Lack of Myoglobin Causes a Switch in Cardiac Substrate Selection

    the Institut fe Herz-und Kreislaufphysiologie (U.F., T.L., A.G., N.A., C.D.F., C.J., J.S.), Heinrich-Heine-Universitt De箂seldorf
    Klinik und Poliklinik fe Nuklearmedizin (M.S.), Universittsklinikum Mester
    Biomedizinisches Forschungszentrum (S.M.), Heinrich-Heine-Universitt De箂seldorf
    Institut fe Pathophysiologie, Zentrum fe Innere Medizin (B.L.), Universittsklinikum Essen, Germany.

    Abstract

    Myoglobin is an important intracellular O2 binding hemoprotein in heart and skeletal muscle. Surprisingly, disruption of myoglobin in mice (myoeC/eC) resulted in no obvious phenotype and normal cardiac function was suggested to be mediated by structural alterations that tend to steepen the oxygen pressure gradient from capillary to mitochondria. Here we report that lack of myoglobin causes a biochemical shift in cardiac substrate utilization from fatty acid to glucose oxidation. Proteome and gene expression analysis uncovered key enzymes of mitochondrial -oxidation as well as the nuclear receptor PPAR to be downregulated in myoglobin-deficient hearts. Using FDG-PET we showed a substantially increased in vivo cardiac uptake of glucose in myoeC/eC mice (6.7±2.3 versus 0.8±0.5% of injected dose in wild-type, n=5, P<0.001), which was associated with an upregulation of the glucose transporter GLUT4. The metabolic switch was confirmed by 13C NMR spetroscopic isotopomer studies of isolated hearts which revealed that [1,6-13C2]glucose utilization was increased in myoeC/eC hearts (38±8% versus 22±5% in wild-type, n=6, P<0.05), and concomitantly, [U-13C16]palmitate utilization was decreased in the myoglobin-deficient group (42±6% versus 63±11% in wild-type, n=6, P<0.05). Because of the O2-sparing effect of glucose utilization, the observed shift in substrate metabolism benefits energy homoeostasis and therefore represents a molecular adaptation process allowing to compensate for lack of the cytosolic oxygen carrier myoglobin. Furthermore, our data suggest that an altered myoglobin level itself may be a critical determinant for substrate selection in the heart. The full text of this article is available online at http://circres.ahajournals.org.

    Key Words: metabolism  -oxidation  glucose  oxygen  heart

    Introduction

    It is well known that red and white muscle are not only characterized by a largely different content of myoglobin (Mb), but also by significant differences in metabolism closely related to their physiological function.1,2 Red muscles exhibit slow twitch speed, are fatigue resistant, and have an aerobic fat-, glucose-, and ketone-based metabolism. In contrast, white muscle fibers are fast contracting anaerobic fibers and easily fatigued because they have few respiratory proteins and metabolize glucose only as far as lactate. Similar to the red skeletal muscle, the heart has a high, enduring energy demand, which under normal conditions is primarily met by metabolism of fatty acids (FAs).3 Nevertheless, in several cardiac diseases, such as ischemic cardiomyopathy, heart failure, hypertrophy, and dilated cardiomyopathy, a reduced oxidation of FAs and an enhanced glucose utilization has been found.4 Interestingly, dilated and ischemic cardiomyopathies have also been reported to be accompanied by a decreased myocardial Mb content.5 However, whether there is more then a mere correlation between muscle Mb level and substrate metabolism has not been explored so far.

    In mice lacking Mb (myoeC/eC), multiple compensatory mechanisms are induced that tend to steepen the oxygen pressure gradient to the mitochondria.6eC8 These include a higher capillary density, reduction in cell width, elevated hematocrit, increased coronary flow, and coronary flow reserve. However, substrate utilization was reported to be preserved in the absence of Mb, although in vitro, a modest increase in lactate utilization in the Mb mutant heart was noted.8 To address the role of Mb in substrate selection of the heart in more detail, we utilized positron emission tomography (PET) using [18F]fluorodeoxyglucose (FDG) and 13C nuclear magnetic resonance (NMR) spectroscopy using [1,6-13C2]glucose and [U-13C16]palmitate to study cardiac intermediary metabolism in wild-type (WT) and Mb-deficient mice generated in our laboratory.7 Furthermore, we analyzed the myocardial protein pattern of WT and myoeC/eC mice by 2-dimensional gel electrophoresis (2D-PAGE) and identified differentially expressed proteins by mass spectrometry. Additionally, we verified whether alterations at the protein level are related to a gene regulatory switch. We found that similar to skeletal muscle, lack of Mb causes a switch in cardiac substrate selection from fatty acid to glucose utilization that is accompanied by a downregulation of key enzymes of the -oxidation pathway.

    Materials and Methods

    Animals

    MyoeC/eC mice were generated in our laboratory by deletion of the essential exon-2 via homologous recombination in embryonic stem cells as described previously.7 Animal experiments were performed in accordance with the national guidelines on animal care and were approved by the local government. Before all metabolic experiments mice were fed with a standard chow diet and received tap water ad libitum.

    Heart Perfusion for Metabolic Analysis

    Preparation of murine hearts and retrograde perfusion at constant pressure of 100 mm Hg was performed essentially as described.9 After hemodynamic and contractile parameters maintained constant under these conditions, hearts were finally switched to buffer containing 5 mmol/L [1,6-13C2]glucose, 0.5 mmol/L [U-13C16]palmitate (the latter bound to 3% essentially fatty acid free albumin; both from Cambridge Isotope Laboratories), and 50 e蘒 insulin. After 25 minutes of perfusion, hearts were freeze-clamped, extracted with perchloric acid (PCA), neutralized, lyophilized, and stored at eC20°C. A more detailed description of the heart perfusion protocol is provided in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.

    Magnetic Resonance Measurements

    Data were recorded on a Bruker DRX 9.4 Tesla WB NMR spectrometer operating at frequencies of 400.1 MHz for 1H and 100.6 MHz for 13C measurements.

    Spectroscopy

    Lyophilized PCA extracts were redissolved in 0.5 mL D2O. Spectra were recorded from a 5-mm 1H/13C dual probe. Acquisition and processing parameters are given in detail in the online data supplement.

    Isotopomer Analysis of Carbon Flow Into the Tricarboxylic Acid Cycle

    The relative contributions of palmitate, glucose, and endogenous sources to the total acetyl-CoA pool entering the tricarboxylic acid (TCA) cycle were determined from isotopomer analysis of glutamate carbons C3 and C4. Consult the online data supplement for a detailed description of the analysis of 13C NMR spectra.

    Imaging

    MRI was performed using a microimaging unit (Mini 0.5, Bruker) as described in the online data supplement.

    High-Resolution PET

    Myocardial glucose transport was noninvasively assessed in vivo by monitoring the uptake of FDG in intact mice, which before PET analysis had been functionally and morphologically characterized by MRI. Mice were anesthetized with isoflurane (1.5%) and kept at 37°C. Each mouse was injected with 10 MBq FDG in 100 e蘈 0.9% saline intravenously. After 60 minutes, mice were positioned on the bed of a submillimeter-resolution PET camera (quad-HIDAC, Oxford Positrons Ltd), and a 15-minute acquisition was initiated. Coronal images were then reconstructed (voxel size 0.25 mm3, 0.7 mm full-width half-maximum). Myocardial FDG uptake was quantified by the ratio between myocardial radioactivity in a region-of-interest encompassing the left ventricular myocardium and the total injected dose (% injected dose, % ID).

    Blood Serum

    Determination of glucose, lactate, and fatty acids in blood serum was performed by the Central Laboratory of the University Hospital De箂seldorf using clinical routine protocols.

    Proteome Analysis

    Sample preparation and 2D-PAGE were essentially performed as previously reported10 and a more detailed description is provided in the online data supplement. SDS-PAGE was performed on an ETTAN-DALT II vertical electrophoresis unit (Amersham Pharmacia Biotech). Equilibrated IPG strips were placed on top of the gel (size of 0.1x25.5x20 cm3) and fixed in place by agarose sealing solution. Twelve gels were run simultaneously (settings: 30 minutes, 30 W; 4.5 hours, 180 W) and either silver-stained or stained by Coomassie blue R250. The gels were digitized and the obtained images analyzed as described in the online data supplement.

    Protein Identification

    Coomassie-stained spots were excised from preparative gels and analyzed by nano spray ESI-MS/MS using a SCIEX Q-STAR system (PE Sciex), as previously described.10

    Expression Analysis

    Expression levels were analyzed by real-time PCR using an ABI SDS 5700 real-time PCR analysis system on reverse-transcribed myocardial RNA isolated from 8 WT and myoeC/eC hearts, respectively. cDNA derived from 100 ng of total RNA was used for each reaction. Signals were amplified using the Taqman based assays on demand (ABI) for PPAR (Mm00440939_m1), short chain enoyl-CoA hydratase (Mm00659670_g1), and short chain acyl-CoA dehydrogenase (Mm00431617_m1) according to the suppliers instructions. Relative expression levels were determined by normalization to transferrin receptor (Mm00441941_m1) and TATA binding protein (Mm00446973_m1) as housekeeping genes.

    Cell Fractionation and Western Analysis of GLUT4 Expression

    Hearts were isolated from WT and myoeC/eC mice and separated into membranous and cytosolic fractions according to published procedures.11 For Western analysis, 10 e of membrane proteins and the corresponding volume fractions of the 30 000g supernatant were analyzed by Western blotting using a polyclonal rabbit anti-GLUT4 antibody (Abcam Ltd; 1:2500) followed by secondary HRPO-coupled goat anti-rabbit antibody (Sigma Heidelberg; 1:5000). Signals were detected by use of an ECL-kit (Amersham Biosciences).

    Statistical Analysis

    All results are expressed as mean±SD. For multiple comparisons, ANOVA followed by the Bonferroni correction was applied. A probability value of less than 0.05 was considered significant. The statistical analysis of the raw data from 2D-PAGE experiments was performed by the program "statistical analysis of microarrays, SAM."12 The parameter " value" was adjusted in order to keep the number of false significant protein spots (90%) below 1. In a nonpaired t test, this translates to approximately P<0.005 for n=12. Additionally, changes in protein expression below 30% were considered to be of low biological relevance.

    Results

    13C NMR Isotopomer Analysis

    For analysis of the contribution of FA and glucose oxidation to TCA cycle turnover, isolated hearts paced at 500 bpm were perfused for 25 minutes with 5 mmol/L [1,6-13C2]glucose and 0.5 mmol/L [U-13C16]palmitate in the presence of 50 e蘒 insulin. Under these conditions, left ventricular developed pressure was equal in both groups (114.4±9.8 mm Hg in WT versus 115.0±10.4 mm Hg in Mb-deficient hearts, n=8). However, despite unrestricted cardiac function, we found myocardial oxygen consumption to be reduced by 7.5% in the transgenic group (15.63±1.43 eol · mineC1 · geC1 in WT versus 14.46±1.37 eol · mineC1 · geC1 in myoeC/eC, n=8; P=0.09). High-resolution 13C NMR spectra of the respective PCA extracts (Figure 1A) showed pronounced differences in the isotopomer pattern of the glutamate carbon C4 of WT (bottom) and myoeC/eC (top) hearts. The sum of the resonances (S+D34) reports the amount of acetyl-CoA derived from [1,6-13C]glucose (entry of [2-13C]acetyl-CoA into the TCA cycle), and the sum of (D45+Q) reflects the amount of acetyl-CoA derived from [U-13C16]palmitate (entry of [1,2-13C2]acetyl-CoA), irrespective of any cycling intermediate or pool sizes.13 Thus, in the example shown in Figure 1A, it is obvious that in Mb-deficient hearts, the amount of carbons incorporated into glutamate that originate from glucose were increased whereas carbons originating from palmitate were decreased compared with WT hearts.

    Quantitative analysis of the 13C NMR spectra (Figure 1B) revealed that glucose utilization was significantly increased in myoeC/eC hearts (38±8% versus 22±5% in WT, n=6; P<0.05), and concomitantly, palmitate utilization was significantly decreased in the Mb-deficient group (42±6% versus 63±11% in WT, n=6; P<0.05), whereas the contribution of endogenous substrates (ie, glycogen or unlabeled glucose and FAs) was similar in both groups. Furthermore, no differences were observed in pool sizes of metabolites (cf. Table I in the online data supplement) and fractional enrichments (data not shown) of alanine, glutamate, and lactate between WT and myoeC/eC hearts.

    In Vivo MRI and PET Analysis

    In order to verify whether the enhanced glucose metabolization found in isolated perfused hearts of myoeC/eC mice can also be observed under in vivo conditions, myocardial glucose transport was noninvasively assessed by monitoring the uptake of FDG in intact mice. Before PET analysis, all mice were initially characterized by MRI, which showed similar values for diastolic and systolic volumes as well as cardiac output in both groups (Figure 2, left, and online Table II). Despite being morphologically and functionally undistinguishable, FDG-PET revealed significant differences between WT and myoeC/eC mice (Figure 2, right) in that myocardial FDG uptake was substantially enhanced in Mb-deficient mice (6.7±2.3%ID versus 0.8±0.5%ID in WT, n=5; P<0.001).

    Analysis of Blood Serum Substrates

    Both 13C NMR and PET data indicate a shift to increased glucose and reduced fatty acid utilization in transgenic hearts. To clarify whether these alterations may have been caused by differences in serum substrate concentrations, we determined the amounts of glucose, lactate, and various FAs (16:0, 16:1, 18:0, 18:1, 20:0, 20:1, 20:2, 20:3, and 20:4) in WT and myoeC/eC mice (n=6). However, there were no significant differences between the 2 groups concerning all parameters analyzed (data are given in online Table III).

    Proteome Analysis

    To investigate whether the observed alterations in cardiac metabolism are related to changes in myocardial protein expression, proteome patterns of WT and myoeC/eC mice were analyzed by 2D-PAGE (see Figure II in the online data supplement for representative gels). Aside from Mb, 21 protein species were found to be differentially expressed when comparing myoeC/eC with WT samples (online Table IV). Noticeably, more than half of the altered proteins are involved in intermediary metabolism (Figure 3), and 9 of these are part of the mammalian mitochondrial -oxidation pathway,14 which is illustrated in the schematic drawing of Figure 4. The first step of the -oxidation spiral is catalyzed by acyl-CoA dehydrogenases (DHs), which lead to the oxidation of acyl-CoA to enoyl-CoA by FAD. Two members of this enzyme family, short-chain and isovaleryl acyl-CoA DH, were reduced in expression by  50%. The next steps of -oxidation are mediated by enoyl-CoA hydratases: two forms of the short-chain FA selective soluble enzyme were decreased in expression by 58% and 33% in myoeC/eC hearts.

    The membrane-bound enoyl-CoA hydratase (selective for long-chain FAs) is part of the -subunit of mitochondrial trifunctional protein (TFP), which combines 3 long-chain (LC) FA selective enzymatic activities: LC enoyl-CoA hydratase and LC 3-hydroxyacyl-CoA DH, both located on the -subunit, as well as LC 3-ketoacyl-CoA-thiolase, located on the -subunit.15 Whereas 2D-PAGE analysis did not reveal a difference in expression of the intact - and -subunits of TFP between the groups, 4 cleavage fragments of -TFP were prominently found on gels of Mb mutant hearts (Figure 3). Similarly, fragments of the -subunit were clearly visible in myoeC/eC samples. Western blot analysis using a polyclonal antibody raised against the -subunit of TFP16 revealed a downregulation of the intact subunit by 30% (n=9 per group, P<0.05). Furthermore, we found the membrane-bound enzyme electron-transferring flavoprotein DH (ETF DH), which feeds the reduction equivalents into the respiratory chain (Figure 4) to be downregulated by 80% in the knockout (Figure 3). On the other hand, the glycolytic enzyme glyceraldehyde 3-phosphate DH was significantly upregulated (+420%) in myoeC/eC hearts (Figure 3). For a more detailed description of the proteome data including the other differentially expressed proteins refer to the online data supplement.

    Because GLUT4 is the major transport system for uptake of glucose into cardiomyocytes,17 we further verified whether the enhanced glucose utilization in Mb-deficient hearts is associated with alterations in overall cardiac GLUT4 expression and/or translocation of this transporter from the cytosol to the plasma membrane. Western blot analysis revealed GLUT4 expression to be significantly increased in both the cytosolic and the membranous fraction of myoeC/eC as compared with WT heart extracts (Figure 5). However, it is noteworthy that the relative raise of the transporter in the plasma membrane (60%) is more pronounced than in the cytosol (20%), which reflects in addition to an increased expression an enhanced translocation of GLUT4 into the membrane.

    Gene Expression

    We further analyzed whether the altered expression of enzymes of FA oxidation relates to a gene regulatory switch. For this purpose, we measured mRNA levels of a subset of proteins that were downregulated in Mb-deficient hearts and, additionally, the expression of the nuclear receptor PPAR (peroxisome proliferator activated receptor ), which has been shown to be a key factor in regulation of several genes involved in -oxidation of fatty acids.18 Transcripts of short-chain acyl-CoA DH and enoyl-CoA hydratase were found to be reduced in myoeC/eC hearts by 40% to 50% (Figure 6), which fits well to the alterations observed at the translated protein level (Figure 3). Furthermore, expression of PPAR was decreased by a similar extent (40%) in the transgenic group (Figure 6, right; WT: 10.5±2.4 AU, myoeC/eC: 6.1±1.0 AU, n=8; P<0.01).

    Discussion

    The results of the present study show that lack of Mb causes a shift from free FA to glucose oxidation in cardiac energy production. Enhanced glucose uptake in myoeC/eC hearts was noninvasively visualized by FDG-PET in vivo and the substrate switch was confirmed by 13C NMR isotopomer studies of isolated hearts. Protein and gene expression analysis demonstrated that the most abundant glucose transporter in the heart, GLUT4, is upregulated whereas important enzymes of mitochondrial -oxidation and the key regulator of genes involved in FA metabolism, PPAR, are downregulated in Mb-deficient hearts. Based on these data and the finding that cardiac structure and function remained fully unchanged in myoeC/eC hearts, it appears that Mb is a critical determinant of cardiac substrate selection.

    Quantitative evaluation of the individual substrate fluxes in WT hearts revealed a utilization of FAs more than glucose in the order of 3:1, which is close to the normal situation in humans.3 In contrast, myoeC/eC hearts used approximately equal amounts of FAs and glucose. This shift in substrate utilization in myoeC/eC hearts resembles the known differences in metabolism of red and white muscle. Early investigations on the enzyme pattern of white (fast) and red (slow) muscles showed that white muscle fibers contain high amounts of glycolytic enzymes, whereas red muscles predominantly express enzymes of the -oxidation pathway.1 The analogy to skeletal muscle is further supported by the observed differences in expression level of -(B)-crystallin in myoeC/eC and WT hearts (online Table IV), which is also similar to that found between white and red muscle.19

    The correlation of myocardial Mb content with the capacity of -oxidation has been described in previous studies under both physiological and pathophysiological conditions. In a recent study of the heterogeneity of cardiac flow and metabolism, we showed that certain areas in the well-perfused dog heart that normally receive <50% of mean myocardial blood flow exhibited reduced Mb levels accompanied by decreased expression of enzymes of the -oxidation pathway and enhanced expression of glycolytic enzymes.10 Studies in canine and bovine models of dilated cardiomyopathy also demonstrated a reduced expression of Mb and concomitant upregulation of glycolytic and/or downregulation of -oxidation enzymes.20,21 Together with the results of the present work, these data suggest a causal relationship between myocytic Mb content and -oxidation of fatty acids. As to the molecular mechanisms involved, several possibilities must be considered by which Mb might directly or indirectly affect muscle substrate selection.

    Mb is generally thought not only to provide the O2 needed for aerobic muscle metabolism and to augment the flow of O2 to the mitochondria but also to buffer intracellular O2 concentrations in response to mitochondrial demand.22 A possible mismatch between O2 requirement and O2 supply because of the lack of Mb could be overcome by a shift from FA oxidation to O2-sparing glucose utilization. It is well known that the complete oxidation of FAs consumes more O2 per mole energy-rich phosphate than the complete oxidation of glucose. On basis of the respective phosphate-to-oxygen ratios (P:O) myocardial O2 consumption (MVO2) can be assumed to increase by 10% when FAs are exclusively utilized as compared with glucose.23 This value, however, may be an underestimation, because it has been recently demonstrated in the in vivo unloaded myocardium that FA usage requires 48% more O2 when compared with glucose.24 MVO2 measurements in the present study have shown an oxygen saving of 7.5% (at a shift of glucose/FA utilization from 1:3 to an equal ratio), which supports the notion of the latter study that the benefit of using glucose may be considerably higher as calculated on basis of the P:O ratios. Thus, a shift toward glucose oxidation will improve the O2 balance in the Mb-deficient heart and may be considered as a molecular adaptation mechanism in the myoeC/eC heart.

    Besides its function in O2 storage and transport, Mb has also been suggested to support ATP generation by cardiac cells under conditions of fully oxygenated Mb: a phenomenon referred to as Mb-mediated oxidative phosphorylation.25 As an underlying mechanism, a preferred uptake of Mb-bound O2 by mitochondria and/or the acceptance of electrons by sarcoplasmic Mb with concomitant reduction of heme iron-ligated O2 to H2O were suggested. However, because an enhanced oxidative phosphorylation would support aerobic oxidation of both glucose and FAs, it is rather unlikely that an impaired Mb-mediated oxidative phosphorylation causes the metabolic shift in hearts lacking Mb.

    In addition to its function as respiratory pigment, Mb of bovine, chicken, and rat muscle was shown to bind FAs.26eC28 Therefore, Mb has been suggested to function as transport protein for FAs in the cytosol working in concert with the well-known fatty acid binding protein, which is generally assumed to be the major player.29 Interestingly, FA binding of Mb depends on its oxygenation in that conformational changes induced by O2 binding favor the interaction of Mb with FAs.28 Although the functional relevance of FA binding to Mb remains to be explored, the simultaneous delivery of O2 and FAs to mitochondria would clearly be advantageous for aerobically working muscle. According to this hypothesis, lack of Mb as putative FA carrier would supply less substrate to the mitochondria, thereby triggering the downregulation of -oxidation to total energy production of myoeC/eC hearts.30

    Because we have previously shown that Mb substantially contributes to NO breakdown in the heart,31 it is conceivable that increased levels of NO as a result of diminished NO degradation may affect cardiac metabolism in Mb-deficient hearts. Indeed, it has been demonstrated that NO is involved in stimulation of glucose uptake and metabolism (eg, via GLUT4 translocation) particularly in skeletal32 but also in heart muscle.33,34 Furthermore, it has also been described that endogenous NO reduces O2 use in excitation-contraction coupling and attenuates cardiac contractility without changing contractile efficiency.35 This could further contribute to improved O2 balance in the Mb-deficient heart.

    It is becoming increasingly evident that protein-protein interactions within the living cell are important in various cellular signal transduction pathways, and that in vivo, many proteins do not work by themselves but, in most cases, by forming a complex or interacting with other proteins, DNA, RNA, or ligands (interactome).36 However, little is known about the interactome of Mb, albeit dynamic docking and electron transfer between Mb and cytochrome b5 have lately been discovered.37 Furthermore, there is recent evidence that the ferric form of neuroglobin, which is homologous to Mb, acts as a heterotrimeric G protein guanine nucleotide dissociation inhibitor thereby shutting off signaling pathways linked to G effectors and favoring G effector pathways leading to protection against neuronal death.38 Because Mb is present in the cytosol in high concentrations (up to 0.5 mmol/L) it is quite conceivable that multiple interactions with other cellular proteins exist, which—when lacking—may cause metabolic rearrangements.

    Taken together the combined effect of Mb’s different biological functions is likely to trigger the changes in intermediary metabolism when Mb is lacking. Decreased mitochondrial O2 availability and increased amounts of bioactive NO may act as complementary players in this process: because lack of Mb results in an enhanced vulnerability to mild or short periods of hypoxic/ischemic conditions,39 it is conceivable that even the unstressed Mb-deficient heart is characterized by a "microhypoxic" environment promoting the expression of hypoxia-responsive genes,8 which have been described to mediate inhibition of expression of the nuclear receptor PPAR.40 The NO-induced stimulation of expression and translocation of GLUT434 together with the recently proposed regulatory "crosstalk" between PPAR signaling and GLUT4 gene expression41 could provide the molecular framework of the observed substrate switch to an increased glucose utilization. Note that, conversely, transgenic mice with cardiac-specific overexpression of PPAR exhibit increased fatty acid uptake and oxidation as well as reciprocal inhibition of glucose uptake and metabolism.42

    Similar shifts in cardiac substrate selection as described in this study for the myoeC/eC mouse have been frequently reported to occur during hypertrophy and several other cardiac diseases (see reviews43,44). It has been postulated that this rearrangement of cardiac energy production contributes to the compensated state during progression to heart failure.43,44 Metabolic remodeling has been as yet seen as part of the reactivation of the fetal gene expression program typically observed during development of left ventricular hypertrophy. However, there is no evidence for an upregulation of atrial natriuretic peptide and skeletal muscle actin or other markers of the fetal gene expression program in myoeC/eC hearts.45 Furthermore, it should be noted that in this and previous studies we and others found no indication for an impaired cardiac function in Mb-deficient mice under normal conditions.6,7 Therefore, the Mb knockout mouse clearly illustrates that metabolic remodeling represents an important compensatory mechanism that can be activated independent of the fetal gene expression program. Thus, this model can be used in future studies to determine the potentially protective role of increased glucose utilization in certain pathological states like ischemia and/or diabetes without interference of mechanisms governed by the reactivation of the fetal gene expression program.

    In summary, our data show that lack of Mb leads to an enhanced glucose and a decreased FA utilization in the mouse heart. Because equimolar production of ATP from glucose consumes less O2 than from FAs, this metabolic switch may be viewed as an additional adaptive mechanism in myoeC/eC hearts. The changes in substrate selection of Mb-deficient hearts resemble the well-known differences in metabolic pattern of red and white muscle. Our data suggest that an altered Mb level by itself is one crucial factor that determines the relative utilization of FAs versus glucose, thus placing Mb in a central stage within the regulatory network that controls cardiac energy production.

    Acknowledgments

    This study was supported by the Biologisch-Medizinisches Forschungszentrum of the Heinrich-Heine-Universitt De箂seldorf, the Sonderforschungsbereich 612 "Molekulare Analyse kardiovaskulrer Funktionen and Funktionsstrungen", Teilprojekt Z2, and in part by the Interdisziplinres Zentrum fe Klinische Forschung (IZKF), Mester, Germany (grant ZPG 4 to M.S.). We thank D. Haubs, S. Ke箂ters, and C. Kirberich for excellent technical assistance, and R. Hoffmann and S. Lauter for their expert help in protein identification by ESI-MS/MS. We thank A. Strauss from the Department of Pediatrics at Vanderbilt University, Nashville, Tenn, for providing us with anti-TFP antibody.

    Both authors contributed equally to this work.

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