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Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats

¹Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, ²Institute of Health and Sport Sciences, and ³Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan

Submitted 8 August 2003 ; accepted in final form 22 December 2003

	ABSTRACT

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Exercise training improves the aging-induced downregulation of myosin heavy chain (MHC) and sarcoplasmic reticulum (SR) Ca²⁺-ATPase, which participate in the regulation of cardiac contraction and relaxation. Thyroid hormone receptor (TR), a transcriptional activator, affected the regulation of gene expression of MHC and SR Ca²⁺-ATPase. We hypothesized that myocardial TR signaling contributes to a molecular mechanism of exercise training-induced improvement of MHC and SR Ca²⁺-ATPase genes with cardiac function in old age. We investigated whether TR signaling and gene expression of MHC and SR Ca²⁺-ATPase in the aged heart are affected by exercise training, using the hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo old, swimming training for 8 wk). Trained aged rats showed improvement in cardiac function. Expression of TR-₁ and TR-₁ proteins in the heart were significantly lower in sedentary aged rats than in sedentary young rats and were significantly higher in trained aged rats than in sedentary aged rats. The activity of TR DNA binding to the transcriptional regulatory region in the -MHC and SR Ca²⁺-ATPase genes and the mRNA and protein expression of -MHC and SR Ca²⁺-ATPase in the heart and plasma 3,3'-triiodothyronine and thyroxine levels were altered in association with changes in the myocardial TR protein levels. These findings suggest that exercise training improves the aging-induced downregulation of myocardial TR signaling-mediated transcription of MHC and SR Ca²⁺-ATPase genes, thereby contributing to the improvement of cardiac function in trained aged hearts.

myosin heavy chain; sarcoplasmic reticulum Ca²⁺-ATPase; swimming training

Myosin heavy chain (MHC), a contractile protein, has two isoforms, and (30). -MHC is associated with high ATPase activity and has a higher contraction velocity in the heart, whereas -MHC is associated with low ATPase activity and has a slower contraction velocity in the heart (30). Although in the heart of normal adult rats 80–90% of the expressed MHC consists of the -form, aging causes a shift of isoform from -MHC to -MHC in the heart (9). In cardiac muscle, the sarcoplasmic reticulum (SR) plays a central role in the contraction and relaxation cycle by regulating the intracellular Ca²⁺ concentration (44). SR Ca²⁺-ATPase is a Ca²⁺ pump that transports Ca²⁺ into the SR lumen through an ATP-dependent mechanism and is primarily responsible for myocardial relaxation (44). Aging decreases expression of SR Ca²⁺-ATPase mRNA in the heart (28). On the other hand, exercise training in aged rats improves the aging-induced decrease in expression of SR Ca²⁺-ATPase mRNA in the heart (47). However, the signaling mechanisms causing to alteration of expression of myocardial -MHC and SR Ca²⁺-ATPase genes with aging and subsequent exercise training are unclear.

Thyroid hormone receptor (TR) binds to thyroid responsive element (TRE) in the promoter regions of several genes, including -MHC and SR Ca²⁺-ATPase in the heart, and regulates the transcription of target genes in the heart (6, 17). TR is the receptor of thyroid hormone and has TR-₁,-₂,-₁, and -₂ isoforms (8, 14, 31). The TR-₂ isoform does not bind thyroid hormone and has a dominant-negative effect on the function of the other receptor isoforms (18, 22). TR-₂ mRNA is not detected in the rat heart (6). TR-₁ and -₁ are expressed in the heart and are functional receptors, which bind thyroid hormone with similar affinity (18, 22, 39). Furthermore, retinoid X receptor (RXR) plays a role in forming heterodimers with TRs to bind to TRE in the promoter regions of target genes and has RXR-, -, and - isoforms (23, 50, 52). It has been reported (27) that aging induced a decrease in mRNA expression of TR-₁, -₁, and RXR- and protein expression of TR-₁ in the heart. However, it is unknown whether exercise training in the aged rat improves aging induced downregulation of the gene and protein levels of myocardial TR. Furthermore, although myocardial TR signaling participates in the transcriptional regulation of -MHC and SR Ca²⁺-ATPase genes, it is unclear whether alterations of TR signaling induce changes in the target genes in the heart.

We hypothesized that exercise training during old age improved aging-induced downregulation of myocardial TR signaling, and the improvement of TR signaling causes changes of -MHC and SR Ca²⁺-ATPase genes in the heart with change of cardiac function. In the present study, we tested our hypothesis by using sedentary young rats (SY group; 4 mo old), sedentary aged rats (SA group; 23 mo old), and swim-trained aged rats (TA group; 23 mo old, swimming training for 8 wk, 5 days/wk, and 90 min/day). We investigated the protein expression of TRs and RXRs and activity of TR DNA binding to TRE in the heart with aging and subsequent exercise training. We also studied the level of mRNA and protein of MHC and SR Ca²⁺-ATPase in the heart with aging and subsequent exercise training.

	METHODS

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Animals and protocol. The experimental protocols were approved by the Committee on Animal Research at the University of Tsukuba. Male 2- and 21-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. Seven 21-mo-old rats were exercised by swimming for 5 days/wk (TA group) in a tank of water at 35–37° C with a surface area of 2,830 cm² and a depth of 60 cm. The animals were swum as a group of three or four rats because the intensity of swimming exercise was significantly raised by any interaction among the rats. The rats swam for 15 min/day for the first 2 days; the swimming time was then gradually increased in 1-wk periods from 15 to 90 min/day. Thereafter, the TA group continued swimming training for 7 wk. Therefore, the TA group received 8 wk of swimming training. Seven 21-mo-old rats (SA group) and seven 2-mo-old rats (SY group) were confined to their cages for 8 wk but were handled daily. Measurements of body weight and echocardiography after swimming training for 8 wk were performed after rats rested 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 and epitrochlearis muscle were removed, rinsed in ice-cold saline, weighed, and frozen in liquid nitrogen. Heart samples were stored at –80° C for determination of the mRNA expression of MHC and SR Ca²⁺-ATPase by RT-PCR analysis, MHC isoforms by electrophoretic separation analysis, protein levels of SR Ca²⁺-ATPase, TR-₁, TR-₁, RXR-, RXR-, and RXR- by Western blot analysis, and activity of TR DNA binding to TRE by gel mobility shift assay. The epitrochlearis muscle was chosen for measurement of citrate synthase activity because a previous study (7) reported that swimming training caused the increase in citrate synthase activity in the epitrochlearis muscle. Therefore, the epitrochlearis muscle samples were also stored at –80° C. SY rats and SA rats were euthanized at the same time point as the TA rats (SY rats: 4 mo old, SA rats: 23 mo old, and TA rats: 23 mo old).

Measurements of two-dimensional echocardiography. On the day of the experiment, the rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip) and transthoracic echocardiography was performed with an echocardiographic system (model SSD-900, Aloka; Tokyo, Japan) equipped with a 7.5-MHz convex scan probe (model VST-987-7.5, Aloka) as described in our previous studies (11). We determined the heart rate (HR), left ventricular (LV) enddiastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV fractional shortening (LVFS), which was calculated according to the following formula: LVFS (%) = [(LVEDD – LVESD)/LVEDD] x 100, LV ejection fraction (LVEF), which was calculated according to the following formula: LVEF (%) = [(LVEDD)³ – (LVESD)³/(LVEDD)³] x 100, and stroke volume (SV), which was calculated according to the following formula (Pombo method): SV = (LVEDD)³ – (LVESD)³.

Muscle oxidative enzyme activity. Citrate synthase activity, a marker of mitochondrial content, was measured in the whole epitrochlearis muscle homogenate using the spectrophotometric method of Srere et al. (41).

RT-PCR to determine levels of mRNA expression in heart. The expression of MHC and SR Ca²⁺-ATPase mRNA in the left ventricle was analyzed by RT-PCR. The expression of -actin mRNA was determined as an internal control. Semiquantitative RT-PCR was performed according to the method described in our previous studies (10, 12).

Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene; Toyama, Japan) according to the method described in our previous studies (10, 12).

Total tissue RNA (10 µg) was primed with 0.05 µg of oligo d(pT)_12–18 and reverse transcribed by avian myeloblastosis virus reverse transcriptase 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: MHC (19), SR Ca²⁺-ATPase (26), and -actin (32). The sequences of the oligonucleotides were as follows: MHC (sense), 5'-GCAGACCATCAAGGACCT-3'; MHC (antisense), 5'-GTTGGCCTGTTCCTCCGCC-3'; SRCa²⁺-ATPase (sense), 5'-CTCAGACAAGACCGGCACACT-3'; SR Ca²⁺-ATPase (antisense), 5'-ACACCTTCTGGAGCACCCTTC-3'; -actin (sense), 5'-GAAGATCCTGACCGAGCGTG-3'; and -actin (antisense), 5'-CGTACTCCTGCTTGCTGATCC-3'

PCR was carried out 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: 15 s at 63° C for MHC, 15 s at 71° C for SR Ca²⁺-ATPase, and 15 s at 72° C for -actin. The extension time was set as follows: 45 s for MHC and 60 s for SR Ca²⁺-ATPase and -actin. The reaction cycles of PCR were performed in a range that demonstrated a linear correlation between the amount of cDNA and the yield of PCR products. The PCR products were found to be of the expected size, as shown by 1.2% agarose gel electrophoresis for SR Ca²⁺-ATPase and -actin. Distinction between -MHC and -MHC was determined by a restriction endonuclease digestion method (10, 15). After PCR in MHC, distinction between -MHC and -MHC was achieved by digestion of 12.5 µl of the PCR reaction mixture with 0.8 units MseI, which recognizes the sequences "TTAA" in a standard reaction buffer at 37° C for 4 h (10). Because the recognition site exists only in the amplicon for -MHC but not in that of -MHC, the reaction yielded fragments of 310 bp for -MHC and 275 + 53 bp for -MHC. The fragments were electrophoresed in 2.0% agarose gel and separated. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme analysis and DNA sequencing. The DNA sequence of each amplicon was perfectly matched to each published sequence.

Semiquantitative analysis of PCR products. We performed semiquantitative PCR analysis to evaluate the expression levels of MHC mRNA, SR Ca²⁺-ATPase mRNA, and -actin mRNA. The amplified PCR products were electrophoresed on a 1.2% or 2.0% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed according to the method described in our previous studies (10, 12). The photographs were scanned (CanoScan model 600, Canon; Tokyo, Japan), and quantification was performed with a computer using MacBAS software (Fuji Film; Tokyo, Japan) according to the method described in our previous studies (10, 12). The cDNAs for the verification of the semiquantitative PCR analysis were prepared from each gene PCR product of rat cDNA. Each PCR product was purified, quantified, and used as a positive-control cDNA. Each PCR product concentration was calculated by assuming that the mass of a nucleotide pair in DNA is 660 Da (38). We performed semiquantitative PCR analysis to evaluate the expression level of MHC mRNA, SR Ca²⁺-ATPase mRNA, and -actin mRNA. To demonstrate that our semiquantitative PCR strategy was valid, serial dilutions of each positive control cDNA were amplified by PCR and quantified by scanner.

Electrophoretic separation analysis for measurement of MHC isoforms in heart. Electrophoretic separation analysis using SDS-polyacrylamide gel was performed by the method described by Talmadge and Roy (45) and Reiser et al. (34) with minor modification. The stacking and separating gels consisted of 4% and 8% acrylamide, respectively, with acrylamide: N,N'-methylene-bis-acrylamide in the ratio of 50:1 (45). The gels were stained with Coomassie brilliant blue and performed scanning to evaluate the isoform of -MHC and -MHC.

Electrophoresis and immunoblot analysis for measurement of SR Ca²⁺-ATPase, TR, and RXR proteins in heart. Western blot analysis of SR Ca²⁺-ATPase protein (37) and TR and RXR proteins (16) was performed according to previous studies with minor modification. Briefly, each sample was separated on SDS-polyacrylamide gel (10%) and then transferred to polyvinylidene difluoride (Millipore; Tokyo, Japan) membranes at 3 mA/cm² for 60 min. The membrane was treated with blocking buffer and 5% skim milk (SR Ca²⁺-ATPase) or 3% skim milk (TR and RXR) in PBS with 0.1% Tween 20 (PBS-T) for 12 h at 4° C. The membrane was probed with polyclonal anti-TR-₁, RXR-, RXR-, and RXR- antibody (1:1,000 dilution with blocking buffer, Santa Cruz Biotechnology) and monoclonal anti-SR Ca²⁺-ATPase (1:500 dilution with blocking buffer, Affinity Bioreagents; Golden, CO) and anti-TR-₁ antibody (1:500 dilution with blocking buffer, Santa Cruz Biotechnology) for 1 h at room temperature, washed with PBS-T three times, and then incubated with a horseradish peroxidase-conjugated secondary antibody, which was an anti-rabbit immunoglobulin (1:5,000 dilution with blocking buffer, Amersham Life Science), and an anti-mouse immunoglobulin (1: 2,000 dilution with blocking buffer, Amersham Life Science) for 1 h at room temperature. After this reaction, the membrane was washed with PBS-T three times. Finally, SR Ca²⁺-ATPase, TR-₁, TR-₁, RXR-, RXR-, and RXR- were detected by an ECL system (Amersham Life Science) and exposed to Hyperfilm (Amersham Life Science).

Gel mobility shift assays. Gel mobility shift assays using the myocardial nuclear extracts were performed by the method described by Ojamaa et al. (33) and Rohrer et al. (35) with minor modification. Briefly, the samples of LV nuclear extracts (15 µg of protein) were incubated with 50,000 cpm ³²P-labeled double-stranded oligonucleotide probes containing the consensus TR binding sequence (5'-GCTGTCCTCCTGTCACCTCCAGA-3') of the promoter region of -MHC and the consensus TR binding sequence (5'-GCCGCGACCGCGTAAGGTCGGGCT-3') of the promoter region of SR Ca²⁺-ATPase at room temperature for 20 min in 10 µl of 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]. 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).

Measurements of plasma thyroid hormone level. The plasma 3,3',5-triiodothyronine (T₃) and thyroxine (T₄) were measured by using radioimmunoassay in duplicate by previously described methods (51).

Statistical analysis. Values are expressed as means ± SE. Statistical analysis was carried out by analysis of variance, followed by Scheffé's F-test for multiple comparisons. P < 0.05 was accepted as significant.

	RESULTS

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Body weight was significantly lower in the TA group than in the SA group (Table 1). LV weight (LVW) in the SA group and TA group was significantly higher than that in the SY group (Table 1). LVW mass index for body weight in the TA group was significantly higher than that in the SA group (Table 1). Citrate synthase activity in the epitrochlearis muscle was significantly lower in the SA group than in the SY group and was significantly higher in the TA group than in the SA group (Table 1). These results suggest that the TA rats exhibited physiological effects of exercise training.

There was no significant difference in LV mass between the SA and TA groups (1.02 ± 0.09 vs. 1.25 ± 0.11 mg). LVEDD was higher in the TA group than in the SA group (Table 2). There was no significant difference in LVESD between the SA and TA groups (Table 2). LVFS and LVEF were significantly lower in the SA group than in the SY group and were significantly higher in the TA group than in the SA group (Table 2). SV was higher in the TA group than in the SA group (Table 2). There was no significant difference in resting HR between the SY and SA groups (Table 2). Resting HR in the TA group was significantly lower than that in the SY and SA groups (Table 2).

To semiquantitatively determine alterations in gene expression of MHC and SR Ca²⁺-ATPase by aging and subsequent exercise training, the relationship between the amount of cDNA and the yield of PCR products was examined. There was a linear correlation between the initial amount of -actin cDNA and the yield of PCR products (r = 0.999). In the cases of MHC and SR Ca²⁺-ATPase, the yield of PCR products was also in proportion to the initial amount of cDNA (r = 0.996 and r = 0.998, respectively). These relationships in control rats were similar to exercise rats. Figure 1A shows typical examples of RT-PCR analysis of -MHC and -MHC in the heart in SY, SA, and TA rats. The mRNA expression of -MHC in the heart was markedly lower in the SA group than in the SY group and was significantly higher in the TA group than in the SA group (Fig. 1B). The mRNA expression of -MHC in the heart was markedly higher in the SA group than in the SY group, and there was no significant difference in mRNA expression of -MHC between the SA and TA groups (Fig. 1B). Therefore, -MHC/-MHC mRNA expression in the heart, which indicates the shift of isoform from -MHC to -MHC in the heart, was markedly higher in the SA group than in the SY group and was significantly lower in the TA group than in the SA group (Fig. 1B).

View larger version (44K): [in this window] [in a new window]   Fig. 1. Expression of -myosin heavy chain (-MHC) mRNA, -MHC mRNA, and -MHC/-MHC mRNA in the heart (left ventricle) of the sedentary young group (SY; n = 7), sedentary aged group (SA; n = 7), and trained aged group (TA; n = 7, swimming training for 8 wk). A: typical examples of RT-PCR analysis are shown for the levels of -MHC and -MHC mRNA. B: results of statistical analysis of the levels of expression of -MHC, -MHC, and /-MHC mRNA, as determined with the use of a densitometer. The expression of -actin mRNA was determined as an internal control. Photos of PCR products were scanned by a densitometer, and the ratios of -MHC and -MHC mRNA to -actin mRNA were calculated. Thus the levels of expression of -MHC and -MHC mRNA were normalized by that of -actin mRNA. Data are expressed as means ± SE.

The mRNA expression of SR Ca²⁺-ATPase in the heart was markedly lower in the SA group than in the SY group and was markedly higher in the TA group than in the SA group (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Expression of sarcoplasmic reticulum (SR) Ca2+-ATPase mRNA in the heart (left ventricle) of the SY group (n = 7), the SA group (n = 7), and the TA group (n = 7, swimming training for 8 wk). Typical examples of RT-PCR analysis are shown for the levels of SR Ca2+-ATPase mRNA (top). Results of statistical analysis of the level of expression of SR Ca2+-ATPase mRNA were determined with a densitometer (bottom). The expression of -actin mRNA was determined as an internal control. Photos of PCR products were scanned by a densitometer, and the ratio of SR Ca2+-ATPase mRNA to -actin mRNA was calculated. Thus the level of expression of SR Ca2+-ATPase mRNA was normalized by that of -actin mRNA. Data are expressed as means ± SE.

Figure 3 shows typical examples of electrophoretic separation analysis of MHC isoforms in the heart in SY, SA, and TA rats. The heart of SY rats mainly expressed isoform of -MHC, whereas the heart of SA rats equally expressed between -MHC and -MHC isoforms (Fig. 3). The heart of TA rats expressed more -MHC isoform than -MHC isoform (Fig. 3). Therefore, -MHC/-MHC expression in the heart, which indicates the shift of isoform from -MHC to -MHC in the heart, was significantly higher in the SA group than in the SY group and was significantly lower in the TA group than in the SA group (Fig. 3).

View larger version (48K): [in this window] [in a new window]   Fig. 3. Expression of -MHC and -MHC isoforms in the heart (left ventricle) of SY (n = 7), SA (n = 7), and TA (n = 7) groups. Left, typical examples of electrophoretic separation analysis are shown for the levels of -MHC and -MHC isoforms. Right, results of statistical analysis of the level of expression of /-MHC, as determined by a densitometer. Data are expressed as means and SE.

The protein expression of SR Ca²⁺-ATPase in the heart was significantly lower in the SA group than in the SY group and was significantly higher in the TA group than in the SA group (Fig. 4).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Expression of SR Ca2+-ATPase protein in the heart (left ventricle) of SY (n = 7), SA (n = 7), and TA (n = 7) groups. Typical examples of Western blot analysis are shown for the levels of SR Ca2+ATPase protein (left). Arrow indicates the immunoblot band for SR Ca2+-ATPase protein. Right, result of statistical analysis of the level of expression of SR Ca2+-ATPase protein determined by a densitometer. Data are expressed as means ± SE.

The protein expression of TR-₁ and TR-₁ in the heart was significantly lower in the SA group than in the SY group and was significantly higher in the TA group than in the SA group (Fig. 5, A and B).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Expression of thyroid hormone receptor (TR)-1 protein (A) and TR-1 protein (B) in the heart (left ventricle) of SY (n = 7), SA (n = 7), and TA (n = 7) groups. Typical examples of Western blot analysis are shown for the levels of TR-1 and TR-1 proteins (left). Arrows indicate the immunoblot band for TR-1 protein and TR-1 protein. Right, results of statistical analysis of the levels of expression of TR-1 and TR-1 proteins determined by a densitometer. Data are expressed as means ± SE.

We performed gel mobility shift assay of myocardial TR DNA binding to examine whether a change in myocardial TR protein affects the activity of TR DNA binding to TREs, which are TR binding domains of the -MHC and SR Ca²⁺-ATPase genes. Figure 4 shows representative films of gel mobility shift assay of myocardial TR DNA binding in the SY, SA, and TA groups. The activity of TR DNA binding was decreased by addition of each unlabeled (competitive) TRE consensus oligonucleotide of -MHC and SR Ca²⁺-ATPase genes in a dose-dependent manner (Fig. 6A). Therefore, these data indicate that this binding occurred specifically with TRE, which is the TR binding domain of the transcriptional regulating regions in the -MHC and SR Ca²⁺-ATPase genes, in the heart. The activity of myocardial TR DNA binding using TRE oligonucleotides of each promoter region in the -MHC and SR Ca²⁺-ATPase genes was lower in the SA group than in the SY group and was higher in the TA group than in the SA group (Fig. 6B).

View larger version (64K): [in this window] [in a new window]   Fig. 6. Typical examples of gel mobility shift assay for the level of TR DNA binding to thyroid response element (TRE), which is the TR binding domain of the transcriptional regulatory region in the -MHC and SR Ca2+-ATPase genes, in the heart (left ventricular nuclear extracts). A: each competitive assay for TR DNA binding of -MHC (lanes 1–4) and SR Ca2+ATPase (lanes 5–8) genes was carried out in the presence of a 50- and 100-fold molar excess of each unlabeled TR oligonucleotide (competitor). Each arrow indicates TR, TR-TRE complex; NS, nonspecific binding; F, free probe. B: activity of TR DNA binding to TRE in heart (left ventricular nuclear extracts) of the SY, SA, and TA groups using each TRE oligonucleotide probe (TRE) of -MHC (left) and SR Ca2+ATPase (right). Lanes 1–3, TRE probe (-MHC): SY, SA, and TA rats, respectively (left); lanes 4–6, TRE probe (SR Ca2+-ATPase): SY, SA, and TA rats, respectively (right).

The protein expression of RXR-, RXR-, and RXR- in the heart was significantly lower in the SA group than in the SY group (Fig. 7, A–C). There was no significant difference in protein expression of RXR- between the SA and TA groups (Fig. 7A). The protein expression of RXR- was significantly higher in the TA group than in the SA group (Fig. 7B). The protein expression of RXR- was significantly lower in the TA group than in the SA group (Fig. 7C).

View larger version (32K): [in this window] [in a new window]   Fig. 7. Expression of retinoid X receptor (RXR)- protein (A), RXR- protein (B), and RXR- protein (C) in the heart (left ventricle) of SY (n = 7), SA (n = 7), and TA (n = 7) groups. Typical examples of Western blot analysis of the levels of RXR-, RXR-, and RXR- proteins (left) are shown. Arrows indicate the immunoblot band for RXR- protein, RXR- protein, and RXR- protein. Right, results of statistical analysis of the levels of expression of RXR-, RXR-, and RXR- proteins determined by a densitometer. Data are expressed as means ± SE.

The plasma T₃ level was significantly lower in the SA group than in the SY group (Table 3). The plasma T₄ level was significantly lower in the SA group than in the SY group and was significantly higher in the TA group than in the SA group (Table 3).

	DISCUSSION

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We demonstrated that exercise training improved the aging-induced decrease in protein expression of TR-₁ and TR-₁, which are one of the transcriptional regulatory factors of the -MHC and SR Ca²⁺-ATPase genes, in the heart. We also demonstrated that exercise training improved the aging-induced decrease in activity of myocardial TR DNA binding to TREs, which are the TR binding domain of the transcriptional regulatory region in the -MHC and SR Ca²⁺-ATPase genes. Furthermore, exercise training improved the aging-induced decrease in mRNA expression of -MHC and SR Ca²⁺-ATPase in the heart, with improvement of the aging-induced reduction of cardiac function. The present study confirmed that expression of MHC isoforms and SR Ca²⁺-ATPase protein in the heart was altered in association with changes in mRNA expression of MHC and SR Ca²⁺-ATPase in the heart. These findings suggest that exercise training improves the aging-induced downregulation of TR signaling in the heart. A previous study (47) has shown that exercise training in aged rat improved aging-induced decrease in expression of SR Ca²⁺-ATPase mRNA in the heart. It has also been reported that aging-induced decrease in mRNA expression of TR-₁ and -₁ and protein expression of TR-₁ in the heart (27). Our data are in accordance with these reports (27, 47). Therefore, it is considered that the alteration in gene and protein expression of MHC and SR Ca²⁺-ATPase in the heart participates in the adaptive mechanism of the change in cardiac function with aging and subsequent exercise training and that myocardial TR signaling-mediated transcriptional regulation participates in the molecular mechanism of the alteration in MHC and SR Ca²⁺-ATPase genes in the heart with aging and subsequent exercise training. Thus this regulation at the molecular level may contribute to beneficial adaptations through which exercise training improves the aging-induced decrease of cardiac function.

It is well known that the aging-induced decrease in cardiac function is improved by exercise training (24, 25, 42). However, the molecular mechanisms for improving cardiac function are unclear. Myocardial TRs bind to TREs in the promoter regions of several genes, including -MHC and SR Ca²⁺-ATPase in the heart, and regulate transcription of target genes in the heart (6, 17). Kinugawa et al. (16) reported that a rat model of pathological cardiac hypertrophy, which has functional abnormalities in the heart, showed a shift of isozyme from -MHC to -MHC mRNA and a decrease in mRNA expression of SR Ca²⁺-ATPase in association with a decrease in mRNA expression of TR-₁ and TR-₁ in the heart. Recently, it has been reported that a lack of TR-₁ (TR-₁^–/– mice) and TR-₁/ (TR-₁^–/–/^–/– mice) caused an increase in mRNA expression of -MHC and a shift of isozyme from -MHC to -MHC protein in the heart, whereas a lack of TR- (TR-^–/– mice) did not change the protein and mRNA expression of MHC in the heart (29). On the other hand, it has been reported that a rat model of hypothyroidism and a pathological cardiac condition showed an improvement of the decrease in protein or mRNA expression of -MHC and SR Ca²⁺-ATPase in the heart and cardiac function by treatment with T₃, which is a ligand for TRs (4, 13). Taken together, these findings suggest that TR signaling regulates target genes encoding -MHC and SR Ca²⁺-ATPase in the heart, and this regulation at the molecular level affects cardiac function. The present study revealed that the myocardial TR-₁ and TR-₁ proteins and myocardial TR DNA binding are altered in association with changes in the gene expression of -MHC and SR Ca²⁺-ATPase, which are the target genes of TR signaling, in the heart during aging and subsequent exercise training. On the basis of the results from past studies plus the present results, it is considered that exercise training improves the aging-induced downregulation of TR signaling, and this alteration of TR signaling participates in the transcriptional regulation of -MHC and SR Ca²⁺-ATPase genes in the heart, thereby causing adaptation of cardiac function to aging and subsequent exercise training. Therefore, it is possible that the TR signaling-mediated molecular regulation in the heart is a candidate for the molecular mechanism of maintenance and/or upregulation of cardiac function in aging and subsequent exercise training.

In the present study, we measured the protein expression of RXRs in the heart in aging and subsequent exercise training because RXRs play a role in forming heterodimers with TRs to bind to TRE in the promoter regions of target genes (23, 50, 52). The present study revealed that the protein expression of RXR-, RXR-, and RXR- in the heart decreased with aging. It has been reported that aging induced decrease in expressions of RXR- mRNA and protein in the heart, whereas mRNA expression of RXR- and - did not change (27). The reason for the discrepancy in results between the present study and the previous study is presently unclear. The present study also showed that exercise training induced an increase in RXR- protein in the aged heart. On the other hand, RXR- protein was not changed by exercise training, and RXR- protein was decreased by exercise training. These findings suggest that exercise training improves the aging-induced decrease in RXR- in the heart. Several studies (23, 50) have shown that TRs and RXRs form heterodimers, which bind to TREs of target genes with high affinity and specificity. Therefore, it is possible that the exercise training-induced improvement of RXR- protein in the aged heart may improve the formation of heterodimers with TRs in the heart, thereby assisting to improve the aging-induced downregulation of myocardial TR signaling, which bind to TRE in the promoter regions of the -MHC and SR Ca²⁺-ATPase genes.

It has been reported that aging caused a decrease in serum or plasma levels of T₃ and T₄ (2, 46), whereas exercise training caused increase and/or unchanged of serum or plasma levels of T₃ and T₄ (36, 49). The present study showed that aging decreased in plasma levels of T₃ and T₄, whereas exercise training improved aging-induced decrease in plasma levels of T₄. Therefore, it is considered that TR activation participates in function and signal pathway for aging and exercise training status.

Aging diminished contraction and relaxation response of the heart to -adrenergic or catecholamine stimulation and increased myocardial working load (21, 42, 43). Several studies (3, 24, 43, 48) also have shown aging-induced reduction of peak early filling rates and diastolic filling volume and aging-induced delay of myocardial contraction and relaxation duration. Furthermore, exercise training improved the aging-induced reduction of cardiac function and performance (24, 42). On the other hand, some studies showed that in a normal aging, resting cardiac function of both animals and humans is unchanged by aging and subsequent exercise training (1, 40). In the present study, SA rats showed a reduction of cardiac function, i.e., a decrease in LVFS and LVEF, whereas TA rats, which received 8-wk swimming training during old age, showed an improvement of cardiac function, i.e., a decrease in resting HR and an increase in LVEDD, LVFS, LVEF, and SV, and developed cardiac hypertrophy. In regard to the difference between our results and several previous results, we calculated LV mass from the echocardiography and investigate to compare this LV mass with measured LVW as an index of reliability of the echocardiographic measurements. Cittadini et al. (5) reported that the echocardiography-calculated LV mass and LVW in the rat was the close correlation (y = 1.03x + 0.05, r = 0.85, P < 0.00001), showing the accuracy of echocardiographic measurements, whereas the echocardiographic measurements in the present study showed a lower but significant correlation between echocardiography-calculated LV mass and LVW (y = 1.06x – 0.46, r = 0.68, P < 0.001) than that in the previous study. Therefore, it is considered that the echocardiographic measurements in the present study may be a relatively lower reliability than in the previous study. The following consideration for analysis by echocardiography is possible. The set of anesthetic doses may affect the hemodynamic status of the animals depending on the amount of fat distribution of the pentobarbital. In the present study, the body weight of rats was markedly increased by aging and was markedly decreased by exercise training. We performed echocardiographic measurements of rats under anesthesia. The anesthetic doses are based on total body weight not lean body weight. Therefore, it is unclear whether the set of anesthetic doses affects hemodynamic status in the aged rats. Furthermore, the present study has the following study limitations. First, the TA rats in the present study experienced dramatic weight loss (27% loss of their body weight in SA rats). Although, in the present study, there may be not only purely a training effect but also is marked other stress related to the swimming, we did not investigate other circulating hormones for this stress. Therefore, it is unclear whether other circulating hormones are changed by this exercise training and these hormones affect the adaptations of cardiac function. Second, the reliance on LVEF as an index of LV contractility may not be ideal because changes in MHC isoforms and probably SR Ca²⁺-ATPase would be more likely to affect the peak first derivative of LV pressure (dP/dt). Although these hemodynamic measurements should be performed to estimate contraction and relaxation response of the heart with aging and subsequent exercise training, we did not investigate the dP/dt in the heart. Furthermore, these findings of cardiac adaptations in rats with aging and subsequent exercise training may not be analogous to those in humans.

In conclusion, we demonstrated that exercise training during old age improved the aging-induced decrease in protein expression of TR-₁ and -₁ and activity of myocardial TR DNA binding to TRE. We also demonstrated that exercise training improved the aging-induced decrease in protein expression of RXR-. These results suggest that exercise training improves the aging-induced downregulation of TR signaling in the heart. This activation of TR signaling pathway in the heart is altered in association with the change in gene and protein expression of MHC and SR Ca²⁺-ATPase in the heart with aging and subsequent exercise training. Therefore, it is considered that myocardial TR signaling-mediated transcriptional regulation participates in the molecular mechanism of the alteration in MHC and SR Ca²⁺-ATPase genes in the heart with aging and subsequent exercise training, thereby contributing to the improvement of cardiac function in trained aged hearts. We propose that regulation at the molecular level mediates TR signaling in the heart and partly contributes to the mechanism of the beneficial adaptive responses to exercise training in old age.

	ACKNOWLEDGMENTS

 
We thank Dr. Wendy Gray for editing our manuscript.

GRANTS

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (00006132, 00006781, 11557047, and 12470147), a grant from University of Tsukuba Research Projects, and a grant from the project of Tsukuba Advanced Research Alliance in University of Tsukuba.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Div., 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.

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