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Am J Physiol Heart Circ Physiol 286: H2243-H2248, 2004. First published January 29, 2004; doi:10.1152/ajpheart.00864.2003
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Myocardial carnitine palmitoyltransferase I expression and long-chain fatty acid oxidation in fetal and newborn lambs

Beatrijs Bartelds,1,2 Janny Takens,1 Gioia B. Smid,1 Victor A. Zammit,3 Carina Prip-Buus,4 Jaap R. G. Kuipers,1 and Feike R. van der Leij1

1Department of Pediatrics, University of Groningen, Beatrix Children's Hospital and Research School Guide, 9713 GZ Groningen, The Netherlands; 2Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2495; 3Cellular Biochemistry, Hannah Research Institute, KA6 5ML Scotland, United Kingdom; and 4Endocrinology Department, Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 567, 75014 Paris, France

Submitted 9 September 2003 ; accepted in final form 27 January 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Carnitine palmitoyltransferase I (CPT I) catalyzes the conversion of acyl-CoA to acylcarnitine at the outer mitochondrial membrane and is a key enzyme in the control of long-chain fatty acid (LC-FA) oxidation. Because myocardial LC-FA oxidation increases dramatically after birth, we determined the extent to which CPT I expression contributes to these changes in the perinatal lamb. We measured the steady-state level of transcripts of the CPT1A and CPT1B genes, which encode the liver (L-CPT I) and muscle CPT I (M-CPT I) isoforms, respectively, as well as the amount of these proteins, their total activity, and the amount of carnitine in left ventricular tissue from fetal and newborn lambs. We compared these data with previously obtained myocardial FA oxidation rates in vivo in the same model. The results showed that CPT1B was already expressed before birth and that total CPT I expression transiently increased after birth. The protein level of M-CPT I was high throughout development, whereas that of L-CPT I was only transiently upregulated in the first week after birth. The total CPT I activity in vitro also increased after birth. However, the increase in myocardial FA oxidation measured in vivo (112-fold) by far exceeded the increase in gene expression (2.2-fold), protein amount (1.1-fold), and enzyme activity (1.2-fold) in vitro. In conclusion, these results stress the importance of substrate supply per se in the postnatal increase in myocardial FA oxidation. M-CPT I is expressed throughout perinatal development, making it a primary target for metabolic modulation of myocardial FA oxidation.

cardiac muscle; gene expression; free fatty acid; cardiac metabolism

LONG-CHAIN FATTY ACIDS (LC-FA) are the prime substrate for energy provision in the myocardium of the adult. The role of LC-FA in myocardial metabolism of the newborn was suggested to be limited based on studies in isolated hearts (3, 14, 29) and homogenized tissue (15). The key enzyme in the regulation of FA oxidation is carnitine palmitoyltransferase I (CPT I), which is an integral protein of the outer mitochondrial membrane, with its catalytic and regulatory domains exposed to the cytosolic aspect. CPT I catalyzes the conversion of LC-acyl-CoA to LC-acylcarnitine, which can be transported across the mitochondrial membrane (24) to be reconverted to LC-acyl-CoA by CPT II for oxidation within the mitochondrial matrix. Two CPT I enzyme forms are known, a liver type (L-CPT I) and a muscle type (M-CPT I). These enzymes are encoded by two different genes, CPT1A and CPT1B, respectively (7, 26). The CPT I enzymes are detergent labile; they catalyze the same reaction, and they share extensive structural resemblance (20). However, they differ in their affinity for carnitine and in their sensitivity to malonyl-CoA inhibition (16). In the rat heart, their expression patterns differ throughout development (8, 9) and under several pathological conditions, as in left ventricular (LV) hypertrophy (4).

We showed previously that glucose and lactate are the prime energy substrates for the myocardium in fetal lambs, whereas in newborn lambs glucose is replaced by LC-FA (5, 6). We also showed that LC-FA are oxidized by the myocardium of the fetus, when supplied (6), and that the contribution of oxidation of LC-FA to myocardial oxygen consumption was the same in fetal as newborn lambs. These results suggest no limitation in the ability of the heart to use LC-FA before or just after birth. So far, most studies have investigated either enzyme expression or activity in isolated hearts or homogenates (3, 9, 14, 15, 29). The aim of this study was to link the perinatal changes in myocardial LC-FA metabolism obtained in vivo with the characteristics of the CPT I enzymes to determine their involvement in the control of the increase in LC-FA oxidation after birth. Therefore, we measured the steady-state level of transcripts of the genes encoding these enzymes, CPT1A and CPT1B, the amount of CPT I protein present in the myocardium, and the CPT I activity in LV tissue from fetal and newborn lambs. We compared these data with previously obtained LC-FA oxidation rates in vivo in the same model (6). Our results provide evidence that substrate supply is a major determinant for LC-FA oxidation around birth.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animals. LV tissue was obtained from fetal and newborn lambs that had been used previously to study myocardial metabolism in vivo (5, 6). Fetal lambs were at 127–134 days of gestation and newborn lambs were 2–16 days of age. Tissues from lambs that were used for experiments with fat infusion were excluded from analysis. After the last experiment, the ewes and lambs were killed with an overdose of intravenous pentobarbital sodium. The heart was excised as quickly as possible, and samples from LV tissue were frozen in liquid nitrogen. The tissue samples were stored at –80°C until further analysis. Surgical and experimental procedures were approved by the animal research committee of the University of Groningen.

CPT I expression. RNA was isolated with RNAzol (Campro Scientific; Veenendaal, The Netherlands) by a standard isolation procedure. Quantitative reverse transcription and competitive PCR was used to measure steady-state levels of mRNA (23). Control fragments for competitive PCR were made with the use of MIMIC primers (Fig. 1, sequences available upon request) for sheep CPT1A and CPT1B. This yielded slightly smaller fragments that retained the primer sites used for analysis. The {beta}-actin competition fragment was a deletion clone (Erase-a-Base, Promega) of a human genomic fragment of a putative actin pseudogene that contains the complete primer sites used for sheep RT-PCR (the nucleotide sequence has been deposited in the EMBL/Genbank/DDBJ databases under Accession No. AJ506991). Reverse transcription was carried out with 2 µg of RNA, 0.02 nmol of the lower primer, 40 units of reverse transcriptase, 0.1 mol/l of DTT, 5 nmol of each dNTP, and 32 units of RNAsin in a final volume of 20 µl at 42°C for 60 min. Competitive PCR with cDNA of the sheep tissue (2 µl of the above reaction), the appropriate control fragment (2 µl of a known concentration), and the appropriate primer set (for CPT1A, CPT1B, and {beta}-actin, respectively) were performed with 5 nmol dNTPs, 50 pmol lower primer, 50 pmol upper primer, 4% DMSO, and 1.7 units DNA polymerase in a final volume of 30 µl. The concentration of the control fragment in the PCR was varied to measure the amount of cDNA of the fragment of interest. The gels were scanned using Pharmacia ImageMaster equipment.



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  		Fig. 1. Use of MIMIC primers to perform competitive RT-PCR. Primers 1 and 2 were used for competitive PCR; region 3 is deleted in the competing fragment with the use of a MIMIC primer, which is a combination of region 4 and primer 2 (sequences available upon request). CPT IA, carnitine palmitoyltransferase IA.

 
Amount of CPT I protein. The proteins present in tissue homogenates were separated by SDS-PAGE [7.5% (wt/vol) gel]. Aliquots (40 µg) of protein were solubilized in a buffer containing 1.5% (wt/vol) SDS. Gels were run with prestained molecular mass markers (Bio-Rad; Veenendaal, The Netherlands). Blotting and detection was performed by standard methods with the use of an enhanced chemiluminescence method (Luminol, Sigma) and exposure times of 0.5–5 min. For L-CPT I, we used rabbit anti-rat L-CPT I (19) as the primary antibody and pig anti-rabbit IgG conjugated to horseradish peroxidase as the secondary antibody. For M-CPT I, we used rabbit anti-mouse M-CPT I antibodies that were raised against a recombinant fragment of M-CPT I comparable with the fragment of recombinant rat L-CPT I. To quantify the amount of CPT I protein present, we measured the efficiency of immunolabeling in samples from the sheep liver (where only L-CPT I is present) and muscle (where only M-CPT I is present) through comparison to labeling with [3H]etomoxir (a kind gift from Novartis), which irreversibly binds to both CPT I enzymes (8). Samples from the sheep liver and muscle were run in four lanes for each tissue and were blotted. Two lanes were immunolabeled, and bands from the other two lanes were excised from the blot, incubated with [3H]etomoxir (specific activity 1.122 Ci/mmol), HS-CoA, ATP, and acyl-CoA synthase (Roche Cat. No. 1002406) in an acyl-CoA synthase buffer according to the manufacturer's protocol for 30 min at 30°C, washed twice, and counted for the specific activity. Comparable surface areas of blot filter without protein showed no detectable etomoxir binding. The efficiency was calculated as the amount in millimoles (measured with specific activity in the [3H]etomoxir incubated lanes) divided by the density per gram of protein (obtained from the immunolabeled lanes).

CPT activity. CPT activity was measured in homogenized tissue using a forward assay as described previously (25). To distinguish the activity of CPT I from CPT II, Triton X-100 (2%) was added to the homogenate before the reaction was started. Triton X-100 solubilizes the mitochondrial membranes and deactivates CPT I (30).

Carnitine content. Carnitine content was measured in homogenized tissue with the use of [14C]acetyl-CoA and carnitine acetyltransferase as described previously (17).

Statistics. Data are presented as means ± SE. Student's t-test and ANOVA with a post hoc Newman-Keuls test were applied when necessary. Statview and SuperAnova for the Macintosh were used as software packages.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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Baseline differences. There were several differences between fetal and newborn lambs in baseline variables that were similar to those described in previous studies (5, 6); that is, body weight, heart rate, arterial pressure, oxygen tension, and pH were higher in newborn lambs than in fetal lambs. As a result, myocardial blood flow and oxygen consumption were higher in newborn than fetal lambs (282 ± 7 vs. 192 ± 7 ml·min–1·100 g LV tissue–1 and 946 ± 20 vs. 371 ± 12 µmol O2·min–1·100 g LV tissue–1, P < 0.001). The concentrations of glucose and fatty acids were also higher in newborn than fetal lambs (5.5 ± 0.0 and 0.35 ± 0.01 µmol/l in newborn lambs vs. 0.9 ± 0.0 and 0.01 ± 0.00 µmol/l in fetal lambs, respectively, P < 0.001).

Gene expression. In the fetal heart, CPT1A as well as CPT1B were expressed as shown by mRNA levels and protein levels (Figs. 2A and 3A). The expression of CPT1A+CPT1B significantly increased in the first week after birth to decrease to levels slightly above the fetal expression levels in the second week (Fig. 2B, top). This increase in expression outweighed the increase in {beta}-actin expression (+33% after birth), and hence the ratio of the expression of CPT I to {beta}-actin also increased in the first week after birth (Fig. 2B, middle; P = 0.09). The ratio of the expression of CPT1A to CPT1B seemed to increase after birth, but the differences were not statistically significant (Fig. 2B, bottom). The protein product of CPT1B, M-CPT I, was constitutively expressed throughout development, whereas the protein product of CPT1A, L-CPT I, transiently increased after birth and declined to low levels in the second week (Fig. 3B). The amount of M-CPT I present in the heart was significantly higher than that of L-CPT I throughout development (Fig. 3B).



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  		Fig. 2. Perinatal myocardial expression of CPT1A and CPT1B in lambs. Gene expression was measured with competitive RT-PCR (METHODS) in homogenized left ventricular (LV) tissue of 5 fetal and 8 newborn lambs (4 in week 1 and 4 in week 2). A: example of a competitive RT-PCR shown after separation by gel electrophoresis. M, marker (506, 396, 344, and 298 bp). The lanes show products from PCR with the same amount of cDNA and increasing amounts of the control fragment (CF). B: amount of RNA present in tissue presented as 106 mRNA molecules/µg total RNA. Data are given as means ± SE. Single-factor ANOVA with a post hoc Newman-Keuls test was used to test for differences.*P < 0.05; {dagger}P = 0.09.

 


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  		Fig. 3. Perinatal myocardial expression of L-CPT I and M-CPT I in lambs. A: representative examples of Western blots of L-CPT I and M-CPT I of homogenized LV tissue. The marker on the right side shows the molecular mass. Above each lane, the age is printed; F, fetal; d, days. B: amount of L-CPT I and M-CPT I in fetal (n = 8 and 5, respectively), 1-wk-old (n = 4 and 6), and 2-wk-old (n = 8 and 9) lambs. Data are given as means ± SE. Single-factor ANOVA with a post-hoc Newman-Keuls test was used to test for differences.*P < 0.05; {dagger}P < 0.05 vs. L-CPT I in the same age group (Student's t-test with Bonferroni correction).

 
CPT activity and carnitine content. The total CPT activity was higher in newborn than fetal lambs (+25%; Fig. 4). The increase in total CPT activity was largely due to an increase in activity of CPT I; the activity of CPT II hardly increased after birth (Fig. 4). The plasma concentrations of carnitine and LC-FA both increased in the first week after birth (Table 1). The myocardial carnitine content significantly increased in the second week after birth (Table 1). The ratio of the carnitine to LC-FA concentration was high in the fetus, reached a nadir in the first week after birth, and increased in the second week after birth (Table 1).



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  		Fig. 4. Perinatal myocardial CPT activity in lambs. CPT activity was measured in homogenized LV tissue of 7 fetal and 9 newborn lambs under standard conditions (METHODS). Data are given as mean ± SE. P values are from Student's t-test. *P < 0.05 vs. fetal lambs; {dagger}P = 0.066 vs. fetal lambs.

 

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  		Table 1. Tissue carnitine and plasma concentrations in fetal and newborn lambs

 
Comparison of LC-FA oxidation in vivo and CPT activity in vitro. The neonatal increases in gene expression (2.2-fold), protein amount (1.1-fold), and CPT activity (1.2-fold) were all considerably lower than the increase in LC-FA oxidation measured in vivo [112-fold (6)]. LC-FA oxidation was measured in fetal lambs during fat infusion and in newborn lambs under physiological conditions with the use of 1-[13C]palmitate as tracer (6). In fetal lambs under physiological conditions, the plasma LC-FA concentration is very low and hence LC-FA oxidation is very low (0.07 ± 0.17 µmol·min–1·100 g LV tissue–1). After birth, LC-FA concentration and oxidation (4.60 ± 1.15 µmol·min–1·100 g LV tissue–1) increased dramatically. However, when the supply of LC-FA in fetal lambs was increased by infusion of a fat emulsion to levels comparable with that of the newborn lamb (to 0.36 ± 0.05 mmol/l) (6), LC-FA oxidation increased as well (to 1.25 ± 0.17 µmol·min–1·100 g LV tissue–1). Although the absolute LC-FA oxidation did not reach newborn levels, the contribution of LC-FA oxidation to myocardial oxygen consumption was the same in newborn lambs as in fetal lambs during fat infusion (83 ± 19% vs. 70 ± 10%, respectively). LC-FA oxidation in newborns was increased 112-fold compared with fetal lambs under physiological conditions and 2.34-fold compared with fetal lambs during fat infusion.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
In this study, we characterized the perinatal changes in gene expression, protein content, and enzyme activity of CPT I in the heart of the lamb, the same model we used to measure myocardial LC-FA metabolism in vivo (5, 6). This study shows that M-CPT I was constitutively expressed throughout development, whereas L-CPT I was transiently upregulated after birth. CPT I gene expression, protein amount, and enzyme activity measured in vitro all increased after birth. However, this increase was lower than that of LC-FA oxidation measured in vivo, stressing the importance of substrate supply per se in the postnatal increase in myocardial LC-FA oxidation.

The sheep model is a unique model to study perinatal changes in both cardiovascular physiology and metabolism because it allows fetal catheterization of, among others, the coronary sinus. Moreover, the genetic information that is available indicates a high level of similarity in nucleotide and protein sequences of the major enzymes involved in FA metabolism. Although it may be argued that sheep are ruminants, suckling lambs can be regarded as monogastric animals (13, 27).

In this study, in tissues from chronically instrumented fetal and newborn lambs that had been used previously for in vivo measurements, we found that the expression of CPT1B mRNA as well as the amount of M-CPT I were high throughout perinatal development. These results suggest that M-CPT I is expressed in large excess relative to the LC-FA supply before birth. Therefore, its presence before birth suggests either a prenatal physiological role other than that strictly related to energy metabolism (e.g., to prevent apoptotic cascades due to intermediates of other fates of LC-FA metabolism) or programmed cardiac preparation for the metabolic switch that occurs shortly after birth. The latter would be clearly different from prenatal expression of CPT I in the rat liver (12). Because peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) is hardly expressed before birth, the fetal expression of CPT1B is probably regulated via a PPAR-{alpha}-independent mechanism, in contrast to the PPAR-{alpha}-mediated downregulation observed during cardiac hypertrophy (4). A candidate route would be through PPAR-{beta}/{delta} mediation because this is ubiquitously expressed and was recently found to be a potent transcriptional activator of the human CPT1B promoter in cardiac myocytes (12). Interestingly, most authors describe a "fetal gene program" (10, 22) as a hallmark of hypertrophic response, and in rodents L-CPT I has been considered to be the fetal enzyme. Similar to what was recently shown to be the case for human (21) and rat (9) hearts, we show that in fetal lambs M-CPT I is constitutively expressed and that the level of expression is higher than that for L-CPT I. Therefore, in the lamb, an enzyme switch does not seem to fully occur, although the enzymatic contribution of L-CPT I may indeed be higher shortly after birth due to the kinetic characteristics of L-CPT I (8). This is probably true for lambs as well, because, when measured in a heterologous yeast expression system, the kinetics of sheep L-CPT I do not dramatically differ from those from other mammals studied thus far (18).

This study confirms earlier findings in rats (8, 9) that showed elevated levels of L-CPT I expression in the first days after birth. The teleological explanation for this phenomenon is that shortly after birth the carnitine supply from the milk has not yet led to sufficient concentrations of myocardial intracellular carnitine to be effectively used by M-CPT I (given its relatively high Km for carnitine) and hence L-CPT I is temporarily expressed (16). In the present study, we indeed found levels of myocardial carnitine in the first week after birth that are comparable with the levels before birth. Within the same tissues as used for the expression measurements, carnitine increases significantly in the second week. Thus L-CPT I expression ceases in the second week after birth, possibly via a signaling route that senses the level of carnitine relative to that of free LC-FA or LC-FA intermediates. Additional indications for such a signaling route exist from studies of carnitine-deficient mice, which showed elevated expression of L-CPT I (23). It should be noted, however, that the tissue carnitine content in our lambs is well above the in vitro measured Km for carnitine throughout perinatal development. We converted the carnitine concentrations of myocardial tissue into carnitine content by assuming a protein content of 18%. Carnitine concentrations range from1,900 to 4,500 µmol/l. The measured Km for ovine CPT I for carnitine in a yeast expression system (18) was 215 ± 9 µM for L-CPT I and 530 ± 35 µM for M-CPT I, respectively. Hence, carnitine does not appear to be the limiting substrate in perinatal myocardial FA oxidation.

CPT I is generally believed to be the key regulatory site of FA oxidation in the myocardium (8, 16, 20, 28). In this study, the postnatal increase in CPT I activity, protein content, and gene expression in vitro was much lower than the increase in the rate of LC-FA oxidation in vivo in the same animals. These data suggest that substrate supply is the major determinant for LC-FA oxidation around birth. In addition to increased substrate supply and CPT I mRNA and protein, CPT I activity in vivo is regulated through malonyl-CoA inhibition, as has been postulated from studies in homogenized cardiac tissue of rabbits (15). Malonyl-CoA is thus thought to be an important site of control of FA oxidation, but it is yet unknown whether it is the control of its synthesis (by acetyl-CoA carboxylase) or its degradation (by malonyl-CoA decarboxylase) that primarily influences myocardial FA oxidation (2, 11, 15). Surprisingly, a null mutation in the gene encoding the cardiac isoform of acetyl-CoA carboxylase does not lead to a disturbed perinatal development (1). These findings suggest that, although malonyl-CoA appears to play an important role in the regulation of myocardial FA oxidation, perinatal development of myocardial FAO is not dependent upon malonyl-CoA synthesis per se.

In conclusion, this study shows that in the myocardium of fetal and newborn lambs CPT1B was constitutively expressed throughout development, making M-CPT I a primary target for metabolic modulation of myocardial LC-FA metabolism. In contrast to M-CPT I, L-CPT I expression was low in the fetus but was transiently upregulated after birth. CPT I gene expression, protein amount, and enzyme activity measured in vitro all increased after birth. However, this increase was lower than that of LC-FA oxidation measured in vivo, stressing the importance of substrate supply per se in the postnatal increase in myocardial LC-FA oxidation.


   NOTE ADDED IN PROOF
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During the preparation of this publication, a relevant and recent study in rabbit hearts was unintendedly omitted from the reference list: Onay-Besikci A, Campbell FM, Hopkins TA, Dyck JR, and Lopaschuk GD. Relative importance of malonyl CoA and carnitine in maturation of fatty acid oxidation in newborn rabbit heart. Am J Physiol Heart Circ Physiol 284: H283–H289, 2003.


   GRANTS
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 METHODS
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REFERENCES
 
This study was supported by The Netherlands Heart Foundation Grants 94.149, 97.093, and 2001.081.


   ACKNOWLEDGMENTS
 
We thank L. IJist at the Amsterdam Medical Centre and F. van der Sluijs at the Research Laboratory Pediatrics, University Hospital Groningen, for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. R. van der Leij, Dept. of Pediatrics, Univ. of Groningen, Research Lab CMCV-2, Hanzeplein 1, NL-9713 GZ Groningen, The Netherlands (E-mail: f.r.van.der.ley{at}med.rug.nl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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