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

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

Ana Cristina Roginski, Alessandro Wajner, Cristiane Cecatto, Simone Magagnin Wajner, Roger Frigério Castilho, Moacir Wajner, Alexandre Umpierrez Amaral PII:

S0925-4439(20)30021-1

DOI:

https://doi.org/10.1016/j.bbadis.2020.165682

Reference:

BBADIS 165682

To appear in:

BBA - Molecular Basis of Disease

Received date:

21 October 2019

Revised date:

8 January 2020

Accepted date:

9 January 2020

Please cite this article as: A.C. Roginski, A. Wajner, C. Cecatto, et al., Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats, BBA - Molecular Basis of Disease(2020), https://doi.org/10.1016/j.bbadis.2020.165682

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© 2020 Published by Elsevier.

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

Ana Cristina Roginski1, Alessandro Wajner1, Cristiane Cecatto1, Simone Magagnin Wajner2, Roger Frigério Castilho3, Moacir Wajner1,4,5, Alexandre Umpierrez Amaral1,4,6* 1

Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências

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Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. 2

Departamento de Medicina Interna, Faculdade de Medicina, Universidade Federal do Rio

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade

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3

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Grande do Sul, Porto Alegre, RS, Brazil.

Estadual de Campinas, Campinas, SP, Brazil. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade

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Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS,

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Brazil.

Departamento de Ciências Biológicas, Universidade Regional Integrada do Alto Uruguai

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e das Missões, Erechim, RS, Brazil.

*Corresponding author: Alexandre Umpierrez Amaral, Departamento de Ciências Biológicas, Universidade Regional Integrada do Alto Uruguai e das Missões, Avenida Sete de Setembro, 1621, CEP 99709-910, Erechim, RS – Brazil. Phone/Fax: +55 54 3520-9000, e-mail: [email protected]

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Journal Pre-proof Abstract 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

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mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA

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markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-

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ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked

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less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly

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inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also

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decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and

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mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed

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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.

Keywords:

propionic

acidemia;

propionic

acid;

maleic

acid;

cardiomyopathy;

mitochondrial functions.

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Journal Pre-proof Abbreviations AA, adipic acid; ANOVA, analysis of variance; BSA, bovine serum albumin; CCCP, carbonyl cyanide m-chloro phenyl hydrazone; CS, citrate synthase; CoA, coenzyme A; CsA, Cyclosporin A; DMEM, Dulbecco’s modified Eagle’s medium; FAU, fluorescence arbitrary

unity;

3OHPA,

3-hydroxypropionic

acid;

α-KGDH,

α-ketoglutarate

dehydrogenase; MA, maleic acid; MDH, malate dehydrogenase; 2MCA, 2-methylcitric

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acid; ΔΨm, mitochondrial membrane potential; mPT, mitochondrial permeability

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transition; PA, propionic acid; PAcidemia, propionic acidemia; RCR, respiratory control

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ratio; SPSS, Statistical Package for the Social Sciences; SUIT, substrate-uncoupler

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inhibitor titration.

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Journal Pre-proof 1. Introduction Propionic acidemia (PAcidemia) is an inherited organic acidemia caused by severe reduction or absent activity of propionyl-CoA carboxylase (EC 6.4.1.3) that catalyzes the carboxylation of propionyl-CoA with bicarbonate producing methylmalonyl-CoA, which is then converted to succinyl-CoA [1-2]. The absence of propionyl-CoA carboxylase activity results in accumulation of propionyl-CoA, which is spontaneously converted to propionic

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acid (PA) that may reach plasma concentration of 5 mM [1, 3]. Other organic acids

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including 3-hydroxypropionic acid (3OHPA), 2-methylcitric acid (2MCA) and maleic acid

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(MA) also accumulate and are excreted in the urine of affected patients [1, 4-6]. MA seems

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to originate from PA and 3OHPA liver metabolism by still unknown mechanisms [7].

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The incidence of this disorder is from 1 in 100,000 to 1 in 150,000 newborns [8]. The clinical spectrum ranges from a severe early-onset with high mortality to a milder late-

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onset forms of the disease both exhibiting elevated morbidity [9]. Early-onset PAcidemic

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patients present progressive encephalopathy in the first days of life manifested as lethargy, seizures, and coma that may progress to a fatal outcome when not diagnosed and treated

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promptly. Patients with late-onset forms may also present acute encephalopathy following metabolic decompensation but usually experience a more insidious onset with the development of multiorgan complications including vomiting, failure to thrive, developmental delay, cardiomyopathy and neurological regression [1-2, 9]. Noteworthy, dilated and hypertrophic cardiomyopathy has been described as a common complication and may occur in the absence of metabolic decompensation or neurocognitive deficits [1012]. Long-term management mainly based on dietary protein restriction and L-carnitine 4

Journal Pre-proof supplementation was shown to reduce mortality but is still inadequate to prevent chronic morbidity [8, 13]. Symptomatology becomes worse during episodes of acute metabolic decompensation that are associated with increased concentrations of the accumulating metabolites, suggesting toxicity of the accumulating endogenous metabolites [9, 14]. Noteworthy, cardiomyopathy has been frequently reported as a serious complication progressing rapidly to death of early [10, 15] and late onset patients [11, 16].

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Regarding to PAcidemia pathophysiology, most reported works refer to the

mitochondrial

bioenergetics

impairment

as

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potential

underlying

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proposed

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mechanisms underlying the neurological alterations. Only recently some studies have

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pathomechanism of cardiomyopathy, but this is still on debate [17-18], implying that more

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studies are necessary to clarify the pathogenesis of the heart damage. The hypothesis that the metabolites accumulating in PAcidemia are mainly responsible for the cardiomyopathy

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is supported by the fact that this lethal manifestation could be improved by liver transplant

19].

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that leads to decreased circulating levels of the propionyl-CoA derivative compounds [10,

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Thus, the present work aimed to investigate the role of PA, 3OHPA, 2MCA and MA on important parameters of mitochondrial bioenergetics and Ca2+ homeostasis in distinct cardiac preparations, including isolated mitochondria and crude homogenates from heart of developing rats, as well as cultured cardiomyocytes. For this purpose a large spectrum of bioenergetics aspects, such as the respiratory parameters state 3 (ADPstimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration, citric acid cycle enzymes activities, ATP production, mitochondrial membrane potential (ΔΨm), matrix NAD(P)H content, swelling and Ca2+ retention capacity were evaluated. 5

Journal Pre-proof 2. Material and methods 2.1. Chemicals and animals Chemicals were purchased from Sigma-Aldrich, except from calcium green-5N (Thermo Fisher Scientific) and 3OHPA (synthesized by Dr. Ernesto Brunet from Universidad Autonoma de Madrid, Spain). MA, PA, 3OHPA, 2MCA (racemic mixture of diastereomers) and adipic acid (AA) were prepared in the technique buffer at the day of the

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experiments.

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Seventy-four 30-day-old (developing) male Wistar rats were used in the

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experiments. The animals were obtained from the breeding colony of Federal University of

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Rio Grande do Sul (UFRGS) and maintained on a 12:12 h light/dark cycle in an air-

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conditioned constant temperature (22 ± 1 °C) colony room, with free access to water and a 20% (w/w) protein commercial chow. The experimental protocol was approved by the

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Ethics Committee for animal research of UFRGS, Porto Alegre, Brazil (no 35344) and followed the National Institutes of Health guide for the care and use of Laboratory animals

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(NIH Publications No. 8023, revised 1996).

2.2. Preparation of isolated mitochondria and crude homogenates from heart of developing rats Mitochondria were isolated from heart of developing rats according to Ferranti and collaborators [20] with some modifications [21]. The final pellet was resuspended in 10 mM HEPES buffer, pH 7.2 without EGTA containing 225 mM mannitol, 75 mM sucrose and 0.1% bovine serum albumin (BSA, fatty acid-free) at an approximate protein concentration of 15 mg . mL-1. Furthermore, heart crude homogenates were prepared in the

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Journal Pre-proof incubation medium used for the respiratory parameters determination at an approximate protein concentration of 3 mg . mL-1, as described by Makrecka-Kuka et al. [22]. Protein concentration was measured by the method of Lowry [23] using BSA as standard.

2.3. Cardiac cell cultures Rat heart-derived cell line (ventricular myoblasts, H9C2, number 0098) was

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obtained from Banco de Células do Rio do Janeiro (BCRJ, Rio de Janeiro, Brazil). Cells

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were grown and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in

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Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine

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serum. Culture medium was changed three times a week. Experiments were performed using cells with 50–60% confluence and between passages 10 and 14 at the time of the

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experiments [24]. New cultures were re-established from frozen stocks every three months.

2.4. Mitochondrial respiratory parameters measured by oxygen consumption

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The rate of oxygen consumption was measured using an OROBOROS Oxygraph-2k

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(Innsbruck, Austria) in a thermostatically controlled (37 °C) and magnetically stirred incubation chamber [25] with modifications [26]. The assay was performed with mitochondrial preparations (0.1 mg protein−1. mL−1 using 2.5 mM pyruvate plus 2.5 mM malate, 5 mM α-ketoglutarate or 5 mM succinate plus 2 μM of the complex I inhibitor rotenone as substrates) and incubated in a buffer containing 0.3 M sucrose, 5 mM KH2PO4, 1 mM EGTA, 0.1 mg . mL-1 BSA, 5 mM MOPS, pH 7.4. MA (0.05-5 mM), PA (5 mM), 3OHPA (5 mM), 2MCA (1 mM) and AA (5 mM) were added to the incubation medium in the beginning of the assay. State 3 (ADP-stimulated) and state 4 (non-phosphorylating) respiration were estimated by addition of 1 mM ADP and 1 μg . mL-1 oligomycin A to the 7

Journal Pre-proof incubation medium, respectively. Then, 1.0 µM CCCP (two pulses of 0.5 µM) was added to induce the uncoupled respiration. In some assays, alamethicin (40 μg/mg protein) [27] or CoA (100 μM) was added to the mitochondrial preparations. The dose of CoA used in the present study was based on its cytosolic concentrations in animal tissues that vary from 20 to 140 μM [28]. Noteworthy, 100 μM CoA was previously demonstrated to stimulate mitochondrial CoA uptake [29-30].

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The means ± standard deviations of the respiratory control ratio (RCR, state 3/state

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4) from controls (absence of organic acids) were 10.6 ± 0.57, 9.17 ± 0.54 and 4.09 ± 0.41

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using pyruvate plus malate, α-ketoglutarate and succinate as substrates, respectively (results not shown), stressing the highly coupled mitochondrial preparations used in the

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experiments. States 3, 4 and uncoupled were expressed as pmol O2 consumed . s-1 . mg

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protein-1.

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Oxygen consumption was also measured in permeabilized H9C2 cells (1.5 million cells.mL-1) and in heart crude homogenates (1 mg tissue.mL-1). Cardiac cells were

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centrifuged, resuspended in the incubation medium and permeabilized with digitonin (8

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µM), as previously described [31]. The substrate-uncoupler inhibitor titration (SUIT) protocol was used in the presence of MA (0.1-5 mM), PA (5.0 mM) or 3OHPA (5 mM) [22]. NADH-linked substrates (5 mM pyruvate, 0.5 mM malate and 10 mM glutamate) were first added to the incubation medium, followed by 500 µM ADP (state 3 respiration), 10 mM succinate and 1 µg.mL-1 oligomycin (state 4 respiration). Next, 1.5 µM CCCP (three pulses of 0.5 µM) was supplemented to induce the uncoupled respiration. Finally, 2 µM rotenone (complex I inhibitor) was used to obtain the uncoupled respiration stimulated by succinate.

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Journal Pre-proof The real-time oxygen fluxes were calculated using DatLab5 (Oroboros Instruments) and expressed as pmol O2 flux . s-1 . mg protein-1 or pmol O2 flux . s-1 . million cells-1.

2.5. α-Ketoglutarate dehydrogenase (α-KGDH), citrate synthase (CS) and malate dehydrogenase (MDH) activities

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The activities of the citric acid cycle enzymes α-ketoglutarate dehydrogenase (αKGDH), citrate synthase (CS) and malate dehydrogenase (MDH) were evaluated in a

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Spectramax M5 microplate spectrofluorimeter using heart mitochondria (α-KGDH: 0.25

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mg protein.mL-1; CS: 0.01 mg protein.mL-1; MDH: 0.0025 mg protein.mL-1) or

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commercially α-KGDH purified from porcine heart (0.012 U.mL-1, K1502, Sigma).

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Samples were pre-incubated at 37 °C for 30 min in the presence of MA (1.0-5.0 mM) or PA (5 mM) before the measurements.

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α-KGDH activity was determined by following NAD+ reduction and expressed as

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nmol NADH . min−1 . mg protein−1 or nmol NADH . min−1 . U−1 [32], whereas MDH activity by measuring NADH oxidation and expressed as nmol NADH oxidized . min−1. mg

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protein−1 [33]. CS activity was assayed by determining DTNB reduction according to Srere [34] and expressed as μmol TNB . min-1. mg protein-1. For the measurements of α-KGDH activity, we used 0.12 mM coenzyme A (CoA) when using isolated heart mitochondria [32], whereas 0.12 or 0.25 mM CoA with the α-KGDH purified from porcine heart.

2.6. ATP production ATP concentrations were determined by the firefly luciferin–luciferase assay system [35] with some modifications [21]. Mitochondrial respiration was sustained by 9

Journal Pre-proof either 2.5 mM pyruvate plus 2.5 mM malate or α-ketoglutarate. The reaction was initiated by addition of 1 mM ADP. MA (1-2.5 mM), PA (5 mM) or 3OHPA (5 mM) were added to the incubation medium containing the heart mitochondria (0.1 mg protein . mL-1) in the beginning of the assay. The luminescence was measured in a SpectraMax I3 microplate spectrofluorimeter. Oligomycin A was used as positive control.

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2.7. Mitochondrial membrane potential (ΔΨm), NAD(P)H content, swelling and Ca2+

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retention capacity

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Measurements of mitochondrial ΔΨm, NAD(P)H content, swelling and Ca2+ retention capacity were performed using a fluorescence spectrophotometer (HITACHI F-

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4500) in a medium containing mitochondria, 150 mM KCl, 5 mM MgCl2, 0.1 mg . mL−1

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BSA, 2 mM KH2PO4, 1 µg . mL-1 oligomycin A, 30 μM EGTA and 5 mM HEPES, pH 7.2,

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at 37 °C with continuous magnetic stirring. The assays were conducted in nonphosphorylating respiring heart mitochondria (0.35 mg protein . mL-1) supported by 2.5

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mM pyruvate plus 2.5 mM malate or 5 mM α-ketoglutarate. MA (0.1-5 mM), PA (5 mM),

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AA (5 mM), CaCl2 (30 µM single or 5 µM successive additions), CCCP (3 µM) and alamethicin (40 μg/mg protein) were added as indicated in the figures. In some experiments cyclosporin A (CsA, 1 µM), ADP (300 µM) and EGTA (500 µM) were used. Statistical calculations utilized the fluorescence changes observed between 50 and 250 seconds after the beginning of the assays. ΔΨm was estimated by following the fluorescence of the cationic dye safranine O (5 μM) at excitation and emission wavelengths of 495 and 586 nm [36-37] and swelling by monitoring light scattering at excitation and emission wavelengths of 540 nm. Matrix NAD(P)H fluorescence was determined at 340 nm excitation and 450 nm emission 10

Journal Pre-proof wavelengths. Mitochondrial Ca2+ retention capacity was determined by measuring extramitochondrial free Ca2+ levels through monitoring the fluorescence of 0.2 μM calcium green-5N at excitation and emission wavelengths of 506 and 532 nm, respectively [38].

2.8. Statistical analysis Results are presented as mean ± standard deviation. Experiments were performed in

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duplicate or triplicate and the mean or median was used for statistical analyses. Data were

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analysed by one-way analysis of variance (ANOVA) or by two-way ANOVA considering

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the factors: 1) MA treatment; 2) CoA or alamethicin supplementation; 3) interaction

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between MA treatment x CoA or alamethicin supplementation. The post-hoc Tukey’s range test was performed when means from three or more groups were compared. Student´s t test

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for unpaired samples was also used to compare means between two different groups.

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Differences between groups were considered significant at P < 0.05. All analyses were

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3. Results

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carried out in an IBM-compatible PC computer using the GraphPad Prism 7.0 software.

3.1. Maleic (MA), propionic (PA) and 3-hydroxypropionic (3OHPA) acids differentially disrupt respiration in heart mitochondria of developing rats We first investigated the effects of MA, PA, 3OHPA and 2MCA on state 3 (ADPstimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration determined by oxygen consumption in isolated heart mitochondria supported by NADHlinked (pyruvate plus malate or α-ketoglutarate) and FADH2-linked (succinate) substrates. MA, at concentrations as low as 0.5 mM, markedly inhibited state 3, state 4 and uncoupled

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Journal Pre-proof respiration using pyruvate plus malate as substrates, whereas PA and 3OHPA provoked milder effects on respiration and only at the highest dose (5 mM) and 2MCA caused no changes at all (Figure 1) (state 3: F(7,24)=45.05, P<0.001; state 4: F(7,24)=24.62, P<0.001; uncoupled: F(7,24)=211.6, P<0.001). Furthermore, MA-induced decrease of ADP-stimulated and uncoupled respiration were more intense in α-ketoglutarate supported mitochondria, differently from PA and 3OHPA inhibitory effects that were similar to those obtained with

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pyruvate plus malate (Figure 2) (state 3: F(7,24)=127.3, P<0.001; state 4: F(7,24)=24.2,

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P<0.001; uncoupled: F(7,24)=42.15, P<0.001).

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In order to test whether the potent effects provoked by MA were specific or a

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general unspecific result of dicarboxylic acids, we performed experiments using AA, which is a dicarboxylic acid structurally related to MA. We found that 5 mM AA did not alter

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state 4, state 3 and uncoupled respiration in α-ketoglutarate-supported heart mitochondria

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(Figure 2), indicating that the marked MA-induced disruption of mitochondrial respiration was selective. It was also verified that PA supplementation to the mitochondrial

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preparations in the absence of exogenous substrates at basal conditions did not increase

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mitochondrial oxygen consumption per se, but instead a significant inhibition was detected (pmol O2 consumed . s-1. mg of protein-1: Control: 114.08 ± 12.27; PA: 70.55 ± 6.44; t(6)=6.283, P<0.001), indicating that under our experimental conditions there was no significant feeding of citric acid cycle by PA through propionyl-CoA carboxylase activity until succinyl-CoA and confirming inhibition of mitochondrial respiration by PA. Of note, MA-induced inhibitory effects on mitochondrial respiration (state 3 and uncoupled respiration) were lower in succinate-respiring mitochondria (state 3: F(5,21)=6.165, P<0.01; uncoupled: F(5,13)=25.14, P<0.001), whereas PA caused no effect and 3OHPA only a mild significant inhibition using this substrate (Figure 3). It was also 12

Journal Pre-proof observed that MA-induced inhibition of succinate-stimulated state 3 respiration was significantly decreased when mitochondria were permeabilized by alamethicin, showing significant influences of MA (F(1,12)=147, P<0.001) and an interaction between MA and alamethicin (F(1,12)=35.61, P<0.001) (two-way ANOVA), implying that MA and succinate may compete for the same mitochondrial transporter (Figure 4).

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of respiration in heart mitochondria of developing rats

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3.2. Coenzyme A (CoA) supplementation attenuates maleic acid (MA)-induced disruption

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Since it has been previously demonstrated that MA may sequester CoA [42], we

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tested whether exogenous CoA (100 μM) addition could prevent MA-induced alterations of

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the respiratory parameters. Statistical analysis by two-way ANOVA considering the separate influences of MA and CoA, as well as their interaction, revealed that CoA

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significantly attenuated the decrease of state 3 and state 4 respiration caused by MA in mitochondria supported by pyruvate plus malate (Figure 5A and 5B) (MA - State 4:

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F(1,12)=72.61, P<0.001; State 3: F(1,12)=1014, P<0.001; CoA - State 4: F(1,12)=5.005, P<0.05;

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State 3: F(1,12)=27.23, P<0.001; interaction between MA and CoA were not significant) and particularly when using α-ketoglutarate as the substrate (Figure 5C and D) (MA: State 4: F(1,12)=262.5, P<0.001; State 3: F(1,12)=449.7, P<0.001; CoA: State 4: F(1,12)=25.60, P<0.001; State 3: F(1,12)=15.35, P<0.01; interaction between MA and CoA: State 4: F(1,12)=25.80, P<0.001; State 3: F(1,12)=24.76, P<0.001). The data suggest that mitochondrial CoA depletion could be partly responsible for MA-induced inhibitory effects on the respiratory parameters.

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Journal Pre-proof 3.3. Maleic acid (MA) markedly inhibits α-ketoglutarate dehydrogenase (α-KGDH) activity in heart mitochondria of developing rats We next tested the effects of MA and PA on the activities of the citric acid cycle enzymes α-KGDH, MDH and CS in heart mitochondria, in order to better clarify the mechanisms involved in the impairment of mitochondrial respiration supported by αketoglutarate and by pyruvate plus malate. It can be seen in figure 6 that MA strongly

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inhibited α-KGDH activity (A) (F(3,12)=20.38, P<0.001) without changing the activities of

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MDH (B) and CS (C), whereas PA did not alter these enzyme activities. We also evaluated

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the effects of MA on α-KGDH purified from porcine heart in the presence of different

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concentrations of CoA (D) and two-way ANOVA statistical analysis demonstrated that MA

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significantly inhibited this enzyme (F(1,20)=151.0, P<0.001) without the influence of CoA concentration, confirming the data obtained in isolated heart mitochondria and indicating

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that this inhibitory effect of MA is not due to CoA depletion. The data suggest that the inhibitory effect caused by MA on α-KGDH may also contribute at least in part to the MA-

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induced decrease of mitochondrial oxygen consumption using α-ketoglutarate as substrate.

3.4. Maleic acid (MA) impairs mitochondrial respiration in crude heart homogenates of developing rats and in digitonin-permeabilized cardiomyocytes Thereafter, we evaluated whether MA, PA or 3OHPA could alter mitochondrial respiration in crude heart homogenates (Figure 7) and in digitonin-permeabilized cardiomyocytes (Figure 8), using the SUIT protocol. These preparations contain all cell machinery and reliably evaluate mitochondrial functionality within an integrated cellular system, better mimicking the in vivo situation. It can be seen that MA at concentrations as

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Journal Pre-proof low as 0.2 mM strongly inhibited state 3 and uncoupled respiration in crude heart homogenates especially when using a cocktail of NADH-linked substrates (pyruvate, malate and glutamate) (state 3: F(5,21)=34.27, P<0.001; uncoupled: F(5,21)=22.63, P<0.001), whereas 3OHPA (5 mM) had a weaker effect and PA (5 mM) caused no changes. When the homogenates were supported by succinate, only MA (1 mM) was able to significantly decrease state 3 and uncoupled respiration (state 3: F(5,21)=7.057, P<0.001; uncoupled:

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F(5,21)=6.948, P<0.001).

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It was also verified that MA (5 mM) significantly decreased state 3 and uncoupled

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respiration in permeabilized cardiomyocytes using pyruvate, malate and glutamate as

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substrates (state 3: t(8)=14.71, P<0.001; uncoupled: t(8)=11.45, P<0.001), but less intense alterations when using succinate as the substrate (uncoupled: t(8)=5.895, P<0.001) (Figure

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8).

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Taken together these data achieved with integrated cellular systems, it is assumed

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that MA is a potent metabolic inhibitor in the heart, as compared to PA and 3OHPA.

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3.5. Maleic (MA) and propionic (PA) acids significantly decrease ATP production in heart mitochondria of developing rats Considering that MA, as well as PA and 3OHPA to a lesser extent, decreased ADPstimulated respiration (state 3), we tested the effects of these organic acids on ATP production in heart mitochondria respiring with α-ketoglutarate (Figure 9A) (F(4,24)=478.9, P<0.001) and pyruvate plus malate (Figure 9B) (F(3,18)=142.6, P<0.001). MA severely reduced ATP production with both substrates, whereas PA caused a less intense effect with

15

Journal Pre-proof pyruvate plus malate and 3OHPA no alterations on ATP synthesis. These data reinforce the marked role of MA disturbing mitochondrial bioenergetics in the heart.

3.6. Maleic (MA) and propionic (PA) acids decrease membrane potential (ΔΨm) and NAD(P)H content and induce swelling in heart mitochondria of developing rats It was also investigated whether MA and PA could compromise other mitochondrial

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functions, including ΔΨm, NAD(P)H content and swelling using isolated heart

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mitochondria in the presence and absence of Ca2+. We first verified that MA, but not PA, significantly decreased ΔΨm (Figure 10A) (F(5,17)=4.617, P<0.01) and NAD(P)H content

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(Figure 11A) (F(2,9)=27.14, P<0.001) in Ca2+-loaded mitochondria supported by pyruvate

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plus malate. Besides, the mitochondrial permeability transition (mPT) inhibitors CsA plus

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ADP totally prevented the reduction of ΔΨm, indicating that these effects were probably due to mPT induction (Figure 10B) (F(2,7)=8.644, P<0.05). Furthermore, 500 μM EGTA

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(Ca2+ chelator) supplementation blocked the dissipation of ΔΨm caused by MA, indicating

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a significant role for Ca2+ in this effect and reinforcing mPT (Figure 10E). MA also

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significantly decreased ΔΨm (Figure 10C) (F(5,20)=39.04, P<0.001) and matrix NAD(P)H content (Figure 11B) (F(4,15)=148.7, P<0.001) in α-ketoglutarate-supported mitochondria, being these effects apparently more severe as compared to those observed with pyruvate plus malate. MA-induced ΔΨm dissipation was attenuated by CsA plus ADP and occurred before and after Ca2+ addition (Figure 10D) (F(4,12)=14.13, P<0.001), indicating that, in addition to mPT pore opening, metabolic inhibition was also involved in these effects. We also observed that MA provoked mitochondrial swelling in α-ketoglutarate respiring Ca2+loaded mitochondria that was fully prevented by CsA plus ADP (Figure 12) (F(4,13)=13.1, P<0.001), further indicating mPT induction by MA. To determine whether MA-induced 16

Journal Pre-proof disturbance of mitochondrial functions was not a general effect caused by dicarboxylic acids, we tested the effects of AA (5 mM) on ΔΨm and verified no alterations on this parameter in pyruvate plus malate-supported mitochondria supplemented by Ca2+ (Figure 10A). Finally, it can be seen in the figure that PA significantly reduced ΔΨm (Figure 10D) and induced swelling (Figure 12) after Ca2+ addition in mitochondria supported by α-

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Figure 12), also suggesting the involvement of mPT.

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ketoglutarate and that these effects were abolished by CsA plus ADP (Figure 10D and

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3.7. Maleic acid (MA) decreases Ca2+ retention capacity in heart mitochondria of

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developing rats

Further experiments testing the role of MA and PA on mitochondrial capacity to

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retain Ca2+ showed that MA markedly reduced this capacity in mitochondria supported by pyruvate plus malate (Figure 13A) or by α-ketoglutarate (Figure 13B). It can be also seen in

4. Discussion

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the figure that PA did not alter this parameter.

PAcidemia is biochemically characterized by predominant accumulation of PA in tissues and biological fluids, as well as 3OHPA, 2MCA and MA to a lesser extent [1, 4-6]. Although the precise intracellular and tissue concentrations of these metabolites are still unknown, we assume that their levels may be similar or even higher than plasma concentrations since they are produced in the mitochondrial matrix and leave mitochondria and the cell towards the circulation. Affected patients usually develop cardiomyopathy

17

Journal Pre-proof during the course of the disease that may be a lethal complication. However, there are no effective therapeutic guidelines to prevent or manage this clinical manifestation since the underlying pathomechanisms of cardiac damage are still unknown [17]. In this scenario, considering that heart is a highly energy dependent tissue, mitochondrial dysfunction may be proposed as a contributor factor for the progression of the cardiomyopathy in the affected patients [39]. This hypothesis is supported by previous reports revealing

of

mitochondrial abnormalities in the heart of PAcidemic patients, decreased myocardial

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levels of coenzyme Q10 [17] and free carnitine [40], as well as reduction of complex I-III

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[40] and III [41] activities in muscular biopsies of patients, and reduced complex I activity

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in the heart of the genetic mice model of PAcidemia [18]. Nevertheless, the precise mechanisms of these morphological and biochemical alterations have not yet been

lP

elucidated. In the present work we demonstrated for the first time that MA, followed by PA

functions.

na

and 3OHPA, with no effect of 2MCA, markedly disrupt crucial heart mitochondrial

ur

We first observed that MA strongly inhibited state 3 (ADP-stimulated), state 4 (non-

Jo

phosphorylating) and uncoupled (CCCP-stimulated) respiration in isolated heart mitochondria supported by pyruvate plus malate or by α-ketoglutarate. Furthermore, these alterations of oxygen consumption were much more intense using α-ketoglutarate as substrate, achieving a complete inhibition at 0.5 mM MA. A possible contributing factor to MA inhibitory effects was shortage of CoA since supplementation of this coenzyme to the medium containing the isolated mitochondria significantly attenuated MA-induced respiratory inhibition, mainly when supported by α-ketoglutarate. Noteworthy, previous observations have shown that MA is able to sequester CoA [42] and that the mitochondrial

18

Journal Pre-proof CoA pool is dependent on the uptake of this coenzyme from the cytosol into the mitochondria [29-30]. The effects caused by MA could be also tentatively attributed at least in part due to the potent inhibition of α-KGDH activity probably not related to CoA depletion, thus impairing α-ketoglutarate oxidation. In this context, our group has previously demonstrated a competitive inhibition of α-KGDH activity by MA using a commercial purified enzyme [43].

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In contrast, MA did not alter CS and MDH activities that metabolize pyruvate and

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malate, which might explain the less intense effects of MA on mitochondrial respiration

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using pyruvate plus malate as compared to that using α-ketoglutarate as substrate. On the

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other hand, MA caused a milder decrease of state 3 and uncoupled respiration in

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mitochondria supported by succinate, and this may have occurred due to a competition between MA and succinate for the same mitochondrial transporter since alamethicin, that

na

permeabilizes mitochondrial membranes, significantly attenuated this effect.

ur

On the other hand, PA and 3OHPA caused a less intense decrease of state 3 and uncoupled respiration in heart mitochondria supported by pyruvate plus malate or by α-

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ketoglutarate, whereas 2MCA caused no effect at all. Inhibition of α-ketoglutarate oxidation in the citric acid cycle probably does not contribute to these alterations, since PA did not change α-KGDH activity. However, we cannot rule out the possibility that PA and 3OHPA could bind CoA and decrease its intramitochondrial pool, since it has been recently demonstrated a significant CoA trapping in PA perfused rat hearts, forming increased concentrations of propionyl-CoA and 3-hydroxylpropionyl-CoA [44]. The differential effects of these metabolites that most accumulate in PAcidemia on the mitochondrial respiratory parameters are in line with the findings that MA severely impaired ATP 19

Journal Pre-proof synthesis, whereas PA mildly compromised this parameter and 3OHPA caused no alterations. Taken together, these data indicate that mitochondrial bioenergetics is markedly compromised by MA in comparison to PA, 3OHPA and 2MCA. We also verified that MA markedly inhibited ADP- and CCCP-stimulated respiration in mitochondria sustained by NADH-linked substrates by using a more physiological cellular milieu consisting of crude heart homogenates and permeabilized

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cardiomyocytes, corroborating with the results obtained with purified mitochondria and

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strengthening the view that MA is a potent mitotoxic compound. Therefore, we presume

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that bioenergetics disruption mainly provoked by MA may be potentially harmful to the

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heart, a high-energy demanding tissue.

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Further novel findings of the present work were that MA severely compromised other mitochondrial functions by decreasing ΔΨm, matrix NAD(P)H content and Ca2+

na

retention capacity, and inducing swelling. Since ΔΨm collapse and mitochondrial swelling

ur

were fully or partially prevented by the classical inhibitors of mPT, CsA [45] and ADP [38] that bind to cyclophilin D and to the adenine nucleotide translocator, respectively, the data

Jo

point to the involvement of mPT pore opening in the MA-mediated effects. It is of note that mPT pore opening provokes non-selective permeabilization that may lead to ΔΨm collapse and mitochondrial swelling, loss of matrix components (Ca2+, Mg2+, glutathione, NADH and NADPH) and release of mitochondrial proapoptotic factors [46-47]. The importance of Ca2+ in the MA-induced mPT was confirmed by the observation that high concentration (500 μM) of the Ca2+ chelator EGTA prevented the dissipation of ΔΨm caused by this organic acid in Ca2+-loaded mitochondria respiring with pyruvate plus malate. On the other hand, it is conceivable that the severe inhibition of mitochondrial 20

Journal Pre-proof respiration caused by MA also contributed to the decrease of ΔΨm, NAD(P)H content and Ca2+ retention capacity in Ca2+-loaded mitochondria supported by α-ketoglutarate, because these effects also occurred before the addition of exogenous Ca2+ and/or were slightly affected by CsA and ADP. As regards to PA, we found that this organic acid also dissipated ΔΨm and provoked swelling in Ca2+-loaded mitochondria and that these effects were prevented by CsA and ADP, suggesting mPT induction.

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In what concerns to the underlying mechanisms of mPT activation, we cannot

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exclude the possibility that it may have occurred due to the MA and PA-induced metabolic

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inhibition, as previously demonstrated for other compounds [48-49]. Furthermore, the

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strong decrease of NAD(P)H content induced by MA in the absence of Ca2+ may also

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precede mPT occurrence, because the oxidized redox state of pyridine nucleotides was demonstrated to play a role on this process [50-51].

na

Otherwise, since cytosolic Ca2+ concentrations maintenance is critical for

ur

cardiomyocyte performance, it is conceivable that mPT induction allied to a deregulation of intracellular Ca2+ concentrations due to reduced mitochondrial Ca2+ retention capacity and

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loss through the mitochondrial membrane may synergistically compromise cardiac physiology and potentially induce cardiomyocyte death [52]. Accordingly, it has been previously shown the involvement of mPT pore opening in the reperfusion-mediated cardiac damage [53]. Furthermore, disruption of Ca2+ homeostasis is considered a key factor of failing cardiomyocytes [54], in addition to impaired ATP synthesis and oxidative stress. In line with this assumption, it has been hypothesized that mPT inhibitors could be beneficial to heart failure treatment preventing loss of cardiomyocytes [55]. In addition, disturbance of Ca2+ homeostasis by MA was previously shown in kidney cell cultures and 21

Journal Pre-proof mitochondrial preparations and proposed to be a contributor pathomechanism to the nephrotoxicity induced by this metabolite in methylmalonic acidemia and PAcidemia [43, 56]. Our in vitro and novel data obtained in isolated heart mitochondria and also in heart crude preparations and permeabilized cardiomyocytes that contain all cell machinery may possibly explain the heart mitochondrial abnormalities encountered in patients myocardial

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biopsies and in the genetic mice model of PAcidemia [17, 40-41]. It is also emphasized that

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the high mitotoxicity presented by MA was selective rather than a general effect caused by

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dicarboxylic acids, since AA, another dicarboxylic acid, did not disturb mitochondrial

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homeostasis even at a high dose (5 mM).

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Although we demonstrated that PA and especially MA are mitotoxic, at the present it is difficult to determine the pathophysiological relevance of our data because we used

na

heart mitochondria, heart homogenates and cardiomyocytes that express propionyl-CoA

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carboxylase, whereas in the disorder this enzyme activity is absent. Furthermore, tissue levels of MA were not yet described in PAcidemic patients, although high urinary excretion

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of this compound has been reported [4]. Importantly, the marked deleterious alterations of mitochondrial functions caused by MA were achieved with concentrations as low as 0.05 mM, whereas disruption of mitochondrial homeostasis provoked by PA occurred with 5 mM, corresponding to the concentrations found in plasma of patients with PAcidemia [3, 57-58].

5. Conclusions In conclusion, we provide novel evidence that MA, and PA to a lesser extent, impair 22

Journal Pre-proof mitochondrial bioenergetics and Ca2+ homeostasis in heart mitochondria, behaving as potent metabolic inhibitors and mPT inductors. The mitochondrial respiratory parameters data demonstrated in isolated mitochondria were confirmed in more physiological and integrated cellular systems, such as heart crude homogenates and permeabilized cardiomyocytes. MA was also demonstrated to inhibit α-KGDH activity in heart mitochondria, which is a critical citric acid cycle enzyme. Furthermore, CoA

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supplementation partially prevented the MA-induced inhibition of mitochondrial

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respiration, implying that CoA depletion contributes at least partly to the severe

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mitochondrial dysfunction caused by this organic acid. In case the present results are

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confirmed in cultured fibroblasts and tissues from affected patients and in heart from the genetic mouse model of PAcidemia, it is tempting to speculate that disruption of

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mitochondrial bioenergetics and Ca2+ homeostasis caused by MA and PA may possibly be

ur

patients with this disease.

na

associated with the cardiomyopathy that is a lethal manifestation commonly affecting

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Declarations of interest: none.

Acknowledgements

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 425914/2016-0), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, 17/2551-0000/800-6), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 17/17728-8) and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção (INCT – EN, 573677/2008-5).

23

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References

Jo

ur

na

lP

re

-p

ro

of

[1] W.A. Fenton, R.A. Gravel, D.S. Rosenblatt, Disorders of Propionate and Methylmalonate Metabolism, In: D. Valle, A.L. Beaudet, B. Vogelstein, K.W. Kinzler, S.E. Antonarakis, A. Ballabio, M.K. Gibson, G. Mitchell (Eds), The Online Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill Inc, New York, 2001. [2] P. Wongkittichote, N. Ah Mew, K.A. Chapman, Propionyl-CoA carboxylase - A review, Mol. Genet. Metab., 122 (2017) 145-152. [3] H. van den Berg, M.T. Boelkens, F.A. Hommes, A case of methylmalonic and propionic acidemia due to methulmalonyl-CoA carbonylmutase apoenzyme deficiency, Acta Paediatr. Scand., 65 (1976) 113-118. [4] T. Bergstrom, J. Greter, A.H. Levin, G. Steen, N. Tryding, U. Wass, Propionyl-CoA carboxylase deficiency: case report, effect of low-protein diet and identification of 3-oxo-2methylvaleric acid 3-hydroxy-2-methylvaleric acid, and maleic acid in urine, Scand. J. Clin. Lab. Invest., 41 (1981) 117-126. [5] T. Ando, K. Rasmussen, W.L. Nyhan, D. Hull, 3-hydroxypropionate: significance of oxidation of propionate in patients with propionic acidemia and methylmalonic acidemia, Proc. Natl. Acad. Sci. U. S. A., 69 (1972) 2807-2811. [6] T. Ando, K. Rasmussen, J.M. Wright, W.L. Nyhan, Isolation and identification of methylcitrate, a major metabolic product of propionate in patients with propionic acidemia, J. Biol. Chem., 247 (1972) 2200-2204. [7] K.A. Wilson, Y. Han, M. Zhang, J. Hess, K.A. Chapman, G.W. Cline, G.P. Tochtrop, H. Brunengraber, G.F. Zhang, Interrelations between 3-hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism, Am. J. Physiol. Endocrinol. Metab., 313 (2017) E413-E428. [8] M.R. Baumgartner, F. Horster, C. Dionisi-Vici, G. Haliloglu, D. Karall, K.A. Chapman, M. Huemer, M. Hochuli, M. Assoun, D. Ballhausen, A. Burlina, B. Fowler, S.C. Grunert, S. Grunewald, T. Honzik, B. Merinero, C. Perez-Cerda, S. Scholl-Burgi, F. Skovby, F. Wijburg, A. MacDonald, D. Martinelli, J.O. Sass, V. Valayannopoulos, A. Chakrapani, Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia, Orphanet J. Rare Dis., 9 (2014) 130. [9] O.A. Shchelochkov, N. Carrillo, C. Venditti, Propionic Acidemia, In: R.A. Pagon, M.P. Adam, H.H. Ardinger, S.E. Wallace, A. Amemiya, L.J.H. Bean, T.D. Bird, N. Ledbetter, H.C. Mefford, R.J.H. Smith, K. Stephens (Eds.), GeneReviews(R), Seattle (WA), 2012 updated 2016. [10] S. Romano, V. Valayannopoulos, G. Touati, J.P. Jais, D. Rabier, Y. de Keyzer, D. Bonnet, P. de Lonlay, Cardiomyopathies in propionic aciduria are reversible after liver transplantation, J. Pediatr., 156 (2010) 128-134. [11] T.M. Lee, L.J. Addonizio, B.A. Barshop, W.K. Chung, Unusual presentation of propionic acidaemia as isolated cardiomyopathy, J. Inherit. Metab. Dis., 32 Suppl 1 (2009) S97-101. [12] A. Laemmle, C. Balmer, C. Doell, J.O. Sass, J. Haberle, M.R. Baumgartner, Propionic acidemia in a previously healthy adolescent with acute onset of dilated cardiomyopathy, Eur. J. Pediatr., 173 (2014) 971-974. [13] J.L. Fraser, C.P. Venditti, Methylmalonic and propionic acidemias: clinical management update, Curr. Opin. Pediatr., 28 (2016) 682-693. 24

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[14] W. Lehnert, W. Sperl, T. Suormala, E.R. Baumgartner, Propionic acidaemia: clinical, biochemical and therapeutic aspects. Experience in 30 patients, Eur. J. Pediatr., 153 (1994) S68-80. [15] C. Perez-Cerda, B. Merinero, P. Rodriguez-Pombo, B. Perez, L.R. Desviat, S. Muro, E. Richard, M.J. Garcia, J. Gangoiti, P. Ruiz Sala, P. Sanz, P. Briones, A. Ribes, M. Martinez-Pardo, J. Campistol, M. Perez, R. Lama, M.L. Murga, T. Lema-Garrett, A. Verdu, M. Ugarte, Potential relationship between genotype and clinical outcome in propionic acidaemia patients, Eur. J. Hum. Genet., 8 (2000) 187-194. [16] K. Ameloot, D. Vlasselaers, M. Dupont, W. Meersseman, L. Desmet, J. Vanhaecke, N. Vermeer, B. Meyns, J. Pirenne, D. Cassiman, C. De Laet, P. Goyens, S.G. MalekzadehMilan, D. Biarent, A. Meulemans, F.G. Debray, Left ventricular assist device as bridge to liver transplantation in a patient with propionic acidemia and cardiogenic shock, J. Pediatr., 158 (2011) 866-867. [17] J. Baruteau, I. Hargreaves, S. Krywawych, A. Chalasani, J.M. Land, J.E. Davison, M.K. Kwok, G. Christov, A. Karimova, M. Ashworth, G. Anderson, H. Prunty, S. Rahman, S. Grunewald, Successful reversal of propionic acidaemia associated cardiomyopathy: evidence for low myocardial coenzyme Q10 status and secondary mitochondrial dysfunction as an underlying pathophysiological mechanism, Mitochondrion, 17 (2014) 150-156. [18] L. Gallego-Villar, A. Rivera-Barahona, C. Cuevas-Martin, A. Guenzel, B. Perez, M.A. Barry, M.P. Murphy, A. Logan, A. Gonzalez-Quintana, M.A. Martin, S. Medina, A. GilIzquierdo, J.M. Cuezva, E. Richard, L.R. Desviat, In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder, Free Radic. Biol. Med., 96 (2016) 112. [19] C. Arrizza, A. De Gottardi, E. Foglia, M. Baumgartner, M. Gautschi, J.M. Nuoffer, Reversal of cardiomyopathy in propionic acidemia after liver transplantation: a 10-year follow-up, Transpl. Int., 28 (2015) 1447-1450. [20] R. Ferranti, M.M. da Silva, A.J. Kowaltowski, Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation, FEBS Lett., 536 (2003) 5155. [21] C. Cecatto, F.H. Hickmann, M.D. Rodrigues, A.U. Amaral, M. Wajner, Deregulation of mitochondrial functions provoked by long-chain fatty acid accumulating in long-chain 3hydroxyacyl-CoA dehydrogenase and mitochondrial permeability transition deficiencies in rat heart--mitochondrial permeability transition pore opening as a potential contributing pathomechanism of cardiac alterations in these disorders, FEBS J., 282 (2015) 4714-4726. [22] M. Makrecka-Kuka, G. Krumschnabel, E. Gnaiger, High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria, Biomolecules, 5 (2015) 1319-1338. [23] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [24] A.V. Kuznetsov, S. Javadov, S. Sickinger, S. Frotschnig, M. Grimm, H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation, Biochimica et biophysica acta, 1853 (2015) 276-284. [25] E. Gnaiger, Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology, Int. J. Biochem. Cell Biol., 41 (2009) 18371845. 25

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[26] C. Cecatto, S. Godoy Kdos, J.C. da Silva, A.U. Amaral, M. Wajner, Disturbance of mitochondrial functions provoked by the major long-chain 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies in skeletal muscle, Toxicol. In Vitro, 36 (2016) 1-9. [27] S.R. Mirandola, D.R. Melo, P.F. Schuck, G.C. Ferreira, M. Wajner, R.F. Castilho, Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake, J. Inherit. Metab. Dis., 31 (2008) 44-54. [28] R. Leonardi, Y.M. Zhang, C.O. Rock, S. Jackowski, Coenzyme A: back in action, Prog. Lipid Res., 44 (2005) 125-153. [29] A.G. Tahiliani, Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient, J. Biol. Chem., 264 (1989) 18426-18432. [30] A.G. Tahiliani, Evidence for net uptake and efflux of mitochondrial coenzyme A, Biochim. Biophys. Acta, 1067 (1991) 29-37. [31] C. Cecatto, A.U. Amaral, J.C. da Silva, A. Wajner, M.O.V. Schimit, L.H.R. da Silva, S.M. Wajner, A. Zanatta, R.F. Castilho, M. Wajner, Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca(2+) homeostasis in the heart, FEBS J., 285 (2018) 1437-1455. [32] L. Tretter, V. Adam-Vizi, Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress, J. Neurosci., 20 (2000) 8972-8979. [33] G.B. Kitto, Intra- and extramitochondrial malate dehydrogenase from chicken and tuna heart, Methods Enzymol., 13 (1969) 106-116. [34] P.A. Srere, Citrate synthase, Methods Enzymol., 13 (1969) 3-11. [35] J.J. Lemasters, C.R. Hackenbrock, Continuous measurement and rapid kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner membrane vesicles determined by firefly-luciferase luminescence, Eur. J. Biochem., 67 (1976) 1-10. [36] K.E. Akerman, M.K. Wikstrom, Safranine as a probe of the mitochondrial membrane potential, FEBS Lett., 68 (1976) 191-197. [37] T.R. Figueira, D.R. Melo, A.E. Vercesi, R.F. Castilho, Safranine as a fluorescent probe for the evaluation of mitochondrial membrane potential in isolated organelles and permeabilized cells, Methods Mol. Biol., 810 (2012) 103-117. [38] A. Saito, R.F. Castilho, Inhibitory effects of adenine nucleotides on brain mitochondrial permeability transition, Neurochemical research, 35 (2010) 1667-1674. [39] S. Kolker, P. Burgard, S.W. Sauer, J.G. Okun, Current concepts in organic acidurias: understanding intra- and extracerebral disease manifestation, J. Inherit. Metab. Dis., 36 (2013) 635-644. [40] R. Mardach, M.A. Verity, S.D. Cederbaum, Clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy, Mol. Genet. Metab., 85 (2005) 286-290. [41] Y. de Keyzer, V. Valayannopoulos, J.F. Benoist, F. Batteux, F. Lacaille, L. Hubert, D. Chretien, B. Chadefeaux-Vekemans, P. Niaudet, G. Touati, A. Munnich, P. de Lonlay, Multiple OXPHOS deficiency in the liver, kidney, heart, and skeletal muscle of patients with methylmalonic aciduria and propionic aciduria, Pediatr. Res., 66 (2009) 91-95. [42] A. Pacanis, T. Strzelecki, J. Rogulski, Effects of maleate on the content of CoA and its derivatives in rat kidney mitochondria, J. Biol. Chem., 256 (1981) 13035-13038. [43] A.C. Roginski, C. Cecatto, S.M. Wajner, F.D. Camera, R.F. Castilho, M. Wajner, A.U. Amaral, Experimental evidence that maleic acid markedly compromises glutamate 26

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

oxidation through inhibition of glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in kidney of developing rats, Mol. Cell. Biochem., 458 (2019) 99112. [44] Y. Wang, B.A. Christopher, K.A. Wilson, D. Muoio, R.W. McGarrah, H. Brunengraber, G.F. Zhang, Propionate-induced changes in cardiac metabolism, notably CoA trapping, are not altered by l-carnitine, Am. J. Physiol. Endocrinol. Metab., 315 (2018) E622-E633. [45] C. Yarana, J. Sripetchwandee, J. Sanit, S. Chattipakorn, N. Chattipakorn, Calciuminduced cardiac mitochondrial dysfunction is predominantly mediated by cyclosporine Adependent mitochondrial permeability transition pore, Arch. Med. Res., 43 (2012) 333-338. [46] A. Rasola, P. Bernardi, Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis, Cell Calcium, 50 (2011) 222-233. [47] A.E. Vercesi, R.F. Castilho, A.J. Kowaltowski, H.C.F. de Oliveira, N.C. de SouzaPinto, T.R. Figueira, E.N.B. Busanello, Mitochondrial calcium transport and the redox nature of the calcium-induced membrane permeability transition, Free Radic. Biol. Med., 129 (2018) 1-24. [48] X.Y. Fan, L. Yuan, C. Wu, Y.J. Liu, F.L. Jiang, Y.J. Hu, Y. Liu, Mitochondrial toxicity of organic arsenicals: membrane permeability transition pore opening and respiratory dysfunction, Toxicol. Res. (Camb), 7 (2018) 191-200. [49] E.N. Maciel, A.J. Kowaltowski, F.D. Schwalm, J.M. Rodrigues, D.O. Souza, A.E. Vercesi, M. Wajner, R.F. Castilho, Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition, J. Neurochem., 90 (2004) 1025-1035. [50] P. Costantini, B.V. Chernyak, V. Petronilli, P. Bernardi, Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites, J. Biol. Chem., 271 (1996) 6746-6751. [51] A.E. Vercesi, The participation of NADP, the transmembrane potential and the energylinked NAD(P) transhydrogenase in the process of Ca2+ efflux from rat liver mitochondria, Arch. Biochem. Biophys., 252 (1987) 171-178. [52] I. Drago, D. De Stefani, R. Rizzuto, T. Pozzan, Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes, Proc. Natl. Acad. Sci. U. S. A., 109 (2012) 12986-12991. [53] E. Murphy, C. Steenbergen, Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury, Physiol. Rev., 88 (2008) 581-609. [54] M. Luo, M.E. Anderson, Mechanisms of altered Ca(2)(+) handling in heart failure, Circ. Res., 113 (2013) 690-708. [55] S.Y. Lim, D.J. Hausenloy, S. Arjun, A.N. Price, S.M. Davidson, M.F. Lythgoe, D.M. Yellon, Mitochondrial cyclophilin-D as a potential therapeutic target for post-myocardial infarction heart failure, J. Cell. Mol. Med., 15 (2011) 2443-2451. [56] A.T. Tuncel, T. Ruppert, B.T. Wang, J.G. Okun, S. Kolker, M.A. Morath, S.W. Sauer, Maleic Acid--but Not Structurally Related Methylmalonic Acid--Interrupts Energy Metabolism by Impaired Calcium Homeostasis, PLoS ONE, 10 (2015) e0128770. [57] F.A. Hommes, J.R. Kuipers, J.D. Elema, J.F. Jansen, J.H. Jonxis, Propionicacidemia, a new inborn error of metabolism, Pediatr. Res., 2 (1968) 519-524. [58] D. Gompertz, C.N. Storrs, D.C. Bau, T.J. Peters, E.A. Hughes, Localisation of enzymic defect in propionicacidaemia, Lancet, 1 (1970) 1140-1143. 27

Journal Pre-proof Figure legends

Fig 1. Effects of maleic (MA), propionic (PA), 3-hydroxypropionic (3OHPA) and 2methylcitric (2MCA) acids on respiratory parameters in pyruvate plus malatesupported heart mitochondria. State 4 (non-phosphorylating) (A), state 3 (ADPstimulated) (B) and uncoupled (CCCP-stimulated) (C) respiration. Pyruvate plus malate (2.5 mM each) were used as substrates. Mitochondrial preparations (0.1 mg protein. mL-1) and MA (0.5-5 mM), PA (5 mM), 3OHPA (5 mM) and 2MCA (1 mM) were added to the

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incubation medium in the beginning of the assays. Controls were performed in the absence of organic acids. Values are means ± standard deviation for four independent experiments

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(N) expressed as pmol O2. s-1. mg of protein-1. One-way ANOVA is described in the text.

-p

*P < 0.05, **P < 0.001, ***P < 0.001 compared to controls (Tukey’s range test).

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Fig 2. Effects of maleic (MA), propionic (PA), 3-hydroxypropionic (3OHPA), 2-

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methylcitric (2MCA) and adipic (AA) acids on respiratory parameters in αketoglutarate-supported heart mitochondria. State 4 (non-phosphorylating) (A), state 3 (ADP-stimulated) (B) and uncoupled (CCCP-stimulated) (C) respiration. α-Ketoglutarate (5

na

mM) was used as substrate. Mitochondrial preparations (0.1 mg protein. mL-1) and MA (0.05-0.5 mM), PA (5 mM), 3OHPA (5 mM), 2MCA (1 mM) and AA (5 mM) were added

ur

to the incubation medium in the beginning of the assays. Controls were performed in the

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absence of organic acids. Values are means ± standard deviation for three to five independent experiments (N) expressed as pmol O2. s-1. mg of protein-1. One-way ANOVA is described in the text. *P < 0.05, ***P < 0.001 compared to controls (Tukey’s range test).

Fig 3. Effects of maleic (MA), propionic (PA) and 3-hydroxypropionic (3OHPA) acids on respiratory parameters in succinate-supported heart mitochondria. State 4 (nonphosphorylating) (A), state 3 (ADP-stimulated) (B) and uncoupled (CCCP-stimulated) (C) respiration. Succinate (5 mM) was used as substrate. Mitochondrial preparations (0.1 mg protein. mL-1) and MA (1-5 mM), PA (5 mM) and 3OHPA (5 mM) were added to the incubation medium in the beginning of the assays. Controls were performed in the absence of organic acids. Values are means ± standard deviation for three to five independent 28

Journal Pre-proof experiments (N) expressed as pmol O2. s-1. mg of protein-1. One-way ANOVA is described in the text. *P < 0.05, ***P < 0.001 compared to controls (Tukey’s range test).

Fig 4. Effects of maleic acid (MA) on state 3 respiration in alamethicin (Ala)permeabilized heart mitochondria supported by succinate. Succinate (5 mM) was used as substrate. Mitochondrial preparations (0.1 mg protein. mL-1), MA (5 mM) and Ala (40 µg/mg protein) were added to the incubation medium in the beginning of the assays. Controls were performed in the absence of organic acids. Values are means ± standard

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deviation for four independent experiments (N) and are expressed as pmol O2. s-1. mg of protein-1. Two-way ANOVA is described in the text. ***P < 0.001 compared to controls.

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P < 0.01 compared to 5 mM MA (Tukey’s range test).

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Fig 5. Effects of coenzyme A (CoA) supplementation on maleic acid (MA)-induced

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decrease of respiratory parameters in heart mitochondria.

State 4 (non-

phosphorylating) (A and C) and state 3 (ADP-stimulated) (B and D) respiration. Pyruvate

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plus malate (2.5 mM each) (A and B) or α-ketoglutarate (C and D) (5 mM) were used as substrates. Mitochondrial preparations (0.1 mg protein. mL-1), MA (0.1 or 2.5 mM) and

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CoA (100 µM) were added to the incubation medium in the beginning of the assays. Controls were performed in the absence of organic acids. Values are means ± standard

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deviation for four independent experiments (N) and are expressed as pmol O2. s-1. mg of

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protein-1. Two-way ANOVA is described in the text. **P<0.01, ***P < 0.001 compared to controls. ##P < 0.01, ###P < 0.001 compared to MA (0.1 and 2.5 mM) (Tukey’s range test).

Fig 6. Effects of maleic (MA) and propionic (PA) acids on the activities of citric acid cycle enzymes. α-Ketoglutarate dehydrogenase (α-KGDH) (A and D), malate dehydrogenase (MDH) (B) and citrate synthase (CS) (C) activities. Heart mitochondrial preparations (A-C) or α-KGDH purified from porcine heart (D) were pre-incubated for 30 min in the presence of MA (1 or 5 mM) or PA (5 mM). Measurement of α-KGDH activity in heart mitochondria was performed in the presence of 0.12 mM CoA (A), whereas for the purified enzyme 0.12 mM or 0.25 mM CoA were used (D). Values are mean  standard deviation of four to six independent experiments performed in triplicate and expressed as 29

Journal Pre-proof nmol NADH . min-1. mg protein-1 (A), nmol NAD+ . min-1 . mg protein-1 (B), μmol TNB . min-1. mg protein-1 (C) and nmol NADH . min-1. U-1 (D). One-way (A-C) or two-way (D) ANOVA are described in the text. **P<0.01, ***P<0.001 compared to controls (Tukey’s range test).

Fig 7. Effects of maleic (MA), propionic (PA) and 3-hydroxypropionic (3OHPA) acids on respiratory parameters in heart homogenates using the substrate-uncoupler inhibitor titration (SUIT) protocol. State 3 (ADP-stimulated) (A and B) and uncoupled

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(CCCP-stimulated) (C and D) respiration. Heart homogenates (1 mg tissue. mL-1) and MA (0.2-1 mM), PA (5 mM) or 3OHPA (5 mM) were added to the incubation medium in the

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beginning of the assays. Pyruvate (5 mM), malate (0.5 mM) plus glutamate (10 mM) (A

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and C) and succinate (10 mM) (B and D) were used as substrates. Controls were performed in the absence of organic acids. Values are means ± standard deviation for four to five

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independent experiments (N) expressed as pmol O2. s-1. mg of protein-1. One-way ANOVA

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is described in the text. *P < 0.05, **P < 0.01, ***P < 0.001 compared to controls (Tukey’s range test).

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Fig 8. Effects of maleic acid (MA) on respiratory parameters in permeabilized cardiac cells using the substrate-uncoupler inhibitor titration (SUIT) protocol. Digitonin (8

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µM) was used to permeabilize cells. State 3 (ADP-stimulated) (A and B) and uncoupled

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(CCCP-stimulated) (C and D) respiration. Cardiac cells (1.5 million cells . mL-1) and MA (5 mM) were added to the incubation medium in the beginning of the assays. Pyruvate (5 mM), malate (0.5 mM) and glutamate (10 mM) (A and C) or succinate (10 mM) (B and D) were used as substrates. Controls were performed in the absence of organic acids. Values are means ± standard deviation for five independent experiments (N) expressed as pmol O2. s-1. million cells-1. ***P < 0.001 compared to controls (Student’s t test for unpaired samples).

Fig 9. Effects of maleic (MA), propionic (PA) and 3-hydroxypropionic (3OHPA) acids on ATP production in heart mitochondria. Experiments were performed in an incubation medium containing heart mitochondrial preparations (0.1 mg protein. mL-1) supported by α30

Journal Pre-proof ketoglutarate (5 mM) (A) or pyruvate plus malate (2.5 mM each) (B). MA (1 or 2.5 mM), PA (5 mM) or 3OHPA (5 mM) were added in the beginning of the assays. Controls were performed in the absence of organic acids. Oligomycin A (Oligo, 1 μg. mL- 1) was used as a positive control. Values are means ± standard deviation for five to six independent experiments (animals) and were expressed as nmol ATP. min-1. mg-1. One-way ANOVA is described in the text. **P < 0.01, ***P < 0.001 compared to controls (Tukey’s range test).

Fig 10. Effects of maleic (MA), propionic (PA) and adipic (AA) acids on membrane

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potential in heart mitochondria. Assays were performed in a reaction medium containing mitochondrial preparations (0.35 mg protein. mL-1) supported by pyruvate plus malate (2.5

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mM each) (A, B and E) or α-ketoglutarate (5 mM) (C and D). Cyclosporin A (CsA, 1 M)

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plus ADP (300 M) were added in the beginning of the assays (panels B and D) and MA (0.1-5 mM), PA (5 mM) or AA (5 mM) 50 seconds afterwards, whereas EGTA (500 M) at

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250 seconds as indicated in panel E. All panels refer to mitochondrial preparations

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supplemented by 30 µM Ca2+, as indicated. Controls were performed in the absence of organic acids. CCCP (3 µM) was added at the end of the assays. Traces are representative

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of three to four independent experiments (N) and were expressed as fluorescence arbitrary units (FAU). One-way ANOVA is described in the text. *P < 0.05, **P < 0.01, ***P <

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0.001 compared to controls (Tukey’s range test).

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Fig 11. Effects of maleic (MA) and propionic (PA) acids on NAD(P)H content in heart mitochondria. Assays were performed in a reaction medium containing mitochondrial preparations (0.35 mg protein. mL-1) supported by pyruvate plus malate (2.5 mM each) (A) or α-ketoglutarate (5 mM) (B). MA (1-5 mM) or PA (5 mM) were added 50 seconds after the beginning of the assays. All panels refer to mitochondrial preparations supplemented by 30 µM Ca2+, as indicated. Controls were performed in the absence of organic acids. CCCP (3 µM) was added at the end of the assays. Traces are representative of four independent experiments (N) and were expressed as fluorescence arbitrary units (FAU). One-way ANOVA is described in the text. ***P < 0.001 compared to controls (Tukey’s range test).

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Journal Pre-proof Fig 12. Effects of maleic (MA) and propionic (PA) acids on swelling in heart mitochondria. Assays were performed in a reaction medium containing mitochondrial preparations (0.35 mg protein. mL-1) supported by α-ketoglutarate (5 mM). Cyclosporin A (CsA, 1 M) plus ADP (300 M) were added in the beginning of the assays, whereas MA (2.5 mM) or PA (5 mM) 50 seconds afterwards. All panels refer to mitochondrial preparations supplemented by 30 µM Ca2+, as indicated. Controls were performed in the absence of organic acids. Alamethicin (Ala, 40 μg/mg protein) was added at the end of the assays. Traces are representative of three to four independent experiments (N) and were

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expressed as fluorescence arbitrary units (FAU). One-way ANOVA is described in the text.

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*P < 0.05, **P < 0.01, ***P < 0.001 compared to controls (Tukey’s range test).

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Fig 13. Effects of maleic (MA) and propionic (PA) acids on Ca2+ retention capacity in heart mitochondria. Assays were performed in a reaction medium containing

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mitochondrial preparations (0.35 mg protein. mL-1) supported by pyruvate plus malate (2.5

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mM each) (A) or α-ketoglutarate (5 mM) (B). MA (5 mM) or PA (5 mM) were added in the beginning of the assays. All panels refer to mitochondrial preparations supplemented by

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successive additions of 5 µM Ca2+ every 2 min, as indicated by the arrows. Controls were performed in the absence of organic acids. CCCP (3 µM) was added at the end of the assays. Traces are representative of three independent experiments (N) and were expressed

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as fluorescence arbitrary units (FAU).

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Journal Pre-proof Declaration of interests X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Declarations of interest: none.

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Journal Pre-proof Highlights Maleic acid (MA) and propionic acid (PA) accumulate in propionic acidemic patients. MA and PA impair mitochondrial respiration in the heart. MA and PA disturb bioenergetics and calcium homeostasis in heart mitochondria.

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Mitochondrial dysfunction may contribute to the cardiomyopathy in these patients.

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