Activation of Peroxisome Proliferator–Activated Receptor-α Protects the Heart From Ischemia/Reperfusion Injury
Background— Peroxisome proliferator–activated receptor-α (PPAR-α) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-α in acute ischemia/reperfusion myocardial injury remains unclear.
Methods and Results— The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-α agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P<0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-α–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-α and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-κB activation in the heart, accompanied by enhanced levels of inhibitor-κBα.
Conclusions— Activation of PPAR-α protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-α agonist could provide an added benefit to patients treated with PPAR-α activators for dyslipidemia.
Received August 28, 2002; de novo received April 9, 2003; revision received July 9, 2003; accepted July 11, 2003.
Peroxisome proliferator–activated receptor-α (PPAR-α) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR-α increases gene transcription by binding as a heterodimer with the retinoid X receptor (RXR) to PPAR response elements in the promoter regions of the target gene. Evidence has emerged that PPAR-α is involved in the regulation of a number of genes encoding fatty acid oxidation (FAO) enzymes.1 More recently, studies have revealed that PPAR-α ligands exert antiinflammatory actions.2
PPAR-α is expressed at a relatively high level in the heart and vasculature and plays a role in maintaining cardiac metabolic homeostasis. However, the role of PPAR-α in the pathogenesis of a variety of heart disorders remains unclear. In PPAR-α–null mice, altered expression of PPAR-α–modulated FAO enzymes led to age-dependent cardiac damage.3 In addition, metabolic stress caused by inhibition of cellular fatty acid flux resulted in massive cardiac and hepatic lipid accumulation and death.4 Furthermore, targeted disruption5 or overexpression6 of PPAR-α–regulated genes encoding FAO enzymes caused development of cardiomyopathy5, left ventricular (LV) dysfunction, or premature death.6 The critical role of PPAR-α and PPAR-α target FAO genes in the human heart was demonstrated by observation of inherited hypertrophic cardiomyopathy and sudden death in infants with inborn errors of myocardial FAO.7 In contrast, studies in rodents have suggested that activation of PPAR-α might be detrimental to the heart. The data have indicated that downregulation of PPAR-α is essential for maintenance of contractile function of the hypertrophic heart, and reactivation of PPAR-α and PPAR-α–regulated genes with a PPAR-α agonist resulted in severe depression of cardiac power and efficiency.8 Mice with cardiac overexpression of PPAR-α exhibited signs of diabetic cardiomyopathy, including ventricular hypertrophy and dysfunction.9 The controversy surrounding the role of PPAR-α in the heart suggests that the function of this transcription factor might not be the same in different cardiac pathologies or different stages of heart disease. It also suggests that additional PPAR-α–mediated effects other than lipid metabolism might be involved.
Ischemia/reperfusion injury occurs as a result of damage to the myocardium after blood restoration after a critical period of coronary occlusion. Myocardial ischemia/reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty, and coronary bypass surgery, which are commonly used to reestablish blood flow and minimize heart damage due to myocardial ischemia. The role of PPAR-α in acute cardiac ischemia/reperfusion injury remains unclear. In the current study, we have investigated the effect of activation of PPAR-α on acute ischemia/reperfusion-induced myocardial infarction and dysfunction.
The study was conducted according to the Guidelines for Care and Use of Experimental Animals. Male CD1 mice (Charles River, Raleigh, NC), PPAR-α−/− mice on an Sv/129 genetic background, or wild-type (PPAR-α+/+) littermates (The Jackson Laboratory, Bar Harbor, Maine) were used. Left anterior descending coronary artery (LAD) occlusion (30 minutes) and reperfusion (24 hours, unless otherwise indicated) were induced by inflating and then deflating a nontraumatic balloon occluder that was fixed on the LAD. The ischemic area and infarct size were determined and quantified.10 GW7647 (EC50=6 nmol/L for PPAR-α and 1.1 μmol/L for PPAR-γ; GlaxoSmithKline Pharmaceuticals)11 at 3 mg · kg−1 · d−1 PO, unless otherwise indicated, was administered for 2 days, and the third dose was given 1 hour before ischemia.
Assessment of Myocardial Function
LV pressure, LV systolic pressure, positive and negative dP/dt, and mean arterial blood pressure were measured as described previously.12
Northern Blot Analysis
Standard Northern blotting was used to investigate mRNA expression of PPAR-α, RXR, acyl-CoA synthetase (ACS), muscle carnitine palmitoyltransferase I (M-CPT I), and long-chain (LCAD) and medium-chain (MCAD) acyl-CoA dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase was used for normalization, as described previously.12
Measurement of Fatty Acid β-Oxidation Activity
FAO activity was measured by the method reported previously, with [1-14C]palmitic acid and [1-14C]lauric acid as the substrates.3 FAO activity was expressed as picomoles per minute per milligram protein.
Assessment of Cardiac Glucose Versus Fat Oxidation
Mice subjected to ischemia/reperfusion were placed in restraining tubes after the jugular vein catheter was implanted. After 1 hour of reperfusion, a continuous [1,6-13C]glucose (25 mg · kg−1 · min−1)/somatostatin (0.15 μg/min) clamp was initiated. All clamps were run for 90 minutes. This period is sufficient for glycolytic and tricarboxylic cycle intermediates to achieve steady-state enrichments. At the end of the clamp experiment, the ischemic myocardium was rapidly removed and prepared for metabolite enrichment measurement as previously described.13 Relative cardiac glucose and fat oxidation in terms of relative substrate contribution to acetyl-CoA oxidation was assessed from the metabolite pool enrichments.14
Measurement of Myeloperoxidase Activity
Myeloperoxidase activity was measured according to a previously described method.12
Multiplexed Serum Cytokine Immunoassays
Standards and serum samples were added to a 96-well, filter-bottom plate. Uniquely fluorescent microspheres (Luminex) with covalently coupled capture antibodies (R&D) were mixed together, added to the 96-well plate, incubated for 4 hours, and then washed. Subsequent 30-minute incubations with biotinylated detection antibody mixture and reporter dye (Molecular Probes) were followed by analysis of resuspended microspheres with a luminometer (Luminex 100). Standard curves were generated from the reporter signals for each cytokine, and results were calculated with a 5-parameter logistic curve fit.
Electrophoretic Mobility Shift Assay for Nuclear Factor-κB
An electrophoretic mobility shift assay was performed according to a previously described method.15 In brief, nuclear extracts from ischemic myocardium were prepared, and gel shift assays with a nuclear factor (NF)-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTC CCAGGC-3′, Promega) were performed. Samples were separated by 5% polyacrylamide gel electrophoresis. Optical density in the region of NF-κB (p65) was quantified by phosphorimaging analysis (PhosphorImager).
Western Blot Analysis
Protein extracts from ischemic myocardium (50 to 100 μg) were fractionated on a 10% polyacrylamide gel under reducing conditions, transferred to nitrocellulose membranes, and probed with various antibodies as stated in the figure legends. After incubation with a secondary peroxidase-conjugated antibody, signals were visualized by chemiluminescence (Amersham).
Measurement of Matrix Metalloproteinases by Zymography
Expression of matrix metalloproteinases (MMPs) was analyzed for gelatinolytic activity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis zymography and quantified as described previously.10
Data are expressed as mean±SEM and were analyzed by 1-way ANOVA, with subsequent post hoc paired comparisons or by unpaired Student’s t test. Differences with a value of P<0.05 were considered statistically significant.
GW7647 Reduces Myocardial Infarction and Improves Myocardial Contractile Function
There was no difference in ischemic area (Figure 1, left side), indicating a comparable degree of ischemic insult between vehicle- and GW7647-treated groups after occlusion of the LAD. At 0.3, 1, and 3 mg · kg−1 · d−1 for 3 days, GW7647 reduced infarct size by 8%, 28%, and 35%, respectively, compared with vehicle (P<0.01 at 1 and 3 mg · kg−1 · d−1, Figure. 1, right side). Wy14643, a fibrate class of PPAR-α agonist, at 5 mg · kg−1 · d−1 for 3 days, also reduced infarct size by 27% (P<0.01). Treatment with GW7647 (3 mg · kg−1 · d−1) also improved myocardial functional contraction at 24 hours after reperfusion. The values of LV systolic pressure, +dP/dt, −dP/dt, and mean arterial blood pressure were enhanced from (percent of sham) 75%, 53%, 50%, and 56% in the vehicle-treated to 85%, 76%, 66%, and 70% in the GW7647-treated group, respectively. (all P<0.05). There was no difference in heart rate between the 2 groups.
Targeted Deletion of PPAR-α Abrogates Cardioprotection by GW7647
As shown in Figure 2, infarct size was larger in PPAR-α−/− mice compared with PPAR-α+/+ mice, although both groups were subjected to a similar degree of ischemic insult. Moreover, the myocardial protective effect of GW7647 was totally abolished in the PPAR-α−/− mice.
Attenuation by GW7647 of Ischemia/Reperfusion-Induced Downregulation of PPAR-α mRNA in Myocardium
Figure 3A illustrates a time course of PPAR-α mRNA levels in myocardium after ischemia/reperfusion, as quantified by Northern blot. A remarkable downregulation of PPAR-α mRNA was observed at 2 to 4 hours and further at 7 and 24 hours after reperfusion in the vehicle group. A significant reduction in PPAR-α mRNA was demonstrated only at 24 hours in the GW7647-treated group. Figure 3B shows a representative Northern blot from ischemic myocardium collected 24 hours after reperfusion. Expression of RXR-α mRNA in the myocardium was changed slightly, and a significant reduction was observed only at 24 hours in the vehicle group.
Attenuation by GW7647 of Reperfusion-Induced Downregulation in Expression of FAO Enzyme Genes and FAO Activity in Myocardium
Ischemia/reperfusion-induced downregulation in expression of PPAR-α–regulated FAO enzyme genes ACS, M-CPT-1, MCAD, and LCAD in the myocardium was similar to the changes in PPAR-α mRNA. That is, a significant reduction was demonstrated at 2 to 4 hours (70% to 80% basal) and further at 7 (50% to 70% basal) and 24 hours in the vehicle group but only at 24 hours in the GW7647 group after reperfusion (Figure 4A). Figure 4B illustrates a representative Northern blot from the myocardium after 24 hours of reperfusion. Similar to PPAR-α expression, reperfusion-induced downregulation in all of these enzyme genes was attenuated by GW7647. Overall FAO activity (in pmol · min−1 · mg−1 protein) for lauric and palmitic acids in ischemic myocardium after 24 hours of reperfusion was reduced from 84±6 and 66±7 in sham to 27±4 and 21±5 in the vehicle group but was reversed to 62±6 and 43±5 in the GW7647 group, respectively (P<0.01 versus vehicle, n=3, performed in triplicate).
Attenuation by GW7647 of Ischemia/Reperfusion-Induced In Vivo Cardiac Metabolism Shift
Fat oxidation composed the majority of relative oxidative metabolism in the hearts of sham mice (Figure 5). By contrast, ischemia/reperfusion resulted in an increase in relative glucose oxidation from 11.9% (sham) to 41.6% (P<0.05), whereas the relative fat oxidation was reduced from 88.1% (sham) to 58.4% (P<0.05). However, this ischemia/reperfusion-induced cardiac metabolism fuel shift was reversed after treatment with GW7647. Relative glucose oxidation decreased to 20.6%, and fat oxidation increased to 79.4% (P<0.05 versus vehicle for both glucose and fat oxidation).
Attenuation by GW7647 of Ischemia/Reperfusion-Induced Elevation in Serum Free Fatty Acid Levels
There was no difference in the basal levels of serum free fatty acids (FFAs) among sham, vehicle, and GW7647 groups (Figure 6). Reperfusion resulted in a significant elevation in FFA levels in the sera collected from the LV in the vehicle compared with the sham group. The peak increase in FFA level at 90 minutes after reperfusion was 1.6-fold over basal. Treatment with GW7647 abrogated the reperfusion-induced elevation in FFA level. In a separate study with PPAR-α−/− mice, ischemia/reperfusion (90 minutes) resulted in a 2.3-fold increase in serum FFA level over basal, and GW7647 had no effect on the elevation in FFA level (data not shown).
Effect of GW7647 on Neutrophil Accumulation in Ischemic Myocardium
Immunohistochemical analysis demonstrated that ischemia/reperfusion resulted in a significant increase in neutrophil accumulation in the heart, frequently concentrated in the subepicardial regions, and that extended into the area of injured myocardium of the LV. Treatment with GW7647 markedly reduced neutrophil infiltration in the heart (data not shown). Myeloperoxidase activity (U/100 mg) in ischemic myocardium after 24 hours of reperfusion was reduced from 1.40±0.22 in the vehicle- to 0.43±0.12 in GW7647-treated mice (P<0.01, n=10).
Inhibition of Ischemia/Reperfusion-Induced Inflammatory Cytokine Production by GW7647
Among the tested cytokines, interleukin-6, KC, and granulocyte colony stimulating factor were found to be substantially increased in blood collected from the LV after reperfusion, and this increase was inhibited by GW7647 (Figure 7A). Tumor necrosis factor-α was elevated moderately but significantly. The peak levels (pg/mL) at 0.5 and 1.5 hours after reperfusion were 36±8 and 71±12 in the vehicle group and 18±9 and 17±6 (P<0.05) in the GW7647 group, respectively.
GW7647 Reduces MMP-9 and MMP-2 Expression in Ischemic Myocardium
The expression of pro- and active isoforms of MMP-9 and of pro-MMP-2 was markedly increased in ischemic myocardium (Figure 7B), and this increase was reduced by 49%, 84%, and 37%, respectively, in the GW7647-treated group versus the vehicle group (all P<0.01, n=6). This was determined by densitometric analysis and further confirmed by Western blotting and immunohistochemical analysis (data not shown).
Effect of GW7647 on Ischemia/Reperfusion-induced NF-κB Activation
Ischemia/reperfusion resulted in activation of NF-κB (enhanced DNA binding activity by 110% versus sham) in myocardium, and this activation was inhibited by 80% in the GW7647-treated group (Figure 8A). Figure 8B depicts a remarkable decrease of inhibitor (I) κBα in the cytoplasmic fractions from ischemic myocardium, and treatment with GW7647 enhanced the levels of IκBα by 77% versus vehicle. Moreover, the level of IκBα in the nuclear fraction was also enhanced by 42% (P<0.05) in the GW7647-treated versus the vehicle group (data not shown).
Previous studies in a mouse cardiac hypertrophic model16 and a rat model of hypoxia17 have found a downregulation in expression of cardiac PPAR-α. However, the effect of acute ischemia/reperfusion on expression of cardiac PPAR-α and PPAR-α–regulated FAO enzymes in vivo had not been reported. In the present study, we have demonstrated a significant downregulation in PPAR-α and FAO enzyme genes after ischemia/reperfusion; this downregulation was significantly attenuated by GW7647. This result was also supported by the ex vivo cardiac FAO assay. Measurement of in vivo myocardial FAO with 13C nuclear magnetic resonance demonstrated that FAO accounted for ≈90% of the energy production in the hearts from the sham group, consistent with the data reported previously.18 However, a significant metabolic shift toward increased glucose oxidation occurred after ischemia/reperfusion. This shift was reversed by GW7647, indicating that activation of PPAR-α enhanced cardiac FAO enzyme activity and fat metabolism.
Downregulation of PPAR-α expression during cardiac hypertrophy or hypoxia, taking days to occur, might be an adaptive process16,17 by which the heart switches metabolic fuel preference from fatty acid to glucose under conditions of chronic ischemia/hypertrophy. This adaptive response appears to be beneficial for the heart by decreasing cardiac oxygen consumption.1 Whether attenuation of acute ischemia/reperfusion-induced downregulation of PPAR-α and FAO by a PPAR-α agonist is beneficial or detrimental to the heart is not known. In an acute cardiac ischemia/reperfusion model, as in this study, the blood supply to the heart is resumed after reperfusion, and the oxygen supply to the heart is therefore no longer rate limiting. This is different from chronic hypertrophic conditions, in which a shift to increased glucose oxidation would be more energy efficient when the oxygen supply is rate limiting. Given that the injured myocardium retains an attenuated level of PPAR-α and FAO enzyme expression after ischemia/reperfusion, the activation of PPAR-α by GW7647 would in theory improve the ability of the heart to use fatty acid as an energy source and thereby favor the maintenance of adequate energy production in the heart. Therefore, a shift back from glucose oxidation to FAO in the heart after reperfusion by GW7647 would be beneficial. Moreover, in GW7647-treated mice, the ischemia/reperfusion-induced elevation of serum FFA levels was almost completely abrogated. Increased FFA levels aggravate the severity of myocardial infarction (for a review, see Van Der Vusse et al19), and a close relation between peak plasma FFA levels and infarct size in patients with acute myocardial infarction has been demonstrated.20 Additionally, high concentrations of palmitate caused apoptosis in myocardial cells.21 Enhanced FAO in peripheral tissues and the heart by GW7647 could diminish the detrimental effect of the accumulated FFAs to the heart. Furthermore, a larger infarct size and a higher serum FFA level in PPAR−/− mice versus wild-type littermates also suggest possible involvement of the decreased myocardial FAO in the increased cardiac response to acute ischemia/reperfusion.
Three important components in reperfusion cardiac injury, accumulation of neutrophils, release of proinflammatory cytokines, and upregulation of MMP-9 and MMP-2 expression in the heart,10,22 were all significantly attenuated by GW7647. The results suggest that activation of PPAR-α might inhibit a common regulatory factor involved in the control of transcription of these proinflammatory mediators. Among the inducible transcription factors involved in reperfusion injury, NF-κB plays a central role.23 In vivo transfer of NF-κB decoy oligodeoxynucleotide reduced myocardial infarction.24 NF-κB binding sites in the promoter region have been demonstrated for a variety of proinflammatory cytokines. Recently, a functional NF-κB binding site was also found in the promoter of the MMP-9 gene, and overexpression of IκBα, an NF-κB inhibitor, inhibited expression of MMP-9 in response to interleukin-1α.25 The data shown in Figure 8A indicate that treatment with GW7647 inhibited the activation of NF-κB induced by ischemia/reperfusion. Our data are in agreement with an in vitro study in which a fibrate inhibited the vascular response by interfering with NF-κB transactivation capacity.26 In the present study, we have also found that GW7647 enhanced cytoplasmic levels of IκBα (Figure 8B). The increase in IκBα protein would halt p65-mediated gene activation and result in acceleration of NF-κB nuclear deactivation. Moreover, treatment with GW7647 enhanced the accumulation of IκBα in the nucleus (data not shown), consistent with data from an in vitro study that showed enhanced nuclear IκBα accompanied by a decrease in NF-κB DNA binding activity.26 Studies have suggested that IκBα, accumulated in the nucleus, is resistant to signal-induced degradation. The presence of IκB in the nucleus contributed to the inhibition of binding of active NF-κB complexes to the κB sites located in the regulatory sequences of various genes.27 Therefore, the enhanced accumulation of IκBα in the cytoplasm as well as the nucleus by the PPAR-α agonist would exert an inhibitory effect on NF-κB activity.
Additional work is necessary to provide further evidence for the direct causal link between enhanced cardiac FAO and cardioprotection by GW7647. For example, it is not clear what contribution the enhanced FAO in the heart alone versus that from peripheral tissues has on lowering serum FFAs. Whether other mechanisms or molecular targets are involved in PPAR-α–related cardioprotection needs to be explored as well.
In conclusion, activation of PPAR-α reduced ischemia/reperfusion-induced myocardial infarction and improved contractile function. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. PPAR-α agonists are currently being used for treatment of hyperlipidemia, and more potent and selective PPAR-α agonists are currently under development. The cardioprotection by PPAR-α agonists in acute ischemia/reperfusion injury could provide additional benefit to these patients. A recent article, published while this manuscript was being prepared, reported that clofibrate and Wy14643 reduced myocardial infarct size in a rat reperfusion model.28
The authors thank Drs Paul G. Lysko and Antoine Bril for their helpful comments.
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