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(Circulation Research. 2005;96:e68.)
© 2005 American Heart Association, Inc.

UltraRapid Communications

Lack of Myoglobin Causes a Switch in Cardiac Substrate Selection

Ulrich Flögel^*, Tim Laussmann^*, Axel Gödecke, Nadine Abanador, Michael Schäfers, Christian Dominik Fingas, Sabine Metzger, Bodo Levkau, Christoph Jacoby, Jürgen Schrader

From the Institut für Herz-und Kreislaufphysiologie (U.F., T.L., A.G., N.A., C.D.F., C.J., J.S.), Heinrich-Heine-Universität Düsseldorf; Klinik und Poliklinik für Nuklearmedizin (M.S.), Universitätsklinikum Münster; Biomedizinisches Forschungszentrum (S.M.), Heinrich-Heine-Universität Düsseldorf; and Institut für Pathophysiologie, Zentrum für Innere Medizin (B.L.), Universitätsklinikum Essen, Germany.

Correspondence to Dr Ulrich Flögel, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany. E-mail floegel{at}uni-duesseldorf.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myoglobin is an important intracellular O₂ binding hemoprotein in heart and skeletal muscle. Surprisingly, disruption of myoglobin in mice (myo^–/–) 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 ß-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 myo^–/– 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 myo^–/– 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 • ß-oxidation • glucose • oxygen • heart

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (myo^–/–), multiple compensatory mechanisms are induced that tend to steepen the oxygen pressure gradient to the mitochondria.^6–8 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 myo^–/– 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 ß-oxidation pathway.

	Materials and Methods

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Animals
Myo^–/– 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 µU insulin. After 25 minutes of perfusion, hearts were freeze-clamped, extracted with perchloric acid (PCA), neutralized, lyophilized, and stored at –20°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 µL 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 Düsseldorf 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 myo^–/– 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 myo^–/– mice and separated into membranous and cytosolic fractions according to published procedures.¹¹ For Western analysis, 10 µg 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µU 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 µmol · min^–1 · g^–1 in WT versus 14.46±1.37 µmol · min^–1 · g^–1 in myo^–/–, 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 myo^–/– (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.

View larger version (21K): [in this window] [in a new window]   Figure 1. A, Representative sections of 13C NMR spectra showing the glutamate C4 isotopomer pattern for WT and myo–/– PCA heart extracts. Hearts 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 µU insulin. D34 indicates doublet because of J34 coupling (34 Hz); D45, doublet because of J45 coupling (51 Hz); Q, quartet (doublet of doublet) because of J345 coupling (J34 34 Hz, J45 51 Hz); S, singlet. B, Analysis of carbon flow into the TCA cycle in WT and myo–/– hearts under the conditions given above. Data are mean±SD (n=6, *P<0.05 vs WT). By definition, FPalmitate+FGlucose+FEndogenous=1.

Quantitative analysis of the ¹³C NMR spectra (Figure 1B) revealed that glucose utilization was significantly increased in myo^–/– 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 myo^–/– hearts.

In Vivo MRI and PET Analysis
In order to verify whether the enhanced glucose metabolization found in isolated perfused hearts of myo^–/– 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 myo^–/– 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).

View larger version (50K): [in this window] [in a new window]   Figure 2. Matched noninvasive functional and metabolic imaging of WT (bottom) and myo–/– (top) mice in vivo. MRI (left panel, coronal slices) shows normal cardiac morphology and function (cf. online Table II) in WT and myo–/–, whereas myocardial glucose uptake (right panel, matching coronal slices) measured by FDG-PET is markedly increased in myo–/– as compared with WT.

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 myo^–/– 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 myo^–/– 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 myo^–/– 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 ß-oxidation pathway,¹⁴ which is illustrated in the schematic drawing of Figure 4. The first step of the ß-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 ß-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 myo^–/– hearts.

View larger version (27K): [in this window] [in a new window]   Figure 3. Differentially expressed proteins in WT vs myo–/– mice related to intermediary metabolism. For the complete list of altered proteins, refer to online Table IV. Data are mean±SD (n=12, *P<0.005, **P<0.0005, ***P<0.00005 vs WT). DH indicates dehydrogenase; ETF, electron transferring flavoprotein; GAP, glyceraldehyde 3-phosphate; HI/HII, hydrogenase isoform I/II; IV, isovaleryl; SC; short chain; TFP, trifunctional protein.

View larger version (71K): [in this window] [in a new window]   Figure 4. Schematic drawing of key steps of mitochondrial ß-oxidation incorporating the observed alterations in protein expression of myo–/– hearts (changes in percent relative to WT hearts). DH indicates dehydrogenase; ETF, electron transferring flavoprotein; IV, isovaleryl; SC; short chain; TFP, trifunctional protein; VLC, very long chain.

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 ß-subunit.¹⁵ Whereas 2D-PAGE analysis did not reveal a difference in expression of the intact - and ß-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 ß-subunit were clearly visible in myo^–/– samples. Western blot analysis using a polyclonal antibody raised against the ß-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 myo^–/– 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 myo^–/– 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.

View larger version (45K): [in this window] [in a new window]   Figure 5. GLUT4 expression in membranous and cytosolic fractions of WT and myo–/– hearts. A, Representative Western blots. B, Densitometric quantification. Data are mean±SD (n=8, *P<0.05 vs WT)

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 ß-oxidation of fatty acids.¹⁸ Transcripts of short-chain acyl-CoA DH and enoyl-CoA hydratase were found to be reduced in myo^–/– 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, myo^–/–: 6.1±1.0 AU, n=8; P<0.01).

View larger version (24K): [in this window] [in a new window]   Figure 6. mRNA levels of selected proteins in hearts of WT and myo–/– mice. Data are mean±SD (n=8, *P<0.05, **P<0.01 vs WT). DH indicates dehydrogenase; H, hydrogenase, PPAR, peroxisome proliferator activated receptor; SC; short chain.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 myo^–/– 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 ß-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 myo^–/– 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, myo^–/– hearts used approximately equal amounts of FAs and glucose. This shift in substrate utilization in myo^–/– 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 ß-oxidation pathway.¹ The analogy to skeletal muscle is further supported by the observed differences in expression level of -(B)-crystallin in myo^–/– 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 ß-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 ß-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 ß-oxidation enzymes.^20,21 Together with the results of the present work, these data suggest a causal relationship between myocytic Mb content and ß-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 myo^–/– 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.^26–28 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 ß-oxidation to total energy production of myo^–/– 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 myo^–/– mouse have been frequently reported to occur during hypertrophy and several other cardiac diseases (see reviews^43,44). 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 myo^–/– 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 myo^–/– 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-Universität Düsseldorf, the Sonderforschungsbereich 612 "Molekulare Analyse kardiovaskulärer Funktionen and Funktionsstörungen", Teilprojekt Z2, and in part by the Interdisziplinäres Zentrum für Klinische Forschung (IZKF), Münster, Germany (grant ZPG 4 to M.S.). We thank D. Haubs, S. Küsters, 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.

	Footnotes

 
*Both authors contributed equally to this work.

Original received May 11, 2004; resubmission received March 3, 2005; revised resubmission received March 24, 2005; accepted March 28, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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