Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Developmental downregulation of the malate-aspartate shuttle has
been observed in cardiac mitochondria. The goals of this study were to
determine the time course of the postnatal decline and to identify
potential regulatory sites by measuring steady-state myocardial mRNA
and protein levels of the mitochondrial proteins involved in the
shuttle. By use of isolated porcine cardiac mitochondria incubated with
saturating concentrations of the cytosolic components of the
malate-aspartate shuttle, shuttle capacity was found to decline by
~50% during the first 5 wk of life (from 921 ± 48 to 531 ± 53 nmol · min1 · mg
protein
1). Mitochondrial
aspartate aminotransferase mRNA levels were greater in adult than in
newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase
in adult cardiac tissue were 224% of levels in newborn tissue, whereas
protein levels were 54% greater in adult myocardium.
Aspartate/glutamate carrier protein levels were also greater in adult
than in newborn tissue. mRNA and protein levels of the
oxoglutarate/malate carrier were increased in newborn myocardium. It
was concluded that 1) myocardial
malate-aspartate shuttle capacity declines rapidly after birth,
2) divergence of mitochondrial
malate dehydrogenase mRNA and protein levels during development
suggests posttranscriptional regulation of this protein, and
3) the developmental decline in
malate-aspartate shuttle capacity is regulated by decreased
oxoglutarate/malate carrier gene expression.
development; energy metabolism; gene expression; oxoglutarate/malate carrier; malate dehydrogenase
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INTRODUCTION |
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THE GLYCOLYTIC METABOLISM of glucose and oxidation of
lactate result in the production of cytosolic NADH. To achieve maximal ATP production from these substrates, NADH must reach the mitochondrial matrix to enter the electron transport chain. In addition, cytosolic NAD+ must be regenerated to
maintain maximal glycolytic flux. It was established by Lehninger (23)
and Purvis and Lowenstein (30) that the inner mitochondrial membrane
was impermeable to NADH. This finding led to the description of several
"NADH shuttle" pathways, including the -glycerophosphate,
fatty acid, malate-citrate, and malate-aspartate shuttles (9). The
basis of the shuttle reactions is the reduction of a suitable cytosolic
intermediate by NADH. The intermediate then enters the mitochondrial
matrix, where NADH is regenerated. In the heart the dominant NADH
shuttle pathway is the malate-aspartate shuttle (33, 36), which was initially described by Borst (5).
As shown in Fig. 1, the reduced intermediate formed in the cytosol as part of the malate-aspartate shuttle is malate, which crosses the inner mitochondrial membrane in exchange for oxoglutarate via the oxoglutarate/malate carrier (OMC). Oxaloacetate (OAA), formed within the mitochondrial matrix as NADH is regenerated, cannot diffuse directly across the inner mitochondrial membrane and must react with glutamate to form aspartate. Aspartate is then exchanged with glutamate via the aspartate/glutamate carrier (AGC) to achieve mass balance across the inner mitochondrial membrane.
The importance of the NADH shuttles in maintaining a continuous supply
of ATP to neonatal myocardium was suggested by observations that the
newborn heart relied predominantly on glucose and lactate as substrates
(15, 25). Oxidation of glucose and lactate generates cytosolic NADH,
which must be oxidized for the glycolytic reactions to continue (26).
Indeed, we found that the capacity of the malate-aspartate shuttle was
nearly three times greater in mitochondria isolated from neonatal than
from adult porcine left ventricle (36). Furthermore, activity of a
second NADH shuttle, the -glycerophosphate shuttle, was found in
newborn cardiac mitochondria but was absent in adult mitochondria (36).
These observations suggested that enhanced capacity of the NADH
shuttles helped maintain the high glycolytic and lactate oxidation flux
rates in the neonatal cardiomyocyte by oxidizing NADH (to
NAD+).
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The goals of the present study were to further characterize the postnatal decline in malate-aspartate shuttle capacity in porcine cardiac mitochondria and to identify potential enzymatic steps of this pathway that are regulated during development. The time course of the biochemical activity of the malate-aspartate shuttle and mitochondrial enzymes was followed over the first 5 wk of postnatal development. On the basis of earlier data that showed significant differences in malate-aspartate shuttle and enzyme activities (36), myocardium from neonatal and adult animals was used for gene expression studies.
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METHODS |
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Animal Preparation and Isolation of Mitochondria
Age-dependent changes in the malate-aspartate shuttle and the soluble mitochondrial matrix enzymes of the shuttle [mitochondrial aspartate aminotransferase (AST) and malate dehydrogenase (mAST and mMDH)] were analyzed in six and four piglets at each time point, respectively. Stable litters were established at birth by decreasing the litter size to eight animals. Groups of two animals from each of three litters were then delivered to the laboratory for study at 0-2, 9-10, 14-16, and 35 days of life. All animals were studied on the day of delivery. Neonatal pigs were anesthetized with 20 mg/kg ketamine, 0.2 mg/kg acepromazine, and 2 mg/kg xylazine given subcutaneously. Once a deep level of sedation was attained, the heart was arrested with a 3- to 5-ml bolus of saturated potassium chloride given rapidly into the left ventricle. The heart was immediately removed through a midline sternotomy and immersed in ice-cold saline. Gene expression studies were performed on hearts from separate 0- to 2-day-old newborn or adult (>5 mo old) pigs. Newborns were handled as described above; adult porcine hearts were obtained from a local abattoir. In both groups of animals, hearts were removed immediately after death. Tissue for RNA studies was immediately frozen in liquid nitrogen and stored atCardiac mitochondria were isolated from the left ventricular free wall of each heart using a modification of the technique described by Saks et al. (34), as described previously (36). The left ventricular free wall of the adult hearts was transported from the slaughterhouse to the laboratory in ice-cold normal saline for processing. All isolation steps were performed at 0-4°C. Briefly, left ventricular free wall was minced with scissors, and ~3 g of either tissue were placed in buffer containing (final concentration in mM) 300 sucrose, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 0.2 EDTA at pH 7.2. Trypsin (2.5 mg) was added to the tissue suspension, which was incubated at 0°C for 15 min. The reaction was stopped with a fivefold excess of trypsin inhibitor dissolved in 20 ml of sucrose buffer with 1 mg/ml bovine serum albumin (BSA). After the fluid was decanted, the tissue was resuspended in 20 ml of buffer with BSA and homogenized with a loose-fitting Potter-Elvehjem homogenizer for six strokes, then with a tight-fitting homogenizer for two strokes. The homogenate was centrifuged three times at 600 g for 10 min at 4°C; each time the supernatant was saved. The mitochondria were pelleted at 8,000 g for 15 min and washed once in buffer with BSA. The final pellet was resuspended in the sucrose buffer without BSA.
Respiratory control ratios (RCR), representing the ratio of the rate of mitochondrial oxygen consumption during state 3 activity (substrate and ADP excess) to the rate of oxygen consumption during state 4 activity (ATP present and ADP depleted), were measured at 37°C with a Clark-type oxygen electrode interfaced to a Gilson OxyGraph. Mitochondria were continuously stirred during these measurements. The mitochondrial suspension (200 µl) was added to 1.5 ml of (final concentration in mM) 130 potassium chloride, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2.5 magnesium chloride, 0.5 EDTA, 5 potassium phosphate, 5 glutamate, and 5 malate at pH 7.2. State 3 activity was measured after addition of 580 µM ADP.
Measurement of Malate-Aspartate Shuttle and Enzyme Activities
The capacity of the malate-aspartate shuttle was determined using the method previously described (10, 36, 39). Briefly, 50 µl of mitochondrial suspension were mixed with 2 ml of (final concentration in mM) 300 mannitol, 10 potassium phosphate, 10 tris(hydroxymethyl)aminomethane (Tris), 10 potassium chloride, 5 magnesium chloride, 2 aspartate, 2 ADP, and 0.14 NADH with 3 U/ml malate dehydrogenase (MDH) and 2 U/ml AST at pH 7.4. Baseline oxidation of NADH was monitored at 340 nm at a constant temperature of 37°C for 4 min (DU-64 spectrophotometer, Beckman Instruments, Fullerton, CA). Malate-aspartate shuttle activity was initiated with the addition of 4 mM malate and 4 mM glutamate (final concentration), and the oxidation of NADH was monitored at 340 nm for 4 min. The difference between the rate of change of absorbance with and without added substrates was normalized to added mitochondrial protein to determine the shuttle capacity.For assay of mMDH and mAST, a portion of mitochondria was resuspended in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.2) with 0.5% Triton X-100. The suspension was incubated for 30 min at 0°C and then sonicated for three 1-min intervals at 140 W in a cup horn filled with ice water. The sample was then centrifuged at 48,000 g for 20 min, and the supernatant was used for mitochondrial enzyme and protein assays.
The assay for mAST was adapted from Bergmeyer (3). Briefly, the reaction catalyzed by mAST between aspartate and oxoglutarate to form glutamate and OAA was coupled to the reaction between OAA and NADH catalyzed by MDH to form malate and NAD+. In a 1.5-ml cuvette, 100 µl of mitochondrial supernatant were diluted in buffer containing (final concentration in mM) 90 Tris, 240 aspartate, 0.11 pyridoxal 5-phosphate, and 0.16 NADH with 1 U/ml MDH and 0.5 U/ml lactate dehydrogenase. Buffer (880 µl) plus mitochondrial suspension was added to the cuvette, and baseline absorbance at 340 nm at 37°C was monitored for 4 min. After the baseline period, 12 mM oxoglutarate was added to the cuvette, and the absorbance at 340 nm was monitored for 4 min.
Activity of mMDH was determined as described by Bergmeyer (3) by measuring the rate of reduction of NAD+ in the presence of malate. Mitochondrial supernatant (100 µl) diluted in buffer was added to a 1.5-ml plastic cuvette. Buffer solution (880 µl) containing (in mM) 90 diethanolamine, 4.5 magnesium chloride, and 2.9 NAD+ at pH 9.2 was added to the cuvette, and baseline absorbance change at 340 nm at 37°C was monitored. Maximal enzyme activity was initiated with the addition of 25 mM malate. Maximal MDH activity was determined by the difference between the rates of change of absorbance with and without added substrate. mAST and mMDH enzyme activities were normalized to added soluble mitochondrial matrix protein.
Preparation of Partial cDNA Clones
Primers for the porcine mAST and mMDH genes were selected from previously published sequences (18, 19). To obtain sufficient DNA for cloning, nested polymerase chain reaction (PCR) primers were prepared for the OMC gene. The OMC gene primers were identified from conserved regions of the bovine and human OMC genes (17, 31). All PCR primers used in this study were designed using the computer software PRIMER (Eric Lander, Massachusetts Institute of Technology) and were synthesized on a DNA synthesizer (model 391, Applied Biosystems, Foster City, CA). Unless stated otherwise, the standard conditions for PCR included 5 ng of template DNA, each PCR primer at 1 µM, 0.25 U of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), deoxynucleotide triphosphates at 200 µM, 1.5 mM Mg2+, 10 mM Tris-Cl (pH 8.3), and 50 mM KCl in a total volume of 100 µl. The thermal cycler parameters were an initial denaturation step at 94°C for 3 min, then 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and finally 72°C for 5 min.Isolation and characterization of cDNA clones. The cDNA products were amplified from RNA isolated from neonatal porcine left ventricular myocardium by reverse transcription (RT) and PCR experiments. Briefly, RT was performed with 1 µg of total RNA using an avian myeloblastoma virus reverse transcriptase (Boehringer Mannheim). The PCR reaction was performed using standard conditions with ~5 ng of cDNA from the RT reaction as the DNA template (total reaction volume = 100 µl). A 1-µl aliquot of the PCR reaction was run out on a 2% agarose gel to verify the size and purity of the amplified product. The PCR product was purified using the QIAquick Kit (Qiagen, Chatsworth, CA), quantitated spectrophotometrically, and ligated into the Xcm I T-tail cloning vector pKRX (38). Recombinant plasmids were identified by PCR using T3 (5'-GCG CAA TTA ACC CTC ACT AAA G) and T7 (3'-GCG TAA TAC GAC TCA CTA TAG) vector primers that flank the site of insertion. Both DNA strands of the cloned products were sequenced at the University of Iowa DNA Core Facility (Dr. David Moser) on an automated DNA sequencer (model 373A, Applied Biosystems, Foster City, CA) using vector primers, Taq DNA polymerase, and fluorescent dye-labeled terminators. The DNA sequences derived from both DNA strands were compared and sequence ambiguities were resolved using the computer software Sequencher 3.0 (GeneCodes, Ann Arbor, MI).
Northern Blot Analyses
RNA isolation from myocardium.
All tissue samples from 0- to 2-day-old newborn and adult porcine left
ventricular myocardium were harvested, immediately frozen in liquid
nitrogen, and stored at 80°C until use. Total RNA was
isolated from ~200 mg of tissue using TRI Reagent (Molecular Research
Center, Cincinnati, OH). The total RNA pellet was then dissolved in
diethylpyrocarbonate-treated water, quantitated spectrophotometrically, and stored at
80°C in ethanol.
Preparation of gene probes. The plasmid containing the respective partial cDNA clones was linearized with EcoR 1. The linearized plasmid (~1 µg) was added to 2.4 µl of diethylpyrocarbonate-treated water, 2 µl of 10× T7 transcription buffer, 2 µl of 100 mM dithiotreitol, 32 U of RNasin, 3 µl of 3.3 mM GTP/CTP/ATP, 6 µl of deoxy-[32P]UTP, and 16 U of T7 RNA polymerase (US Biochemical, Cleveland, OH) and incubated at 37°C for 90 min. At the end of the incubation, 40 U of RNasin and 1 U of deoxyribonuclease were added, and the sample was incubated at 37°C for an additional 10 min. Then 10 µg of tRNA was added as a blocking agent, and the riboprobe was purified by phenol-chloroform extraction followed by ammonium acetate-ethanol precipitation.
Northern blot hybridization.
A 1% agarose-6.3% formaldehyde gel was prepared in 20 mM
3-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate,
and 0.25 mM EDTA at pH 7.0. Approximately 15 µg of total RNA in
loading buffer were applied to each well, and the samples were
electrophoresed overnight in a running buffer of 40 mM
3-(N-morpholino)propanesulfonic acid, 10 mM sodium acetate,
and 0.5 mM EDTA (pH 7.0). An RNA ladder was also run in one lane. The
gel was stained with ethidium bromide and photographed as a reference
for the RNA sizing standards. RNA was then transferred to a 0.45-µm
Nytran membrane (Schleicher and Schuell, Keene, NH). The membrane was
prehybridized for 1 h at 65°C in a solution containing 50%
formamide, 5× SSPE (875 mM sodium chloride, 50 mM sodium
phosphate, 5 mM EDTA), 5× Denhardt's reagent, 0.5% sodium
dodecyl sulfate (SDS), and 200 µg/ml denatured salmon sperm DNA.
Hybridization was performed at 65°C for 12-18 h with the
addition of 2 × 106 cpm/ml
of radiolabeled probe. Four washes were performed at 65°C using
three low-stringency washes (1× SSPE, 0.5% SDS) and one high-stringency wash (0.1× SSPE, 0.5% SDS). Hybridization
signals were quantitated using a beta scanning radioanalytic imaging
system (AMBIS, San Diego, CA). In addition, filters were exposed to
Kodak XAR film at 80°C. Blots were then stripped and
rehybridized with 32P-labeled
probe to the 18S or 28S subunit of ribosomal RNA. Quantitated signals
from the 18S- or 28S-probed blots were used to correct for variable RNA
loading.
Quantitative Immunoblots
Polyclonal antibodies were obtained to the AGC (a generous gift from Drs. Reinhard Kraemer and Klaus Herick, Institut fur Biotechnologie, Juelich, Germany) and mMDH (a generous gift from Dr. Arnold W. Strauss, Washington University, St. Louis, MO). Antibody to the OMC was obtained from rabbits immunized with two polypeptides prepared from conserved regions of published OMC sequences (17, 31) (arginine-48-serine-64 and aspartate-156-glycine-175) and tested by enzyme-linked immunosorbent assay (Research Genetics, Huntsville, AL). Immunoblots were prepared essentially as described previously (8). Briefly, myocardium was homogenized in the presence of protease inhibitors including 5 µg/ml soybean trypsin inhibitor, 20 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride in 50 mM Tris-10 mM EDTA-150 mM NaCl-0.1% mercaptoethanol (pH 7.5) and then sonicated for 20 s. After centrifugation, total protein of the supernatant was quantitated using the Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA). Protein (20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk protein for 1 h and primary antibody incubated overnight at 5°C. After washes with EDTA-0.5% Tween in phosphate-buffered saline, secondary antibody conjugated with horseradish peroxidase was incubated at room temperature for 1 h. Detection of the secondary antibody (SuperSignal Kit, Pierce, Rockford, IL) was performed and used to expose Kodak XAR film at room temperature. Films were digitized, and the difference between protein signals and background was quantitated using NIH Image (Wayne Rasband, National Institutes of Health, Bethesda, MD). Serial protein dilutions were tested with each antibody to ensure that quantitated signals were in the linear range for added protein.Statistical Analyses
Values are means ± SE. Comparison of NADH shuttle capacities and enzyme activities among the four ages of animals was made using analysis of variance. If the overall analysis of variance identified significant differences (P < 0.05), pairwise comparisons were made using Tukey's procedure, with P < 0.05 considered significant. Newborn and adult mRNA and protein levels were compared using an unpaired, two-tailed t-test (with significance at P < 0.05). All analyses were performed using Systat 5.2.1 for the Macintosh (Systat, Evanston, IL). ![]() |
RESULTS |
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Postnatal Malate-Aspartate Shuttle Capacities and Enzyme Activities
RCR values were tested for each group of mitochondria (n = 6 at each age). At all ages, RCR was >12, indicating that the mitochondria were well coupled. Studies of the malate-aspartate shuttle were performed with the intact mitochondria incubated in the presence of saturating concentrations of the cytosolic components of the shuttle. As shown in Fig. 2, a significant decline in capacity of the shuttle occurred over the 35 days of study. Malate-aspartate shuttle capacity in mitochondria from 35-day porcine left ventricle was significantly different from that in mitochondria from 0- to 2- and 9- to 10-day-old animals by Tukey's test, decreasing by ~50% between 0-2 and 35 days of life. Adult values [obtained in previous studies (36)] were not fully achieved by 35 days of life.
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Maximal activities of mMDH and mAST were measured in four animals at each of the four ages. mMDH and mAST activities remained fairly stable over the 5 wk of study (Table 1). Earlier studies demonstrated a significant increase in the maximal activity of both mitochondrial matrix enzymes when 0- to 2-day-old animals were compared with adults (36).
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Partial cDNA Clones of Porcine Enzymes
Partial cDNA clones specific to porcine myocardium were prepared for the mMDH, mAST, and OMC genes. The product sizes of the mMDH and mAST fragments were 417 and 448 base pairs, respectively. Each of these partial cDNA products were identical to the previously published porcine gene sequences (18, 19).The porcine OMC gene had not been previously sequenced. Nested primers designed using homologous regions of the bovine (31) and human (17) sequences resulted in a 260-base pair product. The nucleotide sequence and selected primers are shown in Fig. 3. At the nucleotide level, the gene was 92 and 91% homologous with the bovine and human genes, respectively. The porcine product was 99 and 98% homologous with the bovine and human genes at the amino acid level.
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Gene Expression Studies
The 32P-labeled mAST probe hybridized to a single ~2.7-kb mRNA. Similar to the developmental increase in maximal mAST activity reported previously (36), expression of the mAST gene was significantly greater in adult than in newborn left ventricular myocardium (Fig. 4A). Previous measurements using animals of the same age demonstrated a 54% increase in biochemical activity, and the current study demonstrated a 75% increase in mAST gene expression in adult myocardium compared with newborn.
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By use of the hybridization conditions described above, the 32P-labeled mMDH probe hybridized to a single ~2.2-kb band. Contrary to the developmental increase in maximal mMDH activity (36), steady-state mRNA levels of the mMDH gene were significantly decreased in the adult (Fig. 4B). Whereas maximal mMDH activity increased 40% from the neonatal period to adulthood in the porcine heart, mMDH gene expression decreased ~46% in the adult. The apparent posttranscriptional regulation of the mMDH gene product was further explored by quantitative immunoblot studies using a polyclonal antibody to mMDH. The mMDH band at ~32 kDa is shown in Fig. 5A. Consistent with the maximal mMDH activity measured in isolated mitochondria (36), protein levels were significantly increased in adult myocardial samples compared with newborn. These data demonstrate a divergence between mMDH mRNA levels and tissue protein and enzyme activity levels, suggesting posttranscriptional regulation of the mMDH gene product.
The sequence of the AGC is not known, which precluded preparation of a partial cDNA probe of this protein. A polyclonal antibody prepared against the 68-kDa purified AGC protein from bovine myocardium identified a single band at 68 kDa (Fig. 5B). Quantitation of the immunoblot signal revealed that the amount of AGC protein in the adult porcine myocardium was significantly increased compared with the newborn. Thus, despite a decrease in malate-aspartate shuttle activity in the adult compared with the newborn, levels of the AGC protein were greater in the adult tissue.
A single 1.9-kb mRNA was identified using the 32P-labeled OMC probe. OMC mRNA levels normalized to the 28S rRNA subunit were significantly greater in neonatal than in adult myocardium (Fig. 4C). Similar to the increase in OMC mRNA, OMC protein levels measured by immunoblot were also significantly increased in newborn tissue compared with adult (Fig. 5C). The OMC immunoblots demonstrated a single band at ~62 kDa. The concomitant increase in newborn OMC mRNA and protein levels and malate-aspartate shuttle capacity compared with the adult and the decrease in other shuttle pathway enzymes in the newborn indicated that greater malate-aspartate shuttle capacity in neonatal heart may be regulated at the level of OMC gene expression.
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DISCUSSION |
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High activity of the NADH shuttles is critical in glycolytically active tissues such as the neonatal heart. The regeneration of NAD+ is necessary to maintain maximal aerobic flux as glucose and lactate are oxidized. During the postnatal period, the reliance of the neonatal heart on glucose and lactate diminishes as the metabolism of fatty acid increases (2, 24, 41). The current study demonstrated that the high malate-aspartate shuttle capacity in the newborn porcine heart also decreases after birth, declining ~50% during the first 5 wk of life. Mitochondrial matrix enzyme content of mAST and mMDH, as measured by maximal enzyme activity, remained relatively constant during the first 5 wk. These findings suggested that the regulation of the malate-aspartate shuttle was governed by one or both of the carrier proteins in the inner mitochondrial membrane.
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To explore the potential role of the inner mitochondrial membrane proteins in regulating the malate-aspartate shuttle, a partial cDNA clone of the OMC gene and antibodies to the OMC and AGC proteins were prepared. Although AGC protein levels increased with age, OMC mRNA and protein levels were significantly less in adult than in newborn myocardium. Because whole tissue homogenates were used for the immunoblots, increases in mitochondrial density in adult myocardium (29) may explain the observed increase in AGC protein levels. However, the developmental decline in OMC protein levels per mitochondrion would be accentuated by an increase in mitochondrial number in adult tissue. In parallel with the developmental decline in malate-aspartate shuttle capacity, the observed decrease in OMC gene expression suggests an important regulatory role of the OMC during development.
Age-Related Changes in Shuttle Gene Expression
Previous work has demonstrated that maximal enzyme activity of cardiac mAST and mMDH increases with age (36). In the current study, steady-state levels of mRNA of the mAST gene followed this same pattern, demonstrating an increase in mAST mRNA in adult myocardium compared with the newborn. A concomitant increase in mRNA and maximal enzyme activity during development has been observed for several metabolic intermediates (7, 28).Discordance between mRNA and enzyme levels is less common. However, in the current study, steady-state levels of mMDH mRNA were significantly greater in neonatal myocardium than in adult heart, despite decreased mMDH protein levels in newborn myocardium. Similar divergence between mRNA levels and enzyme content has been demonstrated in the intestinal tract for the alkaline phosphatase and lactase (43, 44). Potential mechanisms of posttranscriptional regulation have been discussed and include changes in the rate of translation of transcribed mRNA or the rate of protein turnover (1). Further studies are needed to determine which of these mechanisms are regulating mMDH during cardiac development.
Regulation of the Malate-Aspartate Shuttle During Development
The malate-aspartate shuttle is nearly three times more active in the neonatal than in the adult heart, a time during which the cardiac myocyte has increased reliance on glucose and lactate as a source of metabolic fuel (25). To maintain maximal glycolytic flux while utilizing glucose and lactate, NADH must be oxidized to NAD+ (16, 26). In adult myocardium the capacity of the NADH shuttles to oxidize cytosolic NADH can be exceeded, resulting in adverse functional consequences. When perfused with glucose and lactate to maximally reduce the cytosol, adult rabbit myocardium demonstrated diminished function and an increase in NADH shuttle cytosolic intermediates (37). These deleterious effects occurred despite enhanced NADH shuttle flux under the same perfusion conditions (45).During the same postnatal period when malate-aspartate shuttle capacity declines (Fig. 2), others demonstrated that fatty acid utilization in the porcine heart increases (2, 41). The enhanced fatty acid metabolism probably reflects greater carnitine acyltransferase I activity (27, 40), which allows the reducing equivalents contained in fatty acids to be delivered directly to the mitochondrial matrix, bypassing cytosolic NADH-generating reactions. The signal(s) that initiates this coordinated metabolic response is not known. It is possible that the fatty acids themselves can induce the metabolic response, since serum fatty acid concentrations in the fetus are known to be low (20). However, numerous hormonal changes also occur at birth. In particular, the surge of thyroid hormone known to occur in the peripartum period may play a role (13, 14) by inducing transcription of metabolic enzymes (6, 12).
The enzymatic step that responds to the developmental cue(s) and regulates myocardial NADH shuttle flux appears to be the OMC, which spans the inner mitochondrial membrane. Expression of the OMC gene at the mRNA and protein level was nearly 2.5 times greater in neonatal myocardium than in adult tissue. Using 13C nuclear magnetic resonance, Yu et al. (46) identified regulation of the malate-aspartate shuttle at the level of the inner mitochondrial membrane. These investigators found the exchange rate between the oxoglutarate and glutamate pools, which is dependent on the OMC and AGC proteins, to be ~20 times less than flux rates through cytosolic AST. The role of the AGC inner mitochondrial membrane protein in regulating the malate-aspartate shuttle appears to be less important, since AGC protein levels increased in adult myocardium. Although AGC flux can be altered by mitochondrial membrane potential and pH (11), these effects would be expected to be limited in our in vitro assay, which exposed neonatal and adult mitochondria to identical conditions.
The OMC gene is encoded in the nucleus and, thus, must be transported to the mitochondrion and incorporated into the inner mitochondrial membrane. Although this complex process is generally guided by a leader sequence on the protein (32), the OMC protein is thought to be primarily translated without the presequence (31). The bovine and human OMC protein has been sequenced, and the 31- to 33-kDa monomer was found to be 314 amino acids in length (17, 31). A functional homodimeric form of the protein has also been described (4), with a molecular weight similar to that found in the current study. There is considerable homology between all the mammalian OMC sequences, including the partial porcine sequence described in the current study. Although isoforms of the OMC gene have not been found (31), variable antigenicity of cardiac and liver OMC proteins has been observed and suggests tissue-specific postprocessing of the protein (47). Of interest are similar structural motifs of the OMC protein, the ADP/ATP translocase, the phosphate carrier, and the uncoupling protein, which indicate a common lineage of these inner mitochondrial membrane proteins (21, 31).
Carrier Protein Regulation and the Tricarboxylic Acid Cycle
OAA and oxoglutarate are intermediates that are shared by the malate-aspartate shuttle and the tricarboxylic acid cycle within the mitochondria. Recognizing these common elements, LaNoue, Williamson, and others (22, 42) explored potential interactions between these two pathways. These elegant investigations identified the importance of oxoglutarate dehydrogenase (orGlutamate and acetyl-CoA compete for OAA within the mitochondria. The first committed step of the tricarboxylic acid cycle is the reaction of acetyl-CoA with OAA catalyzed by citrate synthase to form citrate. As shown in Fig. 1, in the malate-aspartate shuttle, glutamate and OAA react together to form oxoglutarate and aspartate in a reaction catalyzed by mAST. LaNoue and Williamson (22) demonstrated that aspartate efflux from the mitochondria via the AGC was significantly reduced when mitochondria were given acetylcarnitine to metabolize. The acetylcarnitine provided a source of acetyl-CoA for the mitochondria, which channeled OAA through the tricarboxylic acid cycle, rather than toward mAST, to generate aspartate and oxoglutarate. Providing acetylcarnitine to the mitochondria simulates the substrate preference of the adult myocyte, which primarily utilizes fatty acids. Thus it is somewhat incongruous that the maximal reaction velocity of the AGC would be increased in the adult cardiac myocyte. Increased flux through the AGC could deliver more glutamate to the mitochondrial matrix, which would compete with acetyl-CoA generated via fatty acid oxidation for available OAA. As mentioned above, it is possible that, in vivo, maximal flux at the AGC step of the malate-aspartate shuttle is comparable in newborn and adult cardiac myocytes and that the increased AGC protein levels observed by immunoblot reflect increased mitochondrial density in the adult tissue.
Study Limitations
The current study was not able to completely characterize the time course of the postnatal decline in malate-aspartate shuttle capacity. Expression of the genes involved in the malate-aspartate shuttle pathway was measured only at the newborn and adult time points. These time points were chosen on the basis of previous studies in which it was suggested that maximal differences in shuttle capacity (36), and therefore gene expression, would be noted at these times. It has been assumed that gene expression data at these two time points could be used to explain changes in malate-aspartate shuttle capacity data in the first 5 wk after birth, but this has not been tested. In addition, the number of mitochondrial samples studied for mAST and mMDH enzyme activity was small, and significant changes may not have been identified. Thus, although the neonatal-to-adult differences in myocardial malate-aspartate shuttle activity and gene expression are well characterized, these results must be extrapolated to the immediate postnatal period.The malate-aspartate shuttle assay used in the current study tested only the mitochondrial components of the shuttle. Isolated mitochondria were suspended in buffer containing saturating concentrations of the cytosolic components of the shuttle. The relationship between the capacity of the mitochondrial malate-aspartate shuttle reactions measured in the current study and in vivo activity remains to be explored.
Conclusions
The malate-aspartate shuttle provides a crucial link between the cytosol and the mitochondria in glycolytically active hearts. Without the oxidation of NADH produced during glycolysis, flux through these reactions would be impeded. The maintenance of glucose and lactate oxidation is particularly important in the fetal and neonatal hearts, which depend on these substrates for ATP production (24, 25). The current study demonstrated that the capacity of the malate-aspartate shuttle began to decline shortly after birth and decreased by ~50% within 5 wk. An important enzymatic step of the malate-aspartate shuttle, catalyzed by mMDH, was found to have divergent mRNA and protein levels during cardiac development. Studies of the posttranscriptional regulation of the mMDH gene should provide interesting insights into the developmental control of this enzyme. As found in the ![]() |
ACKNOWLEDGEMENTS |
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Funding for this research has been provided by American Heart Association, Iowa Affiliate, Grant-in-Aid IA-95-GS-57 and March of Dimes Basil O'Connor Starter Research Award FY96-0566.
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FOOTNOTES |
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Address for reprint requests: T. D. Scholz, Dept. of Pediatrics, 2852 JPP, University of Iowa, Iowa City, IA 52242.
Received 15 August 1997; accepted in final form 10 November 1997.
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