Peroxisome Proliferator-activated Receptor (PPAR) Gene Profiling Uncovers Insulin-like Growth Factor-1 as a PPARα Target Gene in Cardioprotection

Abstract

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-activated transcription factors and consist of the three isoforms, PPARα, PPARβ/δ, and PPARγ. Considerable evidence indicates the importance of PPARs in cardiovascular lipid homeostasis and diabetes, yet the isoform-dependent cardiac target genes remain unknown. Here, we constructed murine ventricular clones allowing stable expression of siRNAs to achieve specifically knockdown for each of the PPAR isoforms. By combining gene profiling and computational peroxisome proliferator response element analysis following PPAR isoform activation in normal versus PPAR isoform-deficient cardiomyocyte-like cells, we have, for the first time, determined PPAR isoform-specific endogenous target genes in the heart. Electromobility shift and chromatin immunoprecipitation assays demonstrated the existence of an evolutionary conserved peroxisome proliferator response element consensus-binding site in an insulin-like growth factor-1 (igf-1) enhancer. In line, Wy-14643-mediated PPARα activation in the wild-type mouse heart resulted in up-regulation of igf-1 transcript abundance and provided protection against cardiomyocyte apoptosis following ischemia/reperfusion or biomechanical stress. Taken together, these data confirm igf-1 as an in vivo target of PPARα and the involvement of a PPARα/IGF-1 signaling pathway in the protection of cardiomyocytes under ischemic and hemodynamic loading conditions.

Introduction

Long chain fatty acids coordinately induce the expression of a panel of genes involved in cellular fatty acid metabolism in cardiac muscle, thereby promoting fatty acid oxidation in this organ (1). These effects are likely mediated by peroxisome proliferator-activated receptors (PPARs),² members of the nuclear receptor family of ligand-activated transcription factors (2–4). In the presence of ligands, PPARs adopt an active conformation by forming an obligate heterodimer with the retinoid X receptor. Recruitment of additional co-activators provokes binding to peroxisome proliferator response elements (PPRE) in target genes, allowing PPAR-dependent gene expression. Despite the high levels of homologies at the protein level, the three PPAR isoforms, PPARα, PPARβ/δ, and PPARγ, exert different functions relying on differential distribution in distinct tissues, isoform-specific phosphorylation, selective interactions with their specific ligand and co-activators, and, likely, isoform-selective target gene activation (2–4).

Mice overexpressing PPARα in cardiac muscle display increased fatty acid oxidation rates, accumulation of triacylglycerides, a decrease in glucose metabolism, and they eventually develop cardiomyopathy (5, 6). Not surprisingly, mice lacking PPARα have elevated free fatty acid levels as a consequence of inadequate fatty acids oxidation rendering them hypoglycemic as a result of their reliance on glucose (7). Elucidation of the role of PPARγ in cardiac muscle has been hampered by its low abundance and the absence of significant effects of PPAR ligands such as ciglitazone and rosiglitazone on the regulation of target genes (1). Nevertheless, transgenic mice overexpressing PPARγ in the heart displayed increased cardiac free fatty acid uptake without a concomitant reduction in glucose uptake, and they ultimately developed severe heart failure (8).

Less is known about PPARβ/δ function in the heart. Heart muscle-restricted deletion of PPARβ/δ resulted in progressive myocardial lipid accumulation, cardiac hypertrophy, and heart failure (9). Based on the analysis of the PPARβ/δ-null mouse model, PPARβ/δ deficiency leads to multiple developmental and metabolic abnormalities, including frequent embryonic lethality, impaired wound healing, and skin abnormalities due to altered inflammatory responses in the skin (10, 11). Studies in skeletal muscle cells have indicated a role for PPARβ/δ in the regulation of fatty acid oxidation where uncoupling proteins such as UCP-2 and UCP-3 were up-regulated by PPARβ/δ (12, 13). Selective overexpression of PPARβ/δ in the mouse heart provoked an increase in myocardial glucose utilization with no myocardial lipid accumulation and normal cardiac function (14, 15).

Although the above findings suggest that PPARs fulfill a necessary function in myocardial lipid homeostasis and cardiac function, the PPAR isoform-selective gene profiles in cardiac muscle responsible for their pleiotropic biological responses remain ill defined. To circumvent the nonspecificity of PPAR synthetic ligands, we combined PPAR subtype-selective siRNA knockdown with computational PPRE analysis to determine the PPAR isoform-specific endogenous targets in the heart muscle. This screen revealed that PPARα activation induced a unique, antiapoptotic gene ontology that utilized activation of an endogenous insulin-like growth factor-1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K) route. Electromobility shift and chromatin immunoprecipitation assays demonstrated the existence of an evolutionary conserved PPRE consensus-binding site in an igf-1 enhancer. Additionally, PPARα activation in the murine heart, through Wy-14643 administration, provoked induction of igf-1 expression and subsequent protection against ischemia/reperfusion induced apoptosis. Pharmacological inhibition of the IGF-1/PI3K pathway or a null allele for PPARα abrogated this effect. Finally, PPARα-deficient mice displayed impaired induction of igf-1 following ischemic insults or pressure-induced overloading and a higher incidence of cardiomyocyte apoptosis, which contributed to deterioration of cardiac function.

MATERIALS AND METHODS

Animals

PPARα (129S4/Sv Jae-PPARα^tm1Gonz/j)-null mice and background-matched wild-type counterparts were purchased from the Jackson Laboratories. All protocols were performed according to institutional guidelines and approved by local Animal Care and Use Committees. Investigators were blinded to treatment for physiological and biochemical measurements.

Cell Culture and Recombinant Adenoviruses

Isolation and culture of neonatal rat ventricular cardiomyocytes were performed as described before in detail (16, 17). NKL-TAg cells were cultured and mortalized by AdCre infection as described previously (18, 19).

Generation of Stable Cardiac Cell Lines

To generate NKL-TAg clones harboring stably integrated constructs, NKL-TAg cells were transfected using FuGENE 6 reagent (Roche Applied Science), with a vector expressing the PPAR isotype-specific siRNA, pcDNA4/TO-siPPARα, siPPARβ/δ, or siPPARγ, and selected in the presence of 500 μg/μl zeocin to generate stable cell lines.

Histological Analysis and Immunofluorescence Microscopy

For histological analysis, hearts were arrested in diastole, perfusion-fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Paraffin sections were stained with hematoxylin and eosin for routine histological analysis and Sirius red for the detection of fibrillar collagen. Slides were visualized using a Nikon Eclipse E600 microscope or a Zeiss Axiovert 135 (immunofluorescence).

Transthoracic Echocardiography

Echocardiographic measurements were performed on mice anesthetized with isoflurane as described before (20), 8 weeks after starting transaortic banding. In M-mode, the following parameters were obtained: anterior wall thickness in diastole, left ventricular internal diameter in diastole, posterior wall thickness in diastole, anterior wall thickness in systole, left ventricular internal diameter in systole, posterior wall thickness in systole, posterior wall thickness in systole, left ventricular mass, and fractional shortening.

Quantitative RT-PCR

One microgram of total RNA was used as template for Superscript reverse transcriptase II (Promega). For real-time PCR, a Bio-Rad iCycler (Bio-Rad) and SYBR Green were used in combination with specific primer sets designed to detect transcripts (primer sequences available upon request).

Cardiac Ischemia/Reperfusion

Wy-14643 or saline as vehicle was administered as daily intraperitoneal injections (50 mg/kg of body weight) for 4 consecutive days to 2-month-old mice, after which they were anesthetized with isoflurane, intubated, and connected to a rodent respirator (model 683; Harvard Apparatus, South Natick, MA) on room air with a tidal volume of 0.2 ml and a respiratory rate of 180 breaths/min. Following a left thoracotomy, the left anterior descending coronary artery was ligated to induce ischemia for 30 min after which blood flow was reestablished. For the delineation of the area at risk (AAR), 5% Evans blue dye was injected into the apex after the indicated reperfusion times as described previously (21). Hearts were removed and stained with triphenyltetrazolium chloride to quantify infarct sizes. The AAR was expressed as a percentage of the left ventricle (LV) and the infarct area (IA) as a percentage of the AAR.

TUNEL Staining

TUNEL assays were performed as described previously (21), using the In Situ Cell Death Detection TMR-Red Kit (Roche Applied Science) and an antibody against α-actinin (Sigma) and TO-PRO3 (Molecular Probes) on 10-μm frozen, apical cross-sections of hearts.

Flow Cytometry

Flow cytometry was performed on isolated neonatal rat ventricular cardiomyocytes either treated or not with the apoptosis inducer H₂O₂ (500 μm for 3 h). Cells were then washed with ice-cold PBS, trypsinized, and resuspended in binding buffer containing 10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl₂. Cells were double stained with Annexin V conjugated with R-phycoerythrin conjugate (Annexin V-PE; BD Pharmingen) and propidium iodide (2.5 mg/ml, Molecular Probes) and analyzed using the CellQuest^TM Flow Cytometry System from BD Biosciences.

Transverse Aortic Constriction Banding

Transverse aortic constriction (TAC) or sham surgery was performed in WT and PPARα KO mice. The aorta was subjected to a defined, 27-gauge constriction between the first and second truncus of the aortic arch as described in detail previously (22). Pressure gradients between the proximal and distal sites of the TAC were determined by Doppler echocardiography (22).

Statistical Analysis

The results are presented as mean values ± S.E. Statistical analyses were performed using InStat 3.0 software (GraphPad Software) and consisted of ANOVA followed by Tukey's post test when group differences were detected at the 5% significance level.

Supplemental Material

The supplemental table includes an expanded Agilent microarray analysis.

RESULTS

Generation of Cardiac Cell Lines Stably Expressing PPAR Subtype-specific siRNAs

To determine whether the distinct cardiovascular functions of PPAR isoforms are related to activation of distinctive, isoform-selective target gene profiles, we first set up a method to determine PPAR subtype-specific target genes in the cardiomyocyte. To this end, we resorted to the use of a previously developed ventricular muscle cell line, NKL-TAg (18). NKL-TAg cells actively proliferate without apparent senescence, whereas introduction of Cre recombinase results in elimination of a loxP-flanked SV40 large T-antigen (TAg), provoking permanent exit from the cell cycle and cardiogenic differentiation with expression of cardiac markers. By RT-PCR, AdCre-mortalized clones expressed transcripts from the cardiogenic transcription factors nkx2.5 and gata4; cardiac ion channels were expressed regulating the calcium transient (atp2a2 and ryr2) and the transient outward K⁺ current (kncip2). Sarcomeric components were also detected including α-myosin heavy chain (myh6), myosin light chain-1a (myl4), desmin and sarcomeric actin (actc) at levels comparable with parental differentiated NKL-TAg cells and cardiac muscle (Fig. 1a).

FIGURE 1.

Generation of cardiac cell lines stably expressing PPAR subtype-specific siRNAs. a, RT-PCR identification for cardiac markers in NKL-TAg cells compared with adult mouse heart tissue, including the transcript for hypoxanthine guanine phosphoribosyl transferase (hprt). b, real-time RT-PCR quantitative analysis of endogenous pparα, pparβ/δ, pparγ transcripts (encoding PPARα, PPARβ/δ, and PPARγ, respectively), indicating efficient, stable siRNA knockdown of the endogenous transcript. c, Western blot analysis on lysates of NKL-TAg cells stably expressing selected siRNAs showing specific knockdown of the indicated PPAR isoform. d, luciferase measurements on stably transfected siRNA clones, transiently transfected with a mCPT promoter driven reporter as a functional verification of efficient, PPAR-isotype specific knockdown in each clone.

We designed expression vectors expressing PPAR subtype-specific shRNA to generate NKL-TAg clones stably expressing shRNAs against PPARα, β/δ, or γ using zeocin as a selectable marker. To control for siRNA and cell-based variations, we selected two clones each stably expressing siRNA for each PPAR isoform. Western blotting and real-time PCR demonstrated at least 70% knockdown of the endogenous PPAR isoforms in each clone (Fig. 1, b and c). Next, as a functional verification of the clones, parental, wild-type NKL-TAg cells and the siRNA clones were transiently transfected with a luciferase reporter driven by an mCPT promoter harboring a functional PPRE (23). The data show that activation of PPARα, PPARβ/δ, or PPARγ, using their synthetic ligands in the respective PPARα, β/δ, or γ siRNA clones was substantially suppressed with respect to mCPT-Luc activity, indicating functional PPAR isotype-selective knockdown in the selected clones (Fig. 1d).

Subtype-specific Expression Profile of PPAR Target Genes in Cardiac Muscle

Following this proof-of-principle that our PPAR isoform-specific siRNA clones adopt a cardiac muscle fate and provide stable knockdown of each respective PPAR subtype, we designed experiments to profile endogenous PPAR isoform-specific target genes. Thereto, in differentiated wild-type NKL-TAg cells and their corresponding siRNA clones, PPARα, β/δ, or γ, were activated using the specific synthetic ligands, Wy-14643, GW501516, or rosiglitazone, respectively, for 24 h. Agilent mouse chips harboring 22,000 transcripts were used to identify the early gene expression pattern evoked by PPAR isoform-specific activation. As depicted in Fig. 2a, in this setup genes that were differentially expressed in the respective PPAR isotype siRNA clones were considered ligand-activated but nonspecific for PPAR activity. Accordingly, these genes were subtracted from the expression profiles obtained in the corresponding ligand-activated, wild-type NKL-TAg cells. Furthermore, for each PPAR isoform only genes differentially regulated in both siRNA clones were considered.

FIGURE 2.

Genome-wide expression profile of PPAR target genes in a cardiomyocyte-like cell line. a, schematic representation of the genome-wide array setup. b, cardiac RNA collected from three separate siRNA clones and three separate wild-type cell lines and subjected to expression profiling using Agilent 22k mouse whole genome microarray slides. In the heat map representation of the resulting expression profiles, colors represent gene expression levels as indicated, with black equal to 0 (no change), bright red equal to 3.0 (3.0-fold increased expression), and bright green equal to −3.0 (−3.0-fold decreased expression). c, microarray expression profiles depicted in a Venn diagram format, indicating the number of overlapping target genes among the three PPAR isoforms. d, RT-PCR validation of differentially expressed PPAR isoform-specific target genes in stably transfected siRNA clones. e, RT-PCR validation of microarray results for mRNA levels of indicated genes in ligand-stimulated neonatal rat cardiomyocytes.

502, 620, and 277 genes (0.23, 0.28, and 0.13% of all genes present on the arrays) displayed a -fold change ≥2 following PPARα, β/δ, or γ activation, respectively, and were considered to be differentially expressed (p < 0.01) (Fig. 2, b and c). Detection of upstream PPREs using Fatigo matrices revealed that >80% of the reported genes contained at least one evolutionary conserved PPRE, a direct repeat of the consensus half-site motif (AGGNCA) spaced by a single nucleotide (DR-1). The resulting expression profiles were depicted in a heat map and in a Venn diagram format (Fig. 2, b and c).

Next, to validate the gene profiles for each PPAR isoform, we performed RT-PCR to verify differential expression in (non)stimulated wild-type cells and siRNA clones (Fig. 2d). For PPARα activation, acyl-CoA thioesterase 1 (cte1) and insulin-like growth factor-1 (igf-1) genes were up-regulated following Wy-14643 stimulation, but not in the siRNA clones, which further validates our unbiased array approach (Fig. 2d). Likewise, for PPARβ/δ, aldehyde oxidase 1 (aox1) and muscle and heart-type fatty acid binding protein 3 (fabp3) genes were specifically induced following GW5015160 stimulation, but not in the PPARβ/δ siRNA clones (Fig. 2d). Finally, rosiglitazone induced lipoprotein lipase (lpl) gene expression and down-regulation of baculoviral IAP repeat-containing 5 (birc5, also known as survivin), but not in PPARγ siRNA clones (Fig. 2d).

Strikingly, we found that PPARβ/δ and PPARγ shared a more closely related and overlapping number of transcripts than PPARα in the cardiomyocyte. In addition, whereas PPARβ/δ and PPARγ showed considerable resemblance in their target genes, PPARα also regulated a distinct ontology profile of target genes in the cardiomyocyte (Fig. 2e). Gene ontology classifications of the target genes revealed for all the three PPAR isoforms an overrepresentation of genes involved in metabolism regulation, cell cycle, adhesion, apoptosis, and immune response (supplemental table). By RT-PCR we verified two target genes for each of the expression areas indicated in the Venn diagram (Fig. 2, c and e). Conclusively, using an unbiased array approach and including a setup with stringent criteria, we have defined the specific target genes and gene ontologies for each PPAR isoform in cardiac muscle cells.

PPARα Induces igf-1 Gene Expression

As expected, gene ontology classifications revealed an overrepresentation of target genes involved in metabolism regulation (60%) Interestingly, PPARα profiling also displayed a relative overrepresentation of genes involved in apoptosis and cellular survival. In line, it has been reported that activation of PPARα by its respective ligands in cardiac muscle can attenuate cardiomyocytic apoptosis and heart muscle injury (24). By RT-PCR, we confirmed differential regulation of this gene ontology by PPARα activation in primary neonatal rat cardiomyocytes. The results indicate a down-regulation of proapoptotic target genes such as caspase-3 (casp3) and caspase 7 (casp7) and the up-regulation of the antiapoptotic target gene igf-1 upon Wy-14643 stimulation (Fig. 3a). Igf-1 has been identified as a potent growth factor providing protection against apoptosis and prolongs cell survival in several cell types, including cardiac muscle (25, 26), suggesting a plausible genetic mechanism for the potential antiapoptotic function of PPARα in the heart.

FIGURE 3.

PPARα induces an antiapoptotic profile in cardiomyocyte with igf-1 as direct target gene. a, RNA collected from three separate isolations of primary rat cardiomyocytes treated with PPARα agonist, Wy-14643, or left untreated. Depicted are RT-PCR analyses of apoptosis related PPARα isoform-specific target genes. b and c, comparison between the igf-1 genomic regions in mouse and human. Percentage conservation of a 5′ 10.0-kb genomic region upstream of igf-1 first exon is shown along with a schematic presentation of a ±2.5-kb 5′-flanking region in mouse igf-1 and location of potential PPREs conserved in human, mouse, and rat. d, gel mobility shift assay performed using fluorescent probes of a PPARα consensus sequence in the mCPT gene or the PPRE-like site from the igf-1 promoter. e, ChIP assays performed on differentiated NKL-TAg cells either treated or not with Wy-14643 for 24 h to activate PPARα. Soluble chromatin was immunoprecipitated with a specific antibody for PPARα. Associated DNA was purified, and RT-PCR analysis was performed using specific primers to the igf-1 promoter flanking the PPRE or a noncoding genomic region 3′ of the pfkl gene as a control.

To define the PPARα responsiveness of igf-1 mechanistically, we searched for evolutionary conserved enhancers that regulate in vivo transcription of the igf-1 gene. Comparison of genomic sequences across species using rVISTA revealed that a 2.5-kb genomic region immediately upstream of the first exon of igf-1 was conserved between human and mouse, apart from discontinued more distal regions that also displayed high cross-species conservation (Fig. 3b). A potential PPRE in the igf-1 enhancers was nearly identical and conserved in human, mouse, and rat (Fig. 3c). To confirm the binding of PPARα to the igf-1 enhancers, we performed electromobility shift assays and demonstrate that a probe including one putative element (igf-1 PPRE) formed a retardation complex with ligand-activated PPARα (Fig. 3d, fourth lane). The specificity of binding was studied by competition assays. Addition of excess unlabeled igf-1 PPRE eliminated the specific complex (Fig. 3d, fifth lane), whereas a labeled, mutated probe could not inhibit complex formation between PPARα with the wild-type probe (Fig. 3d, sixth lane).

To confirm in vivo binding of PPARα to the igf-1 enhancer, we performed chromatin immunoprecipitation (ChIP) assays. Differentiated wild-type NKL-TAg cells were either or not treated with Wy-14643 for 24 h to activate PPARα. The resultant soluble chromatin fractions were immunoprecipitated using a PPARα-specific antibody, and associated DNA was purified. Using primers specific for the igf-1 promoter flanking the PPRE, a PCR product was detected in input material and in PPARα-immunoprecipitated chromatin (Fig. 3e). This association was specific for PPARα because enrichment was not obtained in unstimulated differentiated wild-type NKL-TAg cells or when using primers to an unrelated intergenic region, in this case a fragment 20 kb upstream of pfkl (Fig. 3e). When using an antibody for H4-acetylated histone as a positive control, enrichment of the PCR product was obtained, indicating the open character of chromatin at this locus. Taken together, these results indicate that PPARα regulates the igf-1 gene by direct transcriptional activation and unambiguously show the presence of endogenous PPARα protein on the proximal igf-1 promoter in vivo, establishing igf-1 as direct target gene of PPARα signaling in cardiac muscle.

PPARα Inhibits Apoptosis via an IGF-1/PI3K Pathway in Vitro and in Vivo

Accumulating evidence suggests that oxidative stress triggers cardiac cell death and the pathogenesis of a number of cardiovascular diseases, such as ischemic heart disease, heart failure, and atherosclerosis (27). Although other growth factors seem unable to suppress apoptosis of cardiac muscle cells at pharmacological levels, IGF-1 has been observed to have profound antiapoptotic properties even at physiological concentrations (26). Activation of the IGF-1 receptor in cardiac muscle leads to activation of the PI3K axis, which also has been linked to antiapoptotic responses and protection from cardiac reperfusion injury in vivo (28). To test whether the antiapoptotic effect of PPARα in cardiomyocytes is mediated via a functional IGF-1/PI3K pathway, primary rat cardiomyocytes were left untreated or were treated with Wy-14643 for 24 h and subjected to H₂O₂ to induce apoptosis. To detect early loss of apoptosis-associated alterations in membrane asymmetry of phospholipids and signs of nuclear apoptosis, cells were stained with Annexin V and propidium iodide and analyzed using flow cytometry. H₂O₂ treatment produced a nearly 50% increase in the proportion of Annexin V/propidium iodide-positive cells compared with control conditions, whereas activation of PPARα profoundly reduced H₂O₂-induced apoptosis (Fig. 4a). Cells co-treated with LY294002, a selective PI3K inhibitor, significantly reduced Wy-14643-induced rescue of H₂O₂-induced apoptosis (Fig. 4a), implicating the downstream activation of the PI3K pathway following PPARα activation in cardiomyocyte survival signaling.

FIGURE 4.

PPARα inhibits apoptosis via an IGF-1/PI3K pathway in vitro and in vivo. a, representative pictures from flow cytometry experiments on primary rat cardiomyocytes, untreated or stimulated with Wy-14643 with co-treatments with H₂O₂ (500 μm for 2 h) or LY294002 (10 μm) 30 min prior to H₂O₂ addition. Percentage of Annexin V-PE/PI double-positive population is shown in the lower corner, demonstrating that fewer apoptotic cells were found only after activation of PPARα in combination with an intact PI3K pathway. b, real-time PCR analysis for igf-1, which is increased after Wy-14643 stimulation in wild-type hearts but not in PPARα KO (n = 3/group). c, representative image of cross-sections of 2–3-month-old wild-type and PPARα KO mice hearts subjected to ischemia/reperfusion, stimulated either with Wy-14643 or vehicle. In red, the AAR is depicted, in white the IA, and in blue, the areas of the heart not perfused by the left anterior descending coronary artery. Scale bars, 5 mm. d, AAR, expressed as S.E. (error bars) of percentage of total LV. The value was comparable in wild-type and PPARα mice. The IA/AAR ratio demonstrated significant differences between wild-type and PPARα KO only after Wy-14643 stimulation (n = 5 and n = 6, respectively). e, representative image of TUNEL labeling of wild-type and PPARα KO hearts stimulated either with Wy-14643 or vehicle and subjected to ischemia/reperfusion. Scale bars, 0.2 mm. f, bar graph indicating mean ± S.E. (error bars) of the percentage of TUNEL-positive cardiomyocytes after ischemic insult (n = 3/group), demonstrating Wy-14643-dependent rescue in wild-type hearts but not in PPARα KO.

To test whether Wy-14643 stimulation also leads to protection of heart muscle apoptosis in vivo, cohorts of 2-month-old wild-type and pparα-null mice were treated with the PPARα agonist Wy-14643 or vehicle for 4 consecutive days. Next, mice were subjected to transient occlusion of the left anterior descending coronary artery for 30 min (ischemia) followed by 24-h reperfusion to provoke widespread cardiac cell death. After the reperfusion period, the left anterior descending coronary artery was religated, and hearts were perfused with Evans blue dye, removed, and incubated in triphenyltetrazolium chloride to quantify AAR and IA, respectively. The AAR/total LV area ratio did not differ among the four groups (Fig. 4, c and d), indicating that there were no genotype-dependent differences in the perfused areas among the experimental groups. Interestingly, the infarct area was substantially reduced following Wy-14643 treatment in wild-type mice, whereas Wy-14643 had no effect in pparα-null mice, indicating the obligate requirement of PPARα in Wy-14643-mediated cardioprotection (Fig. 4, c and d).

To establish whether the Wy-14643-mediated reduction in infarct size was related to reduction in programmed cardiac muscle cell death, the levels of apoptotic cells within the viable LV and septum were assessed by TUNEL labeling. As indicated by the numbers of TUNEL/α-actinin/DAPI triple-positive myocytes, Wy-14643 treatment significantly reduced the incidence of apoptotic cell death in wild-type mice but not in pparα-deficient mice (Fig. 4, e and f). Finally, real-time RT-PCR indicated that igf-1 transcripts were up-regulated after Wy-14643 treatment in wild-type and not in pparα-null mice, demonstrating that Wy-14643-mediated cadioprotection was accompanied by in vivo induction of ifg-1 in a PPARα-dependent manner (Fig. 4b). Combined, these data confirm that PPARα activation in cardiac muscle provides protection against cardiac muscle apoptosis via activation of an IGF-1/PI3K pathway.

PPARα Protects against Pressure Overload-induced Myocardial Cell Death

Biomechanical stress activates signaling cascades, including oxidative stress, predisposes cardiac muscle to late-onset apoptosis (21, 29–31), and provokes progressive LV remodeling and heart failure (29, 30). To determine whether igf-1 induction downstream of PPARα activation also protects from apoptosis in response to biomechanical stress, TAC was performed on cohorts of wild-type and pparα-deficient mice. To ensure equal loading conditions on all experimental groups, pressure gradients were measured noninvasively (Fig. 5b). Eight weeks after pressure overload, gross morphology showed no differences between sham-operated wild-type and pparα-deficient mice (Fig. 5, a and c). Both experimental groups showed substantial cardiac enlargement upon biomechanical stress (heart weight/body weight ratios 9.4 ± 0.8 and 8.9 ± 0.7 mg/g, respectively; not significant.; Fig. 5, a and c), indicating that the cardiac hypertrophic response to pressure overload is not affected in the absence of PPARα.

FIGURE 5.

PPARα protects against pressure overload-induced myocardial cell death. a, representative gross morphology of hearts dissected from 4 month-old-mice of the indicated genotypes after 8 weeks of sham or TAC surgery, demonstrating profound cardiac enlargement after pressure overload. Scale bars, 5 mm. b, pressure gradients across the transverse aorta measured noninvasively to validate the TAC procedure. c, heart weight/body weight ratios of 4 month-old wild-type and PPARα KO mice after 8 weeks of sham or TAC surgery, indicating a significant increase in cardiac mass after pressure overload (n = 5/group) N.S., not significant; **, p < 0.01; error bars, ±S.E. d, representative histological images of hearts from mice of indicated genotypes after 8 weeks of sham or TAC surgery. Scale bars, 0.2 mm. Hematoxylin and eosin-stained images reveal remarkable myocyte hypertrophy and myofiber disarray in mice subjected to TAC surgery. Sirius red staining indicates massive interstitial and perivascular fibrosis in hearts of wild-type and PPARα KO mice subjected to TAC surgery. e, representative M-mode images of wild-type and PPARα KO mice after 8 weeks of sham or TAC surgery, indicating decreased contractility and increased LV internal dimensions after pressure overload. The time scale and size indication are provided in the M-mode images. f, bar graph representations of fractional shortening (FS) and LV internal diameter at systole (LVIDs), indicating increased functional and geometrical deterioration after TAC in PPARα KO mice compared with wild-type mice subjected to TAC (n = 5–8/group). g, quantitative RT-PCR analysis of nppa (atrial natriuretic factor), nppb (brain natriuretic peptide) and myh7 (β-myosin heavy chain) α-ska (α-skeletal actin) in hearts of wild-type and PPARα KO mice after 8 weeks of sham or TAC surgery. Error bars are mean ± S.E. of n = 3. h, quantitative RT-PCR analysis of igf-1 in hearts of wild-type and PPARα KO mice after 8 weeks of sham or TAC surgery, indicating significant up-regulation of igf-1 transcripts in wild-type mice after pressure overload. i, representative image of TUNEL labeling of wild-type and PPARα KO hearts after 8 weeks of sham or TAC surgery. Scale bars, 0.2 mm. j, bar graph indicates mean ± S.E. (error bars) of the percentage of TUNEL-positive cardiomyocytes in hearts of wild-type and PPARα KO mice subjected to sham or TAC surgery (n = 3/group), showing enhanced apoptosis in PPARα KO hearts subjected to pressure overload.

Using M-mode echocardiography, we studied pressure overload-induced hemodynamic behavior at 8 weeks after TAC. Representative images of M-mode recordings of all experimental groups are displayed in Fig. 5d. A more pronounced increase in LV internal diameter (Fig. 5, d and e) and a proportional decrease in systolic contractility were evident in pparα-deficient mice compared with wild-type mice following pressure overload (Fig. 5e), indicative of accelerated progressive LV dilation and heart failure.

Reactivation of fetal gene expression is a hallmark of pathological hypertrophy and heart failure. To this end, transcripts levels for nppa (atrial natriuretic factor), nppb (brain natriuretic peptide), myh7 (β-myosin heavy chain) and acta1 (α-skeletal actin) were analyzed and found significantly up-regulated upon pressure overload in pparα-null mice compared with their wild-type counterparts (Fig. 5f). Next, hematoxylin and eosin- and Sirius red-stained cardiac sections did not show signs of histopathology in sham operated wild-type and pparα-deficient mice (Fig. 5c). In contrast, cardiomyocyte hypertrophy, myocyte disarray, and extensive areas of interstitial and perivascular fibrosis were evident in both pressure-overloaded wild-type and pparα-deficient hearts, but these changes were more pronounced in pressure overloaded pparα-null hearts (Fig. 5c). Pressure overload predisposes loss of excessively stressed myocytes in the hypertrophied heart, further fueling the histopathological changes and functional deterioration of the failing heart. TUNEL labeling showed a significant increase of TUNEL/α-actinin/DAPI-positive myocytes after pressure overload in pparα-deficient hearts compared with wild-type hearts (Fig. 5, h and i), indicating that the pparα-deficient myocardium has a higher incidence of stress-induced myocyte apoptosis. Finally, real-time RT-PCR demonstrated that igf-1 was significantly up-regulated in the pressure-overloaded wild-type heart, but not in pparα knock-out mice, revealing a PPARα-dependent activation of igf-1 upon pressure overload (Fig. 5g). Taken together, these data confirm igf-1 as a genuine in vivo target of PPARα during pressure overload and the involvement of a PPARα-IGF-1 signaling pathway in the protection of cardiomyocytes under increased hemodynamic loading conditions.

DISCUSSION

Microarray profiling and genome wide identification of PPREs by stimulation with isoform-selective synthetic ligands have suggested the existence of a considerable number of PPAR-regulated genes, which have not previously been described as PPAR target genes (32, 33). Although useful, selective activation of PPAR isoforms by their synthetic ligands is seriously hampered by their overlapping specificity (34). These ligands have been shown to differentially induce unspecific activation of other PPAR isoforms, or even act PPAR-independent (34). In the present study, by combining PPAR subtype-selective siRNA knockdown with microarray profiling and genome-wide identification of PPREs, we determined isoform-selective target gene profiles of PPARs in a cardiomyocyte-like cell type.

Genome-wide predictive analyses previously revealed that up to 4.5% of the eukaryote genome may contain predicted PPREs, illustrating the wide range of target genes for the PPAR family members (35). By bioinformatics analysis, >80% of identified target genes from our microarrays were found to contain a conserved PPRE, further lending support to the notion that we identified valuable cardiac PPAR target genes. As expected, many identified genes were related to energy/lipid metabolism, e.g. acyl-CoA dehydrogenase, medium chain (Acadm), cytochrome P450, family 4, subfamily b, polypeptide 1 (Cyp4b1), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), β-subunit (Hadhb), pyruvate dehydrogenase kinase, isoenzyme 4 (Pdk4), acetyl-CoA acyltransferase 2 (Acaa2), carnitine acetyltransferase (Crat), fatty acid-binding protein 4, adipocyte (Fabp4), and fatty acid-binding protein 3, muscle and heart (Fabp3).

Apart from well known target genes, our data also uncovered a series of novel target genes, regulated by one single PPAR isoform or co-regulated by two or even all three PPAR isoforms in cardiac muscle (see supplemental table). For instance, PPARα activation has been shown to repress cytokine-induced activation of a number of inflammatory genes through negatively interfering with NF-κB transcriptional activity (36). Although the influence of PPARα activation on the different components of the NF-κB signaling pathway has not been fully explored, we found that PPARα activation represses the expression of nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (NF-κB2), suggesting a novel mechanism for the anti-inflammatory contribution of PPARα in the cardiomyocyte.

Numerous studies have identified decreased cardiac energy levels and flux as a consistent feature of heart failure and have focused considerable attention on metabolic modulations as a therapeutic modality for heart failure (37–39). However, reactivation of PPARα by a selective agonist in the heart in different models of heart failure has yielded diverse effects. Rats treated with a PPARα agonist and subjected to pressure overload-induced hypertrophy developed contractile dysfunction (40), whereas a rat model of postinfarction heart failure showed enhanced LV hypertrophy but with no apparent deterioration of cardiac function (41). These results indicate that the different etiologies of heart failure respond differently on PPARα activation, probably due to diverse regulation of cardiac substrate metabolism. Indeed, using a pacing-induced heart failure dog model, chronic fenofibrate treatment restored cardiac metabolism without altering the progression toward decompensated heart failure (42).

Apoptotic loss of myocardium itself can increase hemodynamic stress through ventricular dilation and wall thinning and is therefore hypothesized to play an important role in the downward functional spiral that ultimately leads to heart failure (43, 44). Although our approach has yielded a significant amount of interesting PPAR subtype-specific target genes in the cardiomyocyte, we noted that PPARα regulates a subset of genes that suggest activation of survival pathways and antagonizes apoptotic cardiomyocyte cell death, compared with the gene profiles regulated by PPARβ/δ and PPARγ. The results indicated down-regulation of proapoptotic target genes such as caspase 3 (casp3) and caspase 7 (casp7) and the up-regulation of the antiapoptotic target gene insulin-like growth factor-1 (igf-1) upon Wy-14643 stimulation.

An emerging body of evidence suggests that PPARα agonists indeed can protect the heart from apoptosis (45). Here, we show that the splice isoform 1 for igf-1 was significantly up-regulated after PPARα activation through direct transcriptional activation, as shown by ChIP and electromobility shift assays. Igf-1 has been shown to decrease myocyte apoptosis after myocardial infarction in mice (46) and in ischemia/reperfusion injury in rats and cultured rat myocytes (47). Our findings also demonstrate that PPARα activation in the mouse heart, through Wy-14643 administration, resulted in up-regulation of igf-1 expression and subsequent protection against ischemia/reperfusion-induced apoptosis.

Apart from stimuli secondary to direct massive ischemic myocardial injury, primary hemodynamic overload, as observed under conditions of chronic hypertension and aortic stenosis, also leads to cardiomyocyte loss through apoptosis. TAC demonstrated an increase in igf-1 expression in wild-type hearts, but this induction of igf-1 was abrogated in mice lacking PPARα, indicating the requirement of the PPARα isoform in regulating igf-1 in vivo. Taken together, this study mechanistically links PPARα activation, induction of igf-1 gene expression, cardiomyocyte dropout, ventricular remodeling, and functional deterioration in the genesis of heart failure.

In conclusion, by combining large scale gene expression analysis using microarrays and computational approaches, we revealed PPAR subtype-specific target genes in the heart. Primary analysis of these results present a novel mechanism linking PPARα signaling in the heart muscle and protection against apoptotic cell death, suggesting a pharmaceutical value for select activation of the PPARα isoform in certain forms of heart disease.