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Am J Physiol Heart Circ Physiol 283: H1750-H1760, 2002. First published July 18, 2002; doi:10.1152/ajpheart.01051.2001
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Vol. 283, Issue 5, H1750-H1760, November 2002

Aging-induced decrease in the PPAR-alpha level in hearts is improved by exercise training

Motoyuki Iemitsu1, Takashi Miyauchi1, Seiji Maeda1, Takumi Tanabe2, Masakatsu Takanashi1, Yoko Irukayama-Tomobe1, Satoshi Sakai1, Hajime Ohmori2, Mitsuo Matsuda2, and Iwao Yamaguchi1

1 Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, 2 Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptor (PPAR)-alpha , a transcriptional activator, regulates genes of fatty acid (FA) metabolic enzymes. To study the contribution of PPAR-alpha to exercise training-induced improvement of FA metabolic capacity in the aged heart, we investigated whether PPAR-alpha signaling and expression of its target genes in the aged heart are affected by exercise training. We used hearts of sedentary young rat (4 mo old), sedentary aged rat (23 mo old), and swim-trained aged rat (23 mo old, training for 8 wk). The mRNA and protein expression of PPAR-alpha in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats and was significantly higher in the swim-trained aged rats compared with the sedentary aged rats. The activity of PPAR-alpha DNA binding to the transcriptional regulating region on the FA metabolic enzyme genes, the mRNA expression of 3-hydroxyacyl CoA dehydrogenase (HAD) and carnitine palmitoyl transferase-I, which are PPAR-alpha target genes, and the enzyme activity of HAD in the heart altered in association with changes of the myocardial PPAR-alpha mRNA and protein levels. These findings suggest that exercise training improves aging-induced downregulation in myocardial PPAR-alpha -mediated molecular system, thereby contributing to the improvement of the FA metabolic enzyme activity in the trained-aged hearts.

peroxisome proliferator-activated receptor-alpha ; swimming training; aged rat; fatty acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE HEART is known for its ability to produce energy from fatty acids (FA) because of its important beta -oxidation equipment, but it can also derive energy from several other substrates, including glucose and lactate (10, 23). On a physiological condition, FA is considered to account for 60-70% of oxygen consumption for energy production in the heart (23). However, FA metabolic capacity in the heart is reduced by aging (19, 32). It has been reported that exercise training improved an aging-induced decrease of FA metabolic capacity in the heart (7, 18, 25, 32). However, the mechanisms for improving FA metabolic capacity in the heart by exercise training are unclear.

Peroxisome proliferator-activated receptor (PPAR)-alpha is a member of the nuclear receptor transcription factor superfamily and is mainly expressed in the heart, liver, and kidney (2, 13, 29). The recent studies indicated that PPAR-alpha plays a critical role in the expression of genes involved in FA metabolism (13). PPAR-alpha heterodimerizes with the retinoid X receptor (RXR-alpha ) to bind to peroxisome proliferator-response elements (PPRE) in the upstream regions of a number of genes involved in metabolic homeostasis (13, 29). PPAR-alpha regulates target genes encoding FA metabolic (beta -oxidation) enzymes and FA transporters such as FA binding protein, carnitine palmitoyl transferase-I (CPT-I), acyl-CoA synthase, 3-hydroxyacyl CoA dehydrogenase (HAD), apolipoproteins, and so on, suggesting that PPAR-alpha plays an important role in FA metabolic homeostasis (2, 5, 17, 29). However, it is unknown whether the aging and subsequent exercise training affect the levels of myocardial PPAR-alpha mRNA and protein and subsequently induce a change of its target enzymes activities of the FA metabolic pathway in the heart.

Because the expression of many genes involved in FA metabolism (beta -oxidation) is regulated by the PPAR-alpha , we hypothesized that myocardial PPAR-alpha participates in a molecular mechanism that exercise training improves the aging-induced decrease of FA metabolic capacity. Therefore, we investigated whether the mRNA and protein expression of PPAR-alpha in the rat hearts is decreased by aging, and whether the aging-induced change in the levels of PPAR-alpha mRNA and protein is improved by exercise training. We also studied whether gene expression of FA metabolic enzymes in the heart alters in association with change of the PPAR-alpha mRNA and protein by aging and subsequent exercise training. Furthermore, we investigated whether the aging-induced decrease of FA metabolic capacity (enzyme activity) in the rat hearts is improved by exercise training. In the present study, we tested our hypothesis by using the sedentary young rat (Sedentary-young group; 4 mo old), sedentary aged rat (Sedentary-aged group; 23 mo old), and swim-trained aged rat (Trained-aged group; 23 mo old, swimming training for 8 wk, 5 days/wk, 90 min/day). We also determined the levels of mRNA expression of PPAR-alpha , HAD, and CPT-1 in the heart of the rats with chronic heart failure (CHF), which is a pathological condition.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and protocol. The experimental protocols were approved by the Committee on Animal Research at the University of Tsukuba. Male 21-mo-old and 2-mo-old Wistar rats were obtained from the Institute for Animal Reproduction (Ibaraki, Japan) and cared for according to the Guiding Principles for the Care and Use of Animals based on the Helsinki Declaration of 1964. These rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. Nine 21-mo-old rats were exercised by swimming for 5 days/wk (Trained-aged group) in a tank of water at 35°C with a surface area of 2,830 cm2 and a depth of 60 cm. The rats swam for 15 min/day for the first 2 days, then the swimming time was gradually increased in a 1-wk period from 15 min/day to 90 min/day. Thereafter, the Trained-aged group continued swimming training for 7 wk. Therefore, the Trained-aged group received 8 wk of swimming training. Nine 21-mo-old rats (Sedentary-aged group) and seven 2-mo-old rats (Sedentary-young group) were confined to their cages for 8 wk but were handled daily. The measurements of body weight, hemodynamic parameters, and echocardiography after swimming training for 8 wk were performed after the rats were allowed to rest for 24 h. Therefore, it was considered that there was no acute effect from the most recent bout of exercise. After these measurements, the heart was rapidly excised and washed thoroughly with cold saline to remove contaminating blood, and then the left ventricle was separated from the right ventricle and atria. The left ventricle was weighed and frozen in liquid nitrogen. Heart samples were stored at -80°C for determination of the protein of PPAR-alpha by ECL Western blotting analysis, mRNA expression of PPAR-alpha by RT-PCR analysis, the activity of PPAR-alpha DNA binding to PPRE by gel mobility shift assay, mRNA expression of enzymes in FA metabolic pathway by RT-PCR analysis, enzyme activity in FA metabolic pathway, and myocardial ATP concentration. Sedentary-young rats and Sedentary-aged rats were killed at the same time point as the Trained-aged rats (Sedentary-young rat: 4 mo old, Sedentary-aged rat: 23 mo old, and Trained-aged rat: 23 mo old).

Measurements of hemodynamic parameters and two-dimensional echocardiography. On the day of the experiment, systolic and diastolic arterial pressures and heart rate (HR) of the animals were measured by using a tail-cuff sphygmomanometer (model MK-1030, Muromachi Kikai; Tokyo, Japan). After these measurements, the rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip), and transthoracic echocardiography was performed with an echocardiographic systems (model SSD-900, Aloka; Tokyo, Japan) equipped with a 7.5-MHz convex scan probe (VST-987-7.5, Aloka) as described in our previous papers (20, 33). We determined the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular fractional shortening (LVFS), which was calculated according to the following formula: LVFS (%) = [(LVEDD - LVESD)/LVEDD]×100, and stroke volume (SV), which was calculated according to the following formula (Pombo method): SV = (LVEDD)3 - (LVESD)3.

RT-PCR to determine levels of mRNA expression in heart. The expression of PPAR-alpha , HAD, and CPT-I mRNA in the left ventricle was analyzed by RT-PCR. The expression of beta -actin mRNA was determined as an internal control. Semiquantitative RT-PCR was performed according to the method described in our previous papers (12, 15, 16, 20).

Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene; Toyama, Japan). Briefly, the tissue was homogenized in Isogen (100 mg tissue/1 ml Isogen) with a Polytron tissue homogenizer (model PT10SK/35, Kinematica; Lucerne, Switzerland). The precipitated RNA was extracted with chloroform, precipitated with isopropanol, and washed with 75% (vol/vol) ethanol. The resulting RNA was resolved in diethyl pyrocarbonate-treated water, treated with DNase I (Takara; Shiga, Japan), and extracted again by Isogen to eliminate the genomic DNA. The RNA concentration was determined spectrophotometrically at 260 nm.

Total tissue RNA (10 µg) was primed with 0.05 µg of oligo d (pT)12-18 and reverse transcribed by avian myeloblastosis virus RT using a first-strand cDNA synthesis kit (Life Sciences). The reaction was performed at 43°C for 60 min.

The cDNA was diluted in a 1:10 ratio, and 1 µl was used for PCR. Each PCR reaction contained 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, dNTP at 200 µM each, gene-specific primer at 0.5 µM each, and 0.025 U/µl Taq polymerase (Takara). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following: PPAR-alpha (9), HAD (21), CPT-I (34), and beta -actin (22). The sequences of the oligonucleotides were as follows: PPAR-alpha (sense), 5'-GATGGTTGGTTACACACG-3'; PPAR-alpha (antisense), 5'-CTTGATGGTGGAGTACAG-3'; HAD (sense), 5'-CCTTCCAGATGGCCTTCCTG-3'; HAD (antisense), 5'-GTTGCCAGATAGCGCAGAGC-3'; CPT-I(sense), 5'-GCCTCAACACAGAACACTCATG-3'; CPT-I (antisense), 5'-GTACTTGGAGACGATGTAGAGG-3'; beta -actin (sense), 5'-GAAGATCCTGACCGAGCGTG-3'; and beta -actin (antisense), 5'-CGTACTCCTGCTTGCTGATCC-3'.

PCR was carried out by using a PCR thermal cycler (model TP-3000, Takara). The cycle profile included denaturation for 15 s at 94°C, annealing for each suitable time at each suitable temperature, and extension for each suitable time at 72°C. The annealing time and temperature were set as follows: 20 s at 58°C for PPAR-alpha , 30 s at 60°C for HAD, 15 s at 57°C for CPT-I, and 15 s at 72°C for beta -actin. The extension time was set as follows: 45 s for PPAR-alpha , HAD, and CPT-I, and 60 s for beta -actin. The reaction cycles of PCR were performed in the range that demonstrated a linear correlation between the amount of cDNA and the yield of PCR products. The amplified PCR products were electrophoresed on 1.2% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed. The photographs were scanned by CanoScan 600 (Canon; Tokyo, Japan), and quantification was performed by a computer with MacBAS software (Fuji Film; Tokyo, Japan).

Electrophoresis and immunoblot analysis for measurement of PPAR-alpha protein in heart. Heart tissues were homogenized with 10 vol of 25 mM HEPES, 400 mM KCl, 1 mM EDTA, and 1.5 mM MgCl2 on ice by using a Teflon homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and protein concentration was determined in the supernatant. Protein concentrations were determined by the BCA protein assay reagents (Pierce; Rock ford, IL) with bovine serum albumin as a standard (30). The samples (50 µg of protein) were followed by heat denaturation at 96°C for 7 min with beta -mercaptoethanol and SDS sample buffer (62.5 mM Tris · HCl buffer, pH 6.8, containing 25% glycerol, 2% SDS). Western blot analysis was performed by the method described by Bordji et al. (4) with a minor modification. Briefly, each sample was separated on a SDS-polyacrylamide gel (8%) and then transferred to polyvinylidene difluoride (PVDF, Millipore; Tokyo, Japan) membranes at 1 mA/cm2 for 120 min. After the membrane was treated with blocking buffer, 5% skim milk in PBS contained 0.05% Tween 20 (PBS-T) for 12 h at 4°C. The membrane was probed with polyclonal anti-PPAR-alpha antibody (1:1,000 dilution with blocking buffer, Santa Cruz Biotechnology) for 1 h at room temperature, washed five times with PBS-T, and then incubated with a horseradish peroxidase-conjugated secondary antibody, which is a donkey anti-goat immunoglobulin (1:1,000 dilution with blocking buffer, Santa Cruz Biotechnology), for 1 h at room temperature. After this reaction, the membrane was washed six times with PBS-T. Finally, the PPAR-alpha were detected by ECL system (Amersham Life Science) and exposed to Hyper film (Amersham Life Science).

Gel mobility shift assays. Heart tissues were homogenized with 10 vol of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, 20 mM beta -glycerophosphate, 0.5 mM PMSF, 60 µg/ml aprotinin, and 2 µg/ml leupeptin on ice using a teflon homogenizer. After the addition of Nonidet P-40 to 0.6%, the homogenate was rotated for 30 min at 4°C and was centrifuged at 3,000 g for 10 min at 4°C. The precipitated nuclear fraction resuspended in 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 0.2 mM DTT, 20 mM beta -glycerophosphate, 0.5 mM PMSF, 60 µg/ml aprotinin, 2 µg/ml leupeptin, and 20% glycerol and was centrifuged at 18,000 g for 10 min at 4°C, and the protein concentration was determined in the supernatant. Gel mobility shift assays using in the myocardial nuclear extracts were performed by the method described by Yano et al. (37) and Juge-Aubry et al. (14) with a minor modification. Briefly, the samples of left ventricular nuclear extracts (15 µg protein) were incubated with 50,000 counts/min of a 32P-labeled double-stranded oligonucleotide probe containing the consensus PPAR-alpha binding sequence (5'-TGACCTTTGACCTAGTTTTG-3') of the promoter regions of HAD and CPT-I at room temperature for 20 min, in 10 µl binding buffer, consisting of 10 mM Tris · HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT, 4% glycerol, and 0.05 mg/ml poly[dI-dC]. We designed using mutant PPRE oligonucleotide (5'-TGTGCTTTGTGCTAGTTTTG-3') for competitive experiment. The DNA-protein complexes were electrophoresed on 4% nondenaturing polyacrylamide gel, and the gel was then dried, subjected to autoradiography, and analyzed with a bioimaging analyzer (BAS-5000, Fuji Film).

Metabolic enzyme activity in heart. Heart tissues (50 mg) were homogenized with 20 vol of 175 mM KCl, 2 mM EDTA, and 10 mM glutathione on ice by using a Teflon homogenizer. The homogenate was centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was diluted with 200 vol of 10 mM Tris · HCl (pH 7.0). For determination of enzyme activity of HAD, 50 µl of each sample was incubated for 5 min at 30°C in a 925-µl incubation mixture containing 10 mM Tris · HCl (pH 7.0), 167 mM triethanolamine, 50 mM EDTA, and 4.5 mM NADH. The reaction was initiated by addition of 25 µl of 1 mM acetoacetyl-CoA and then was determined spectrophotometrically at 340 nm for 10 min (3).

Heart tissues (50 mg) were homogenized with 10 vol of 250 mM sucrose, 1 mM Tris · HCl (pH 7.4), and 130 mM NaCl on ice by using a Teflon homogenizer. The homogenate was centrifuged at 9,000 g for 20 min at 0°C, and the pellet was resuspended with homogenate buffer and centrifuged at 600 g for 10 min at 0°C. The supernatant was centrifuged at 8,000 g for 15 min at 0°C, and the pellet was resuspend with 250 mM sucrose. For determination of enzyme activity of citrate synthase (CS), which is a rate-limiting step enzyme of the TCA cycle, 50 µl of each sample were incubated for 2 min at 30°C in a 900-µl incubation mixture containing 100 mM Tris · HCl (pH 8.0), 1 mM 5,5'-dithio-bis [2-nitro benzaic acid], and 10 mM acetyl-CoA. The reaction was initiated by addition of 50 µl of 10 mM oxaloacetate and then was determined spectrophotometrically at 412 nm for 3 min (31). For determination of enzyme activity of cytochrome oxidase (COX), which is a rate-limiting step enzyme of the electron transfer system, the reaction was initiated by addition of 5 µl of each sample with 995 µl of reaction mixture containing 100 mM potassium phosphate buffer (pH 7.0) and 1% ferrocytochrome c and then was determined spectrophotometrically at 550 nm for 2 min (36).

Myocardial ATP concentration. The ATP concentration was measured with ATP bioluminescence assay kit HS-II (Roche Molecular Biochemical; Tokyo, Japan), which contained cell lysis solution preventing ATP degradation.

Animals and protocol in CHF model. We used the left coronary artery-ligated rat model of CHF, which is well established (24, 26-28). To produce rats with CHF, left ventricular free wall myocardial infarction was induced in 7-wk-old male rats (CHF group) according to the method described in our previous papers (26-28). In brief, each rat was anesthetized with ether. A thoracotomy was performed, the heart was rapidly exteriorized, and the proximal portion of the left coronary artery was ligated with a 5-0 silk suture. The heart was then returned to its normal position, and the thorax was closed. Except for coronary arterial ligation, sham-operated rats were produced by an identical procedure (Sham-control group). Mortality in the animals with myocardial infarction was ~67% within the first 24 h. Six months after surgery, hemodynamic parameters were measured under pentobarbital anesthesia. On the day of the experiment, the rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip). A microtip pressure transducer catheter (model SPC-320, Millar Instruments; Houston, TX) was inserted into the right carotid artery. After the arterial blood pressure and heart rate were monitored, the catheter was advanced into the left ventricle for evaluation of the left ventricular pressure. These hemodynamic measurements were recorded with the use of a polygraph system (AP-601G amplifier and WT-687G thermal pen recorder, Nihon Koden; Tokyo, Japan). In addition, the peak positive first derivative of left ventricular pressure ([+dP/dt/P]max) was derived by active analog differentiation of the pressure signal differentiation amplifier (model EQ-601G, Nihon Koden). After these measurements, the heart was removed, weighed, frozen in liquid nitrogen, and stored at -80°C. The mRNA expression of PPAR-alpha and enzymes in the FA metabolic pathway in the left ventricle was analyzed by RT-PCR.

Statistical analysis. Values are expressed as means ± SE. Statistical analysis among Sedentary-young, Sedentary-aged, and Trained-aged groups was carried out by analysis of variance, followed by Scheffé's F-test for multiple comparisons. Furthermore, to evaluate differences between the Sham-control and the CHF groups, Student's t-test for unpaired values was used. P < 0.05 was accepted as statistically significant.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study of aging and subsequent exercise training. Body weight was significantly lower in the Trained-aged group compared with the Sedentary-aged group (Table 1). Left ventricular weight (LVW) in the Sedentary-aged group and Trained-aged groups was significantly higher compared with the Sedentary-young group (Table 1). LVW mass index for body weight (LVW/BW) in the Trained-aged group was significantly higher compared with the Sedentary-aged group (Table 1). Resting HR was significantly lower in the Trained-aged group compared with the Sedentary-aged group, and that in the Sedentary-aged group was significantly higher compared with the Sedentary-young group (Table 1). There were no significant differences in systolic or diastolic blood pressure among the three groups (Table 1).

                              
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  		Table 1.   Body weight, left ventricular weight, and hemodynamic parameters in sedentary-young, sedentary-aged, and trained-aged rats

Figure 1A shows the representative charts of transthoracic M-mode echocardiograms in the Sedentary-young, Sedentary-aged, and Trained-aged groups. LV-EDD was higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 1B). There was no significant difference in LVESD between the Sedentary-aged and Trained-aged groups (Fig. 1B). LVFS was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 1B). SV was higher in the Trained-aged group compared with the Sedentary-aged group (723.0 ± 48.1 vs. 467.4 ± 40.3 µl).


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  		Fig. 1.   A: typical examples of transthoracic M-mode echocardiograms. A two-dimensional short-axis view was recorded at the level of papillary muscle. B result of the statistical analysis of the M-mode echocardiography. Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular fractional shortening (LVFS) in Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups are shown. Data are expressed as means ± SE.

The mRNA expression of PPAR-alpha in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 2).


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  		Fig. 2.   Expression of peroxisome proliferator-activated receptor (PPAR)-alpha mRNA in the heart (left ventricle) of Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups. Typical examples of RT-PCR analysis are shown for the levels of PPAR-alpha mRNA and beta -actin mRNA (top). Bottom: result of statistical analysis of the level of expression of PPAR-alpha mRNA by a densitometer. We determined the expression of beta -actin mRNA as an internal control. Photos of PCR products were scanned by a densitometer, and ratio of PPAR-alpha mRNA to beta -actin mRNA was calculated. Thus the value of expression of PPAR-alpha mRNA was normalized by that of beta -actin mRNA. Data are expressed as means ± SE.

The protein expression of PPAR-alpha in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 3).


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  		Fig. 3.   Expression of PPAR-alpha protein in the heart (left ventricle) of Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups. Left, typical examples of Western blotting analysis of levels of PPAR-alpha protein. Arrow indicates immunoblot band for PPAR-alpha protein (55 kDa). Right, result of statistical analysis of level of expression of PPAR-alpha protein by a densitometer. Data are expressed as means ± SE.

We performed the gel mobility shift assays of myocardial PPAR-alpha DNA binding to examine whether the change of myocardial PPAR-alpha expression affects the activity of PPAR-alpha DNA binding to PPRE, which is the PPAR-alpha -binding domain of FA metabolic enzyme genes such as HAD, CPT-I, and so on. Figure 4A shows the representative film of gel mobility shift assays of myocardial PPAR-alpha DNA binding in the Sedentary-young, Sedentary-aged, and Trained-aged groups. The activity of myocardial PPAR-alpha DNA binding using a PPRE oligonucleotide was lower in the Sedentary-aged group compared with the Sedentary-young group (Fig. 4A, lane 3 vs. lane 4 and B) and was higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 4A, lane 4 vs. lane 5 and B). The activity of PPAR-alpha DNA binding using mutant PPRE oligonucleotide was almost never detected in the three groups (Fig. 4A, lanes 6-8 and Fig. 4B). Therefore, these data indicated that these bindings (these bands: Fig. 4A, lanes 3-5) specifically formed with PPRE, which is a PPAR-alpha -binding domain of the transcriptional regulating region on the FA metabolic enzyme genes, in the heart, but not with PPRE mutant.


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  		Fig. 4.   Activity of PPAR-alpha DNA binding to peroxisome proliferator-response elements (PPRE), which is PPAR-alpha binding domain of the transcriptional regulating region on the fatty acid (FA) metabolic enzyme genes, in the heart (left ventricular nuclear extracts) of Sedentary-young (SY; n = 7), Sedentary-aged (SA; n = 9), and Trained-aged (TA; n = 9, swimming training for 8 wk) groups using PPRE oligonucleotide probe and mutant PPRE oligonucleotide probe (Mutant PPRE). A: typical examples of gel mobility shift assay of the levels of PPAR-alpha DNA binding activity. Arrow indicates PPAR-PPRE complex. Lane 1, PPRE probe; lane 2, mutant PPRE probe; lanes 3-5, PPRE probe, Sedentary-young rat, Sedentary-aged rat, and Trained-aged rat, respectively; Lanes 6-8, Mutant PPRE probe, Sedentary-young rat, Sedentary-aged rat, and Trained-aged rat, respectively. B: result of statistical analysis of the level of PPAR-alpha DNA binding activity in the heart by a densitometer. Mean value of PPAR-alpha DNA binding activity in the PPRE oligonucleotide probe-Sedentary-young rats (lane 3, PPRE-SY) is represented as 100%. Data are expressed as means ± SE. Significant difference, *P < 0.01. Significantly different vs. PPRE-SY, PPRE-SA, and PPRE-TA, respectively, dagger P < 0.001.

The mRNA expression of HAD, which is a key enzyme of the FA metabolic pathway (beta -oxidation), in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 5A). The mRNA expression of CPT-I, which is a key enzyme of the FA metabolic pathway, in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 5B).


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  		Fig. 5.   Expression of 3-hydroxyacyl CoA dehydrogenase (HAD) mRNA (A) and carnitine palmitoyl transferase-I (CPT-I) mRNA (B) in the heart (left ventricle) of Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups. Top: typical examples of RT-PCR analysis for the levels of HAD mRNA, CPT-I mRNA, and beta -actin mRNA. Bottom: results of the statistical analysis of the levels of expression of HAD mRNA and CPT-I mRNA by a densitometer. We determined the expression of beta -actin mRNA as an internal control. Photos of PCR products were scanned by densitometer, and the ratios of HAD mRNA and CPT-I mRNA to beta -actin mRNA were calculated. Thus the values of expressions of HAD mRNA and CPT-I mRNA were normalized by that of beta -actin mRNA. Data are expressed as means ± SE.

Enzyme activity of HAD, which is a key enzyme of beta -oxidation, in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 6).


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  		Fig. 6.   Enzyme activity of HAD in the heart (left ventricle) of Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups. Data are expressed as means ± SE.

Enzyme activity of CS, which is a key enzyme of the TCA cycle, in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 7). Enzyme activity of COX, which is a key enzyme of electron transfer system, in the heart was significantly lower in the Sedentary-aged group compared with the Sedentary-young group and was significantly higher in the Trained-aged group compared with the Sedentary-aged group (Fig. 7).


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  		Fig. 7.   Enzyme activity of citrate synthase (CS, A) and cytochrome oxidase (COX, B) in the heart (left ventricle) of Sedentary-young (n = 7), Sedentary-aged (n = 9), and Trained-aged (n = 9, swimming training for 8 wk) groups. Data are expressed as means ± SE.

Myocardial ATP concentration, which is an index of myocardial energy molecules, was significantly lower in the Sedentary-aged group compared with the Sedentary-young group (1.71 ± 0.08 vs. 2.30 ± 0.23 µmol/g tissue, P < 0.05), and there was no significant difference in myocardial ATP concentration between the Sedentary-young and Trained-aged groups (1.99 ± 0.11 µmol/g tissue, not significant), indicating that the aging-induced decrease of myocardial ATP concentration is improved by exercise training.

Study of chronic heart failure model. There was no significant difference in body weight between the Sham-control (559 ± 8 g; n = 8) and the CHF groups (555 ± 6 g; n = 8). LVW/BW in the CHF group was significantly higher compared with the Sham-control group (Table 2). There was no significant difference in resting HR between the two groups (Table 2). Mean blood pressure and left ventricular systolic pressure in the CHF group was significantly lower compared with the Sham-control group (Table 2). Left ventricular end-diastolic pressure in the CHF group was significantly higher compared with the Sham-control group (Table 2). Left ventricular [+dP/dt/P]max in the CHF group was significantly lower compared with the Sham-control group (Table 2). These data indicated that the CHF rats had developed chronic heart failure.

                              
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  		Table 2.   Hemodynamic parameters in sham-control rats and rats with CHF

The mRNA expression of PPAR-alpha in the heart was significantly lower in the CHF group compared with the Sham-control group (Fig. 8). The mRNA expression of HAD and CPT-I in the heart was significantly lower in the CHF group compared with the Sham-control group (Fig. 8).


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  		Fig. 8.   Expression of PPAR-alpha mRNA (A), HAD, mRNA (B), and carnitine palmitoyl transferase-I (CPT-I) mRNA (C) in the heart (left ventricle) of Sham-control (n = 8) and CHF (n = 8) groups. Top: typical examples of RT-PCR analysis are shown for the levels of PPAR-alpha mRNA, HAD mRNA, CPT-I mRNA, and beta -actin mRNA. Bottom: results of the statistical analysis of the levels of expression of PPAR-alpha mRNA, HAD mRNA, and CPT-I mRNA by a densitometer. We determined the expression of beta -actin mRNA as an internal control. Photos of PCR products were scanned by densitometer, and the ratios of PPAR-alpha mRNA, HAD mRNA, and CPT-I mRNA to beta -actin mRNA were calculated. Thus the values of expressions of PPAR-alpha mRNA, HAD mRNA, and CPT-I mRNA were normalized by that of beta -actin mRNA. CHF, chronic heart failure. Data are expressed as means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrated for the first time that exercise training improved the aging-induced decrease of mRNA and protein expression of PPAR-alpha , which is a transcriptional regulator of FA metabolic enzyme genes, in the heart. We also demonstrated that exercise training improved the aging-induced decrease of activity of myocardial PPAR-alpha DNA binding to PPRE, which is PPAR-alpha binding domain of the transcriptional regulating region on the FA metabolic enzyme genes. Therefore, it is considered that the aging-induced decrease of the transcriptional regulation in myocardial FA metabolic enzyme gene expressions is improved by exercise training. Furthermore, the present study showed that exercise training improved the aging-induced decrease of mRNA expression of HAD and CPT-I, which are target genes of PPAR-alpha , in the heart, and that the gene expression of HAD and CPT-I altered in association with changes of the mRNA and protein levels of PPAR-alpha in the heart. Therefore, we consider that the myocardial PPAR-alpha participates in the regulation of gene expression of FA metabolic enzymes in the heart for aging and subsequent exercise training. Furthermore, we demonstrated that exercise training improved the aging-induced decrease of enzyme activity of HAD, CS, and COX in the heart and myocardial ATP concentration. These results suggest that the aging-induced decrease of energy production capacity from FA metabolic pathway in the heart was improved by exercise training. Taken together, it is considered that the changes of HAD and CPT-I mRNA levels in the heart are attributed to the mRNA and protein levels of PPAR-alpha , thereby causing to the change of the HAD activity in the heart. Therefore, we consider that PPAR-alpha participates in the regulation of cardiac FA metabolic capacity for aging and subsequent exercise training. Thus this regulation in a molecular level may contribute to adaptive responses of FA metabolic capacity in the heart for aging and subsequent exercise training.

By echocardiography observation in the present study, Trained-aged rats showed an improvement of aging-induced decrease of LVFS, and SV in the Trained-aged rats was higher than that in the Sedentary-aged rats. These results suggest that the aging-induced decrease in cardiac function is improved by exercise training. The present study also revealed that the exercise training induced an improvement of aging-induced decrease of HAD, CS, and COX activities in the heart and myocardial ATP concentration. Therefore, these results indicate that the change of energy production capacity from FA metabolic pathway in the heart for aging and subsequent exercise training is accompanied with the change of cardiac function. Furthermore, the present study revealed that exercise training improved the aging-induced decreases of mRNA and protein expression of PPAR-alpha and mRNA expression of FA metabolic enzyme in the heart. Therefore, it is possible that the changes in myocardial energy production capacity from FA metabolic pathway and a molecular regulation of PPAR-alpha and FA metabolic enzyme genes partly contribute to the improvement of cardiac function by exercise training in the aged rats.

Energy production capacity from the FA metabolic pathway in the heart is reduced by aging (19, 32). It has been reported that an aging-induced decrease of energy (FA) metabolic capacity in the heart is improved by exercise training (7, 18, 32). However, the mechanisms for improving FA metabolic capacity in the heart are unclear. PPAR-alpha binds to PPRE present in the upstream regions of a number of genes involved in the FA metabolic pathway and regulates transcription of target genes (13, 29). In the present study, the model rats of CHF, of which mRNA expression of PPAR-alpha is downregulated in the heart, caused a decrease in mRNA expression of HAD and CPT-I, which are target genes of PPAR-alpha . It has been reported that the lack of PPAR-alpha (PPAR-alpha -/- mice) caused decreases of enzyme activity and protein and mRNA expression in FA metabolic enzymes in the mouse heart (1, 35). On the other hand, it has recently been reported that cardiac-specific overexpression of PPAR-alpha caused an increase in gene expression and enzyme activity of CPT-I by the increase in gene and protein expressions of PPAR-alpha in the mouse heart, thereby inducing increases in FA uptake and utilization in the mouse heart (8). Therefore, PPAR-alpha regulates target genes encoding FA metabolic (beta -oxidation) enzymes such as CPT-I, acyl-CoA oxidase, HAD, and so on (1, 2, 35). The present study revealed that the decrease of mRNA expression of CPT-I and HAD with the decrease of mRNA and protein expression of PPAR-alpha in the aged heart is improved by exercise training. The present study also showed that exercise training improved the aging-induced decrease of activity of myocardial PPAR-alpha DNA binding to PPRE, which is PPAR-alpha binding domain of the transcriptional regulating region on the FA metabolic enzyme genes. Furthermore, the HAD activity alters in association with changes of gene expression of HAD and PPAR-alpha in the heart. On the basis of results from past studies plus the present results, it is considered that the PPAR-alpha signaling in the heart regulates gene expression in FA metabolic enzymes and that this regulatory system participates in a mechanism of adaptations of FA metabolic capacity in the heart for aging and subsequent exercise training. Therefore, it is possible that PPAR-alpha plays a critical role in a maintenance of cardiac energy and FA metabolic homeostasis for aging and subsequent exercise training.

The mechanism underlying the improvement of aging-induced decrease in PPAR-alpha expression (both mRNA and protein levels) in the heart by exercise training remains to be elucidated. The following reports, which examined effects of chronic motor nerve stimulation or exercise training on expression of PPAR-alpha in the skeletal muscle, would provide useful suggestion for the above question. Cresci et al. (6) reported that chronic motor nerve stimulation in dogs increased a protein expression of PPAR-alpha in the skeletal muscle and also increased expression of its target gene of FA metabolic enzymes. Furthermore, it has been reported that endurance training for 12 wk in women increased a protein expression of PPAR-alpha in the skeletal muscle with increased FA oxidation capacity (11). Therefore, in the skeletal muscle, it is considered that some factors, which stimulate an increase of PPAR-alpha expression, are activated by exercise training. As discussed above, because Cresci et al. (6) reported that the stimulation of nerves induces an increase in expression of PPAR-alpha in the skeletal muscle, the stimulation of nerves may be one of the causal factors in the increase of PPAR-alpha expression in the skeletal muscle by exercise training. The present study demonstrated that, in the heart, chronic exercise training improved the aging-induced decrease in mRNA and protein expression of PPAR-alpha in rats. Therefore, because exercise induces activation of nerves in the heart, it is possible that the stimulation of nerves in the heart by exercise may be one of the causal factors in the exercise training-induced increase of myocardial PPAR-alpha expression. Furthermore, an alternative hypothesis is that the exercise training-induced alterations of mechanical factors and metabolic factors in the heart may participate in the mechanism by which exercise training improves the myocardial PPAR-alpha expression, because exercise induces an integrated physiological response on the heart, e.g., an increase in mechanical stress and an activation of metabolism in the heart. The present study also showed that, in the heart, exercise training improved the aging-induced decrease in the activity of PPAR-alpha DNA binding to PPRE, which is PPAR-alpha binding domain of the transcriptional regulating region on HAD gene and CPT-I gene, and improved the aging-induced decrease in mRNA expression of HAD and CPT-I and the activity of FA metabolic enzyme. Thus the present study showed that, in the heart, improvement of aging-induced decrease in PPAR-alpha expression by exercise training may participate in the improvement of aging-induced decrease in FA metabolic capacity (enzyme gene expression and enzyme activity) by exercise training. Nevertheless, the precise mechanism by which exercise training improves the aging-induced decrease in PPAR-alpha expression and activity in the heart remains to be elucidated.

Acceleration of myocardial FA uptake and oxidation is accompanied with the increase of FA enzyme activity and gene expression, such as CPT-I (8). The present study revealed that the exercise training improves the aging-induced decrease of mRNA expression of HAD and CPT-I in the heart. The present study also showed that the exercise training improves the aging-induced decrease of enzyme activity of HAD, CS, and COX, which are main enzymes involved in the FA metabolic pathway (beta -oxidation, TCA cycle, and electron transfer system) and the aging-induced decrease of myocardial ATP concentration. These results suggest that, in the Trained-aged rats, swimming training caused the improvement of aging-induced declination in energy production capacity from FA metabolic pathway in the heart. Alternatively, the following consideration is also possible. The present study determined CS and COX activities as one of enzymes in the energy production pathway of the FA metabolism, whereas CS and COX activities are also indexes of oxidative metabolic capacity. Therefore, it is considered that the exercise training-induced improvement of CS and COX activities in the aged heart is caused by PPAR-alpha -mediated pathway or other transcriptional factor-mediated pathways. If it is mediated by such other transcriptional pathways, exercise training would stimulate other transcription pathways in addition to PPAR-alpha -mediated pathway. However, the precise mechanism is not known.

The present study showed that the heart of the rats with CHF, which is a pathological condition, represented a decrease in mRNA expression of HAD and CPT-I with a downregulation of mRNA expression of PPAR-alpha . It has been reported that PPAR-alpha gene expression is downregulated in the heart of some pathological conditions, which induces a decrease in mRNA expression of main enzymes in FA metabolic pathway (2). Our findings are in accordance with this report.

In conclusion, we demonstrated that the exercise training improves the aging-induced decrease of mRNA and protein expression of PPAR-alpha in the heart. We also demonstrated that exercise training improves the aging-induced decrease of activity of myocardial PPAR-alpha DNA binding to PPRE, which is a PPAR-alpha binding domain of the transcriptional regulating region on the FA metabolic enzyme genes. The present study showed that the exercise training improves the aging-induced decrease of mRNA expression of CPT-I and HAD, which are target genes of PPAR-alpha , in the heart. Furthermore, the present study demonstrated that the exercise training improves the aging-induced decrease of activity of HAD, CS, and COX and cardiac function. These results suggest that the aging-induced decrease of energy production capacity from FA metabolic pathway in the heart is improved by exercise training and that the mRNA and protein of PPAR-alpha , myocardial PPAR-alpha DNA binding and the mRNA of HAD and CPT-I alter in association with change of the HAD activity in the heart. Therefore, it is considered that the PPAR-alpha signaling in the heart regulates a gene expression of FA metabolic enzymes and that this regulatory system participates in a mechanism of adaptations of FA metabolic capacity in the heart for aging and subsequent exercise training. We propose that the regulation of PPAR-alpha in the heart participates in the change of cardiac FA metabolic capacity for aging and subsequent exercise training.


    ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (00006781, 11480003, 11557047, 12470147, and 12670646), a grant from University of Tsukuba Research Projects, and a grant from the project of Tsukuba Advanced Research Alliance in the University of Tsukuba.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Division, Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan (E-mail: t-miyauc{at}md.tsukuba.ac.jp).

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.

July 18, 2002;10.1152/ajpheart.01051.2001

Received 5 December 2001; accepted in final form 1 July 2002.


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