Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

AIM: To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. METHODS: Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. RESULTS: The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or beta1-adrenoceptor (beta1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac beta-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. CONCLUSION: db/db mice show impaired cardiac functional reserve capacity during maximal beta-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism.

Connective tissue growth factor and cardiac fibrosis.

Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart.

Myofibroblast progenitor cells are increased in number in patients with type 1 diabetes and express less bone morphogenetic protein 6: a novel clue to adverse tissue remodelling?

AIMS/HYPOTHESIS: Growth factor imbalance and endothelial progenitor cell dysfunction are well-known elements of the inappropriate response to injury in human and experimental diabetes. We hypothesised that in diabetes the outgrowth of myofibroblast progenitor cells (MFPCs) is also altered and that this relates to aberrant gene expression of growth factors involving members of the TGF-beta/bone morphogenetic protein (BMP) superfamily. SUBJECTS AND METHODS: MFPCs were cultured from peripheral blood mononuclear cells of patients with type 1 diabetes and control subjects. Microarray analysis, quantitative PCR and ELISA were used to identify differentially regulated TGF-beta/BMP superfamily genes in diabetes- and control-derived MFPC. Possible effects of BMP6 on TGF-beta-induced gene expression were examined in cultured renal fibroblasts (TK173 cells). RESULTS: Blood from diabetic patients yielded higher numbers of MFPCs than blood from control subjects (1.6-fold increase, p<0.05), involving increased proliferation and decreased apoptosis. BMP6 mRNA and protein were downregulated in MFPCs derived from patients with diabetes (3.9- and 1.8-fold decrease, respectively, p<0.05). Furthermore, an inverse correlation was observed between BMP6 mRNA level and the number of MFPCs in patients with diabetes (r=-0.85, p<0.05). In TK173 cells, BMP6 antagonised the TGF-beta-induced expression of the genes encoding plasminogen activator inhibitor-1 and connective tissue growth factor (70 and 50% reduction, respectively). CONCLUSIONS/INTERPRETATION: Considering the importance of BMP6 in processes such as angiogenesis and its novel anti-TGF-beta effects, we propose that the excess numbers of BMP6-deficient MFPCs may favour adverse tissue remodelling in patients with diabetes, both numerically and by inappropriate orchestration of their microenvironment.

Heat shock proteins and cardiovascular pathophysiology.

In the eukaryotic cell an intrinsic mechanism is present providing the ability to defend itself against external stressors from various sources. This defense mechanism probably evolved from the presence of a group of chaperones, playing a crucial role in governing proper protein assembly, folding, and transport. Upregulation of the synthesis of a number of these proteins upon environmental stress establishes a unique defense system to maintain cellular protein homeostasis and to ensure survival of the cell. In the cardiovascular system this enhanced protein synthesis leads to a transient but powerful increase in tolerance to such endangering situations as ischemia, hypoxia, oxidative injury, and endotoxemia. These so-called heat shock proteins interfere with several physiological processes within several cell organelles and, for proper functioning, are translocated to different compartments following stress-induced synthesis. In this review we describe the physiological role of heat shock proteins and discuss their protective potential against various stress agents in the cardiovascular system.

HSP70-mediated acceleration of translational recovery after stress is independent of ribosomal RNA synthesis.

HSP70 is known to protect cells against stressful events. In the present study, the hypothesis was investigated that elevated HSP70 levels protect RNA polymerase I during stress, leading to decreased inhibition of ribosomal RNA (rRNA) synthesis and accelerated recovery of protein translation after stress. To this end, transcriptional and translational activity was studied in H9c2 cells during recovery after a severe heat treatment (SHT, 1 h 45 degrees C) in the presence of elevated HSP70 levels. The latter was achieved by heat pretreatment or by adenovirus-mediated hsp70 gene transfer. Rates of transcription and translation were determined by measuring cellular 3H-labelled uridine and leucine incorporation, respectively. The two types of pretreatment did not affect basal rates of transcription and translation, immediately before SHT. During SHT, both transcriptional and translational rates dropped to less than 10% of basal levels in pretreated as well as non-pretreated cells. Two and four h after SHT, both transcriptional and translational rates were significantly higher in HSP70-overexpressing cells compared to non-pretreated cells. However, immediately after SHT, transcription rates were similarly depressed in non-pretreated and pretreated cells, showing that increased levels of HSP70 did not protect RNA polymerase I activity during SHT. Thus, the HSP70-mediated acceleration of translational recovery is not preceded in time by an enhanced recovery of rRNA synthesis. Therefore, the HSP70-mediated early recovery of protein synthesis after heat stress is independent of rRNA synthesis.

Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins.

Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cells via passive diffusion and/or carrier-mediated transport. Kinetic studies of FA uptake by cardiac myocytes and the use of membrane protein-modifying agents have suggested the bulk of FA uptake is due to a protein component. Three membrane-associated FA-binding proteins were proposed to play a role in FA uptake, a 40-kDa plasma membrane FA-binding protein (FABPpm), an 88-kDa FA translocase (FAT/CD36), and a 60-kDa FA transport protein (FATP). In cardiac and skeletal myocytes the intracellular carrier for FA is cytoplasmic heart-type FA-binding protein (H-FABP), which likely transports FA from the sarcolemma to their intracellular sites of metabolism. A scenario is discussed in which FABPpm, FAT/CD36, and H-FABP, probably assisted by an albumin-binding protein, cooperate in the translocation of FA across the sarcolemma.

Co-expression in rat heart and skeletal muscle of four genes coding for proteins implicated in long-chain fatty acid uptake.

It has been suggested that specific membrane-associated and cytoplasmic proteins cooperate in the uptake of long-chain fatty acids by cardiac and skeletal muscle cells. A prerequisite for this hypothesis would be the co-occurrence of these proteins in muscle. Thus, we studied the possible co-expression in rat muscles of the genes coding for the integral membrane proteins fatty acid transport protein (FATP) and fatty acid translocase (FAT), the membrane-associated plasmalemmal fatty acid-binding protein (FABPpm) and the cytoplasmic heart-type fatty acid-binding protein (H-FABPc). The transcripts of the four proteins were assessed in heart and skeletal muscles of adult Wistar rats, in isolated cells and cell lines from rat heart and also in rat heart during development and upon streptozotocin-induced diabetes. All four genes showed high expression levels in heart, somewhat lower in red skeletal muscle (soleus) and appreciably lower in white skeletal muscle (extensor digitorum longus). FATP, FAT and H-FABPc showed a 3- to 5-fold increase in mRNA expression during maturational growth of the heart, while the FABPpm expression remained virtually constant. In the heart, streptozotocin-diabetes induced a slight, but statistically not significant, increase in the expression of all four genes. In conclusion, this study shows the co-expression of FATP, FAT, FABPpm and H-FABPc in rat muscles. This finding supports the possible cooperation of these proteins in the uptake of long-chain fatty acids by muscle cells.

Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.

A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes.

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131. 4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 microgram/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart ( approximately 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (<0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7+/-0.7 mg/g of protein on the day before birth to 3.6+/-0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P<0.05) 2-4-fold increase of FAT was seen in heart (from 4.2+/-2.3 to 11.0+/-5.7 mg/g of protein), soleus (from 0.6+/-0.1 to 1.4+/-0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3+/-0.1 to 1. 2+/-0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Stable transfection of fatty acid translocase (CD36) in a rat heart muscle cell line (H9c2).

Fatty acid translocase (FAT/CD36) is a membrane protein putatively involved in the transmembrane transport of long-chain fatty acids. We tested the hypothesis that expression of this protein in H9c2, a rat heart cell line normally not expressing FAT, would increase cellular palmitate uptake. We were able to stably transfect H9c2 cells with FAT, yielding 15 cell lines showing varying levels of FAT expression. The uptake and metabolism of palmitate was first studied in the non-transfected H9c2 cells and in two FAT-transfected cell lines. In each case, uptake of palmitate was found to be linear in time for at least 30 min and the uptake rate was saturable with increasing palmitate concentrations. Using conditions under which the maximal capacity of intracellular palmitate handling was not fully utilized, we tested 7 out of 15 FAT-transfected cell lines with varying FAT expression levels. No significant correlation was found between the level of FAT expression and the rate of palmitate uptake. In conclusion, we found that palmitate uptake by H9c2 cells occurs mainly by passive diffusion. Fatty acid translocase (FAT) transfection did not significantly increase the palmitate uptake rate, raising the possibility that H9c2 cells lack a protein (or set of proteins) that acts as an obligatory partner of FAT in long-chain fatty acid transport from the extracellular compartment to the cytoplasm.

Transport of long-chain fatty acids across the muscular endothelium.

Both skeletal and cardiac muscle cells rely heavily on the oxidation of long-chain fatty acids to utilize chemically stored energy for contractile work. Under normal conditions fatty acids are continuously supplied from the microvascular compartment to the contracting myocytes. Exogenous fatty acids are transported to muscle tissue via the blood either complexed to albumin or covalently bound in triacylglycerols forming the neutral lipid core of circulating lipoproteins such as chylomicrons or very low-density lipoproteins. The first barrier met by fatty acids on their way from the vascular compartment to the myocytes is the endothelium constituting the capillary wall. After dissociation of the albumin-fatty acid complex or release from the triacylglycerol core of lipoproteins, fatty acids most likely transverse the endothelium by crossing the luminal membrane, the cytosol, and subsequently the abluminal wall of the endothelial cell. Transfer through the interendothelial clefts or lateral diffusion within the phospholipid bilayer of the endothelial plasmalemma should be considered as inconsequential. The mechanism responsible for transmembrane movement of fatty acids is incompletely understood, although recent findings suggest the involvement of a number of membrane-associated proteins. Kinetic studies have revealed that interaction of the albumin-fatty acid complex with the endothelial membrane may accelerate the dissociation of the complex, which facilitates the uptake of fatty acids by the endothelium. Albumin-binding proteins (ABP) might be instrumental in this interaction. Moreover, plasmalemmal fatty acid-binding protein (FABPpm), fatty acid translocase (FAT) and fatty acid-transport protein (FATP) are putatively involved in transmembrane movement of the fatty acid molecules. Diffusion through the endothelial cytosol might be facilitated by a cytoplasmic fatty acid-binding protein, the type of which may be related to the epithelial fatty acid-binding protein (E-FAPBc).

Molecular mechanism of cellular uptake and intracellular translocation of fatty acids.

The molecular mechanism of the transport of long-chain fatty acids across cellular membranes and the necessity and precise functioning of specific proteins in this process are still unclear. Various alternative mechanisms have been proposed. Studies with artificial phospholipid bilayers support the concept that fatty acids may enter and traverse the plasma membrane without the involvement of proteins. On the other hand, a number of membrane-associated fatty acid-binding proteins (FABPs) have been described which putatively function as acceptors for fatty acids released from albumin or from lipoproteins. Albumin binding proteins located at the outer cell surface could play an additional role in the delivery of fatty acids. The subsequent transmembrane translocation of fatty acids could take place by a membrane protein acting as a translocase, or by simple diffusion of fatty acids through either the phospholipid bilayer or a pore or channel formed by one or more membrane fatty acid transporters. At the inner side of the plasma membrane, the fatty acid is bound to a cytoplasmic FABP, which serves to buffer the intracellular aqueous fatty acid concentration. The direction of fatty acid migration through the plasma membrane most likely is governed by the transmembrane gradient of fatty acid concentration, assisted to some extent and in selected tissues by co-transport of sodium ions. The intracellular transport of fatty acids from the plasma membrane to the sites of metabolic conversion (oxidation, esterification) or subcellular target (signal transduction) is greatly facilitated by cytoplasmic FABPs. In conclusion, cellular uptake and intracellular translocation of long-chain fatty acids is a multi-step process that is facilitated by various membrane-associated and soluble proteins. The mechanism of cellular uptake of fatty acids probably involves both a passive and carrier-mediated transmembrane translocation.

Uptake and metabolism of palmitate by isolated cardiac myocytes from adult rats: involvement of sarcolemmal proteins.

The precise mechanism of uptake of long-chain fatty acids (FA) by cardiac myocytes is incompletely understood. We examined the involvement of sarcolemmal proteins in the initial uptake of FA by isolated rat cardiac myocytes, and the relation between initial uptake and metabolism. Cardiac myocytes were incubated in the presence of 90 microns [1-14C]palmitate complexed to 300 microns bovine serum albumin (BSA), presenting a physiologically relevant condition. During initial palmitate uptake (3 min), 56% of the intracellularly sequestered palmitate was esterified, and an additional 21% converted into oxidation intermediates. Varying the palmitate/BSA molar ratio revealed saturation kinetics with the apparent Km for cellular palmitate uptake (435 micro M) to be comparable to those for esterification (465 micro M) and oxidation (222 micro M). Varying the BSA concentration at a fixed palmitate/BSA molar ratio also showed saturation of uptake at increasing concentrations, with an apparent Km for BSA of 23 micro M. Changes in palmitate metabolism induced by changes in glucose utilization were accompanied by identical effects on palmitate uptake. Addition of lactate also inhibited both oxidation and uptake of palmitate, but had no effect on esterification. Virtually complete inhibition of palmitate oxidation by etomoxir inhibited palmitate uptake for 50%, while decreasing esterification by 33%. In the presence of phloretin and trypsin, palmitate uptake and metabolism were inhibited 76-88%, and in the presence of sulfo-N-succinimidyloleate by 53%. It is concluded that a) the bulk of sarcolemmal palmitate translocation occurs by membrane-associated FA-binding proteins, most likely assisted by albumin binding proteins without regulatory function, and b) palmitate uptake is most likely driven by its rapid intracellular metabolic conversion.

Role of membrane-associated and cytoplasmic fatty acid-binding proteins in cellular fatty acid metabolism.

A number of membrane-associated and cytoplasmic fatty acid-binding proteins (FABPs) are now being implicated in the cellular uptake and intracellular transport of long-chain fatty acids (FA). These proteins each have the capacity of non-covalent binding of FA, are present in tissues actively involved in FA metabolism, and are upregulated in conditions of increased cellular FA metabolism. To date, five distinct membrane FABPs have been described, ranging in mass from 22 to 88 kDa and each showing a characteristic tissue distribution. Evidence for involvement in cellular fatty acid uptake has been provided for several of them, because it was recently found that isolated cell lines transfected with 88-kDa putative fatty acid translocase (FAT; homologous to CD36) or with 63-kDa fatty acid-transport protein show an increased rate of FA uptake. The (at least nine) FABPs of cytoplasmic origin belong to a family of small (14-15 kDa) lipid binding proteins, all having a similar tertiairy structure but differing in binding properties and in tissue occurrence. The biological functions of the various FABPs, possibly exerted in a concerted action among them, comprise solubilization and compartmentalization of FA, facilitation of the cellular uptake and intracellular trafficking of FA, and modulation of mitosis, cell growth, and cell differentiation. In addition, the FABPs have been suggested to participate in and/or modulate FA-mediated signal transduction pathways and FA regulation of gene expression, and to prevent local high FA concentrations thereby contributing to the protection of cells against the toxic effects of FA. In conclusion, long-chain fatty acids are subject to continuous interaction with multiple proteins, which interplay influences their cellular metabolism.

Release of proteins from isolated neonatal rat cardiomyocytes subjected to simulated ischemia or metabolic inhibition is independent of molecular mass.

This study addressed the question whether the molecular mass of proteins influences their release from isolated rat neonatal cardiomyocytes subjected to simulated ischemia (SI) or metabolic inhibition (MI). During these interventions cellular ATP content and the relative releases of several proteins, ranging in molecular mass from 15 to 140 kDa, were determined. After 180 min of normoxia, cellular ATP content was about 90% of the initial value, and cellular protein loss was about 1%. During either SI (180 min) or MI (120 min) the cellular ATP content decreased to less than 5% of the initial value. After 180 min of SI the release of soluble cytoplasmic proteins from the cells had increased to about 35%, and after 120 min of MI to about 90%. There were no major differences in the release pattern of four cytoplasmic proteins, during both SI and MI. A soluble mitochondrial and a partly mitochondrial protein, however, showed delayed release patterns. These data indicate that the release of proteins from damaged isolated neonatal rat cardiomyocytes is not related to the molecular mass of the proteins. It is concluded that protein release from damaged cardiomyocytes is not a sieving process in which small proteins are preferentially lost. In contrast, our data suggest that sarcolemmal disruption is a relatively fast process resulting in the simultaneous release of all soluble cytoplasmic proteins, irrespective of their molecular mass.

Membrane-associated and cytoplasmic fatty acid-binding proteins.

A number of cellular fatty acid-binding proteins are being implicated in the uptake and intracellular transport of long-chain fatty acids by parenchymal cells. Having been a topic of research for more than 20 years, cytoplasmic fatty acid-binding proteins now are assigned various pivotal functions in intracellular fatty acid transport and metabolism. More recently several membrane-associated fatty acid-binding proteins have been identified and these proteins are thought to function in the transmembrane transport of fatty acids. In this review, a short summary is provided of the latest developments in this research area.

Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein.

BACKGROUND: Myoglobin and fatty acid-binding protein (FABP) each are useful as early biochemical markers of muscle injury. We studied whether the ratio of myoglobin over FABP in plasma can be used to distinguish myocardial from skeletal muscle injury. METHODS AND RESULTS: Myoglobin and FABP were assayed immunochemically in tissue samples of human heart and skeletal muscle and in serial plasma samples from 22 patients with acute myocardial infarction (AMI), from 9 patients undergoing aortic surgery (causing injury of skeletal muscles), and from 10 patients undergoing cardiac surgery. In human heart tissue, the myoglobin/FABP ratio was 4.5 and in skeletal muscles varied from 21 to 73. After AMI, the plasma concentrations of both proteins were elevated between approximately 1 and 15 to 20 hours after the onset of symptoms. In this period, the myoglobin/FABP ratio was constant both in subgroups of patients receiving and those not receiving thrombolytics and amounted to 5.3 +/- 1.2 (SD). In serum from aortic surgery patients, both proteins were elevated between 6 and 24 hours after surgery; the myoglobin/FABP ratio was 45 +/- 22 (SD), which is significantly different from plasma values in AMI patients (P < .001). In patients with cardiac surgery, the ratio increased from 11.3 +/- 4.7 to 32.1 +/- 13.6 (SD) during 24 hours after surgery, indicating more rapid release of protein from injured myocardium than from skeletal muscles. CONCLUSIONS: The ratio of the concentrations of myoglobin over FABP in plasma from patients with muscle injury reflects the ratio found in the affected tissue. Since this ratio is different between heart (4.5) and skeletal muscle (20 to 70), its assessment in plasma allows the discrimination between myocardial and skeletal muscle injury in humans.

Putative membrane fatty acid translocase and cytoplasmic fatty acid-binding protein are co-expressed in rat heart and skeletal muscles.

A membrane protein (FAT) homologous to CD36 has recently been implicated in the binding and transport of long-chain fatty acids (FA). Expression of this protein in rat heart, skeletal muscles and in isolated cardiac cells was studied. Changes in expression during development of the heart were also examined. Expression of FAT was compared to that of the cytoplasmic fatty acid-binding protein (H-FABP) to determine whether coexpression, indicative of related biological functions, could be demonstrated. FAT and H-FABP mRNAs showed a similar muscle tissue distribution and similar cellular localization in the heart. During development, heart mRNA levels for both proteins were upregulated in the same way. In conclusion, expression of FAT and H-FABP in muscle tissues and cell-types with high FA metabolism and the upregulation of mRNA levels associated with heart development, when FA utilization increases, support the suggested role of both proteins in FA metabolism.