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Am J Physiol Heart Circ Physiol 294: H936-H943, 2008. First published December 14, 2007; doi:10.1152/ajpheart.00870.2007
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Overexpression of pyruvate dehydrogenase kinase 4 in heart perturbs metabolism and exacerbates calcineurin-induced cardiomyopathy

Guixiang Zhao,1 Nam Ho Jeoung,6 Shawn C. Burgess,5 Kimberly A. Rosaaen-Stowe,5 Takeshi Inagaki,1 Shuaib Latif,2 John M. Shelton,3 John McAnally,1 Rhonda Bassel-Duby,1 Robert A. Harris,6 James A. Richardson,3 and Steven A. Kliewer1,4

Departments of 1Molecular Biology, 2Internal Medicine, 3Pathology, and 4Pharmacology and 5Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas; and 6Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 25 July 2007 ; accepted in final form 3 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The heart adapts to changes in nutritional status and energy demands by adjusting its relative metabolism of carbohydrates and fatty acids. Loss of this metabolic flexibility such as occurs in diabetes mellitus is associated with cardiovascular disease and heart failure. To study the long-term consequences of impaired metabolic flexibility, we have generated mice that overexpress pyruvate dehydrogenase kinase (PDK)4 selectively in the heart. Hearts from PDK4 transgenic mice have a marked decrease in glucose oxidation and a corresponding increase in fatty acid catabolism. Although no overt cardiomyopathy was observed in the PDK4 transgenic mice, introduction of the PDK4 transgene into mice expressing a constitutively active form of the phosphatase calcineurin, which causes cardiac hypertrophy, caused cardiomyocyte fibrosis and a striking increase in mortality. These results demonstrate that cardiac-specific overexpression of PDK4 is sufficient to cause a loss of metabolic flexibility that exacerbates cardiomyopathy caused by the calcineurin stress-activated pathway.

fatty acid; hypertrophy; transgenic mice

THERE IS A GROWING AWARENESS that systemic perturbations in energy metabolism such as those that occur in diabetes mellitus and other forms of metabolic disease contribute to cardiovascular disease (9). The healthy heart is a metabolic omnivore capable of switching between fatty acids and carbohydrates to match energy demands with dietary and physiological conditions (30, 31). Under conditions of pressure overload and cardiac hypertrophy, increased carbohydrate oxidation is part of the adaptive response to increased workload (21). However, in diabetes the metabolic flexibility of the heart is diminished, and it becomes more reliant on fatty acids for energy (10, 21, 30, 31). This may contribute to functional derangements by causing oxidative stress and the accumulation of harmful lipid intermediates (10, 21, 30, 31).

In heart and other tissues, competition between fatty acids and carbohydrates occurs at the level of pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA, thereby linking glycolysis to the Krebs cycle and ATP production. PDH is active when glucose oxidation prevails for the generation of energy and repressed when glucose is in short supply, such as during fasting (14). Regulation of PDH is accomplished by its interconversion between an active, nonphosphorylated form and an inactive, phosphorylated form. Phosphorylation and inactivation of PDH is mediated by a family of four PDH kinases (PDK1-4) (14, 29). Expression of one of these isozymes, PDK4, is rapidly and markedly induced in heart and other tissues in response to various metabolic stimuli, including fasting and a high-fat diet (28, 33, 34, 36). Transcription of the PDK4 gene is induced directly by the transcription factors FoxO1, which is repressed by insulin, and peroxisome proliferator-activated receptor (PPAR)-{alpha}, which is activated by fatty acids (12, 20, 35). Thus PDK4 functions as a metabolic switch, altering substrate utilization to favor fatty acids under physiological conditions where fatty acid concentrations are elevated and insulin is reduced. There is also evidence that excessive PDK4 expression can contribute to metabolic imbalances: in a study of insulin-resistant subjects, PDK4 mRNA levels in skeletal muscle positively correlated with fasting plasma insulin concentrations in response to oral glucose and negatively correlated with insulin-mediated glucose uptake (23). PDK4 is also overexpressed in heart and other tissues in diabetic rodents (36). Thus increased PDK4 activity may contribute to the impaired glucose oxidation that occurs in diabetic patients.

To test whether PDK4 overexpression causes metabolic inflexibility in the heart, we have generated transgenic mice that express PDK4 under the control of the {alpha}-myosin heavy chain (MHC) gene promoter. Hearts from the PDK4 transgenic mice have decreased PDH activity and a corresponding increase in fatty acid oxidation. Although no overt cardiomyopathy was seen in the PDK4 transgenic mice, a pathological synergy was observed when the PDK4 transgene was cointroduced into hearts with a constitutively active form of calcineurin (CnA), a calcium-activated serine/threonine phosphatase that causes cardiac hypertrophy (25). These data demonstrate that PDK4 overexpression is sufficient to cause metabolic inflexibility and exacerbate cardiomyopathy caused by other stress-induced pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice. The {alpha}-MHC-PDK4 transgene consists of a 5.5-kb segment of the {alpha}-MHC gene promoter, a 1.3-kb mouse PDK4 cDNA, and the 0.6-kb human growth hormone (hGH) gene polyadenylation signal. Two independent lines were established. Mice were genotyped by PCR on DNA samples extracted from mouse tails. PDK4/CnA double transgenic mice were generated by breeding {alpha}-MHC-PDK4 mice with {alpha}-MHC-CnA mice (25). All animal experiments were approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.

RNA preparation and real-time quantitative PCR analysis. Mice were killed, and the hearts were dissected into left and right ventricles. Samples were flash-frozen in liquid nitrogen and stored at –80°C until RNA preparation. Total RNA was extracted with Qiagen fibrous RNA minikits. All real-time quantitative PCR primers were designed with Primer Express Software (Applied Biosystems, Foster City, CA) based on GenBank sequence data. Primer sequences are available on request. For reverse transcriptase reactions, 2 µg of total RNA was reverse transcribed into cDNA with Superscript II RNA polymerase. Each real-time quantitative PCR reaction contained 25 ng of cDNA, each primer at 150 nM, and 5 µl of SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 10 µl. Each sample was performed in triplicate on an Applied Biosystems Prism 7900HT Sequence Detection System, and the relative mRNA levels were calculated by the comparative threshold cycle method with cyclophilin as an internal control.

Western blot analysis. To prepare total heart protein, frozen hearts were thawed on ice and homogenized in 500 µl of muscle lysis buffer [mM: 140 NaCl, 10 Tris·HCl (pH 8.1), 1 CaCl2, and 1 MgCl2 with 10% glycerol, 1% NP-40, and miniComplete proteinase inhibitor cocktail (Roche)] with a Polytron homogenizer. The homogenate was incubated on ice for 30 min with agitation and then spun down at 10,000 rpm for 10 min at 4°C. For mitochondrial protein, mice were killed by cervical dislocation and heart samples were removed quickly and rinsed with chilled PBS. The heart was minced with scissors in ice-cold MR buffer H1 [mM: 225 mannitol, 75 sucrose, 10 MOPS buffer (pH 7.2), and 1 EGTA (pH 7.3) with 0.5% fatty acid-free BSA; final pH = 7.2] and rinsed repeatedly with ice-cold MR buffer H1. The minced heart was homogenized in 10 ml MR buffer H1 with a precooled Dounce. The homogenate was spun down for 10 min at 700 g at 4°C to remove unbroken cells and nuclei. The supernatant was then spun down for another 10 min at 6,600 g (4°C) to pellet mitochondria. Mitochondrial pellets were homogenized in muscle lysis buffer. One microgram of total or mitochondrial protein from hearts of PDK4 transgenic mice or control littermates was run on a 10% acrylamide/bis (37.5:1) gel. PDK4 protein was detected with a previously described rabbit anti-mouse PDK4 antibody (17).

Pyruvate and lactate measurements. Mice were killed under pentobarbital sodium anesthesia, and hearts were removed and immediately freeze-clamped in liquid nitrogen. To extract pyruvate and lactate, frozen heart tissue was pulverized in liquid nitrogen and the powdered tissue was mixed with ice-cold 3 M perchloric acid. The tissue-perchloric acid mixture was incubated with agitation for 10 min at 4°C and then homogenized using a polytron homogenizer. After centrifugation at 5,000 g for 10 min at 4°C, the supernatant was collected and adjusted to pH 6 with 2 M KHCO3. Pyruvate and lactate concentrations were measured by modification of previously described procedures (3, 13). For the pyruvate assay, 50 µl of perchloric acid extract was mixed with 1.4 ml of β-NADH solution (0.13 mM in 100 mM sodium phosphate buffer, pH 7.5). After addition of 50 µl of lactate dehydrogenase solution (10 mg/ml in 10 mM Tris-Cl-0.1 M EDTA buffer, pH 7.5), absorbance at 340 nm (A340) was measured immediately and again at 10 min. Pyruvate concentrations were calculated from the change in A340 and a standard curve derived from reactions performed with serial dilutions of sodium pyruvate (Sigma). For the lactate assay, 90 µl of perchloric acid extract was mixed with 820 µl of hydrazine-glycine buffer (3.8 g glycine, 8.3 ml hydrazine hydrate, H2O added to 100 ml, pH 9.0) and 70 µl of β-NAD solution (30 mg/ml in H2O). After addition of 20 µl of lactate dehydrogenase solution (10 mg/ml in 10 mM Tris-Cl-0.1 M EDTA buffer, pH 7.5), A340 was measured immediately and again at 1 h. Lactate concentrations were calculated from the change in A340 and a standard curve derived from reactions performed with serial dilutions of L-(+)-lactate (Sigma).

Echocardiography. Noninvasive echocardiograms were obtained on unsedated mice. Transthoracic echocardiographic examination was performed by using a General Electric Vivid7 Pro machine equipped with a 12-mHz transducer. Motion mode (M mode) and two-dimensional echo images were obtained in the parasternal short-axis view. Fractional shortening was calculated from the M-mode images as the left ventricular end-diastolic dimension (LVEDD) minus the left ventricular end-systolic dimension (LVESD) divided by the LVEDD. Measurements were made in 8-wk-old mice.

Histology and morphometric analysis. Mice were euthanized via pentobarbital overdose. Hearts were quickly removed and rinsed in PBS buffer lacking calcium and magnesium. After weighing, hearts were incubated in room temperature Krebs-Henseleit solution (mM: 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 glucose) as a relaxant for 30 min and then transferred to Carson's buffered formalin solution for overnight fixation at 4°C. Heart specimens were paraffin processed, and the resulting sections were stained with hematoxylin and eosin or Masson's trichrome. For quantification of fibrosis, TIFF images encompassing the right and left ventricles and septum from a minimum of three mice/group were captured at x2.5 magnification with an Optronics Microfire digital charge-coupled device camera (Optronics, Goleta, CA) with Picture Frame 2.0 imaging software. These images were separated into RGB channels, and fibrotic area and total area were quantified with NIH Image J software. For quantification of centrilobular congestion and necrosis, liver sections from a minimum of three mice/group and three sections/mouse were stained with hematoxylin and eosin, and images were captured and quantified with Image J software as described above.

Substrate utilization assay. Isolated Langendorff mouse heart perfusions (1) were carried out on fed 10-wk-old male wild-type and PDK4 transgenic mice. Hearts were perfused retrogradely with Krebs-Henseleit buffer containing 23.7 µU/l insulin and a mixture of U-13C-labeled free fatty acids (FFA; 0.38 mM algal mix containing 11.6% palmitoleic, 39.1% palmitic, 14.6% linoleic, and 34.8% oleic acids bound to 2% albumin) and [1,6-13C2]glucose (8.2 mM). Nonrecirculating buffer was oxygenated with a thin-film oxygenator with a 95:5 mixture of O2 and CO2 and pumped into a water-jacketed, all-glass perfusion apparatus maintained at 37°C. Excised hearts were cannulated at the aorta and attached to the perfusion apparatus. Spontaneously beating hearts were perfused for 60 min, during which time heart rate, developed pressure, and oxygen consumption were monitored. Heart rate was monitored with an open-ended cannula placed across the mitral valve in the left ventricle and attached to a pressure transducer and was typical for Langendorff-perfused mouse hearts (300–350 beats/min). To measure oxygen consumption, perfusate samples collected into a gas-tight syringe from just above the aorta and below the pulmonary artery were immediately injected into a blood gas analyzer for measurement of PO2 (Instrumentation Laboratory, Lexington, MA). The PO2 difference and a separate measure of coronary flow were used to evaluate myocardial O2 consumption. After the perfusion period, the hearts were freeze-clamped and stored at –80°C until extraction with perchloric acid. Proton-decoupled 13C-NMR spectra of heart extracts were acquired at 150 MHz in a 5-mm broad-band probe with a 45° pulse, a 1.5-s acquisition time, and a 1.5-s postacquisition delay. A bilevel WALTZ-16 sequence was used for broad-band proton decoupling. 13C-NMR multiplets of tissue extract glutamate were measured and used to determine the relative oxidation of [U-13C]FFA, [1,6-13C2]glucose, and unlabeled endogenous substrates (e.g., triglycerides) (24, 38).

PDH and PDK activity measurements. PDH activity was assayed by coupling the generation of acetyl-CoA with the acetylation of 4-aminoazobenzene-4'-sulfonic acid (2) with recombinant arylamine N-acetyltransferase as described in detail recently (16). Briefly, hearts were removed from the mice within 10 s, freeze-clamped at the temperature of liquid nitrogen, and frozen until being used for assays. Frozen hearts were pulverized and extracted with a buffer containing Triton X-100 and protease inhibitors. The supernatant obtained by centrifugation was assayed immediately for PDHa activity with a SpectraMax190 microplate reader (Molecular Devices, Sunnyvale, CA); PDHt activity was measured after complete dephosphorylation of the complex with recombinant pyruvate dehydrogenase phosphatase 1 (16). PDHa designates the activity of PDH before activation by its phosphatase, i.e., the actual activity of the complex as it existed in the tissue before extraction and PDHt the activity of the complex after complete activation by dephosphorylation with its phosphatase, i.e., the maximum activity of the complex. PDK activity was measured by determining the rate of PDHt inactivation by ATP as previously described (16). Since inactivation of PDH by phosphorylation is a pseudo-first-order reaction (19), rates of kinase activity are expressed as apparent first-order rate constants (min–1) calculated from least-squares linear regression analysis of ln(inactivation by ATP) against time of incubation (18).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Metabolic consequences of PDK4 overexpression in heart. To investigate the effects of chronic PDK4 overexpression in the heart, a cDNA encoding full-length mouse PDK4 was inserted downstream of the {alpha}-MHC gene promoter (Fig. 1A) and two independent {alpha}-MHC-PDK4 transgenic mouse lines were generated. Both PDK4 transgenic strains had comparable increases in PDK4 mRNA and protein concentrations (Fig. 1, B and C, and data not shown). Analysis of the intracellular localization of PDK4 showed that it was correctly targeted to the mitochondria (Fig. 1C).


Figure 1
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  		Fig. 1. Generation and characterization of pyruvate dehydrogenase kinase (PDK)4 transgenic mice. A: schematic representation of the {alpha}-MHC-PDK4 transgene construct, which includes the {alpha}-myosin heavy chain ({alpha}-MHC) promoter linked to the mouse (m) PDK4 coding region and the human growth hormone (hGH) intron and polyA tail. B: PDK4 mRNA levels were measured in left ventricle (LV) and right ventricle (RV) of samples pooled from wild type (WT) and PDK4 transgenic (Tg) mice (n = 5/group). C: PDK4 protein levels were measured by Western blot analysis using PDK4 antiserum and either mitochondrial (Mito) or total protein prepared from pooled heart samples of wild-type and PDK4 transgenic mice (n = 4/group).

 
PDK activity was increased approximately sixfold in hearts of PDK4 transgenic mice, and there was a corresponding decrease in PDH activity (Fig. 2, A and B). To determine the effect of PDK4 overexpression on substrate utilization, hearts were isolated and perfused with [1,6-13C2]glucose and [U-13C]long-chain fatty acid. As predicted, NMR isotopomer analysis of heart extracts from PDK4 transgenic mice showed a significant decrease in glucose oxidation and a corresponding increase in long-chain fatty acid oxidation (Fig. 2, C and D). Oxygen consumption trended lower in hearts from PDK4 transgenic mice compared with control mice [16 ± 7 vs. 20 ± 4 µmol·min–1·g dry wt–1], but did not reach significance. These data demonstrate that overexpression of PDK4 in heart is sufficient to alter substrate utilization.


Figure 2
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  		Fig. 2. Altered substrate utilization in PDK4 transgenic mice. A: PDK activity was measured in heart extracts prepared from either wild-type or PDK4 transgenic mice (n = 8/group). *P < 0.01. B: active and total pyruvate dehydrogenase levels (PDHa and PDHt, respectively) were measured in heart extracts prepared from either wild-type or PDK4 transgenic mice (n = 8/group) and are shown on left. Percent PDH activity for hearts of wild-type and PDK4 transgenic mice is shown on right. *P < 0.05 compared with equivalent wild-type group. C: 13C-NMR spectra (150 MHz) of the C4 of glutamate from wild-type (n = 8/group) and PDK4 transgenic (n = 4/group) heart extracts. The quartet due to C4 coupling with both C3 and C5 (Q) and the doublet due to C4-C5 coupling (D45) report relative oxidation of free fatty acids (FFA), while the doublet due to C4-C3 coupling (D34) reports relative glucose oxidation. D: relative oxidation rates of [1,6-13C2]glucose (glucose), [U-13C]long-chain fatty acids (LCFA), and unlabeled endogenous substrates (Endo). *P < 0.01.

 
PDK4 overexpression exacerbates calcineurin-induced cardiomyopathy. Despite their altered metabolism, hearts from 8-wk-old PDK4 transgenic mice showed no overt abnormalities or hypertrophy (Fig. 3) or changes in left ventricular function as measured by echocardiography (Table 1). Metabolic alterations are among a number of stress-induced events that contribute collectively to cardiovascular disease. To examine whether metabolic changes in PDK4 transgenic mice exacerbate preexisting cardiomyopathy caused by other stress-related pathways, PDK4 transgenic mice were crossed with transgenic mice expressing a constitutively active form of the calcineurin catalytic subunit (CnA), also under the control of the {alpha}-MHC promoter (25). Calcineurin is a calcium-dependent serine/threonine phosphatase that regulates cardiac growth during development and disease (32). The {alpha}-MHC-CnA model was chosen because it is a noninvasive and robust model of cardiac hypertrophy with a well-defined pathological progression (25, 27). Thus it avoids some of the variability associated with surgical models of cardiac pressure overload. Mice bearing both the CnA and PDK4 transgenes did not have increased cardiac hypertrophy compared with the single CnA transgenic animals (Fig. 3A). However, PDK4/CnA double transgenic mice did have pronounced multifocal cardiomyocyte necrosis, fibrosis, and mineralization in all chambers of the heart (Fig. 3, B–D). The fibrosis was much more prevalent in the double transgenic mice than in the CnA transgenic mice (Fig. 3D). Similar results were obtained with PDK4/CnA double transgenic mice generated with two independently derived PDK4 transgenic lines (data not shown).


Figure 3
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  		Fig. 3. Cardiac fibrosis occurs in PDK4/constitutively active calcineurin (CnA) double transgenic mice. A: ratio of heart weight to body weight is shown for wild-type and PDK4, CnA, and PDK4/CnA transgenic mice (n = 5/group). *P < 0.01 compared with wild-type group. B: hematoxylin and eosin-stained heart sections derived from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice. Arrowheads indicate representative regions of fibrosis and necrosis. C: Masson's trichrome-stained heart sections derived from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice. Bar = 100 µm. D: quantification of trichrome staining in heart sections from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice (n = 3 or 4 mice analyzed/group). *P < 0.05 compared with wild-type group.

 

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  		Table 1. Echocardiogram data

 
Decreased PDH activity is predicted to result in an increase in pyruvate concentrations. Consistent with this hypothesis, pyruvate concentrations were significantly increased in hearts from PDK4 transgenic mice but not CnA transgenic animals (Fig. 4). These levels were further increased in PDK4/CnA double transgenic mice (Fig. 4). Lactate concentrations showed a similar trend (Fig. 4). These changes demonstrate that there are strong metabolic perturbations in the hearts of PDK4/CnA double transgenic mice.


Figure 4
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  		Fig. 4. Pyruvate and lactate concentrations were measured in hearts from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice (n = 4/group). The presence of different letters indicates statistical significance (P < 0.05) between groups. HW, heart weight.

 
The expression of a panel of genes involved in metabolism and cardiac remodeling was analyzed in the hearts of wild-type, PDK4 transgenic, CnA transgenic, and PDK4/CnA double transgenic mice. In PDK4 transgenic mice, the only gene other than PDK4 that changed significantly was β-MHC (Fig. 5). While these data are consistent with the lack of any evidence of cardiomyopathy in the PDK4 transgenic mice, they do not reflect the changes that occur in metabolic flux in these animals. Thus they highlight the limitations of using gene expression data to assess metabolic status. In contrast to what was seen in PDK4 transgenic mice, expression of most of the genes was altered in CnA transgenic mice (Fig. 5). The changes included increased mRNA levels of natriuretic peptide precursor type A (ANF), natriuretic peptide precursor B (BNP), β-MHC, and skeletal muscle actin-{alpha}1 (ACTA1) and decreased mRNA levels of PDK2, PDK4, {alpha}-MHC, PPAR-{alpha}, sarco(endo)plasmic reticulum Ca2+-transport ATPase isoform 2a (SERCA2a), phospholamban (PLB), glucose transporter-4 (GLUT4), carnitine palmitoyltransferase 1b (CPT1b), malonyl-CoA decarboxylase (MCD), and uncoupling proteins (UCP)2 and UCP3. The changes in mRNA levels of ANF, β-MHC, PPAR-{alpha}, GLUT4, and CPT1b in hearts from CnA transgenic mice are similar to those previously reported (4). A similar pattern of altered gene expression also occurs in pressure overload-induced cardiac hypertrophy (21, 30).


Figure 5
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  		Fig. 5. Gene expression in left ventricles from wild-type and transgenic mice. mRNA levels of the indicated genes were measured with total RNA prepared from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice (n = 5/group). ANF, natriuretic peptide precursor type A; BNP, natriuretic peptide precursor B; ACTA1, skeletal muscle actin-{alpha}1; PPAR-{alpha}, peroxisome proliferator-activated receptor-{alpha}; SERCA2a, sarco(endo)plasmic reticulum Ca2+-transport ATPase isoform 2a; PLB, phospholamban; GLUT, glucose transporter; CPT1b, carnitine palmitoyltransferase 1b; MCD, malonyl-CoA decarboxylase; UCP, uncoupling protein. For PDK4, inset shows the difference in PDK4 mRNA levels between wild-type and CnA transgenic mice.

 
Gene expression in the PDK4/CnA double transgenic mice generally followed one of two distinct patterns compared with wild-type and CnA transgenic mice. Expression of the first group of genes, including PDK2, {alpha}-MHC, SERCA2a, PLB, PPAR-{alpha}, GLUT4, CPT1b, MCD, and UCP3, was decreased in the CnA single transgenic mice and further decreased in the PDK4/CnA double transgenic animals (Fig. 5). Since several of these gene products are involved in fatty acid and carbohydrate metabolism, the changes are consistent with generalized metabolic dysfunction as previously hypothesized for CnA transgenic mice (4). A second set of genes, including BNP, β-MHC, and ACTA1, were most highly expressed in the CnA transgenic mice, with lower expression levels in PDK4/CnA transgenic animals (Fig. 5). Although no synergy between the PDK4 and CnA transgenes was seen in the gene expression studies, the cumulative effect of all these changes may contribute to the metabolic perturbations and cardiomyopathy in the double transgenic mice.

Given the changes in heart histology and metabolite profile in the PDK4/CnA transgenic mice (Figs. 3 and 4), we next investigated whether the double transgenic animals have compromised cardiac function beyond that previously reported for CnA mice (25, 27). Although PDK4/CnA double transgenic mice had a significantly reduced heart rate compared with the single transgenic mice, no additional deterioration in left ventricular function was detected by echocardiography performed on 8-wk-old double transgenic mice compared with CnA transgenic mice (Table 1). However, the CnA transgenic mice already have severe left ventricular dysfunction that may mask any further deterioration (22, 25, 27). Importantly, longevity studies showed that double transgenic mice have a marked increase in mortality compared with single transgenic animals: at 130 days of age all of the double transgenic mice had died, whereas 60% of the CnA transgenic mice were still alive (Fig. 6A). No deaths occurred in either wild-type or PDK4 transgenic mice (data not shown). Histology performed on liver sections from PDK4/CnA double transgenic mice revealed centrilobular congestion consistent with heart failure that was not seen in either the PDK4 or CnA single transgenic animals (Fig. 6, C and D). Moreover, gross histological examination of hearts from several dead PDK4/CnA double transgenic mice revealed prominent mural thrombi that were located in all cardiac chambers (Fig. 6B and data not shown). It is concluded that the metabolic alterations caused by PDK4 overexpression exacerbate cardiomyopathy and increase mortality caused by activation of the calcineurin stress-response pathway.


Figure 6
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  		Fig. 6. Increase in mortality in PDK4/CnA double transgenic mice. A: Kaplan-Meier survival curves are plotted for CnA ({circ}), and PDK4/CnA ({triangleup}) transgenic mice. No deaths occurred in the wild-type and PDK4 groups (data not shown) (n = 12 male, 12 female mice/group). P < 0.0001 for CnA and PDK4/CnA transgenic groups compared with wild-type mice. B: cross sections of hearts from PDK4/CnA double transgenic mice showing ventricular mural thrombosis. C: hematoxylin and eosin-stained sections of liver derived from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice. Bar = 50 µm. D: quantification of centrilobular congestion in liver sections from wild-type and PDK4, CnA, and PDK4/CnA transgenic mice (n = 3–5 mice analyzed/group). *P < 0.05 compared with wild-type group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PDK4 plays a crucial role in dictating substrate utilization throughout the body (29). Its importance in whole body energy homeostasis is underscored by the recent description of global PDK4-knockout mice, which have a range of metabolic defects during starvation including lower concentrations of blood glucose and gluconeogenic intermediates in liver and a faster rate of pyruvate oxidation in muscle (17). In this report, we demonstrate that selective overexpression of PDK4 in heart results in significant changes in energy metabolism, including increased cardiac fatty acid utilization and decreased carbohydrate consumption with a corresponding increase in pyruvate concentrations. These data demonstrate that overexpression of PDK4 is sufficient to markedly alter substrate utilization in the heart.

Despite the change in their cardiac substrate utilization profile, the PDK4 transgenic mice showed no evidence of overt cardiomyopathy. These findings highlight the remarkable metabolic flexibility of the heart. However, overexpression of PDK4 in the context of the activated calcineurin stress-response pathway resulted in profound cardiomyopathy that included fibrosis and necrosis that were not seen in the CnA single transgenic mice (Fig. 3, C and D). Moreover, the PDK4/CnA double transgenic mice showed a pronounced increase in mortality (Fig. 6A). The pathological synergy between PDK4 and CnA is reminiscent of that seen by combining banding-induced overload with either streptozotocin-induced diabetes or PPAR-{alpha} reactivation (6–8, 26, 37). Our findings are consistent with the hypothesis that disturbances in contractile function can be compensated for by changes in metabolism and vice versa, but that simultaneous disruption of both energy-producing and energy-consuming pathways causes cardiac maladaption and failure (5).

While the impact of the CnA transgene on substrate utilization has not been established, cardiac hypertrophy is generally associated with an increased reliance on carbohydrate metabolism to meet energy demands (30, 31). CnA transgenic mice had decreased expression of PDK4, PDK2, and several genes involved in fatty acid oxidation including PPAR-{alpha}, CPT1b, and MCD (Fig. 5). However, CnA transgenic mice also had decreased expression of GLUT4 (Fig. 5), suggesting a more generalized metabolic dysfunction as previously reported (4). While the changes in gene expression that were observed in the PDK4/CnA double transgenic mice are generally consistent with those seen in other models of cardiac hypertrophy (21, 30), it is important to note that changes in mRNA levels may not accurately reflect changes in either the levels or activities of the proteins they encode.

Transcription of PDK4 is stimulated in heart and other tissues by the nuclear fatty acid receptor PPAR-{alpha} (15, 28, 34). Two recent studies showed that increased PPAR-{alpha} activity in heart can cause cardiomyopathy (11, 37). Finck et al. (11) showed that selective overexpression of PPAR-{alpha} in heart caused ventricular hypertrophy and systolic dysfunction. Young et al. (37) showed that PPAR-{alpha} mRNA levels decrease in the heart of mice subjected to pressure overload, suggesting that downregulation of PPAR-{alpha} is part of the adaptive response of the hypertrophic heart. Similar effects on PPAR-{alpha} expression were observed in the CnA transgenic mice (Fig. 5). Importantly, Young et al. (37) showed that ligand-mediated reactivation of PPAR-{alpha} caused contractile dysfunction and heart failure in the pressure overload model. Among the genes induced by PPAR-{alpha} reactivation was PDK4 (37). While multiple PPAR-{alpha} target genes undoubtedly contribute to the cardiomyopathy observed in both the Finck et al. (11) and Young et al. (37) studies, our results demonstrate that overexpression of PDK4 alone is sufficient to cause metabolic inflexibility and to exacerbate preexisting cardiomyopathy caused by chronic activation of the calcineurin signaling pathway.

Limitations of study. The results presented in this article highlight the important role that PDK4 plays in determining substrate utilization in heart and, furthermore, demonstrate that PDK4 overexpression in heart increases the already elevated mortality seen in CnA single transgenic mice. Nevertheless, there are several important limitations of these studies. First, the perfusates in the heart perfusion studies did not include ketone bodies, lactate, or pyruvate. Thus the analysis of cardiac metabolism in the PDK4 transgenic mice was limited to glucose and fatty acid catabolism. Second, the only gene other than PDK4 that changed significantly in our analyses of PDK4 transgenic mice was β-MHC. While these data are consistent with the lack of any evidence of cardiomyopathy in the PDK4 transgenic mice, they do not reflect the changes that occurred in metabolic flux in these animals. Thus they highlight the inherent limitations of using mRNA measurements to assess metabolic status. A related limitation is that only PDH activity was measured in the PDK4 transgenic mice. Clearly, altering PDK4 could have secondary effects on the expression and activity of proteins other than PDH that could contribute to metabolic inflexibility and, ultimately, cardiomyopathy. Finally, we have not been able to determine the cause of the increased mortality of the PDK4/CnA double transgenic mice. PDK4/CnA double transgenic mice had prominent cardiomyocyte fibrosis and necrosis and hepatic centrilobular congestion consistent with heart failure, and postmortem cardiac examination revealed mural thrombosis. The extensive interstitial fibrosis raises the possibility of reentrant arrhythmias as a potential etiology of sudden death in these animals. However, additional studies will be required to determine the precise cause of death.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Welch Foundation (SAK) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47844 (R. A. Harris).


   ACKNOWLEDGMENTS
 
We thank Angela Milde and Charles Storey for technical assistance and Drs. Craig Malloy, Dan Garry, and Hesham Sadek for discussion and support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Kliewer, Depts. of Molecular Biology and Pharmacology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041 (e-mail: steven.kliewer{at}utsouthwestern.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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