The FASEB Journal • Research Communication
Inhibition of cardiac PGC-1␣ expression abolishes ER agonist-mediated cardioprotection following trauma-hemorrhage Ya-Ching Hsieh, Mashkoor A. Choudhry, Huang-Ping Yu, Tomoharu Shimizu, Shaolong Yang, Takao Suzuki, Jianguo Chen, Kirby I. Bland, and Irshad H. Chaudry1 Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA
Key Words: hemorrhagic shock 䡠 heart 䡠 estrogen receptor 䡠 mitochondria
patients following major surgery (1– 8). Previous studies have shown that prolonged depression of cardiovascular functions occurs in males following trauma-hemorrhagic shock despite fluid resuscitation (9 –11). However, administration of 17-estradiol (E2) in male or ovariectomized female animals following trauma-hemorrhage (T-H.) restored the depressed cardiovascular functions (12–14). The estrogen-mediated events were believed to be regulated by the estrogen receptor (ER). This suggestion was supported by our previous study which showed that administration of estrogen along with ICI 182,780, a selective ER antagonist, abolished the salutary effects of E2 on cardiac function following trauma-hemorrhage. Estrogen action is mediated by two ER subtypes designated ␣ and  (15–17). In recent years, much effort has been invested in the development of ER␣and ER-specific agonists and antagonists. Several such compounds have been developed, and this has permitted dissection of the specific functions of each receptor. One ER␣-specific agonist, 4,4⬘,4⬙-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT), is 410-fold more potent in binding to ER␣ than ER (18), whereas 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) binds to ER with an affinity 72-fold higher than to ER␣ (19). Both ligands induce expression of an estrogen response element (ERE)-luciferase reporter gene with potencies similar to that of E2; hence, they are considered to be ER␣- and ER-selective agonists, respectively. There is evidence suggesting that ER␣ and ER differ in their tissue distribution, transcriptional activities and phenotypes in knockout animals (20). Recent knockout studies have suggested that ER mediates gender-specific response to ischemia-reperfusion injury (21), development of cardiac hypertrophy (22), and myocardial infarction (23), thereby demonstrating ER’s importance in the heart under stress conditions. A previous study from our laboratory suggests that
Despite numerous advances in intensive care medicine, ischemia/reperfusion injury, sepsis, and organ dysfunction leading to multiple organ failure remain the major cause of death in trauma patients as well as in
1 Correspondence: Center for Surgical Research, University of Alabama at Birmingham, 1670 University Blvd, Volker Hall, Rm. G094, Birmingham, Alabama 35294-0019 USA. E-mail:
[email protected] doi: 10.1096/fj.05-5549com
ABSTRACT PGC-1␣ (peroxisome proliferator-activated receptor [PPAR␥] coactivator-1␣) activates PPAR␣ and mitochondrial transcription factor A (Tfam), which regulate proteins, fatty acid and ATP metabolism (i.e., FAT/CD36, MCAD, and COX I). Recently we found that the salutary effects of estradiol (E2) on cardiac function following trauma-hemorrhage (T-H) are mediated via estrogen receptor (ER). In this study we tested the hypothesis that ER-mediated cardioprotection is induced via up-regulation of PGC-1␣ through PPAR␣ or Tfam-dependent pathway. Male rats underwent T-H and received ER␣ agonist propylpyrazole-triol (PPT), ER agonist diarylpropionitrile (DPN), E2, or vehicle. Another group was treated with antisense PGC-1␣ oligonucleotides prior to administration of DPN. E2 and DPN treatments attenuated the decrease in cardiac mitochondrial ATP, abrogated the T-H-induced lipid accumulation, and normalized PGC1␣, PPAR␣, FAT/CD36, MCAD, Tfam, and COX I after T-H. In contrast, PPT administration did not abrogate lipid accumulation. Moreover, in PPT-treated animals mitochondrial ATP remained significantly lower than those observed in DPN- or E2-treated animals. Prior administration of antisense PGC-1␣ prevented DPN-mediated cardioprotection and increase in ATP levels and Tfam but not in PPAR␣ following T-H. These findings suggest that the salutary effects of E2 on cardiac function following T-H are mediated via ER up-regulation of PGC-1␣ through Tfam-dependent pathway.—Hsieh, Y.-C., Choudhry, M. A., Yu, H.-P., Shimizu, T., Yang, S., Suzuki, T., Chen, J., Bland, K. I., Chaudry, I. H. Inhibition of cardiac PGC-1␣ expression abolishes ER agonist-mediated cardioprotection following trauma-hemorrhage. FASEB J. 20, 1109 –1117 (2006)
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E2-mediated restoration of cardiac function following trauma-hemorrhage is due in part to ER-dependent up-regulation of PGC-1␣ (peroxisome proliferator-activated receptor (PPAR)-␥ coactivator-1␣) (24). The nuclear coactivator PGC-1␣, known for its role in cellular energy metabolism, regulates a number of genes required for lipid metabolism and ATP production by activating transcription factor PPAR␣ and mitochondrial transcription factor A (Tfam), respectively (24 – 28). It is well known that lipids produce ATP through mitochondrial fatty acid (FA) -oxidation. The PPAR␣ regulates genes involved in lipid transport and mitochondrial FA -oxidation, including FAT/CD36, and medium chain acyl-coenzyme A dehydrogenase (MCAD) (29 –31). The mitochondrial transcription factor, Tfam, transactivates mitochondrial DNA (mtDNA)encoded gene cytochrome c oxidase subunit I (COX I) that is required for mitochondrial ATP production (24, 32). Although our recent study has shown that the salutary effect of E2 on cardiac function following T-H is mediated via ER, it remains unknown whether the effect of ER on cardiac function following T-H is mediated via up-regulation of PGC-1␣ and whether its downstream effect is through PPAR␣ or Tfam-dependent pathway. To test this paradigm, rats that received PPT (ER␣ agonist), DPN (ER agonist), or E2 following trauma-hemorrhage were examined for cardiac PGC-1␣, mitochondrial ATP contents, lipid accumulation, PPAR␣, Tfam, FAT/CD36, MCAD, and COX I protein levels. Moreover, a group of DPN-treated trauma-hemorrhage rats was pretreated with antisense PGC-1␣ oligonucleotides to determine whether inhibition of PGC-1␣ expression prevents DPN-mediated attenuation of cardiac dysfunction and increases in mitochondrial ATP levels, Tfam, and PPAR␣ expressions.
MATERIALS AND METHODS Rat trauma-hemorrhagic shock model Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were fasted overnight but were allowed free access to water before the experiments. Trauma-hemorrhage and resuscitation was then carried out as described previously (10, 12, 13, 24). Briefly, rats were anesthetized by isoflurane inhalation and a 5-cm midline laparotomy performed to induce soft-tissue trauma. The abdominal wound was then closed in layers and polyethylene (PE-50, Becton Dickinson & Co., Sparks, MD) catheters were placed in both femoral arteries and the right femoral vein and subsequently tunneled through to the dorsal surgical and the incision sites closed. The rats were then placed into a Plexiglas box (21⫻9⫻5 cm) in a prone position and allowed to awaken after which they were bled rapidly within 10 min to a mean arterial pressure (MAP) of 35– 40 mmHg. Hypotension was maintained until the animals could no longer maintain a MAP of 35 mmHg unless some fluid in the form of Ringer’s lactate solution was administered. This time was defined as maximal bleed-out (MBO). Following the MBO, MAP was maintained between 35 and 40 mmHg until 40% of the MBO vol was returned in the form of Ringer’s lactate solution (⬃90 min from the onset 1110
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of bleeding). The rats were then resuscitated with four times the vol of MBO with Ringer’s lactate over 60 min. Following resuscitation, the catheters were removed and the wounds were closed. Sham-operated animals underwent the surgical procedures except that neither hemorrhage nor resuscitation was carried out. The animals were sacrificed at 24 h after the end of resuscitation or sham operation. In the treatment group, ER␣ agonist propylpyrazole-triol (PPT; 5 g/kg), ER agonist diarylpropionitrile (DPN; 5 g/kg) (Tocris Cookson, Ballwin, MO), E2 (50 g/kg) (Sigma, Saint Louis, MO), or vehicle was administered subcutaneously (s.c.) at the middle of resuscitation. In another group of trauma-hemorrhage rats treated with DPN, sense (5⬘-TCA GGA GCT GGA TGG C-3⬘) or antisense (5⬘-GCC ATC CAG CTC CTG A-3⬘) PGC-1␣ phosphothionate-modified oligonucleotides (20 nmoles/kg BW) (Invitrogen, Carlsbad, CA) was given intraperitoneally (i.p.) with 2 doses (24 h and 4 h before the traumahemorrhage). These oligonucleotides have been tested in previous studies and the antisense was capable of reducing the expression of PGC-1␣ by ⬃60% in pancreatic islets and ⬃70% in skeletal muscle (33,34). All the experiments were carried out in adherence to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Western blot analysis The heart tissues were homogenized in lysis buffer comprised of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.5 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 1% Triton X-100, 0.5% Nonidet P-40, and 1 g/mL of aprotinin. Tissue lysates were centrifuged at 17,000 g for 10 min. An aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC Protein Assay, Bio-Rad Laboratories, Hercules, CA). Protein aliquots were mixed with 4x lithium dodecyl sulfate sample buffer and were electrophoresed on 4 –12% SDS-polyacrylamide gels and transferred electrophoretically onto nitrocellulose paper. The membranes were immunoblotted with PGC-1␣, FAT/CD36, PPAR␣, Tfam (Santa Cruz Biotechnology, Santa Cruz, CA), MCAD (Abcam Inc, Cambridge, MA), COX I (Molecular Probes, Eugene, OR), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Abcam Inc) antibodies. This was followed by the addition of horseradish peroxidase-conjugated secondary antibody (Ab). After the final wash, membranes were probed using enhanced chemiluminescence (Amersham, Piscataway, NJ) and autoradiographed. GAPDH was used as a loading control. Isolation of mitochondria Following sacrifice, the hearts were removed, minced and homogenized gently in 0.25 mol/L sucrose, 0.5 mmol/L EGTA, HEPES 3 mmol/L, pH 7.2 at the ratio of 10 ml/g of heart using a motor-driven grinder. The suspension was centrifuged at 800 g for 10 min at 4°C. The supernatant was centrifuged twice at 9500 g for 10 min and the pellet fraction enriched mitochondria was collected and stored at ⫺70°C for measurements of ATP levels. Mitochondrial ATP levels determination ATP content was measured by the ATP Bioluminescence Assay kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, the mitochondrial pellet was suspended in lysis reagent supplied in the kit. The suspensions were pipetted, vortexed, and the protein concen-
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isopropanol, and stained with 0.5% Oil Red O for 60 min; sections were then rinsed briefly again as above, and counterstained with in Gill’s hematoxylin for 1 min. Finally, sections were washed in water, then mounted in Aquamount. Determination of cardiac function At 24 h after trauma-hemorrhage or sham operation, the animals were anesthetized with pentobarbital sodium (30 –50 mg/kg i.p.). Cardiac output (CO) was determined using the indocyanine green (ICG) dilution technique (35). A 2.4-Fr fiber-optic catheter (Hospex Fiberoptics, Chestnut Hill, MA) was inserted into the concentration of aortic arch for continuous measurement of ICG concentration by using an in vivo hemoreflectometer (Schwarzer-Picker International, Munich, Germany). A silicone rubber catheter was inserted into the
Figure 1. Effects of PPT (ER␣ agonist), DPN (ER agonist) and 17-estradiol (E2) on protein levels of peroxisome proliferator-activated receptor (PPAR␥) coactivator-1␣ (PGC1␣) in heart after sham operation or trauma-hemorrhage (T-H). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values pooled from 6 animals in each group and are shown as mean ⫾ sem in the bar graph. Data are compared by one-way ANOVA and Tukey’s test. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2. trations measured (Bio-Rad Laboratories, Hercules, CA). The samples were kept on ice until measurements were performed. For determination of ATP, 100 l of luciferase reagent was added to the standards or 1 mg of mitochondrial proteins and the measurement performed with a Luminometer AutoLumat LB953 (Berthold, Wildbad, Germany). Electrophoresis mobility shift assay (EMSA) A double-stranded oligonucleotide probe containing a tandem repeat of the consensus sequencing binding site for PPRE (5⬘- CAA AAC TAG GTC AAA GGT CA-3⬘) was radiolabeled with [␥-32P] dCTP and purified in a sephadex MicroSpin G-25 column (Amersham, Piscataway, NY). The gel shift reaction typically contained 5 g of nuclear extract, 10% (v/v) glycerol, 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 1 g poly(dI-dC), and 5–20 ⫻ 104 cpm of 32P-labeled consensus oligonucleotide with a total vol of 20 l. The reaction was incubated for 30 min at room temperature and separated on 6% nondenaturing polyacrylamide gels in 0.5⫻ TBE buffer (45 mM Tris borate, pH 8.0, 1 mM EDTA). To characterize the binding complexes of PPAR␣, reactions were preincubated for 45 min at room temperature with Ab-recognizing PPAR␣ (Santa Cruz). Competition was performed by addition of 80-fold excess nonradioactive double-stranded oligonucleotide competitors at the same time radioactive probes were introduced. Oil red O staining Heart tissue was sectioned using a cryostat. Ten-m frozen sections were stained with Oil Red O to demonstrate lipid deposition. Sections were rinsed in water, and then 60% ER UP-REGULATES PGC-1␣ AFTER TRAUMA-HEMORRHAGE
Figure 2. Effects of PPT (ER␣ agonist), DPN (ER agonist), and 17-estradiol (E2) on (A) cardiac mitochondrial ATP contents and (B) lipid accumulation in cardiomyocytes after sham operation or T-H. Values are means ⫾ sem of 6 animals in each group. Data are compared by one-way ANOVA and Tukey’s test. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2. 1111
right atrium. ICG (50 l, 1 mg/ml) was injected via the right jugular vein catheter. The concentration of ICG was recorded using a computer-assisted data acquisition program (Asystant⫹, Asyst Software, Rochester, NY). After the measurement of CO, the right carotid artery was recannulated with PE-50 tubing, and after measurement of BP, the catheter was advanced into the left ventricle and connected to a heart performance analyzer (DigiMed) to monitor and record positive and negative first derivatives of pressure (⫾dP/ dtmax). CO and stroke vol (SV) were calculated according to standard equations. Statistical analysis All data are presented as mean ⫾ se. One-way ANOVA and Tukey’s test were employed for the comparison among groups, and differences were considered significant at P ⬍ 0.05.
RESULTS PGC-1␣ protein levels As shown in Fig. 1, PGC-1␣ decreased significantly (P⬍0.05) in rats receiving vehicle- or PPT- (ER␣ ago-
nist) following trauma-hemorrhage compared to the sham-operated group. DPN (ER agonist) as well as E2 administration following trauma-hemorrhage, however, normalized PGC-1␣ protein levels to sham values. Mitochondrial ATP levels and lipid deposits in cardiomyocytes In order to evaluate the PGC-1 downstream effects, we measured mitochondrial ATP levels and lipid deposits in cardiomyocytes. The results show that mitochondrial ATP levels decreased significantly (P⬍0.05) in vehicleor PPT-treated trauma-hemorrhage group compared to sham-operated group (Fig. 2A). DPN as well as E2 administration following trauma-hemorrhage, however, attenuated the decrease in mitochondrial ATP levels. Moreover, Oil red O staining was performed to observe lipid deposits. The result showed abundant intracytoplasmic microdroplets of lipid deposited in cardiomyocytes in vehicle-treated trauma-hemorrhage group (Fig. 2B). DPN or E2 administration following trauma-hemorrhage, however, abrogated the traumahemorrhage-induced lipid accumulation in cardiomyo-
Figure 3. Effects of PPT (ER␣ agonist), DPN (ER agonist), and 17-estradiol (E2) on (A) peroxisome proliferator-activated receptor (PPAR)␣ protein levels, (B) PPAR␣ DNA-binding activity, (C) FAT/CD36 protein levels, and (D) medium chain acyl-coenzyme A dehydrogenase (MCAD) in heart after sham operation or trauma-hemorrhage (T-H). Values are mean ⫾ sem of 6 animals in each group. Data are compared by one-way ANOVA and Tukey’s test. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2. 1112
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Fat/CD36 and MCAD (Figs. 3C and 3D) protein levels decreased significantly (P⬍0.05) in vehicle-treated rats following trauma-hemorrhage compared to the shamoperated group. DPN or E2 administration following trauma-hemorrhage, however, increased the PPAR␣ protein levels and DNA-binding activity as well as Fat/CD36 and MCAD protein levels. Although PPT administration following trauma-hemorrhage also increased PPAR␣, CD36, and MCAD levels, the levels of these intermediates were significantly lower than those observed after DPN treatment. Tfam and its target, mitochondrial COX I protein levels Tfam and mitochondrial COX I are key regulators for mitochondrial ATP production. The results showed
Figure 4. Effects of PPT (ER␣ agonist), DPN (ER agonist), and 17-estradiol (E2) on (A) mitochondrial transcription factor A (Tfam) and (B) mitochondrial cytochrome c oxidase subunit I (COX I) protein levels in heart after sham operation or trauma-hemorrhage (T-H). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values pooled from 6 animals in each group and are shown as mean ⫾ sem in the bar graph. Data are compared by one-way ANOVA and Tukey’s test. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2.
cytes. In contrast, treatment of rats with PPT following trauma-hemorrhage did not completely abolish the lipid accumulation. Moreover, mitochondrial ATP levels also remained significantly lower than those observed in DPN- or E2-treated animals following trauma-hemorrhage. PPAR␣ and its targets, fat/CD36 and MCAD protein levels PPAR␣, Fat/CD36, and MCAD are involved in lipid metabolism, which is the major source of ATP production. The result showed that PPAR␣ protein levels and DNA-binding activity (Figs. 3A and 3B) as well as ER UP-REGULATES PGC-1␣ AFTER TRAUMA-HEMORRHAGE
Figure 5. Effect of treatment with antisense PGC-1␣ oligonucleotide on cardiac PGC-1␣ protein levels in (A) sham operation and (B) DPN (ER agonist)-treated trauma-hemorrhage (T-H) rats. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values pooled from 6 animals in each group and are shown as mean ⫾ sem in the bar graph. Data are compared by one-way ANOVA and Tukey’s test. Shamoperated and DPN-treated T-H groups pretreated with either sense (S) or antisense (AS) PGC-1␣ oligonucleotide. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2. 1113
levels were not affected by prior administration of antisense PGC-1␣ olignucleotide in DPN-treated trauma-hemorrhage rats (Fig. 6). Suppression of PGC-1␣ protein expression abolishes DPN-mediated attenuation of cardiac function
Figure 6. Effect of inhibition of PGC-1␣ expression on cardiac peroxisome proliferator-activated receptor (PPAR)␣ protein levels by treatment with antisense PGC-1␣ oligonucleotide in DPN (ER agonist)-treated T-H rats. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values pooled from 6 animals in each group and are shown as mean ⫾ sem in the bar graph. Data are compared by one-way ANOVA and Tukey’s test. Sham-operated and DPN-treated T-H groups pretreated with either sense (S) or antisense (AS) PGC-1␣ oligonucleotide. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2.
Cardiac output (CO), stroke vol (SV), ⫹dP/dtmax, and ⫺dP/dtmax were significantly decreased (P⬍0.05) following trauma-hemorrhage in vehicle-treated rats compared to sham-operated animals (Fig. 8). Administration of DPN following trauma-hemorrhage restored all of the above parameters except ⫺dP/dtmax to levels comparable to those of the sham-operated group. Prior administration of antisense PGC-1␣ oligonucleotide, however, prevented DPN-mediated restoration of CO, SV, ⫹dP/dtmax, and ⫺dP/dtmax following trauma-hemorrhage compared to administration of sense olignucleotide.
that Tfam (Fig. 4A) and COX I (Fig. 4B) protein levels decreased significantly (P⬍0.05) in vehicle- and PPTtreated rats following trauma-hemorrhage compared to the sham-operated group. DPN as well as E2 administration following trauma-hemorrhage, however, normalized the Tfam and COX I protein levels to sham values. Suppression of PGC-1␣ protein levels abolished DPN-upregulated Tfam and ATP levels but not PPAR␣ To evaluate whether DPN-mediated salutary effects on cardiac function following trauma-hemorrhage are via up-regulation of PGC-1␣ and whether PGC-1␣ downstream effects are regulated through PPAR␣ or Tfamdependent pathway, a group of DPN-treated traumahemorrhage rats was injected with 2 doses (24 h and 4 h before the trauma-hemorrhage) of phosphorothioatemodified sense or antisense oligonucleotide specific for PGC-1␣. As shown in Fig. 5A, administration of antisense PGC-1␣ olignucleotide decreased cardiac PGC-1␣ protein levels in sham-operated rats by 53% (P⬍0.05) compared to sham-operated rats treated with sense olignucleotide. The DPN-mediated up-regulation of PGC-1␣ expression following trauma-hemorrhage was also abolished by administration of antisense PGC-1␣ olignucleotide prior to trauma-hemorrhage (Fig. 5B). Moreover, administration of antisense PGC-1␣ oligonucleotide abrogated DPN-mediated up-regulation in Tfam protein levels and mitochondrial ATP levels (Figs. 7A and 7B; P⬍0.05). However, PPAR␣ protein 1114
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Figure 7. Effect of inhibition of PGC-1␣ expression on cardiac (A) mitochondrial transcription factor A (Tfam) protein levels and (B) mitochondrial ATP contents by treatment with antisense PGC-1␣ oligonucleotide in DPN (ER agonist)-treated T-H rats. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values pooled from 6 animals in each group and are shown as mean ⫾ sem in the bar graph. Data are compared by one-way ANOVA and Tukey’s test. Sham-operated and DPN-treated T-H groups pretreated with either sense (S) or antisense (AS) PGC-1␣ oligonucleotide. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN and T-H-E2.
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Figure 8. Effect of inhibition of PGC-1␣ protein levels on cardiac output (CO, A), stroke vol (SV, B), positive dP/dtmax (C), and negative dP/dtmax (D) by treatment with antisense PGC-1␣ oligonucleotide in DPN (ER agonist)-treated T-H rats. Values are mean ⫾ sem of 6 animals in each group. The results were compared by one-way ANOVA and Tukey’s test. Sham-operated and DPN-treated T-H groups pretreated with either sense (S) or antisense (AS) PGC-1␣ oligonucleotide. *P ⬍ 0.05 vs. sham; #P ⬍ 0.05 vs. T-H-DPN-Sense.
DISCUSSION The findings of this study reveal that the administration of E2 as well as DPN (ER agonist) but not PPT (ER␣ agonist) following trauma-hemorrhage normalized cardiac PGC-1␣ levels, attenuated the decrease in cardiac mitochondrial ATP levels, abrogated the trauma-hemorrhage-induced lipid accumulation in cardiomyocytes, and normalized PGC-1␣ downstream effects PPAR␣, FAT/CD36, MCAD, Tfam, and COX I levels. However, inhibition of PGC-1␣ expression in DPN-treated trauma-hemorrhage rats by antisense oligonucleotide abolished the DPN-mediated cardioprotection and the increase in Tfam expressions. PPAR␣, on the other hand, was not affected in antisense oligonucleotidetreated trauma-hemorrhage rats. We further found that administration of ER␣ agonist PPT following traumahemorrhage also increased PPAR␣, FAT/CD36, and MCAD levels, but the levels of these intermediates were significantly lower than those observed following administration of DPN or E2. Moreover, PPT-treated trauma-hemorrhage rats did not completely abrogate cardiomyocytes lipid accumulation and the mitochondrial ATP levels also remained significantly lower than those observed in DPN- or E2-treated trauma-hemorER UP-REGULATES PGC-1␣ AFTER TRAUMA-HEMORRHAGE
rhage animals. These findings therefore suggest that the salutary effects of E2 on cardiac function following trauma-hemorrhage are mediated via ER up-regulation of PGC-1␣ through Tfam-dependent pathway. It is well known that different subtypes of ER play different roles in each organ. Recent knockout studies have indicated that ER mediates gender-specific response to ischemia-reperfusion injury (21), development of cardiac hypertrophy (22), and myocardial infarction (23). These findings suggest that ER might play a predominant role in the heart under stress conditions. Indeed, our recent findings indicate that ER plays a major role in mediating the salutary effects of E2 on cardiac function following trauma-hemorrhage (36). We also found that E2 upregulated cardiac PGC-1␣ through ER following trauma-hemorrhage (24). In this study, we explored whether the beneficial effect of ER on cardiac function following traumahemorrhage is mediated via the PGC-1␣ and whether PGC-1␣ downstream effect is through PPAR␣ or Tfamdependent pathway. The nuclear coactivator PGC-1␣ plays an important role in regulating cellular lipid storage and ATP production via activating transcription factor PPAR␣ and Tfam (24 –26). The PPAR␣ regulates FAT/CD36 and MCAD expressions, both of which are key enzymes involved in lipid metabolism (29, 30). Tfam, an essential factor for mtDNA transcription and replication, transactivates mtDNA-encoded gene COX I which is required for mitochondrial oxidative phosphorylation and ATP production (24, 32). In this study, we found that although administration of PPT following traumahemorrhage increased PPAR␣, FAT/CD36, and MCAD levels, the levels of these intermediates remained significantly lower than those observed after DPN or E2 treatment. Moreover, administration of PPT did not completely abrogate the trauma-hemorrhage-induced lipid accumulation in cardiomyocytes and did not restore mitochondrial ATP levels, suggesting that the levels of intermediates such PPAR␣, CD36, and MCAD are not sufficient for lipid metabolism or to produce sufficient ATP for improving cardiac function in PPTtreated trauma-hemorrhage rats. On the other hand, administration of E2 as well as DPN following traumahemorrhage upregulated cardiac PGC-1␣ protein levels, attenuated the decrease in cardiac mitochondria ATP levels, and abolished the trauma-hemorrhageinduced lipid accumulation in cardiomyocytes as well as normalized PGC-1␣ downstream signaling molecules PPAR␣, CD36, MCAD, Tfam, and COX I. These results indicate that administration of ER agonist and E2 following trauma-hemorrhage are equally effective in restoring the above parameters following trauma-hemorrhage. Moreover, the findings that ER agonist DPN and E2 up-regulates PGC-1␣ and PGC-1␣ downstream signaling molecules following trauma-hemorrhage suggests that up-regulation of PGC-1␣ is responsible for the salutary effect of ER on cardioprotection following trauma-hemorrhage. To evaluate the role of PGC-1␣ in ER agonist 1115
DPN-mediated attenuation of cardiac function following trauma-hemorrhage, we utilized an antisense PGC-1␣ oligonucleotide that produces a significant reduction of cardiac PGC-1␣ expression. This oligonucleotide has been utilized in other studies and it produces reproducible and effective inhibition of PGC-1␣ expression (33, 34, 37). In this study, we observed that administration of antisense PGC-1␣ oligonucleotide in rats suppressed cardiac PGC-1␣ protein levels by 52% in sham-operated group. However, inhibition of PGC-1␣ expression in DPN-treated trauma-hemorrhage rats by antisense oligonucleotide treatment abolished the DPN-mediated cardioprotection. These results suggest that PGC-1␣ plays a major role in ER-mediated cardioprotection following trauma-hemorrhage. More specifically, our study also indicated that the suppression of PGC-1␣ in DPNtreated trauma-hemorrhage rats abolished DPN-increased Tfam and mitochondrial ATP levels without affecting PPAR␣ levels after inhibition of PGC-1␣ in PPN-treated DPN rats following trauma-hemorrhage. This suggests that the protective effect of PGC-1␣ on cardiac function in DPN-treated trauma-hemorrhage rats is through Tfam but not through PPAR␣ pathway. Studies have reported that PPAR␣ can be regulated by estrogen receptor-related receptor (ERR) (38). Therefore, it can be suggested that DPN-mediated PPAR␣ following trauma-hemorrhage is due to ERR rather than PGC-1␣. However, the precise mechanism by which DPN up-regulates PPAR␣ pathway remains to be determined. It has been reported that PGC-1␣ expression can be induced by transcription factor CREB (cyclic-AMP response element binding). PGC-1␣ mRNA levels were reduced in the CREB knockout mice and sequence analysis of the mouse PGC-1␣ promoter revealed a full consensus CREB binding site (26, 39). Studies have also shown that E2 increased the enhancer activity of CREB binding and CREB protein levels (40, 41). Moreover, the effects of E2 are through both ER␣ and ER to increase CREB phosphorylation (42). Furthermore, ER antagonist ICI 182,780 was able to completely block the increase in CREB phosphorylation induced by E2 in hippocampal cell line (42). However, it remains to be determined whether ER-mediated-PGC-1␣ up-regulation following trauma-hemorrhage is through CREB phosphorylation. In summary, E2 as well as DPN, but not PPT administration following trauma-hemorrhage attenuated the decrease in mitochondrial ATP levels and abrogated the trauma-hemorrhage-induced lipid accumulation in cardiomyocytes. E2 and DPN administration following trauma-hemorrhage also normalized cardiac PGC-1␣, PPAR␣, CD36, Tfam, and COX I levels under those conditions. Moreover, the findings that pretreatment of trauma-hemorrhage rats with antisense PGC-1␣ oligonucleotide prevented DPN-mediated cardioprotection and abolished the increases in mitochondrial ATP levels as well as Tfam but not in PPAR␣ suggest that the salutary effects of E2 on cardiac function following trauma-hemorrhage are mediated via ER up-regula1116
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tion of PGC-1␣ through Tfam but not PPAR␣-dependent pathway. This work was supported by NIH grant R37 GM-39519.
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