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induces an alteration of cardiac functions
1Laboratoire de Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, UMR-Centre National de la Recherche Scientifique (CNRS) 7034, Faculté de Pharmacie, Université Louis Pasteur (ULP), Illkirch, France, 2Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS, Institut National de la Santé et de la Recherche Médicale, ULP; and 3Institut Clinique de la Souris, Illkirch, France
Submitted 18 October 2004 ; accepted in final form 30 January 2006
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ABSTRACT |
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(PPAR) plays a major role in the control of cardiac energy metabolism. The role of PPAR on cardiac functions was evaluated by using PPAR knockout (PPAR –/–) mice. Hemodynamic parameters by sphygmomanometric measurements show that deletion of PPAR did not affect systolic blood pressure and heart rate. Echocardiographic measurements demonstrated reduced systolic performance as shown by the decrease of left ventricular fractional shortening in PPAR –/– mice. Telemetric electrocardiography revealed neither atrio- nor intraventricular conduction defects in PPAR –/– mice. Also, heart rate, P-wave duration and amplitude, and QT interval were not affected. However, the amplitude of T wave from PPAR –/– mice was lower compared with wild-type (PPAR +/+) mice. When the myocardial function was measured by ex vivo Langendorff's heart preparation, basal and 
-adrenergic agonist-induced developed forces were significantly reduced in PPAR-null mice. In addition, Western blot analysis shows that the protein expression of 1-adrenergic receptor is reduced in hearts from PPAR –/– mice. Histological analysis showed that hearts from PPAR –/– but not PPAR +/+ mice displayed myocardial fibrosis. These results suggest that PPAR-null mice have an alteration of cardiac contractile performance under basal and under stimulation of 1-adrenergic receptors. These effects are associated with myocardial fibrosis. The data shed light on the role of PPAR in maintaining cardiac functions.
cardiomyopathy; peroxisome proliferator-activated receptor-; cardiac 1-adrenergic receptor protein expression
(PPAR) is a member of the nuclear hormone receptor superfamily that is activated by several fatty acids. PPAR is predominantly expressed in tissues exhibiting a high rate of fatty acid catabolism, such as liver, heart, kidney, and muscle. In the cardiovascular system, PPAR is expressed in cardiac and smooth muscle cells, endothelial cells, and monocyte/macrophage cells (9, 11). PPAR regulates fatty acid transport, esterification, and oxidation via the transcriptional activation of genes encoding key enzymes involved in these processes (1).
In the normal adult heart, mitochondrial fatty acid oxidation accounts for the majority of energy production (2). Alterations of the latter often lead to cardiomyopathy, such as those occurring in human genetic defects for mitochondrial enzymes involved in fatty acid oxidation (10). PPAR plays a major role in the control of cardiac energy metabolism (8). Indeed, the cardiac expression of genes encoding fatty acid transport and oxidation enzymes is reduced in PPAR knockout (PPAR –/–) mice (6). The dysregulation of PPAR signaling contributes to the pathogenesis of a variety of diseases, such as cardiac hypertrophy and heart failure (8).
We have also shown that PPAR agonists exert a cardioprotective effect against ischemia-reperfusion-induced injury (14). However, the main role of PPAR in the homeostasis of cardiovascular functions is not fully understood yet.
Therefore, the aim of the present study was to evaluate the role of PPAR in the regulation of cardiac functions. Cardiac contractility, electrical activity, and cardiac 1-adrenergic receptor protein expression were studied. We report here that PPAR knockout mice have defects of cardiac contractile performance, reduction of cardiac 1-adrenergic receptor protein expression, a weak myocardial fibrosis, and reduction of T-wave amplitude.
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MATERIALS AND METHODS |
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Animal experiments were performed in accordance with guidelines of the European Community and the French government concerning the use of animals. This study was performed in male 17- to 19-wk-old PPAR –/– mice (25% C57BL/6, 75% SV-129 strain) and their wild-type (PPAR +/+) littermates. PPAR –/– mice were generated by the group of Gonzalez as previously described (12). Mice were housed at 20°C with a 12:12-h light-dark cycle and allowed free access to food and water.
In Vivo Experiments
Blood pressure measurements. Systolic blood pressure (SBP) and heart rate (HR) were measured in the morning in trained conscious mice by indirect tail-cuff sphygmomanometer.
Echocardiographic examination. Transthoracic echocardiography was performed using an ATL-HDI 5500 ultrasound machine equipped with a 12.5-MHz imaging transducer. Mice were anesthetized with ketamine (50 mg/kg ip), shaved, and placed on a heating pad in a left lateral position. After a two-dimensional (2-D) image was obtained, measurements were recorded on the M-mode tracings in parasternal short-axis view at the level close to the papillary muscles. The left ventricular end-systolic diameter (LVESD), the left ventricular end-diastolic diameter (LVEDD), and the HR were measured. The percentage of LV fractional shortening [(LVEDD – LVESD)/LVEDD] x 100 was then calculated. The measurements were made by using the leading edge method of the American Society of Echocardiography. After the echocardiography, mice were killed, hearts were excised, and the left ventricle was isolated and weighed.
Ex Vivo (Isolated Perfused Heart)
PPAR +/+ and –/– mice were anesthetized with ketamine (100 mg/kg ip) and medetomidine (50 µg/kg ip) and heparinized (500 U/kg ip). The heart was rapidly excised, cannulated, and perfused in the retrograde mode according to Langendorff. Perfusion pressure was maintained constant at 80 cmH2O with Krebs-Henseleit solution containing (in mM) 118 NaCl, 24 NaHCO3, 4 KCl, 1.2 MgCl2, 1 NaH2PO4, 2.5 CaCl2, 0.5 Na2EDTA, and 10 glucose, gassed with 95%O2-5% CO2 at 37°C and pH 7.4. During the stabilization period, the heart was stretched to a diastolic force of 1 g, and the preparation was equilibrated for 30 min. Then -adrenergic agonist was perfused at increased concentrations (0.1–30 nM). A sensor tension placed at heart apex and connected to a computer via an amplifier and Mac Lab (ADI, Castle Hill, Australia) allows the measurement of the systolic and diastolic force from which the cardiac developed force was calculated as the difference between systolic force and diastolic force. Results are expressed as increase in developed force (g).
Coronary flow was determined by collecting and weighing 1-min samples of the effluent that drip from the heart. The cardiac parameters developed force (g), HR (beats/min), and coronary flow (ml/min) were monitored and recorded at baseline conditions and continuously during the perfusion of isoproterenol. During the experiments, diastolic force remained stable, so the changes of developed force and the systolic force were of the same values.
Western Blotting
Mice were anesthetized with ketamine (100 mg/kg ip) and medetomidine (50 µg/kg ip) and killed. Hearts were homogenized in cold lysis buffer, and 40 µg of total proteins from the supernatant fraction were loaded onto 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with rabbit 1-adrenergic receptor polyclonal antibody (Abcam, Cambridge, UK). Bound antibodies were detected with a secondary peroxidase-conjugated anti-rabbit IgG (Promega; Charbonnières, France). The bands were visualized by using the enhanced chemiluminescence system (ECL Plus, Amersham; Buckinghamshire, UK) and quantified by densitometry. Results were expressed as the percentage of staining compared with PPAR +/+, which was taken as 100%.
Tissue Preparation for Histological Analysis
On removal, hearts from both wild-type and PPAR-null mice were rinsed with PBS and fixed for 24 h in 10% formalin at room temperature. They were processed routinely in paraffin. Serial 5-µm-thick sections from the middle level of both ventricles were stained with picric acid-Sirius red staining for collagen.
In bright-field microscopy, sections had a pale pink background and collagen was stained in red. Collagen fibers were detected by polarized light microscopy. Under these conditions, type I collagen fibers were stained from yellow to orange, whereas type III collagen fibers appeared green.
For morphological evaluation, a Leica DMLB light microscope with cool-snap camera was used. The quantification of yellow and orange collagen brightness was evaluated by using Adobe Photoshop 6.0 software.
ECG
ECGs were recorded with the use of a telemetry system (Data Sciences, St. Paul, MN). This system consists of implantable radio-frequency transmitters (TA10ETA-F20, 3.6 g) and a receiver placed under the cage of each animal. The data were recorded at 1,000 Hz. Seven days before measurements, mice were anesthetized with ketamine (100 mg/kg ip) and medetomidine (50 µg/kg ip). The transmitter was implanted subcutaneously; the negative lead was positioned and sutured at the right shoulder, and the positive lead was sutured toward the lower left chest. The ECG was measured continuously during 24 h. All analyses were performed on recordings obtained at the beginning of each hour of the 24-h recordings (20 different cardiac cycles per mice). The RR interval, PR interval, QRS interval, P wave duration, and P- and T-wave amplitudes were determined automatically with the Emka ECG analysis software. All analyses of QT interval were controlled by the same operator.
Statistical Analysis
Results are expressed as means ± SE of n experiments, where n represents the number of mice. Statistical evaluation was carried out by using two-way ANOVA followed by multiple Bonferroni test or Student's t-test. In all cases, a P value <0.05 was considered to be significant.
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RESULTS |
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–/– Mice
SBP and body weight.
SBP, HR, and body weight of PPAR –/– mice (106.5 ± 0.85 mmHg, 540 ± 17 beats/min, 30 ± 1.7 g; n = 6, respectively) were not significantly different from the values measured in wild-type mice (107.8 ± 1.87 mmHg, 552 ± 23 beats/min, 32 ± 0.7 g; n = 6).
Echocardiographic evaluation.
To evaluate the effects of PPAR deletion on cardiac structure and function, transthoracic echocardiograms were analyzed. The results show that the calculated percentage of LV fractional shortening, an indicator of systolic cardiac function, was significantly reduced in hearts from PPAR –/– mice (Fig. 1) without significant modification of LVEDD and LVESD (Table 1). HR during the echocardiography is significantly enhanced in PPAR –/– mice (Table 1) (476 ± 24 vs. 362 ± 19 beats/min, P < 0.01). LV mass calculated from the echocardiographic parameters or measured after necroscopy were not different between PPAR –/– and wild-type mice. Calculated LV mass was 112 ± 5 and 126 ± 8 mg (n = 6) and measured LV mass was 108 ± 7 and 115 ± 8 mg in hearts from PPAR +/+ and PPAR –/– mice, respectively.
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–/– mice was significantly reduced (
25%) compared with that obtained in wild-type animals (P < 0.05, Fig. 2A). HR and coronary flow were not significantly different between the PPAR –/– and PPAR +/+ mice (Table 1).
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–/– compared with PPAR +/+ mice (Fig. 2B). There was no difference in the increase of HR and coronary flow induced by isoproterenol between the two groups of mice (data not shown). Western Blot Analysis
The 64-kDa 1-adrenergic receptor protein was expressed in hearts from PPAR –/– and PPAR +/+ mice (Fig. 3). This expression was significantly reduced in hearts from PPAR –/– mice (Fig. 3).
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The area of myocardial fibrosis was identified by the collagen birefringence correlated with collagen yellow or orange brightness viewed with polarized light.
The heart from wild-type mice seemed to be histologically normal, without collagen red staining in bright-field microscopy (Fig. 4A). The same sections observed with polarized light did not show yellow or orange brightness corresponding to collagen type I infiltration in cardiac tissue (Fig. 4C). We used as positive control (PC in Fig. 4C) the orange collagen staining of the wall of a cardiac vessel in the same section of wild-type mice. It is interesting to note that hearts from PPAR-null mice showed weak fibrosis compared with PPAR wild-type mice, as evidenced by red collagen staining in sections observed in bright-field microscopy (Fig. 4B) and yellow or orange collagen brightness in the same sections observed with polarized light (Fig. 4D). The quantification of yellow and orange collagen brightness was significantly enhanced in heart sections from PPAR-null mice compared with PPAR wild-type mice (P < 0.05; Fig. 4E).
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ECG analysis in PPAR knockout mice revealed neither atrioventricular nor intraventricular conduction defects (similar PR intervals, QRS duration) (Table 2). Also, HR, P-wave duration and amplitude, and QT interval were not altered in PPAR knockout mice (Table 2). The most striking difference was a reduced T-wave amplitude in PPAR knockout mice compared with that of PPAR wild-type mice (Fig. 5, Table 2).
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DISCUSSION |
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plays a major role in the control of cardiac energy metabolism (8), and PPAR agonist exerts a cardioprotective effect against ischemia-reperfusion injury (14); it was then important to describe the cardiac phenotype of PPAR knockout mice. We report, in the present study, that PPAR deletion induced an alteration of cardiac contractile performance without change in blood pressure and HR.
The alteration of cardiac contractile performance in the PPAR –/– mice detected in vivo by echocardiography was confirmed ex vivo on isolated perfused heart. This alteration might be due to a dysregulation of the fatty acid oxidation. Indeed, PPAR has been reported to regulate cardiac lipid and energy metabolism, by controlling genes encoding enzymes of the mitochondrial fatty acid -oxidation pathway (8), which provides the major source of energy to the heart muscle (2). The cardiac expression of genes for enzymes involved in fatty acid transport and -oxidation is decreased in PPAR –/– mice (6). The rate of fatty acid oxidation in isolated working hearts from PPAR –/– mice is significantly reduced and glucose utilization is increased to compensate for the reduction of energy production (4). Moreover, the rate of ATP concentration in cardiomyocytes is reduced in PPAR knockout mice (15). The deletion of PPAR induced a low rate of -oxidation (7, 15). The defects in cardiac contractile performance observed in PPAR –/– mice might be due to a decrease of energy production in cardiomyocytes as a consequence of the reduced rate of fatty acid oxidation. In our isolated heart experiments, the hearts were perfused without fatty acids but in the presence of glucose as the only source of energy. The results that we present here are in discordance with a previous study of Campbell et al. (4). These authors, using an isolated working heart preparation, have shown no difference in cardiac function between PPAR–/– and wild-type mice. It should be noted that Campbell et al. (4) used fatty acids as substrate in their experiments and have shown that fatty acid oxidation was decreased in PPAR –/– hearts. In the present study, the perfusion medium did not contain fatty acids and contained glucose as substrate. Under these conditions, the energy was produced by glycolysis and glucose oxidation. Even though glucose might partially prevent the absence of fatty acids, it cannot prevent the alteration of cardiac contractile performance observed ex vivo. Moreover, our in vivo measurements of physiological cardiac parameters using echocardiography showed decreased systolic performance. These data suggest the existence of cardiac defect in PPAR-null mice independently of the presence of fatty acids in the medium. They rather support the hypothesis that this defect is a consequence of cardiomyocyte alteration on PPAR deletion, like cardiac lipid accumulation and myocardial fibrosis (15). With regard to the former, the deletion of PPAR induced reduction of the rate of fatty acid oxidation and abnormal lipid accumulation in myocardium (7). The lipid accumulation might induce a long-term decrease in cardiac contractile performance. Indeed, lipid accumulation is associated with cardiac hypertrophy followed by the development of LV dysfunction (5). With regard to the latter, the present work showed that PPAR-null mice but not wild-type mice displayed myocardial fibrosis, which might participate in the alteration of cardiac contractile function in this strain. The alteration in cardiac performance observed in PPAR –/– mice could also be a consequence of the absence of PPAR from extracardiac tissue. Indeed, in our previous study (14), we found that PPAR agonist improved endothelial function in wild-type but not in PPAR-null mice, suggesting a role of PPAR in the regulation of vascular vasodilatation that probably has an impact for cardiac performance.
Although PPAR deletion induced changes in cardiac contractile performance, the present study shows that the deletion of PPAR does not affect HR. Indeed, we did not find any difference in heart rate using the tail-cuff sphygmomanometry, the telemetry, and the isolated heart preparation between PPAR –/– and PPAR +/+ mice. In conscious mice, by two different methods (telemetry and tail-cuff sphygmomanometry), we confirmed that PPAR gene deletion did not modify HR.
We also observed that hearts from PPAR-null mice displayed reduced contractile force in response to -adrenergic stimulation ex vivo. These defects can be due to either changes in 1-adrenergic receptor expression or an alteration of the transduction pathway, leading to cardiac contractility. The positive inotropic effect in response to isoprenaline results from phosphorylation of voltage-dependent Ca2+ channel by protein kinase A on the increase of adenylyl cyclase activity after -adrenergic stimulation. In the present study, we found a reduction of 1-adrenergic receptor protein expression in hearts from PPAR –/– mice. However, we cannot answer whether the change in 1-adrenergic receptor is a direct or indirect effect of PPAR target gene.
It might also be possible that the alteration of contractile force in response to -adrenergic stimulation in PPAR –/– mice is the consequence of cardiomyocyte damage induced by PPAR gene deletion, like myocardial fibrosis (15), as discussed above.
In our study, ECG measurements show reduced T-wave amplitude in PPAR –/– mice. T-wave amplitude is an indicator of ventricle repolarization’s state. The repolarization of ventricles depends on K+ efflux due to activation of several voltage-gated K+ channels. Two types of voltage-gated K+ channels play a major role in determining repolarization: transient outward (IT0) and delayed rectifier (IK) current (13). The expression and the activity of these channels could be modified by the changes in fatty acid content of the plasma membrane of cardiomyocytes. Indeed, fatty acids have been reported to inhibit IT0 density in rat ventricular myocytes (3), and PPAR –/– mice show a low rate of -oxidation and an abnormal accumulation of medium- and long-chain fatty acids in the myocardium (7, 15). The defect of the T-wave amplitude might result from an alteration in K+ channel activity that is linked to an excess of fatty acids and subsequent changes in the content of membrane phospholipids in PPAR-null mice.
In conclusion, the present study reports that deletion of PPAR gene induced defects of cardiac contractile performance, reduction of the protein expression of 1-adrenergic cardiac receptor, and reduction in T-wave amplitude in association with myocardial fibrosis. These results shed light on the role of PPAR in the maintenance of cardiovascular homeostasis within the physiological range.
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ACKNOWLEDGMENTS |
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knockout mice (Mol Cell Biol 15: 3012–3022, 1995). |
FOOTNOTES |
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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.
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REFERENCES |
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(PPAR-) in the control of cardiac malonyl-CoA levels: reduced fatty acid oxidation rates and increased glucose oxidation rates in the hearts of mice lacking PPAR- are associated with higher concentrations of malonyl-CoA and reduced expression of malonyl-CoA decarboxylase. J Biol Chem 277: 4098–4103, 2002. (PPAR) in the control of cardiac lipid metabolism. Prostaglandins Leukot Essent Fatty Acids 60: 339–343, 1999.[CrossRef][Web of Science][Medline]- deficient mice. J Clin Invest 102: 1083–1091, 1998.[Web of Science][Medline] (PPAR) signaling in the gene regulatory control of energy metabolism in the normal and diseased heart. J Mol Cell Cardiol 34: 1249–1257, 2002.[Web of Science][Medline] isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15: 3012–3022, 1995.[Abstract] protects against myocardial ischaemic injury and improves endothelial vasodilatation. BMC Pharmacol 2: 10–20, 2002.[CrossRef][Medline] associated with age-dependent cardiac toxicity. J Biol Chem 275: 22293–22299, 2000.This article has been cited by other articles:
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