Energetics of heart mitochondria during acute phase of Trypanosoma cruzi infection in rats

ht.

Pergamon

J. Biochem.

Cell Bid. Vol. 27, No. I I, pp. 1183-l 189, 1995

Copyright 0 1995 Else&r Science Ltd Printed in Great Britain. All rights reserved 1357-2725195 $9.50 + 0.00

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Energetics of Heart Mitochmdria of Trypaaomna cruzi Infection in Rats Sf3RGIO A. UYEMURA,’

SERGIO

ALBUQUERQUE;

Acate Phstse CARLOS

CURT13*

’ Departamento de An&es Clinicas, Bromatokjgicas e Toxicoldgicas, ‘Departamento de Cihtcias da Satide, and ‘Departamento de Fisica e Quimica, Faculdade de Cihcias Farmacthticas-USP, Au. CaSp s/No, 14040-903 Ribeirdo Preto, S.P., Brazil The energetica of heart mitochondria was studii in the acute phase of Trypunosoma cruti infection in rats. Wiiar rata were infected with 2 X 105 trypomaat@te forms of the Y strain of T. crrrrj, and heart mitochombia and submitochondrial particka isdated after 7 and 25 days of infection. Uitrastructure of mitochondria seemed to be preserved, but cytochrome c Ieveis were signiticantly depressed. Respiratory control ratios (RCR) were decreased for gktamate and succhtate oxidations, as a consequence of hthiiition of respiration in state 3 and/or of sthurdation of respiration in state 4. SthnuIation of hydrolytic activity of FoFl-ATPase by energiaation of mitochondria was approx. 2-fold higher in relation to controls. Mitocbondriai ATP conceWation remained constant. In con&shut, during the acute phase of T. cruzi infection in rats there is an energy hnpairment at the level of heart mitochondria, but their tdtr a&nctnre and ATP concentration seem to be preserved, the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoFl-ATPase. Keywords: phorylation

Trypumosomu crrrzi Chagasic cardiomyopathy FoFl-ATPase ATP

Mitochondria

Oxidative

phos-

Znt. J. Biochem. Cell Biol. (1995) 27, 1183-l 189

INTRODUCTION

Chagasic cardiomyopathy is the most severe and frequent manifestation of Chagas’ disease, a pathological process induced by human infection with Trypanosoma cruzi (Chagas, 1909; Cancado and Chuster, 1985). In spite of the well accepted neurogenic and autoimmune hypothesis, the pathogenesis of chagasic cardiomyopathy is not well understood (Kiiberle, 1968; Teixeira, 1980; Oliveira, 1985; Rossi and Santana, 1990; Junqueira et al., 1992). More recently, the microcirculation of the heart has been postulated to play a significant role in this process via a mechanism of ischemiareperfusion (Rossi and Carobrez, 1985; Rossi, 1990). The energetic state of the myocardium is mainly supported by oxidative phosphorylation, *To whom all correspondence should Received 3 November 1994; accepted

be addressed. 12 June 1995. 1183

in which FoFl-ATPase (mitochondrial ATP synthase) synthesizes ATP coupled to the respiratory electron flow in the inner membrane of mitochondria (for reviews see Boyer et al., 1977; Brown, 1992). Under physiological conditions the heart is fully aerobic and oxidative phosphorylation provides over 90% of the cellular energy required for contractile function (for a review see Harris and Das, 1991). Ischemiareperfusion and related pathological situations affect the energetics of heart mitochondria, impairing the synthesis of ATP and stimulating hydrolytic activity of FoF 1-ATPase (Jennings and Ganote, 1976; Trump et al., 1976; Jennings et al., 1978; Rouslin, 1983). In previous studies by our group on the during isoenergetics of mitochondria proterenol-induced cell injury of the myocardium we reported impairment of respiration and alterations in the kinetic properties of FoFlATPase (Curti et al., 1990; Uyemura and Curti, 1991). The drug-induced process is similar in

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several aspects to that occurring in chagasic cardiomyopathy. Thus, we have standardized experimental T. cruzi infection of rats to study the energetics of heart mitochondria in the acute phase of the disease. Structural and functional aspects were analyzed following 7 and 25 days of infection, which coincided with the parasitemic peak and the disappearance of circulating parasites from peripheral blood, respectively. Energy impairment at the heart mitochondria level may be involved in the acute phase of chagasic cardiomyopathy. MATERIALS

Standardization

AND

METHODS

of T. cruzi infection in rats

Male Wistar rats weighing approx. 100 g were inoculated i.p. with 2 x lo5 trypomastigote forms of the Y strain of T. cruzi obtained from blood of infected mice. Circulating parasites in peripheral blood were detected by microscopic examination (Brener, 1962). After 7 (ch-7) or 25 (ch-25) days, the infected rats (groups of 6) were killed by cervical dislocation to cause immediate death, and the ventricles were rapidly excised. Control groups were non-infected rats, maintained under the same conditions. Electrocardiograms (ECG) were recorded with a FUNBEC electrocardiograph (Model ECG-3), according to Bestetti et al. (1987); histopathological analysis was performed according to Junqueira et al. (1992). For electron microscopy, tissue was fixed with glutaraldehyde and OsO,, and ultrathin sections were stained with uranyl acetate and lead citrate (Chan et al., 1970). Isolation of cardiac mitochondria submitochondrial particles

and coupled

Mitochondria (M, Mch-7, Mch-25) and submitochondrial particles were isolated as described previously (Uyemura and Curti, 1992), with slight modifications. The ventricles were homogenized for 10 set in 250 mM sucrose, 2 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.2 (homogenization medium), using a high-speed shearing homogenizer (5 ml medium/heart). The homogenate was centrifuged at 770g for 5 min and the supernatant obtained was centrifuged at 9750g for 10 min. The pellet was suspended in EDTA-free homogenization medium to the original volume and centrifuged at 9750g for 10 min. This step was repeated once and the final pellet was suspended

to a concentration of 20 mg of mitochondrial protein/ml. Giemsa staining was used to determine the absence of parasites in the mitochondrial preparation, which was used for the experiments with energized mitochondria within one hour. To isolate submitochondrial particles, the final pellet of mitochondria was frozen and stored at - 20°C. After 48 hr, it was thawed and suspended in the homogenization medium to a concentration of 20 mg protein/ml, and supplemented with 1 mM glutamate, 1 mM ATP and 10 mM MgCl, . The mitochondrial suspension was sonicated 5 times for 10 set at 30 set intervals, using 80 W. The suspension was centrifuged at 9750g for 10 min and the submitochondrial particles in the supernatant were isolated by centrifugation in a Sorval SV-80 vertical rotor for 1 hr at 15,000 r.p.m., using a discontinuous gradient containing 1 ml of 0.5 M sucrose and 1 ml of 2.0 M sucrose in 5 mM Tris-HCl, pH 7.4. All steps for mitochondrial and submitochondrial isolation were performed at 4°C. All solutions were prepared with glassdistilled deionized water. Mitochondrial protein was determined according to Murphy and Kies (1960), using bovine serum albumin (BSA) as a standard. Oxygen consumption assays

Oxygen consumption was analyzed polarographically at 30°C with an oxygraph equipped with a Clark oxygen electrode (Gilson Medical Electronics, U.S.A.), and the respiratory parameters were determined as previously described (Chance and Willians, 1956). Oxidizable substrates were either 7.5 mM glutamate or 10 mM succinate/5 PM rotenone in 1.5 ml of medium containing 250 mM sucrose, 100 mM KCl, 1 mM EDTA, 1 mg BSA/ml and 10 mM potassium phosphate in 10 mM Tris-HCl, pH 7.4. The amount of protein in each test was 2 mg, and state 3 respiration was initiated by the addition of MgADP in the range of 30&600 nmol. Determination

of cytochrome content

Mitochondria in 20 mM potassium phosphate, pH 7.4, were solubilized with Triton X100 (2% final concentration) to a final concentration of 2 mg of mitochondrial protein/ml. The difference between the spectra of dithionite-reduced and ferricyanide-oxidized cytochromes were recorded with a Beckman spectrophotometer Model DU-70. Cytochrome

Heart mitochondria

concentrations were calculated described (Williams Jr, 1964).

energetics in chagasic rats

as previously

NADH oxidation NADH oxidation by coupled submitochondrial particles was monitored at 340 nm, at 30°C. The reaction was started by the addition of particles (200 pg of protein) to a medium containing 250 mM sucrose and 0.2 mM NADH in 50 mM Tris-HCl, pH 7.4 (final volume of 1 ml). Mitochondrial ATP determination Mitochondrial ATP levels were monitored using the luciferin-luciferase system (1243- 102 ATP monitoring kit Bio-Orbit) in a Bio-Orbit Luminometer Model 1250. Mitochondria were used in the range of 0.2-2.0mg of protein. A TPase assays Mitochondrial ATPase activity was determined by assaying phosphate liberated by MgATP hydrolysis, using the method of Heinonen and Lahti (1981). The energization medium contained 100mM sucrose, 80 mM KC1 and 5 mM succinate/5 PM rotenone in 50 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) -KOH, pH 7.4. Mitochondria (0.6 mg of protein) were incubated with the energization medium, and the enzymatic reaction was started at 37°C by the addition of 3 mM MgATP plus 2 ,uM carbony1 cyanide p-trifluorometoxyphenylhydrazone (FCCP) in order to block further energization (final volume of 1 ml). Hydrolysis was allowed to proceed for 5 min and stopped with 0.5 ml of 30% (w/v) trichloroacetic acid (TCA). Statistical analysis The Kruskal-Wallis or Mann-Whitney test was used for statistical evaluation of mean values for experimental and control animals. The levels of significance were taken as P < 0.05 or P cO.01.

RESULTS

Standardization of T. cruzi infection in rats The circulating parasites in peripheral blood attained maximum numbers (about 40 x lo6 forms) 7 days after inoculation of the rats with 2 x lo5 trypomastigote forms of the Y strain of T. cruzi, and were completely suppressed as

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early as 15 days after infection. The death rate was approx. 50% for ch-25. Compared to the controls, 71.9% of ch-7 and 64.9% of the surviving ch-25 showed at least one alteration in the electrocardiographic parameters (Table 1). These values were lower when less than 2 x lo5 forms were used for the infection, and did not change significantly with the use of 2.5 x lo5 forms. Histopathological analysis of heart tissue showed an intense parasitism of the myofibers of group ch-7, in association with foci of granulocytic inflammatory infiltrates and presence of mononuclear cells. A prominent reduction in the parasitism of cells was observed for ch-25; the foci of inflammatory infiltrate showed predominantly mononuclear cells, and occasional areas of myocardial necrosis were observed (not shown). Structural and functional studies Electron microscopic analysis of Mch-7 and Mch-25 did not reveal appreciable alterations, except for a low degree of swelling. The protein profile of mitochondria and submitochondrial particles, analyzed by polyacrylamide gel electrophoresis in the presence of SDS, was also unchanged (not shown). The respiratory parameters of mitochondria are presented in Fig. 1. The rate of state 3 respiration in Mch-25 decreased significantly both for glutamate and succinate oxidations, and only for glutamate oxidation in Mch-7. Although not statistically significant, stimulation

Table 1. Changes in the electrocardiographic parameters of Trypanosoma cruzi-infected rats ECG parameters ch-7 (%) ch-25 (“0) No changes ICD LAD and ICD ICD and pathological Q Wave IAD Abnormal J point LAD IA RAD, ICD and IAD LAD, ICD and abnormal J point 64

28.1 21.9 12.5 15.7 3.1 3.1 12.5 3.1 0 0

35.1 11.8 11.8 5.9 11.8 0 r.9 0 1.9 I I.8

ch-7 Trypanosoma cruzi-infected rats, and the 34 ch-25 surviving from the previous stage were used to determine the relative percentage of changes in electrocardiographic parameters. Control groups, consisting of a similar number of non-infected animals, showed no ECG changes. The conditions of inoculation and ECG records are described in “Materials and Methods”. LAD = left axis deviation; RAD = right axis deviation; IA = indeterminate axis; ICD = intraventricular conduction delay; IAD = intraatrial conduction delay.

Sergio A. Uyemura ef al.

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(A)

(B)

Glutamate

Succinate

Fig. 1. Respiratory parameters of heart mitochondria (2.0 mg of protein) energized with 7.5 mM glutamate (A) or 10 mM succinate/5 PM rotenone (B) at 30°C. The medium (final volume of 1.5 ml) contained 250 mM sucrose, 100 mM KCl, 1 mM EDTA, 1 mg BSA/ml and IOmM potassium phosphate in 10 mM Tri-HCl, pH 7.4. State 3 respiration was initiated by the addition of MgADP in the range of 30@-600nmo1. Data are means & SEM of five experiments with different preparations. *P < 0.05, **P < 0.01 vs respective controls. Rates are expressed in mg of protein.

observed in the respiration of state 4. Thus, the decrease of RCR values (respiratory control ratio) was a consequence of inhibition of respiration in state 3 and/or of stimulation of respiration in state 4. The ADP: 0 ratios, which were close to 3.0 and 2.0 respectively for glutamate and succinate oxidations in the controls, did not change significantly (data not shown). The contents of mitochondrial cytochrome c and ATP, as well as the rates of NADH oxidation by coupled submitochondrial particles are presented in Table 2. A progressive depression in the cytochrome c levels of Mch-7 and Mch-25 was observed, but there were no significant alterations in content of cytochromes a, b or c, (not shown). The rate of NADH

oxidation by coupled submitochondrial particles of Mch-7, but not of Mch-25, was significantly inhibited. The mitochondrial ATP levels of Mch-7 and Mch-25 remained constant. Fresh mitochondria were energized with succinate, and the activity state of FoFl-ATPase was evaluated at various time intervals by assaying its hydrolytic activity (Fig. 2). Enzyme stimulation of control mitochondria was 20% after 2 min of energization, while stimulation of about 50% was observed after 4 min of energization of Mch-7, and stimulation of about 60% was observed after 5 min of energization of Mch-25. 2.0 1.5

M r

$ 1.0 L-7 $ 2.0 r

Mch-7

were

I

I

I

0

2.5

5.0

Time of energization (min) Fig. 2. Influence of energization time on MgATP hydrolysis by FoFl-ATPase of mitochondria in the presence of 5 mM succinate/5 PM rotenone in a medium containing 100 mM sucrose, 80 mM KC1 and 50 mM Hepes-KOH, pH 7.4 (final volume of 1 ml) at 37°C. Energization was started by the addition of mitochondria (0.6mg of protein), and the enzymatic reaction by addition of 3 mM MgATP plus 2 PM FCCP. Data are means k SEM of three experiments with different preparations. Non-stimulated ATPase activities were 0.36, 0.31 and 0.27pmol phosphate/min/mg protein for M (M-7 and M-25), Mch-7 and Mch-25, respectively.

Table 2. Mitochondrial parameters in Trypanosoma cruzi-infected rats Cytochrome c ATP NADH* (nmol/mg protein) (nmol/mg protein) (nmol/min/mg protein) 0.335 + 0.048 0.47 f 0.07 167 + 0.008 M-7 0.253 + 0.05St 0.44 + 0.07 73 & 0.0143 Mch-7 M-25 0.335 + 0.036 0.68 + 0.02 193 + 0.002 Mch-25 0.220 Io.099t 0.55 + 0.02 196 z 0.002 Values are means & SEM of three determinations with different preparations. *NADH oxidation by coupled submitochondrial particles. tP < 0.05, $P < 0.01 vs respective controls.

Heart mitochondria

energetics in chagasic rats

DlSCUSSlON

The infection of Wistar rats with the Y strain of T. cruzi causes irregular injury to the myocardium associated with intense myocarditis (Andrade and Andrade, 1979). Also, it is known that changes in electrocardiographic parameters are associated with Chagas’ heart disease in rats (Meira de Oliveira et al., 1986; Bestetti et al., 1987). In our studies, electrocardiographic, parasitological and histopathological analyses were used to characterize the T. cruzi infection. Our results and data from the literature indicate that infection with 2 x lo5 forms of the Y strain of T. cruzi is an appropriate model. In addition, the parasitological and histopathological features of ch-7 and ch-25 resemble those of the acute and initial chronic process respectively (Milei et al., 1992; Rossi and Mengel, 1992). We propose ch-7 and ch-25 as an early and a late stage in the acute phase of T. cruzi infection in rats, respectively. Several pathophysiological situations such as ischemia-reperfusion and other conditions of oxygen depletion affect the mitochondria, impairing the production of energy (for review see Piper et al., 1994). During the acute phase of T. cruzi infection in rats, the ultrastructure of heart mitochondria seems to be preserved, as indicated by the absence of damage such as membrane rupture and calcium overload. The profile of mitochondrial protein was qualitatively maintained. However, the content of cytochrome c, which is only loosely bound to the inner membrane of mitochondria, was significantly reduced. This finding was also reported for mitochondria isolated from ischemic tissue, and has been ascribed to a progressive loss or denaturation of this component (Piper et al., 1985, 1994). RCR values provide a measure of mitochondrial function by showing how the oxidation of respiratory substrates proceed in close association with ATP production (Toth et al., 1990). Therefore, a decrease in this index indicates that the main energy source of the myocardium was impaired during the acute phase of infection; this mitochondrial dysfunction might be an early event in the course of chagasic cardiomyopathy. The effect is interesting in that it impairs mitochondrial function affecting RCR values, but it is not critical to ADP:O ratios, another index reflecting the efficiency of mitochondrial ATP production. Impairment of mitochondrial function is also indicated by the inhibition of NADH oxidation by coupled submitochondrial

II87

particles observed in the early stage of the acute phase of infection, but reversed during the late stage. It has been reported that NADH dehydrogenase of the respiratory chain is early and reversibly impaired in the hypoxic and ischemic myocardium (Piper et al., 1985, 1994). FoFl -ATPase reversibly catalyses the synthesis and hydrolysis of ATP in mitochondria. Under normal respiratory conditions, the proton electrochemical gradient generated by the electron flux in the inner membrane of mitochondria is used to drive the synthesis of ATP by the enzyme (for a review see Hatefi, 1985). In &hernia and related pathological situations, when the electron flux is low, FoFl-ATPase catalyses net hydrolysis of ATP, requiring a regulatory mechanism to minimize ATP depletion (Harris and Das, 1991; Piper et al., 1994). In vitro, FoFl-ATPase shows a reversible activity transition induced by the energization of mitochondria, which is ascribed to dissociation of a natural inhibitor protein (Tuena de Gomez-Puyou et al., 1980). Thus, association of this inhibitor with the enzyme seems to be favored in pathology with energetic implications, and the degree of association may presumably be assessed by determining how ATPase activity is stimulated by the energization of mitochondria. It is significant that the enzyme stimulation of Mch-7 and Mch-25 was higher than in controls; possibly denoting a more effective interaction of the inhibitor with the enzyme in order to preserve ATP levels under conditions of impaired mitochondrial function. Indeed, in spite of an unfavorable energetic situation, mitochondrial ATP levels were not significantly depressed in the early or the late stage of acute infection. This is important since ATP is essential for cell survival, and factors affecting its level can lead to lethal injury to the cell (Jennings et al., 1978). In conclusion, during the acute phase of T. cruzi infection in rats there is an energy impairment at the level of heart mitochondria, but their ultrastructure and ATP concentration seem to be preserved; the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoFl-ATPase. In addition, some similarities with the mitochondrial findings associated with ischemia suggest that a similar mechanism may be operating in the infected animals. Studies are now in progress to investigate other aspects of mitochondrial function

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Sergio A. Uyemura et al.

during this phase, as well as during the chronic phase of T. cruzi infection. Acknowledgements-Supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paul0 (Grant 92/2916-4), and Conselho National de Desenvolvimento Cientifico e Tecnologic0 (Grant 300403/86-O and 400075/93-8). The authors thank Dr Sergio B. Garcia, Mirian P. A. Toldo, Lucimara Z. Pinto and Ana C. M. Polizello for technical support, and Dr Ana I. Assis Pandochi and Dr Zuleika Rothschild for reviewing the manuscript. REFERENCES

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