Expression of the ADP/ATP Carrier and Expansion of the Mitochondrial (ATP + ADP) Pool Contribute to Postnatal Maturation of the Rat Heart

Eur. J. Biochem. 241, 895-900 (1996) 0 FEBS 1996

Expression of the ADP/ATP carrier and expansion of the mitochondrial (ATP + ADP) pool contribute to postnatal maturation of the rat heart Peter SCHONFELD I. Lorenz SCHILD' and Ralf BOHNENSACK'

' Institut of Biochemistry, Otto-von-Guericke-Universitat Magdeburg, Germany ' Institut of Clinical Chemistry, Department Pathological Biochemistry, Otto-von-Guericke-Universitat Magdeburg, Germany

(Received 8 August 1996) - EJB 96 1187/6

The role of the ADP/ATP carrier (AAC), a key protein of the mitochondrial ATP-generating system, is not clear during postnatal rat heart development. To elucidate this role, the phosphorylating respiration (state 3), the activity and the content of AAC, the size of the exchangeable mitochondrial (ATP + ADP) pool and the control of AAC over respiration at state 3 were measured in mitochondria isolated from rat hearts at various postnatal ages.

There was a 5-fold increase in the AAC activity from newborn to aged rat hearts, which was paralleled by a 1 .S-fold increase in state 3 respiration. At birth, the AAC and the F,F,-ATP synthase exerted about 80 % of the control over phosphorylating respiration (state 3 : flux control coefficients 0.39 -C 0.04 and 0.38 t 0.08). The strong increase in the AAC activity was partly caused by the doubling of the protein content. In addition, the turnover number of AAC increased by a factor of 2.5 due to the expansion of the (ATP + ADP) pool from 3.420.9 to 10.62 1.5 nmol . mg protein-'. The data strongly indicate that the increase in the AAC activity is an essential step in the postnatal maturation of rat heart mitochondria.

Keywords: ADP/ATP carrier; adenine nucleotide ; F,F, -ATP synthase; heart mitochondria; maturation.

At birth the mammalian heart is functionally not full devel- oped. Quantitative ultrastructural analysis of the neonatal myo- cardium revealed a low number of small-sized mitochondria, a sparse sarcoplasmatic reticulum, less myofibrillar protein, and the absence of t-tubules compared with adults [l-41. During perinatal development there is an increase in the contractility and a shift from glycolytic to oxidative metabolism in heart muscle [5]. For liver it has been demonstrated that the energy-linked functions of mitochondria in neonatal tissue are in an unmatured state (for reviews see [6, 7]), as indicated by a low oxidative phosphorylation efficiency, a low rate of phosphorylating respiration (state 3) and a high passive proton permeability of the inner mitochondrial membrane [S]. During the postnatal maturation the concentration of matrix adenine nucleotides increased due to a net uptake of adenine nucleotides from the cytosol and the expression of key proteins of the oxidative phosphorylation is stimulated [6, 71. One of the key proteins is the ADP/ATP carrier (abbreviated here as AAC), a protein (=30 kDa) intrinsic to the inner mitochondrial membrane catalyzing the 1:1 exchange of cytosolic ADP and matrix ATP in the process of oxi-

Correspondence to P. Schonfeld, Institut of Biochemistry of the Otto-von-Guericke-Universitat Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany

F a : f 4 9 391 67 15898. E-mail: [email protected] Abbreviations. AAC, ADP/ATP carrier; RCR, respiratory control

ratio. Enzymes. F,,F,-ATP synthase (EC 3.6.1.34); citrate synthase (EC

4.1.3.7); lactate dehydrogenase (EC 1.1 .I .27); pyruvate dehydrogenase (EC 1.2.4.1); creatine kinase (EC 2.7.3.2).

dative phosphorylation [9]. AAC has a low turnover number [lo], so its enrichment in the inner mitochondrial membrane is essential to fulfill the increased ATP demand of the postnatal tissue.

Earlier studies with mitochondria from liver and brain revealed, that the AAC has significant control over phosphorylating respiration in the newborn state [ll-131. The role of the adenine nucleotide translocation in the cellular energy metabolism during perinatal heart development is not clear. Therefore, we studied the changes in the AAC activity with rat heart mitochondria at different postnatal ages and its interrelationship to the expression pattern of the AAC protein, the pool of the exchangeable mitochondrial adenine nucleotides and the control of the AAC over phosphorylating respiration (state 3).

MATERIALS AND METHODS Chemicals. [2,3-3H]ADP was obtained from NEN-DuPont.

Atractyloside, carboxyatractyloside, oligomycin A, pyruvate, malate and ADP were from Sigma. Protein assay reagent (BCA) was from Pearce. Goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase were from Dianova GmbH. ECLTM western blotting kit was from Amersham Life Science.

Biological material. Heart mitochondria were prepared from fetal, newborn, postnatal and adult Wistar rats. Adult females (mean mass 18Og) were caged with males overnight. Fetuses were obtained from pregnant anaesthetized rats by cesarean sec- tion. The body mass of the fetuses was used to control their age [14]. Fetuses and puppies used in these studies were of mixed

896

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Schonfeld et al. ( E m J. Biochem. 241)

'c E 600 - e n .-

p

v E

,E 400 -

0 - 0

c 200

e A

- .- * .- cl

ri? o u t - - J o -5 0 5 10 120

Age (days)

Fig. 1. Developmental change of state 3 respiration and respiratory control ratio. Rat heart mitochondria of various perinatal ages were incubated as described in Materials and Methods in the presence of S mM pyruvate/S mM malate. Mitochondria from adult rat hearts are designated 120. State 3 respiration was started b,y addition of 0.5 mM ADP. Respiratory control ratios (A) were calculated from state 3 respiration and the rate of respiration (A) measured after phosphorylation of added ADP. Values are given as mean ? SD of four or five experiments obtained with separate mitochondrial preparations. Where no error bars are shown, the SD falls within the size of the symbols.

sex. After decapitation, the heart was immediately dissected, put in trypsin-containing medium, stirred for 20 min, and finally ho- mogenized by an Ultra Turrax homogenizer (twice for 20 s). Mitochondria were prepared from homogenate by standard differential centrifugation procedure [ 151. The protein content in the mitochondrial stock suspension was determined by the biuret method or with the bicinchoninic acid assay (BCA) in the presence of 0.2 % sodium dodecyl sulfate [16]. The purity of mitochondria was similar in all preparations as indicated by the activity ratio of citrate synthaseAactate dehydrogenase, the former as a mitochondria1 marker enzyme and the latter as a marker for cytosolic contamination (newborn state: ratio = 14?6, n = 6; adult state: ratio = 27 5 11, n = 6).

Oxygen uptake and flux control coefficient. State 3 respiration and the respiratory control ratio (RCR) were measured polarographically in a water-jacketed chamber maintained at 30°C. The medium contained 110 mM mannitol, 60 mM KCl, 60 mM Tris, 10 mM potassium phosphate, 5 mM pyruvate, 5 mM malate and 0.5 mM NaZEDTA, pH 7.4. The flux control coefficient of the adenine nucleotide translocase (C$Ac) at state 3 respiration was estimated by measuring the effects on the mitochondrial oxygen uptake produced by successive additions of carboxyatractyloside 1171. Titration of F,,F,-ATP synthase was performed by a 4-min incubation of the mitochondrial suspension with increasing concentrations of oligomycin A before state 3 respiration was adjusted by addition of ADP. From the obtained sigmoidal inhibitor-titration curves the flux control coefficients (C$,4,., CJ,u, ,, ,,n,,,d\c) were estimated by non-linear regression as described in 1181.

ADPIATP carrier activity and (ATP+ADP) pool size. The activity of the adenine nucleotide translocase was measured at 0°C using the atractyloside-inhibitor stop technique 1191. The forward exchange reaction was initiated by addition of ['HIADP (specific activity 2300 dpmhmol ADP) to 0.1 mM. From the uptake of ['HH]ADP by mitochondria during a 20-s incubation the translocation activity was calculated. Nonspecific binding of ['HIADP to mitochondria was estimated by incubation of mitochondrial samples with 0.3 mM atractyloside prior addition of

A h -

0 500 1000 1500 2000

Carboxyatractyloside (pmolmg protein")

0.4

0.2

00

F,F,-ATPsynthase

AAC-

I

-5 0 5 10 I20

Age (days)

Fig. 2. Control of the adenine nucleotide translocase over state 3 respiration. Mitochondria were incubated as in Fig. 1. To adjust stationary state 3 respiration, the standard incubation medium was supplemented with 5 mM ADP. (A) Representative titration experiments with carboxyatractyloside. For titration of the ADP/ATP carrier, incremental amounts of carboxyatractyloside were added and changes in respiration wa5 re- corded with a rate meter. For titration of the F,,F,-ATP synthase, mitochondria were first incubated in separate experiments with oligomycin (4 min) and statc 3 respiration was initiated thereafter by addition of 5 mM ADP. (B) Developmental changes of the flux control coefficients of AAC or F,]F,-ATP synthase. The values given are the mean ? SD of two-four separate mitochondrial preparations.

['HIADP. The size of the exchangeable adenine nucleotide pool (ATP + ADP) was estimated from the uptake of [.'H]ADP after a 2-min incubation period.

Quantification of the ADP/ATP carrier. The content of the AAC in isolated mitochondria was measured by an assay based on the high-affinity binding of ['Hlatractyloside (specific activity 220 d p d n m o l ) to the AAC as described in 1201. In order to correct the data for adherent ['Hlatractyloside in the pellct, each incubation was repeated in the presence of 0.1 mM atractyloside. This blank reactivity was subtracted from the total radioactivity measured in absence of atractyloside.

SDSPAGE, western blotting and immunostaining. Before solubilizing the rat heart mitochondria by detergent, they were loaded for 10 min with 10 pM carboxyatractyloside at 0°C to protect them against proteolysis 121 1. The loaded mitochondria were then pelleted by centrifugation and the pellet was solubilized in sample buffer (60.6 g Tris, 7.7 g dithiothreitol, 4 g SDS,

Schiinfeld et al. (Eur .I. Biochem. 241) 897

Z I C + 4 A

al X

I n a $ 3 0

0 0 20 40 60 80 100 120

Time (s)

Fig. 3. Developmental changes of the activity of adenine nucleotide translocation. The ['HIADP uptake by mitochondria was measured as described in Materials and Methods using the standard incubation medium. The translocation activities were calculated from the [ 'HIADP uptake measured after 10 s of incubation. Developmental changes in translocation activity are shown in the insert. The data represent the mean value -C SD of 3-6 separate mitochondria1 preparations.

0.1 g NaN,, 0.2 g bromophenol blue/l; pH 6.8) supplemented with 1 mM phenylmethylsulfonyl fluoride as protease inhibitor. Electrophoresis was performed on a continuous 11 % polyacrylamide slab gel with Tridglycine running buffer (3.025 g Tris, 14.4 g glycine and 1 g SDW pH 7.4). The stacking gel contained 3 % polyacrylamide. The lanes were loaded with 2O-pl aliquots of solubilized mitochondria. After separation, the protein bands were visualized by Coomassie blue staining or trans- ferred to nitrocellulose sheet by electroblotting as described in the manufacturer's protocol (Gibco). The nitrocellulose sheet was blocked with a bovine serum albumin buffer (SO mM Tris/ HCI, 200 mM NaCI, 4 O/o BSA, pH 7.4) at room temperature for 2 h. Then, the sheet was washed four times with medium containing 50 mM Tris/HCI, 200 mM NaC1, 0.1 % BSA, 0.05 % Tween 20, pH 7.4. The immunostaining was performed with polyclonal rabbit anti-AAC antisera raised against the AAC protein of bovine heart (a generous gift of Dr Schagger, Universitat- sklinikum, Frankfurt/Main, Germany, and Dr H. A. C. M. Bentlage, University Hospital, Nijmegen, The Netherlands) by incubating the nitrocellulose sheets with antisera diluted in 0.01 M sodium phosphate, 0.25 M NaCl, pH 7.4; 1 :20000) at room temperature for 1 h. The blots were incubated with a 1 : 1000 dilution of the F(ab'), fragment of goat anti-rabbit IgG ( H f L) conjugated to horseradish peroxidase, washed, incubated with an ECL kit (Amersham) and exposed to Agfa curix films.

RESULTS

Respiration and energy coupling. Using the isolation procedure described in Materials and Methods, we obtained suspen- sions of rat heart mitochondria with high functional integrity from adults and also from newborns. As shown in Fig. 1 , the respiratory control ratios (calculated from the rate of respiration induced by ADP and after complete phosphorylation to ATP) were between 4- 9 depending on the developmental stage. The immature state of oxidative phosphorylation in rat heart mito-

g20Lt- 0 0 5 10 120

Age (days)

Fig.& SDSPAGE and western blotting analysis of rat heart mitochondria from various postnatal ages. Lysates of rat heart mitochondria (20 pg) obtained from newborns (lane I), I-, 4- and 11-days old neonates (lane 2,3,4), adult rats (lane 5 ) and a crude extract of AAC protein (lane 6) were subjected to SDYPAGE. AAC protein extracts were prepared from adult rat heart mitochondria (dissolved in medium containing Triton X-100) based on the hydroxyapatite separation procedure essentially as described in [22]. Top: Coomassie-blue-stained gel ; middle, gel transfei-red to nitrocellulose membranes and probed with polyclonal anti-AAC protein antibodies ; bottom, quantification by laser densitometric scanning (Pharmacia LKB UltroScan XL) of the Coomas- sie-blue-stained bands (*) or immunoreactive signals (M). The peak areas were integrated using the GSXL densitometer software. The data are the mean value 2 SD fo three or four mitochondrial preparations.

chondria isolated from newborn puppies was reflected by the RCR, which was only 50% of the adult value (Fig. 1). More- over, postnatal maturation of rat heart mitochondria resulted i n a 1.5-fold enhancement of state 3 respiration (Fig. 1). The maximal uncoupled respiration [obtained by stepwise addition of car- bonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) at state 3) was always 10-20% above state 3 respiration (data not shown) indicating that the phosphorylating system limits the activity of the respiratory chain in state 3. Nearly full capacity of state 3 respiration and maximal respiratory control ratios wcre found at the end of the first week of extrauterine live.

Control over respiration. A significant inhibition of state 3 respiration by titrating the AAC with the irreversible inhibitor carboxyatractyloside was observed in mitochondria from newborn rats (Fig. 2A). The flux control coefficients were estimated from the titration curves. The results suggest an important control of the AAC over respiration in the newborn state (C$Ac = 0.39*0.04; Fig. 2B). The control of the F,,F,-ATP synthase over state 3 respiration was also estimated. For this purpose, state 3 respiration was titrated with oligomycin. As shown in Fig. 2B, the F,F,-ATP synthase exerted a similar control over state 3 respiration in the newborn heart (C~~,.Prvn,,r.,,e - -

898 Schonfeld et al. ( E m J. Biochem. 241)

- - :s 1.0 0 g 0.0

- 0 E S 0.6

B 8 0.4 0

..4

2 0.2

-4 6; (ATP + ADP)

-5 0 5 10 120

Age (days)

Fig. 5. Developmental changes of adenine nucleotide translocase and the exchangeable (ATP+ADP) pool. The content of the AAC protein in rat heart mitochondria was measured by means of high-affinity binding of ['H]atractyloside to the AAC as described in Materials and Meth- ods. The size of the exchangeable (ATP + ADP) pool was estimated from the uptake of ['HIADP in rat heart mitochondria during a 120-s incubation period. The data are the mean value-CSD of 3-6 separate preparations

0.8 - ,- fn v 3 0.6

5 0.4 z a

Fig. 6. Turnover number of the adenine nucleotide translocase as function of the exchangeable adenine nucleotide pool size. The data were from Figs 3 and 5.

0.38 f 0.08) as the AAC did. Both proteins control about 80 % oxidative phosphorylation in the newborn rat heart. The control of both proteins ceases during mitochondrial maturation (CkAr = 0.03t0.01 and CAvPc y,,,,, d,c = 0.16?0.07 for adult).

Translocation activity. To compare the developmental change in the control of the AAC over state 3 respiration with that of the AAC activity, we measured the rate of ['HIADP uptake by mitochondria. Experiments with mitochondria from 1 -day-old, 4-day-old and adult rat heart showed that during a 10-s incubation the rate of [XJADP uptake was approximatelly linear (Fig. 3). The insert clearly shows that the decline in the control of AAC over state 3 respiration was paralleled by an increase (5-fold) in the translocation activity from birth to adult.

AAC expression and pool size. To gain deeper insight into the developmental changes of AAC activity, the enrichment of the AAC in mitochondria as well as the expansion of the exchangeable mitochondrial adenine nucleotide pool (ATP + ADP) were studied. Fig. 4 shows the developmental increase of the AAC in mitochondria as indicated by Coomassie blue staining and immunoreactivity after electrophoretic separation of the proteins

in mitochondrial lysates. The AAC protein band was identified within the protein patterns by applying the isolated AAC protein to electrophoresis (lane 6). In newborns the AAC is one of the main mitochondrial proteins (Fig. 4, top). There is a similar increase in the protein content and in the immunoreactivity of AAC during development (Fig. 4, bottom). For quantification of the AAC protein content we applied the ['H]atractyloside binding assay [20]. Fig. 5 shows that there was a doubling of the AAC in rat heart mitochondria from birth to adult. During the same developmental period the exchangeable adenine nucleotide pool size increased threefold. The role of the developmental change in the (ATP + ADP) pool size for the postnatal increase in the translocation activity is clearly seen when the turnover number (mol ADP exchanged/mol AAC) was plotted versus the (ATP + ADP) pool size (Fig. 6). As can be seen, a linear rela- tionship exits between the turnover number and the (ATP + ADP) pool size.

DISCUSSION The postnatal maturation of oxidative phosphorylation is an

important cellular event to circumvent the insufficient mitochondria] ATP supply found in the tissues of newborn mammals. In the present study, maturation of oxidative phosphorylation in rat heart mitochondria can be clearly seen from the improvement in the coupling efficiency between mitochondrial electron trans- port and phosphorylation (RCR in Fig. 1) and the control of the AAC over respiration (C&.c in Fig. 2B) during development from birth to adult. Both parameters are independent of the protein content, so that the observed changes are not due to alter- ations in the content of protein contaminations in the mitochondrial preparation. In contrast to liver mitochondria [I 2, 23 - 251, the maturation process of oxidative phosphorylation pro- ceeded much more slowly in rat heart mitochondria. So, full respiratory control is achieved at the end of the first extrauterine week, whereas the same process is finished in liver mitochondria already a few hours after birth.

The development of mitochondrial energy metabolism has been studied mostly with mitochondria from rat and rabbit heart. Both mammals belong in the group of so-called non-precocial animals 1271, for which a lack i n visual perception and co-ordi- nated motor activity was found up to the end of the second postnatal week. Therefore, it is a surprising finding that the oxidative phosphorylation in rabbit heart mitochondria is completely de- veloped at birth [26], but not so in rat heart mitochondria.

Our study demonstrates that the activity of AAC increased strongly in rat heart mitochondria, fivefold from birth to adult (insert in Fig. 3). In previous studies with liver mitochondria, the increase in the AAC activity was attributed to the postnatal increase in the matrix adenine nucleotide concentration 123, 281. The important role of the matrix adenine nucleotides for the development of the adenine nucleotide translocation in heart mitochondria is clearly seen, when the activity was compared to the AAC protein content (Fig. 6). Such a plot shows that a linear dependency exits between AAC activity and the (ATP + ADP) pool size. However, assuming linearity between the AAC activity and the (ATP + ADP) pool size as suggested from 123, 281, a threefold expansion of the (ATP + ADP) pool size cannot account for the fivefold increase found in the translocation activity. A similar discrepancy between the increase in the translocation activity and the expansion exits in liver mitochondria [23]. Therefore, the postnatd enhancement in the AAC activity must be ascribed to developmental changes in both the matrix adenine nucleotides and the AAC protein content.

The AAC and the F,F,-ATP synthase were found to exert the main control over respiration in the neonatal heart (Fig. 2 B).

Schonfeld et al. (Eul: J. Biochem. 241) 899

The control of both enzymes amounted to 80 % of the total control over state 3 respiration in rat heart mitochondria from newborns, whereas all other proteins share the residual 20%. The slow down in the control of AAC over respiration during further postnatal development is ascribed to a switch in the control to the respiratory chain, phosphate carrier and substrate dehydroge- nases [29]. Further indication of the importance of the ADP/ATP exchange in the striated muscle tissue of newborns comes from the demonstration of a switch in the AAC isoform expression during perinatal development [30, 311. Three isoforms of the AAC (AAC1, AAC2 and AAC3) have been identified in mammals (shown for human, ox, mouse), which are expressed in a tissue-specific manner. The isoforms probably have different ki- netic properties [30, 311. In the late prenatal age, the three tran- scripts of the AAC isoforms were found in comparable amounts in muscle tissue, but at the end of postnatal development one of them, AAC1, is predominantly expressed. It is belived, that this isoform permits rapid ADP/ATP exchange across the inner mitochondria] membrane [30, 311.

The data presented here demonstrate clearly that changes in the matrix adenine nucleotides and in the AAC protein are important factors in the postnatal maturation of oxidative phosphorylation. On the other hand, enrichment of the F,F,-ATP synthase [24, 251, of the respiratory chain complexes [32, 331, of the hydrogen-supplying enzymes (citric acid cycle enzymes [ 321, pyruvate dehydrogenase complex [ 341, P-oxidation enzymes [35]), the mitochondrial creatine kinase [36] in mitochondria and the reduction of the passive proton permeability of the inner mitochondrial membrane [8] also contribute to this process. Interestingly, the reduction in the passive proton permeability of the inner mitochondrial membrane during postnatal development has been attributed to the interaction of the AAC with the matrix adenine nucleotides [37]. The rapid decline of the control exerted by AAC and F,F,-ATP synthase in the newborn rat heart indicates that other enzymes dominate the mitochondrial maturation process in the later period of heart development. Complex IV of the respiratory chain is an example for a late-developing enzymatic complex within the system of oxidative phosphorylation in rat heart mitochondria [33].

We wish to thank Mrs H. Goldammer for her skillful help in per- forming the experiments and are grateful to Dr S. No11 (Dresden) for preparation and purification of ['H]atractyloside. We thank Dr H. Schagger (Frankfurt) and Dr H. Bentlage (Nijmegen) for their generous gift of anti-AAC sera.

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Expression of the ADP/ATP Carrier and Expansion of the Mitochondrial (ATP + ADP) Pool Contribute to Postnatal Maturation of the Rat Heart