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European Journal of Heart Failure 2006 8(3):290-294; doi:10.1016/j.ejheart.2005.09.003
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© 2005 European Society of Cardiology

Cardiac PPAR{alpha} expression in patients with dilated cardiomyopathy

Michael Schuppa, Ulrich Kintschera, Jens Fielitzb,d, Jennifer Thomasd, Reinhard Preglac, Roland Hetzerc, Thomas Ungera and Vera Regitz-Zagrosekc,d,*

a Center for Cardiovascular Research (CCR), Institute of Pharmacology and Toxicology, Charité-Universitätsmedizin Berlin CCM, Hessischestr. 3-4, 10115 Berlin, Germany
b Department of Molecular Biology, University of Texas Southwestern Medical Center Dallas, TX 75390-9148, United States
c German Heart Institute, Department of Cardiothoracic and Vascular Surgery Augustenburger Platz 1, 13353 Berlin, Germany
d Center for Cardiovascular Research (CCR), Cardiovascular Disease in Women, Charité-Universitätsmedizin Berlin, CCM Hessischestr. 3-4, 10115 Berlin, Germany

* Corresponding author. Center for Cardiovascular Research (CCR), Cardiovascular Disease in Women, Charité-Universitätsmedizin Berlin CCM, Hessischestr. 3-4, 10115 Berlin, Germany. Tel.: +49 30 450 525172; fax: +49 30 450 525972. E-mail address: vera.regitz-zagrosek{at}charite.de


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a central regulator of myocardial fatty acid (FA) metabolism implicated in the pathogenesis of heart failure.

Aims: To characterize PPAR{alpha} regulation in human dilated cardiomyopathy (DCM), we studied the expression of cardiac PPAR{alpha}, cardiac carnitine palmitoyl-transferase I (CPT-1), a major PPAR{alpha} target gene, and of the cardiac glucose transporter GLUT-4 in patients with DCM.

Methods: Left ventricular biopsies were taken from patients with DCM (n=16) and control subjects (n=15), and mRNA expression was quantitated using real-time PCR (SYBR®Green) and protein expression was measured by Western immunoblotting.

Results: Left ventricular PPAR{alpha} mRNA levels were significantly increased in the DCM group compared to the control group (136±25.4% vs. control, p<0.01). Consistently, DCM patients had a significantly higher cardiac CPT-1 mRNA expression (147±51% vs. control, p<0.05) compared to the control group. Cardiac GLUT-4 expression was similar in both groups.

Conclusion: Elevated cardiac PPAR{alpha} levels followed by an induction of cardiac CPT-1 expression may result in increased fatty acid metabolism for cardiac energy production in DCM, suggesting a specific cardiac metabolic program in human DCM compared to other types of cardiomyopathy.

Key Words: Peroxisome proliferator-activated receptor alpha • Dilated cardiomyopathy • Fatty acids • Cardiac metabolism

Received March 11, 2005; Revised July 20, 2005; Accepted September 6, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The peroxisome proliferator-activated receptor alpha (PPARP) is a nuclear hormone receptor, which functions as a transcriptional regulator in tissues with high metabolic activity including liver, heart, kidney and skeletal muscle [1,2]. Activated by its ligands as endogenous, lipid-derived molecules (long-chain FAs (fatty acid), eicosanoids, leukotrienes) or synthetic, hypolipidemic fibrates, PPARP regulates the expression of genes involved in every step of FA utilization including FA-uptake/-esterification (e.g.FATP1), mitochondrial transport (e.g. CPT-1), and mitochondrial β-oxidation (e.g. MCAD) [3,4]. Recently, PPARP has been described as a key regulator of FA-metabolism in the heart, being substantially involved in the determination of substrate utilization during the development of cardiac dysfunction [5].

Alterations in cardiac energy substrate metabolism have been implicated in the pathogenesis of cardiac dysfunction and heart failure. The normal adult heart relies primarily on FA-oxidation as the preferred substrate for ATP production [5]. Myocardial substrate utilization in the disease state is highly dependant on disease aetiology and systemic substrate availability. In hypertrophic cardiomyopathy glucose is the preferred substrate for ATP production, whereas the heart still relies on FA-oxidation in diabetic cardiomyopathy [6-9]. Conflicting results have been documented when comparing animal and human studies. Animal studies have demonstrated a switch in substrate from FA to glucose during cardiac failure [10,11]. In contrast, a number of studies in humans have shown that patients with heart failure exhibit increased rates of myocardial FA-uptake [12,13]. Taken together, one may hypothesize that different forms of cardiomyopathy have distinct species-specific metabolic programs. Knowledge about these programs in humans could facilitate the development of specific therapeutic strategies to treat cardiac failure.

To characterize the metabolic program in human dilated cardiomyopathy (DCM), we studied the regulation of PPARP, CPT-1 and GLUT-4 expression in left ventricular biopsies from patients with DCM and healthy control subjects.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Patients
Left ventricular myocardial samples from 16 patients with DCM and 15 control subjects were analyzed for mRNA and/or protein expression. Tissue samples from patients with DCM were obtained at orthotopic heart transplantation. The control group was composed of donor hearts that were not used for technical/logistic reasons. Clinical features of the patients/subjects are presented in detail in Table 1. Written informed consent was obtained from all patients. The study followed the rules for investigation of human subjects, as defined in the Declaration of Helsinki.


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  		Table 1 Characteristics of normal controls and DCM-patients

 
2.2. Quantitative real-time PCR
Real-time PCR was performed as previously described with SYBR®Green fluorescence (Applied Biosystems) for detection [14]. To correct for potential variances between samples in mRNA extraction or RT-efficiency, the mRNA content of PPARP, CPT-1, and GLUT-4 was normalized to the expression of the stably expressed reference gene GAPDH in the same sample.

2.3. Western immunoblotting
After tissue homogenization, Western immunoblotting including protein isolation, electrophoresis, and blotting were performed as previously described [15]. Blots were incubated with a specific antibody recognizing GLUT-4 (1:1000 dilution, Santa Cruz, CA, USA, sc-7938) or GAPDH (1:1000 dilution, Chemicon, Temecula, CA, MAB374). Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution). The peroxidase reaction was developed using an enhanced chemiluminescence detection system (ECL, Amersham Corp.). Band intensity was analyzed by densitometry.

2.4. Statistical analysis
Results are expressed as mean±standard deviation (SD) or scatter graph. ANOVA and t-test were performed for statistical analysis as appropriate. Statistical significance was designated at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Patient characteristics
LV-samples of sixteen patients with DCM and fifteen control subjects were analyzed for cardiac mRNA and protein expression. The mean LV-EF in the DCM group was 19.8±6.7%, whereas the control subject had normal systolic LV-function (mean LV-EF: >55%) (Table 1). Consistently, left ventricular end-diastolic diameters (LV-EDD) were prominently elevated in the DCM group (mean LV-EDD 8.1±1.2 cm) compared to normal diameters in the control group (mean LV-EDD 6.3±1.3 cm). The control group had no cardiac history and no concomitant disease. In the DCM group two patients had a history of stroke, and four patients had hypercholesterolemia, none had diabetes.

3.2. Cardiac PPARP and CPT-1 mRNA expression
Myocardial PPARP mRNA expression was significantly increased by 136±25.4% (p<0.01) compared to control subjects (Fig. 1A). To evaluate whether enhanced PPARP expression resulted in a stimulation of genes involved in FA-oxidation, expression of the mitochondrial FA-transport molecule CPT-1, a major PPARP target gene, was analyzed. Corresponding to an increased rate of FA-oxidation, CPT-1 mRNA expression was significantly higher in samples from DCM patients compared to controls (147±51% vs. control, p<0.05) (Fig. 1B).


Figure 1
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  		Fig. 1 Cardiac PPARP and CPT-1 mRNA expression. Total RNA was isolated from left ventricular myocardial samples (Control subjects: n=15; DCM-patients: n=16 patients). Expression of mRNA was measured by real-time PCR with SYBR®Green fluorescence. GAPDH was used as a housekeeping gene. Mean values (±SD) as a percentage of the control group are presented. *=p<0.05 vs. control; **=p<0.01 vs. control. (A) Relative expression of PPARP mRNA. (B) Relative expression of CPT-1 mRNA.

 
3.3. Cardiac GLUT-4 mRNA and protein expression
To examine whether the increase in genes involved in FA-oxidation in DCM is associated with a decrease in glucose utilization, mRNA and protein expression of the glucose transport molecule GLUT-4 in LV-samples from DCM patients and healthy subjects was measured. No difference in myocardial GLUT-4 mRNA or protein expression between DCM and controls was observed (Fig. 2A and B), suggesting the absence of a counterregulatory decrease in glucose uptake in DCM.


Figure 2
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  		Fig. 2 Cardiac GLUT-4 mRNA and protein expression. (A) Total RNA was isolated from left ventricular myocardial samples (Control subjects: n=15; DCM-patients: n=16 patients). Expression of mRNA was measured by real-time PCR with SYBR®Green fluorescence. GAPDH was used as a housekeeping gene. Scatter graph data are expressed as percentage of the mean value of the control group. Relative expression of GLUT-4 mRNA is shown. (B) Protein was isolated from left ventricular myocardial samples. GLUT-4 and GAPDH protein expression was measured by Western immunoblotting as described in the Methods. Representative immunoblots are shown from n=5 control subjects and n=5 DCM patients. Densitometry results are presented as a percentage of the mean value of the control group±SD, n.s.: non-significant.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study demonstrates that cardiac PPARP levels are elevated in patients with DCM, followed by an induction of cardiac CPT-1 expression. This metabolic gene expression may result in increased fatty acid metabolism for cardiac energy production in DCM, suggesting a specific cardiac metabolic program in human DCM compared to other types of cardiomyopathy.

PPARP is the major transcriptional regulator of most cardiac FA import and oxidation genes, identifying this nuclear receptor as a molecular marker for the rate of myocardial FA-oxidation [5]. Controversial results have been reported in studies investigating the expression or activity of PPARP in different forms of cardiomyopathy in animals. Experiments in rodent models of pressure overload studying hypertrophic cardiomyopathy have shown that activity of PPARP gene regulatory pathways is reduced at several levels, suggesting a low rate of FA-oxidation in hypertrophic cardiac dysfunction [6,8]. In contrast, cardiac PPARP activity is increased in diabetic rodent models including streptozotocin-induced diabetes and obese db/db mice [7,9]. Differences between the hypertrophied failing myocardium and diabetes-induced cardiac dysfunction may result from systemic substrate availability. Free FA plasma levels are elevated in rodent models of diabetes. Since in the present study none of the DCM patients had diabetes, it is unlikely that systemic substrate availability accounts for the differences in cardiac PPARP expression.

To clarify the processes involved in cardiac PPARP upregulation in DCM patients, data in humans has to be considered. In humans, several studies have investigated cardiac substrate utilization during heart failure, using positron emission tomography (PET). Dávila-Román and colleagues examined patients with idiopathic dilated cardiomyopathy, and demonstrated decreased cardiac fatty acid metabolism and increased glucose utilization, suggesting decreased myocardial PPARP activity in these patients [16]. However, PPARP activity was not directly assessed in this study. Furthermore, the mean LV-EF from the seven patients in this study with idiopathic DCM was 27±8%, which is higher than in the present study. Our study included end-stage heart failure patients with a mean LV-EF of 19.8±6.7%. This difference may account for the changes in PPARP regulation, suggesting that in advanced stage heart failure additional substrate switches occur.

In another study performed in humans, twelve patients with heart failure were investigated by PET [13]. Myocardial fatty acid uptake rates were higher than expected for the normal heart, which is in contrast to Dávila-Román but consistent with our results. In addition, Tanaka and colleagues showed elevated PPARP mRNA expression levels in eight DCM patients with decompensated heart failure compared to normal controls [17]. Taken together, it appears that DCM patients in the advanced stages of cardiac failure have increased expression of myocardial PPARP together with a higher rate of cardiac FA-metabolism. This cardiac substrate utilization might be specific to end-stage DCM.

The focus of the present study was the characterization of cardiac PPARP expression and related target genes involved in FA-metabolism in human DCM. PPARP and CPT-1 were upregulated in the myocardium of DCM which may result in an increased rate of FA-oxidation. Recently, it has been reported that enhanced cardiac FA-oxidation is associated with an attenuation of myocardial glucose utilization [13]. Therefore, we hypothesized that in addition to the induction of FA-oxidative pathways in the DCM-group, cardiac utilization of glucose might be suppressed. GLUT-4 is an important mediator of cardiac glucose-uptake. Differences in GLUT-4 mRNA and protein expression were not detected in DCM samples compared to healthy controls suggesting the absence of a counterbalancing regulation of cardiac glucose metabolism. However, since we did not directly assess glucose uptake/oxidation, it is possible that effects on myocardial glucose metabolism may have been missed. Therefore, it is essential that future studies investigate the regulation of glucose metabolism in human DCM.

A potential mechanism for increased PPARP expression and FA-oxidation in end-stage DCM might be a long-term adrenergic activation, which is typical during progressive cardiac failure. Cardiac FA-utilization is enhanced by catecholamines, and blockade of cardiac β1-receptors results in a marked decrease of FA-metabolism and an improvement in myocardial energy efficiency [18,19]. Recently, it has been shown that norepinephrine is capable of inducing PPAR{gamma} mRNA and protein expression in neuronal cells, suggesting a direct link between adrenergic activation and regulation of PPAR gene expression [20]. Whether cardiac PPARP is directly regulated via β1-adrenergic receptor stimulation requires further investigation.

In conclusion, patients with end-stage DCM present with a specific cardiac metabolic program, which involves increased cardiac PPARP and CPT-1 levels, and increased fatty acid metabolism for cardiac energy production. Given the controversial results from animal experiments, further clinical studies are required to identify the specific metabolic programs involved in other cardiomyopathies. With knowledge of these programs, pharmacological manipulation of cardiac metabolism, and in particular regulation of cardiac PPARP activation, may provide a promising therapeutic and highly specific tool to improve cardiac function.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
U.K., T.U., and V.R-Z. are supported by the Deutsche Forschungsgemeinschaft (GK 754).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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