Peroxisome Proliferator-Activated Receptor γ Activators Inhibit Cardiac Hypertrophy in Cardiac Myocytes
Background Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. PPARγ mRNA is present in cardiac myocytes; however, whether PPARγ affects cardiac hypertrophy remains unknown.
Methods and Results We investigated the effects of PPARγ 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 PPARγ activators troglitazone and 15-deoxy-Δ12-14-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 PPARγ activators inhibited this induction. Furthermore, PPARγ activators inhibited mechanically induced activation of nuclear factor (NF)-κB. Pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, inhibited strain-induced [3H]leucine uptake (−50% at 100 μmol/L, P<0.05).
Conclusions These results demonstrate that PPARγ activators inhibit cardiac hypertrophy in cardiac myocytes and suggest that PPARγ activators may regulate cardiomyocyte hypertrophy at least partially through the NF-κB pathway.
Received May 7, 2001; revision received June 22, 2001; accepted July 2, 2001.
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-1β (IL-1β), tumor necrosis factor-α, 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, PPARα, PPARδ, and PPARγ.3 PPARγ is activated by the natural ligand 15-deoxy-Δ12,14-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-α expression at the transcriptional level in part by preventing nuclear factor (NF)-κB activity in cardiac myocytes. Barger et al9 reported that PPARα is deactivated during cardiac hypertrophic growth, leading to diminished capacity for myocardial lipid and energy homeostasis. However, it remains unclear whether PPARγ participates in cardiac hypertrophy in cardiac myocytes.
In this study, we investigated the effects of PPARγ activators on cardiac hypertrophy in cultured neonatal rat cardiac myocytes. We found that PPARγ 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 PPARγ pathway participates in cardiac hypertrophy.
Human recombinant IL-1β 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. [α-32P]dCTP (3000 Ci/mmol), [γ-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 PPARγ 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.
NRVM were subjected to 0% or 4% cyclic mechanical strain in the presence or absence of PPARγ 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).
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 [α-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-κB binding (5′-TCAACAGAGGGGACTTTCCGAGGCCA-3′) was labeled with [γ-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-κB/DNA binding, antibodies (Santa Cruz Biotechnology Inc) against the p65 subunits of NF-κB 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-κB-bound radioactivity.
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.
Inhibitory Effects of PPARγ Activators on Myocyte Hypertrophy Induced by Mechanical Strain
We initially investigated whether myocyte hypertrophy induced by mechanical strain was affected by the PPARγ 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 PPARγ 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). PPARγ ligands (10 μmol/L) by themselves did not affect the myocyte surface area (n=100 cells, P=NS, Figure 2).
Inhibitory Effects of PPARγ 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). PPARγ activators (10 μmol/L) by themselves did not affect basal levels of total cell protein (n=4, P=NS, Figure 3).
Inhibitory Effects of PPARγ 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). PPARγ activators (10 μmol/L) by themselves did not affect basal levels of [3H]leucine incorporation. (n=4, P=NS, Figure 4).
Inhibitory Effects of PPARγ Activators on [3H]Leucine Incorporation Induced by Angiotensin II and Phenylephrine
Next, we investigated whether the blocking effects of PPARγ 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).
Effects of PPARγ Activators on BNP mRNA Expression Induced by Mechanical Strain
We investigated whether the PPARγ 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 PPARγ activators, troglitazone, and 15d-PGJ2 (10 μmol/L), inhibited this effect. PPARγ activators (10 μmol/L) by themselves did not affect BNP mRNA expression.
Effects of PPARγ Activators on NF-κB Activation by Mechanical Strain
Because activation of NF-κB 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-κB. The addition of IL-1β (10 ng/mL) also induced the activation of NF-κB in the absence of strain. Both PPARγ activators (10 μmol/L), troglitazone, and 15d-PGJ2 completely inhibited the activation of NF-κB induced by mechanical strain. The shifted complexes were specific for NF-κB because they were supershifted in the presence of antibody to the NF-κB subunit and disappeared with excess unlabeled oligonucleotide. In addition, we investigated whether NF-κB pathway participates in cardiac hypertrophy. Pyrrolidine dithiocarbamate, an inhibitor of NF-κB 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-κB pathway may be involved in the inhibitory effects of PPARγ activators on cardiac hypertrophy in cardiac myocytes.
In the present study, PPARγ 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-κB by cyclic mechanical strain in cardiac myocytes. In addition, PPARγ activators inhibited an increase in protein synthesis induced by angiotensin II or phenylephrine. These findings suggest that the PPARγ pathway may regulate the molecular response to hypertrophic stimuli in the heart.
PPARγ is expressed predominantly in adipose tissue, whereas PPARα is present in liver, kidney, and muscle.16 Previous studies16,17 have shown that PPARα is expressed in variable amounts between individuals, whereas PPARγ is expressed at a low level in adult rat or adult human heart. Recently, it was reported that expressions of PPARα and PPARγ were similar in neonatal rat cardiac myocytes.8 PPARγ 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, PPARγ 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 PPARγ 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 PPARγ-dependent pathway, ligand-activated PPARγ 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-κB.19 In the present study, both PPARγ activators completely inhibited the activation of NF-κB by mechanical strain, and pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, partially but significantly inhibited the hypertrophic response induced by 4% mechanical strain. These findings suggest that PPARγ activators may regulate cardiomyocyte hypertrophy at least partially through the NF-κB pathway. However, we cannot exclude a role for NF-κB as a mechanism, because pyrrolidine dithiocarbamate has many effects that could affect hypertrophy. In addition, further experiments will be necessary to investigate whether PPARγ 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 PPARγ 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, PPARγ activators inhibited the myocyte hypertrophy induced by mechanical strain, angiotensin II or phenylephrine in neonatal rat cardiac myocytes. It is therefore likely that PPARγ activators may have preventive effects on pathological myocyte hypertrophy.
In summary, our data showed that PPARγ activators inhibit cardiac hypertrophy in neonatal rat cardiac myocytes. These data suggest that the PPARγ pathway may be involved in cardiac hypertrophy or remodeling, at least in part, by antagonizing the binding activity of NF-κB.
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.
Presented in part at the 73rd Scientific Sessions of the American Heart Association, New Orleans, La, November 12 to 15, 2000 and published in abstract form (Circulation. 2000;102[suppl II]:II-70).
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