currently difficult to separate the biological function of fumarate from malate under certain circumstances. However, in other cases this can be easily performed. This physiological complexity notwithstanding it remains possible that the engineering of fumarate metabolism may provide opportunities to improve plant growth and performance.     

Growth of Pseudomonas fluorescens with Sodium Maleate as a Carbon Source

The growth of Pseudomonas fluorescens (ATCC 11150) on sodium maleate was observed, and the composition of the media was monitored in batch culture. Utilization of the maleate as the sole organic carbon source proceeded by stepwise conversion to fumarate and then to malate. Respiration rates of P. fluorescens on maleate, mixtures of maleate and fumarate salts, and mixtures of maleate and malate salts were measured. Complex effects, possibly due to a change in metabolic mechanism, were observed.     

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05–5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca²⁺ retention capacity, and caused swelling in Ca²⁺-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.