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(Circulation. 2001;104:1670.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Peroxisome Proliferator-Activated Receptor Activators Inhibit Cardiac Hypertrophy in Cardiac Myocytes

Keiji Yamamoto, MD; Ruri Ohki, MD; Richard T. Lee, MD; Uichi Ikeda, MD; Kazuyuki Shimada, MD

Department of Cardiology (K.Y., R.O., U.I., K.S.), Jichi Medical School, Minamikawachi-Machi, Tochigi, Japan, and the Cardiovascular Division (R.T.L.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Keiji Yamamoto, MD, Department of Cardiology, Jichi Medical School, Minamikawachi-Machi, Tochigi, Japan 329-0498. E-mail kyamamoto{at}jichi.ac.jp

	Abstract

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Background— Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. PPAR mRNA is present in cardiac myocytes; however, whether PPAR affects cardiac hypertrophy remains unknown.

Methods and Results— We investigated the effects of PPAR activators on cardiac hypertrophy in neonatal rat cardiac myocytes. Cyclic 4% biaxial mechanical strain caused enlargement of cardiac myocytes (1.3-fold versus control, P<0.0001), but the PPAR activators troglitazone and 15-deoxy-^12-14-prostaglandin J₂ (15d-PGJ₂) (10 µmol/L) inhibited this effect (troglitazone, -72%, P<0.0005; 15d-PGJ₂, -88%, P<0.0002). Total cell protein was increased by mechanical strain (control, 164.3 µg/dish; strain, 265.5, P<0.0002), and this effect was inhibited by troglitazone and 15d-PGJ₂ (troglitazone, -61%, P<0.005; 15d-PGJ₂, -72%, P<0.001). [³H]Leucine uptake was also increased by mechanical strain (1.9-fold versus control, P<0.002), and this increase was inhibited by troglitazone and 15d-PGJ₂ (troglitazone, -52% at 10 µmol/L, P<0.01; 15d-PGJ₂, -70% at 10 µmol/L, P<0.005). An increase in [³H]leucine uptake induced by angiotensin II or phenylephrine was significantly inhibited by troglitazone and 15d-PGJ₂. Mechanical strain induced mRNA expression for brain natriuretic peptide, but PPAR activators inhibited this induction. Furthermore, PPAR activators inhibited mechanically induced activation of nuclear factor (NF)-B. Pyrrolidine dithiocarbamate, an inhibitor of NF-B activation, inhibited strain-induced [³H]leucine uptake (-50% at 100 µmol/L, P<0.05).

Conclusions— These results demonstrate that PPAR activators inhibit cardiac hypertrophy in cardiac myocytes and suggest that PPAR activators may regulate cardiomyocyte hypertrophy at least partially through the NF-B pathway.

Key Words: hypertrophy • stress • myocytes

	Introduction

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Increased cardiac work load leads to cardiac hypertrophy, an independent risk factor of cardiac morbidity and mortality that can progress to clinical heart failure.¹ Studies performed in animal models of hypertrophy and cardiac myocytes have shown that mechanical strain and other factors, including norepinephrine, angiotensin II, endothelin-1, insulin-like growth factor-I, interleukin-1ß (IL-1ß), tumor necrosis factor-, leukemia inhibitory factor, and cardiotropin-1 are potential stimuli for myocyte hypertrophy.² Mechanical overload is a common clinical stimulus of cardiac hypertrophy, and deformation of cardiac myocytes increases specific genes, protein synthesis, and cell size.

Peroxisome proliferator-activated receptors (PPARs) are a family of 3 nuclear hormone receptors, PPAR, PPAR, and PPAR.³ PPAR is activated by the natural ligand 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂)⁴ as well as the synthetic ligand thiazolidinedione (troglitazone).⁵ The thiazolidinediones decrease blood pressure in a hypertensive rat model⁶ and inhibit neointimal formation of balloon-injured vessels in rats.⁷ Recently, Takano et al⁸ demonstrated that PPAR activators inhibit tumor necrosis factor- expression at the transcriptional level in part by preventing nuclear factor (NF)-B activity in cardiac myocytes. Barger et al⁹ reported that PPAR is deactivated during cardiac hypertrophic growth, leading to diminished capacity for myocardial lipid and energy homeostasis. However, it remains unclear whether PPAR participates in cardiac hypertrophy in cardiac myocytes.

In this study, we investigated the effects of PPAR activators on cardiac hypertrophy in cultured neonatal rat cardiac myocytes. We found that PPAR activators inhibit cardiomyocyte hypertrophy induced by mechanical strain as well as angiotensin II or phenylephrine in neonatal rat cardiac myocytes. These results suggest that the PPAR pathway participates in cardiac hypertrophy.

	Methods

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Materials
Human recombinant IL-1ß was provided by Otsuka Pharmaceutical Co. Troglitazone was a gift from Sankyo Co, Ltd. Fibronectin was purchased from Life Technologies, Inc. 15d-PGJ₂ was purchased from Cayman Chemical. [-³²P]dCTP (3000 Ci/mmol), [-³²P]dATP (3000 Ci/mmol) and [³H]leucine (50 Ci/mmol) were purchased from Amersham Pharmacia Biotech KK. All other chemicals were purchased from Sigma.

Culture of Neonatal Rat Ventricular Myocytes
Neonatal rat ventricular myocytes (NRVM) from 1-day-old Sprague-Dawley rats were isolated by previously described methods.¹⁰ The ventricles were excised from the rat, cut into several pieces, and incubated overnight at 4°C in 1 mg/mL of 1:300 trypsin in Hanks’ balanced salt solution (HBSS, Life Technologies, Inc). The ventricular tissue was then digested with 1 mg/mL of collagenase type II (239 U/mg, Worthington Biochemicals) in HBSS, centrifuged twice at 50g to remove less dense cells such fibroblasts, and then plated. The cells were cultured at 37°C, 5% CO₂ in DMEM (BioWhittaker) containing 7% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin (PS).

This investigation was performed according to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication No. 85–23, revised 1996).

Mechanical Strain Device and Preparation of Cells
Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that produces controlled cellular strain as well as visualization of cells.¹¹

For the preparation of NRVM to be subjected to mechanical strain, autoclaved membrane dishes were coated with 2 µg/mL of fibronectin in 13 mL of HBSS for 6 to 12 hours at 4°C and then washed twice with 10 mL of PBS. NRVM were plated on the coated membrane dish at a density of 2 000 000 cells/dish in 13 mL of DMEM containing 7% FCS and incubated 48 hours. NRVM were then made quiescent by washing with 10 mL of HBSS twice and incubating with 10 mL of DMEM containing 1% insulin, transferrin, selenium media supplement (ITS; Sigma), and PS. All experiments were performed on NRVM that had been serum-starved for 24 hours.

Cardiac Myocyte Surface Area
The myocyte surface area was measured by the method of Simpson.¹² Cell images captured by video camera (Nikon) were traced and analyzed with NIH Image 1.56. The area was then doubled to account for the surface portion in contact with the dish. All cells from randomly selected fields in 2 or 3 dishes were examined for each condition. We measured 100 cells in each condition.

Incorporation of [³H]Leucine
NRVM were subjected to 0% or 4% cyclic mechanical strain in the presence or absence of PPAR activators with 1.0 µCi/mL [³H]leucine for 24 hours. The medium was aspirated and the cells were washed twice with ice-cold PBS and once with 10% trichloroacetic acid (TCA; Sigma) and fixed for 45 minutes at 4°C with 10% TCA. After washing twice with cold 95% ethanol, radioactivity incorporated into the TCA-precipitable material was determined by liquid scintillation counting after solubilization in 0.15N NaOH.

Protein Content
NRVM were subjected to 0% or 4% cyclic mechanical strain in the presence or absence of PPAR activators for 48 hours. The cells were washed twice with PBS and then treated with 10% TCA as described above. The precipitates were dissolved in NaOH (0.15N). The protein content was measured by the Bio-Rad DC protein assay (Bio-Rad Laboratories).

Northern Analysis
Total RNA was isolated by the guanidinium thiocyanate and phenol chloroform method.¹³ Purified RNA (1 µg) was used for the synthesis of cDNA with a reverse-transcriptase polymerase chain reaction system (Stratagene). Synthesis of the cDNAs was performed by polymerase chain reaction with Taq polymerase (Perkin-Elmer). The primer set for the synthesis of the 387-base pair rat brain natriuretic peptide (BNP) cDNA probe contained the 5'-TTTTCCTTAATCTGTCGCCG-3' sense and 5'-AGAGCTGGGGAAAGAAGAGC-3' antisense oligonucleotides. This rat BNP cDNA was radiolabeled by the random priming method with [-³²P]dCTP and the Klenow fragment of DNA polymerase (Stratagene). For Northern blotting, 15 µg of total RNA was loaded on a 1.0% formaldehyde gel, transferred to a nylon membrane (Stratagene), and UV cross-linked with a UV Stratalinker (Stratagene). The probe was hybridized with QuikHyb solution (Stratagene) at 68°C for 1 hour. Normalization of RNA for equal loading was carried out by rehybridizing the blots with a glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA probe (Clontech).

Electrophoretic Mobility Shift Assay
Nuclear extracts from cardiac myocytes were prepared by 3 washes of the cell layer in ice-cold PBS; the cells were scraped off the tissue culture dish, resuspended, and sedimented by centrifugation. The cell pellet was lysed in a buffer composed of 20 mmol/L HEPES-KOH (pH 7.9), 0.35 mmol/L NaCl, 20% glycerol, 1% NP-40, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 10 µg/mL leupeptin, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L PMSF by incubation on ice for 30 minutes. After centrifugation, the supernatant containing the protein fraction was frozen at -80°C. For electrophoretic mobility shift assays, a double-strand oligonucleotide representing the consensus sequence for NF-B binding (5'-TCAACAGAGGGGACTTTCCGAGGCCA-3') was labeled with [-³²P]dATP by use of T₄ polynucleotide kinase. The labeled probe was separated from unincorporated nucleotide with a Sephadex G-50 column (Amersham Pharmacia Biotech KK). Ten micrograms of nuclear extract was incubated in 10 µL of binding buffer containing 5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, 250 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 0.25 mg/mL poly(dI-dC), and 20% glycerol for 10 minutes at room temperature. ³²P-labeled oligonucleotide probe (50 000 to 200 000 cpm) was then added, and the reaction mixture was incubated for 20 minutes at room temperature. Immediately after binding, the protein/DNA complexes were separated from unbound oligonucleotide by electrophoresis on a native 5% polyacrylamide gel in Tris-HCl-EDTA buffer. Autoradiography was performed with the dried gels and Hyperfilm (Amersham Pharmacia Biotech KK). For testing of specificity of NF-B/DNA binding, antibodies (Santa Cruz Biotechnology Inc) against the p65 subunits of NF-B were added to the proteins, resulting in further retardation of electrophoretic mobility, or a 160-fold molar excess of unlabeled oligonucleotide was added to the binding reaction, leading to a decrease in NF-B-bound radioactivity.

Statistical Analysis
Data are expressed as mean±1 SD. Statistical analysis was performed by 1-way ANOVA, with comparison of different groups by Fisher’s protected least significantly difference test. A value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Inhibitory Effects of PPAR Activators on Myocyte Hypertrophy Induced by Mechanical Strain
We initially investigated whether myocyte hypertrophy induced by mechanical strain was affected by the PPAR activators troglitazone and 15d-PGJ₂. As shown in Figures 1 and 2, cyclic 4% biaxial mechanical strain at a frequency of 1 Hz significantly caused enlargement of cardiac myocytes (1.3±0.1-fold versus control, n=100 cells, P<0.0001). The PPAR activators troglitazone and 15d-PGJ₂ (both 10 µmol/L) significantly inhibited this effect (troglitazone, -72%, n=100 cells, P<0.0005; 15d-PGJ₂, -88%, n=100 cells, P<0.0002). PPAR ligands (10 µmol/L) by themselves did not affect the myocyte surface area (n=100 cells, P=NS, Figure 2).

View larger version (138K): [in this window] [in a new window]   Figure 1. PPAR activators inhibit mechanically induced increase in myocyte size: Representative living cardiac myocytes. A, Myocytes subjected to no mechanical strain (control). B, Myocytes subjected to 4% cyclic mechanical strain. C, Myocytes subjected to 4% cyclic mechanical strain in the presence of troglitazone (10 µmol/L). D, Myocytes subjected to 4% cyclic mechanical strain in the presence of 15d-PGJ2 (10 µmol/L). NRVM were plated on 2 µg/mL of fibronectin in DMEM containing 7% FCS for 24 hours. After serum deprivation for 24 hours, myocytes were exposed for 48 hours to 0% or 4% cyclic mechanical strain (1 Hz) in the presence or absence of PPAR activators. PPAR activators were applied to myocytes 30 minutes before mechanical strain. Bar=100 µm.

View larger version (22K): [in this window] [in a new window]   Figure 2. Inhibitory effects of PPAR activators on myocyte enlargement induced by mechanical strain. Myocytes were exposed for 48 hours to 0% (open bars) or 4% (closed bars) cyclic mechanical strain (1 Hz) in the presence or absence of PPAR activators (10 µmol/L). PPAR activators were applied to myocytes 30 minutes before mechanical strain. Cell size results are expressed as relative surface area standardized to mean surface area of control cells in each experiment. Bar graphs with error bars represent mean±SD (n=100). *P<0.0001 vs control; P<0.0005 vs exposed to strain; P<0.0002 vs exposed to strain.

Inhibitory Effects of PPAR Activators on Mechanically Induced Increase in Protein Content
Total cell protein was also significantly increased by 4% cyclic mechanical strain (control, 164.3±11.9 µg/dish, n=4; strain, 265.5±18.0, P<0.0002), and this was inhibited by troglitazone and 15d-PGJ₂ (10 µmol/L) (troglitazone, 204.0±14.5 µg/dish, -61%, n=4, P<0.005; 15d-PGJ₂, 192.3±14.7, -72%, n=4, P<0.001). PPAR activators (10 µmol/L) by themselves did not affect basal levels of total cell protein (n=4, P=NS, Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Inhibitory effects of PPAR activators on mechanically induced increase in protein content. Myocytes were exposed for 48 hours to 0% (open bars) or 4% (closed bars) cyclic mechanical strain (1 Hz) in the presence or absence of PPAR activators (10 µmol/L). Protein content was then measured. PPAR activators were applied to myocytes 30 minutes before mechanical strain. Bar graphs with error bars represent mean±SD (n=4). *P<0.0002 vs control; P<0.005 vs exposed to strain; P<0.001 vs exposed to strain.

Inhibitory Effects of PPAR Activators on [³H]Leucine Incorporation Induced by Mechanical Strain
As shown in Figure 4, [³H]leucine uptake was significantly increased by 4% mechanical strain (1.9±0.2-fold versus control, n=4, P<0.002, Figure 4), and this was inhibited by troglitazone and 15d-PGJ₂ in a concentration-dependent manner (0.1 to 10 µmol/L) (troglitazone, -52% at 10 µmol/L, n=4, P<0.01; 15d-PGJ₂, -70% at 10 µmol/L, n=4, P<0.005). PPAR activators (10 µmol/L) by themselves did not affect basal levels of [³H]leucine incorporation. (n=4, P=NS, Figure 4).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effects of PPAR activators on protein synthesis induced by mechanical strain. Myocytes were exposed to 0% (open bars) or 4% (closed bars) cyclic mechanical strain (1 Hz) in the presence or absence of PPAR activators (10 µmol/L) with 1.0 µCi/mL [3H]leucine for 24 hours. PPAR activators were applied to myocytes 30 minutes before experiments. Protein synthesis results are expressed as relative cpm/dish standardized to mean cpm of control cells in each experiment. Bar graphs with error bars represent mean±SD (n=4). *P<0.002 vs control; P<0.05 vs exposed to strain; P<0.02 vs exposed to strain; P<0.01 vs exposed to strain; ¶P<0.005 vs exposed to strain.

Inhibitory Effects of PPAR Activators on [³H]Leucine Incorporation Induced by Angiotensin II and Phenylephrine
Next, we investigated whether the blocking effects of PPAR activators are also observed with hypertrophic growth-induced angiotensin II or phenylephrine. [³H]Leucine uptake was increased by angiotensin II (0.1 µmol/L) (1.6±0.2-fold versus control, n=4, P<0.01, Figure 5A) or phenylephrine (50 µmol/L) (1.7±0.2-fold versus control, n=4, P<0.005, Figure 5B), and this increase was inhibited by troglitazone and 15d-PGJ₂ in a concentration-dependent manner (0.1 to 10 µmol/L) (angiotensin II: troglitazone, -60% at 10 µmol/L, n=4, P<0.01; 15d-PGJ₂, -74% at 10 µmol/L, n=4, P<0.005; phenylephrine: troglitazone, -56% at 10 µmol/L, n=4, P<0.02; 15d-PGJ₂, -67% at 10 µmol/L, n=4, P<0.01).

View larger version (21K): [in this window] [in a new window]   Figure 5. A, Effects of PPAR activators on protein synthesis induced by angiotensin II. NRVM were treated with angiotensin II (0.1 µmol/L, closed bar), PPAR activators (0.1 to 10 µmol/L), or diluent without angiotensin II (open bars) and coincubated with 1.0 µCi/mL [3H]leucine for 24 hours. PPAR activators were applied to myocytes 30 minutes before experiments. Protein synthesis results are expressed as relative cpm/dish standardized to mean cpm of control cells in each experiment. Bar graphs with error bars represent mean±SD (n=4). *P<0.01 vs control; P<0.05 vs angiotensin II; P<0.02 vs angiotensin II; P<0.01 vs angiotensin II; ¶P<0.005 vs angiotensin II. B, Effects of PPAR activators on protein synthesis induced by phenylephrine. NRVM were treated with phenylephrine (50 µmol/L, closed bars), PPAR activators (0.1 to 10 µmol/L), or diluent without phenylephrine (open bars) and coincubated with 1.0 µCi/mL [3H]leucine for 24 hours. PPAR activators were applied to myocytes 30 minutes before experiments. *P<0.005 vs control; P<0.05 vs phenylephrine; P<0.02 vs phenylephrine; P<0.01 vs phenylephrine.

Effects of PPAR Activators on BNP mRNA Expression Induced by Mechanical Strain
We investigated whether the PPAR ligands affect the expression of BNP mRNA, a marker for cardiac hypertrophy¹⁴ that is induced by mechanical strain. As shown in Figure 6, 4% cyclic mechanical strain induced mRNA expression for BNP in cardiac myocytes, but both PPAR activators, troglitazone, and 15d-PGJ₂ (10 µmol/L), inhibited this effect. PPAR activators (10 µmol/L) by themselves did not affect BNP mRNA expression.

View larger version (41K): [in this window] [in a new window]   Figure 6. Effects of PPAR activators on induction of BNP mRNA expression by mechanical strain. Myocytes were exposed for 6 hours to 0% or 4% cyclic mechanical strain (1 Hz) in presence or absence of PPAR activators (10 µmol/L). PPAR activators were applied to myocytes 30 minutes before mechanical strain. Total RNA was isolated and analyzed by Northern blotting with 32P-labeled BNP (upper panel) and GAPDH (lower panel) cDNA probes. Two separate experiments yielded similar results.

Effects of PPAR Activators on NF-B Activation by Mechanical Strain
Because activation of NF-B may participate in cardiac hypertrophy or remodeling,¹⁵ electrophoretic mobility shift assays were performed with the use of radiolabeled oligonucleotides. As shown in Figure 7, 4% mechanical strain caused the activation of NF-B. The addition of IL-1ß (10 ng/mL) also induced the activation of NF-B in the absence of strain. Both PPAR activators (10 µmol/L), troglitazone, and 15d-PGJ₂ completely inhibited the activation of NF-B induced by mechanical strain. The shifted complexes were specific for NF-B because they were supershifted in the presence of antibody to the NF-B subunit and disappeared with excess unlabeled oligonucleotide. In addition, we investigated whether NF-B pathway participates in cardiac hypertrophy. Pyrrolidine dithiocarbamate, an inhibitor of NF-B activation, inhibited an increase in [³H]leucine uptake induced by 4% mechanical strain (-50% at 100 µmol/L, n=4, P<0.05, Figure 8). These findings suggest that the NF-B pathway may be involved in the inhibitory effects of PPAR activators on cardiac hypertrophy in cardiac myocytes.

View larger version (17K): [in this window] [in a new window]   Figure 7. Electrophoretic mobility shift assay showing effects of PPAR activators on activation of NF-B by mechanical strain. Myocytes were exposed for 1 hour to 0% or 4% cyclic mechanical strain (1 Hz) in the presence or absence of PPAR activators (10 µmol/L) or IL-1ß (10 ng/mL). PPAR activators were applied to myocytes 30 minutes before mechanical strain. Nuclear extracts (10 µg) from cardiac myocytes were incubated with 32P-labeled NF-B consensus oligonucleotide. Binding of activated NF-B to oligonucleotide was visualized by autography after separation by nondenaturing polyacrylamide gel electrophoresis. Specificity was determined by addition of p65 antibody (supershift) or unlabeled (cold) NF-B oligonucleotide to the nuclear extracts. Two separate experiments yielded similar results.

View larger version (20K): [in this window] [in a new window]   Figure 8. Effects of pyrrolidine dithiocarbamate on protein synthesis induced by mechanical strain. NRVM were subjected to 0% (open bar) or 4% cyclic mechanical strain (closed bars) in the presence or absence of pyrrolidine dithiocarbamate (10 to 100 µmol/L) with 1.0 mCi/mL [3H]leucine for 24 hours. Pyrrolidine dithiocarbamate were applied to myocytes 30 minutes before experiments. Protein synthesis results are expressed as relative cpm/dish standardized to mean cpm of control cells in each experiment. Bar graphs with error bars represent mean±SD (n=4). *P<0.01 vs control; P<0.05 vs exposed to strain.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, PPAR activators such as troglitazone and 15d-PGJ₂ inhibited increases in cell size, protein content, protein synthesis and BNP mRNA expression, and the activation of NF-B by cyclic mechanical strain in cardiac myocytes. In addition, PPAR activators inhibited an increase in protein synthesis induced by angiotensin II or phenylephrine. These findings suggest that the PPAR pathway may regulate the molecular response to hypertrophic stimuli in the heart.

PPAR is expressed predominantly in adipose tissue, whereas PPAR is present in liver, kidney, and muscle.¹⁶ Previous studies^16,17 have shown that PPAR is expressed in variable amounts between individuals, whereas PPAR is expressed at a low level in adult rat or adult human heart. Recently, it was reported that expressions of PPAR and PPAR were similar in neonatal rat cardiac myocytes.⁸ PPAR can be activated by a number of ligands including thiazolidinedione (troglitazone), 15d-PGJ₂, PGJ₂, oxidized LDL, 13-oxidized octadecadienoic acid, and nonsteroidal anti-inflammatory drugs.¹⁸ PPARs act as transcription factors on ligand-induced heterodimerization with the common nuclear receptor binding partner, the retinoid X receptor (RXR). When combined as a PPAR:RXR heterodimer, PPAR ligands and 9-cis retinoic acid can act synergistically on PPAR responses. Different dimers of RXR induce specific responses by binding highly specific sequences in the promoter regions of the various genes. In the present study, PPAR ligands troglitazone and 15d-PGJ₂ inhibited increases in cell size as well as protein content and protein synthesis induced by mechanical strain, angiotensin II, or phenylephrine in rat cardiac myocytes. These findings suggest that PPAR pathway may play an important role in cardiac hypertrophy.

A greater understanding of the transcriptional regulation that directs cardiac hypertrophy will be critical for implementing novel and more effective therapeutic strategies in the future. In the PPAR-dependent pathway, ligand-activated PPAR positively regulates gene expression through binding to specific DNA sequence (PPAR response element)³ or inhibiting other gene expression in part through antagonism of the activities of other transcription factors, such as NF-B.¹⁹ In the present study, both PPAR activators completely inhibited the activation of NF-B by mechanical strain, and pyrrolidine dithiocarbamate, an inhibitor of NF-B activation, partially but significantly inhibited the hypertrophic response induced by 4% mechanical strain. These findings suggest that PPAR activators may regulate cardiomyocyte hypertrophy at least partially through the NF-B pathway. However, we cannot exclude a role for NF-B as a mechanism, because pyrrolidine dithiocarbamate has many effects that could affect hypertrophy. In addition, further experiments will be necessary to investigate whether PPAR activators affect other transcription factors such as signal transducers and activators of transcription (STAT) and nuclear factor of activated T cells-3 (NF-AT3).

It is controversial whether PPAR activators such as troglitazone can prevent cardiac hypertrophy. Bell et al²⁰ reported that troglitazone did not initiate cardiomyocyte growth directly in adult rat cardiac myocytes and could inhibit protein kinase C–mediated growth mechanisms. Ghazzi et al²¹ reported that the left ventricular mass index of diabetes mellitus patients treated with troglitazone was not statistically or clinically different from baseline after 48 weeks. In our study, PPAR activators inhibited the myocyte hypertrophy induced by mechanical strain, angiotensin II or phenylephrine in neonatal rat cardiac myocytes. It is therefore likely that PPAR activators may have preventive effects on pathological myocyte hypertrophy.

In summary, our data showed that PPAR activators inhibit cardiac hypertrophy in neonatal rat cardiac myocytes. These data suggest that the PPAR pathway may be involved in cardiac hypertrophy or remodeling, at least in part, by antagonizing the binding activity of NF-B.

	Acknowledgments

 
This study was supported by the Ministry of Education, Science, Sports, and Culture of Japan (12670686), the Jichi Medical School Young Investigator Award, and the Kanae Foundation for Life and Socio-Medical Science. We thank Toshiko Kanbe for technical assistance.

Received May 7, 2001; revision received June 22, 2001; accepted July 2, 2001.

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