AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H687-H693, 2006. First published March 31, 2006; doi:10.1152/ajpheart.00926.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/H687    most recent
00926.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Molavi, B.
Right arrow Articles by Mehta, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Molavi, B.
Right arrow Articles by Mehta, J. L.

Cardioprotective effects of rosiglitazone are associated with selective overexpression of type 2 angiotensin receptors and inhibition of p42/44 MAPK

Behzad Molavi, Jiawei Chen, and J. L. Mehta

Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas

Submitted 21 August 2005 ; accepted in final form 24 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Current evidence points to renin-angiotensin system as a key mediator in ischemia-reperfusion injury. Rosiglitazone, a peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) ligand, has recently been shown to confer cardioprotection against ischemia-reperfusion in animal models. We sought to examine the expression of ANG II receptors during PPAR-{gamma}-mediated cardioprotection. Male Sprague-Dawley rats (nondiabetic) were fed either regular rat chow (control diet group, n = 9) or rosiglitazone-rich diet (rosiglitazone-rich diet group, n = 9) and were subjected to 1 h of myocardial ischemia followed by 1 h of reperfusion. A third group of rats had only thoracotomy and pericardiotomy and served as a sham control group (n = 9). Hemodynamics, infarct size, and expression of ANG II type 1 and type 2 receptors (AT1 and AT2) were measured in all groups. There was a 58% reduction of infarct size in the rosiglitazone-rich diet group (P < 0.01 vs. control diet group). Increased myocardial expression of AT1 receptors in the ischemic-reperfused myocardium was attenuated in the rosiglitazone-rich diet group (P < 0.05 vs. control diet group). Importantly, myocardial AT2 mRNA and protein expression were significantly increased (by >100-fold) in the rosiglitazone-rich diet group (P < 0.05). These changes were accompanied by inhibition of p42/44 MAPK in the rosiglitazone-rich diet group, while the Akt1 expression, believed to mediate insulin sensitization, remained similar in all three groups. The cardioprotective effects of rosiglitazone against myocardial ischemia-reperfusion injury are independent of its insulin-sensitizing properties and are associated with significant overexpression of AT2 receptors along with inhibition of p42/44 MAPK.

peroxisome proliferator-activated-{gamma} receptors; angiotensin receptors; ischemia-reperfusion injury; rosiglitazone

THE IMPLEMENTATION of immediate reperfusion strategies to salvage the ischemic myocardium has reduced mortality and morbidity in the setting of acute coronary syndromes (8) but has also brought the phenomenon of ischemia-reperfusion injury into focus. This phenomenon can present clinically as acute congestive heart failure or malignant ventricular arrhythmias after successful revascularization.

Previous studies have shown that ANG II type 1 (AT1) receptors are overexpressed during ischemia-reperfusion and exert deleterious effects on myocardial contractility (37, 38). Although a few studies have failed to show any cardioprotective effects by angiotensin receptor blockers, the bulk of current evidence suggests that AT1 receptor blockade is associated with cardioprotection (28, 33). In addition, AT1 receptor blockade during ischemia-reperfusion injury in rat hearts is associated with cardioprotection, increased AT2 expression and signaling through inositol 1,4,5-trisphosphate (IP3) pathway activation (22). The IP3 pathway has also been described to be a component of insulin signaling and glucose utilization in the cardiomyocytes (11). In fact, some studies indicate that ANG II receptor activation leads to insulin resistance in vasculature through IP3 pathway inhibition (9, 18, 31). In addition, recent clinical trials of renin-angiotensin-aldosterone system (RAAS) modulation in coronary artery disease and heart failure suggest a significant reduction (14–30%) in incidence of diabetes with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (26, 27, 41). Therefore, a cross-talk between RAAS and glucose utilization pathways appears to exist.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family (2, 29). There are three categories of PPARs: {alpha}, {gamma}, and {delta} (beta) (17). The cardioprotective effects of PPAR-{gamma} agonists, which are currently used to treat Type 2 diabetes, against ischemia-reperfusion injury have been recently demonstrated in diabetic and nondiabetic animals (40, 42). We sought to explore the molecular aspects of the cardioprotective effects of PPAR-{gamma} ligand activation against ischemia-reperfusion injury with particular regard to changes in ANG II receptor expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ischemia-Reperfusion Experiments

Male Sprague-Dawley rats (nondiabetic) were fed either regular rat diet (control diet group, n = 9) or the PPAR-{gamma} ligand rosiglitazone-rich diet (rosiglitazone, 3 mg·kg–1·day–1 for 7 days; n = 9) and were subjected to ischemia-reperfusion. Rosiglitazone is a highly selective PPAR-{gamma} agonist and has been shown to increase PPAR-{gamma} transcriptional activity in a dose-dependent manner by up to 35-fold (1, 15). A third group of rats was subjected to only thoracotomy and pericardiotomy and served as a sham control group (n = 9). The study protocol was reviewed and approved by the animal care and use committee at the Central Arkansas Veterans Healthcare System. The methodology for the induction of ischemia-reperfusion injury has been described earlier (5). Briefly, rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg), incubated, and mechanically ventilated on a positive-pressure respirator with room air. Body temperature was maintained at 36–37°C with a heating blanket. A small plastic catheter was placed into the left ventricle via the right carotid artery with the other end of the catheter attached to a transducer. The transducer was connected to a physiological probe (Grass 7400, AstroMed) to monitor contractility as measured by positive and negative first derivative of left ventricular pressure (±dP/dt). Arterial pressure was monitored by placing a catheter in the right common iliac artery. Heart rate was recorded by surface ECG. A left thoracotomy was performed via the fifth intercostal space to expose the heart. The main left coronary artery (LCA) was ligated 2–3 mm proximal to the origin of first diagonal branch with a 6-0 silk suture. Induction of ischemia was indicated by elevation of ST segment on ECG and cyanosis of anterior wall of the left ventricle. Hearts of rats on both the control diet and the rosiglitazone-rich diet were subjected to 60 min of ischemia followed by 60 min of reperfusion (release of ligature). In the sham control group of rats, thoracotomy and pericardiotomy were performed, and silk thread was passed around LCA without ligation. Blood samples for determination of plasma glucose levels were withdrawn at the time of the procedure in all three groups of rats.

Determination of Infarct Size

At the end of ischemia-reperfusion, the hearts were quickly removed and mounted on a Langendorff apparatus and then flushed with saline for 60 s. The LCA was reoccluded with a snare, and Evans blue dye was infused into the perfusate to mark the area at risk. The heart was then frozen and cut into 2-mm transverse slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in 0.2 M PBS buffer (pH 7.4) for 5 min (32). The area of infarcted tissue (TTC-negative tissue) and the area at risk were determined by planimetry.

Measurement of Plasma Glucose Levels

Plasma glucose levels were quantitatively measured by the glucose oxidase method by using a Beckman Glucose Analyzer-2 machine.

Immunohistochemical Localization of ANG II Receptors

Immunohistochemical characterization of AT1 and AT2 receptors was made in formalin-fixed samples (n = 4) in each group. (Seven to ten discontinuous slides were used in every heart.) A biotin-streptavidin detection system with diaminobenzidine as the chromogen (Santa Cruz Biotechnology, Santa Cruz, CA) was used for immunohistochemical localization of AT1 and AT2 receptors. The primary antibody for AT1 was rabbit polyclonal IgG (200 µg/ml), which recognizes the NH2-terminal extracellular domain and is reactive against rat AT1 receptors (Santa Cruz Biotechnology). The primary antibody for AT2 receptors was a rabbit polyclonal IgG (200 µg/ml), which recognizes the COOH-terminal domain of AT2 receptor (amino acids 221–363) and is reactive against rat AT2 receptors (Santa Cruz Biotechnology).The primary rabbit polyclonal antibodies to AT1 and AT2 were subsequently diluted 1:50. Rabbit serum was used instead of primary antibody for the negative control specimens. A second anti-rabbit antibody was used with the biotin-streptavidin detection system.

Real-Time PCR for ANG II Receptor Gene Expression

RNA extraction and cDNA preparation. The methodology for RNA extraction has been described earlier (6). Briefly, 500 mg of myocardial tissue was homogenized in 300 µl of warmed Ultraspec RNA extraction reagent (Biotecx Laboratories, Houston, TX). The samples were then centrifuged and mixed with chloroform and subsequently isopropyl alcohol to fully precipitate RNA. The samples were subsequently centrifuged at 15,000 rpm, and the resulting pellets were washed twice with 70% ethanol. After brief centrifugation, the remaining fluid was drained, and the pellets were allowed to dry for 5 min. The pellets were subsequently rehydrated with diethyl pyrocarbonate water, and the resultant RNA was quantitated by spectrophotometry and light absorption at 260 nm (A260) and 280 nm (A280) wavelength. The A260/A280 ratio of 1.8–2.0 was considered optimal. A high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) was used to produce cDNA copies of myocardial RNA. In brief, 50 µl of RNA solution was mixed with 50 µl of 2x RT master mix (Applied Biosystems-Foster City, CA) containing 2x RT buffer, 2x dNTP mixture, 2x random primers, multiScribe RT at 50 U/µl, and RNase-free water. The solution was mixed and incubated at 25°C for 10 min and 37°C for 2 h.

RT-PCR for AT1 and AT2 receptors. Applied Biosystems (Foster City, CA) 7700 Sequence Detection System and TaqMan probes were used for real-time PCR (RT-PCR). The AT1 (reference sequence: AK087228 [GenBank] ), AT2 (reference sequence: AK086334 [GenBank] ) and ribosomal 18S (control) primers and TaqMan probe, as well as the TaqMan Universal Master Mix, were purchased form Applied Biosystems. The cDNA templates (9 µl in each tube) were mixed with 10 µl of TaqMan Universal Master Mix and 1 µl of 20x working stock of AT1, AT2, or ribosomal 18S genes, bringing the final volume to 20 µl. The PCR conditions were 50°C for 2 min and 95°C for 10 min repeated for 40 cycles. Thereafter, the data were processed by using the accompanying software, and amplification plots for fluorescence responses were obtained for AT1 and AT2 genes. The ribosomal 18S gene expression was used as the housekeeping gene to normalize the data.

Western Blot Analyses for AT1, AT2, p42/44 MAPK, and Akt1

Monoclonal mouse antibody to p42/44 MAPK was purchased from Cell Signaling Technology (Beverly, MA). This antibody detects endogenous levels of p44 and p42 MAP kinase (ERK1 and ERK2) phosphorylated at threonine 202 and tyrosine 204. This antibody does not cross-react with the corresponding phosphorylated residues of either stress-activated protein kinase/JNK or p38 MAP kinase and therefore selectively binds the activated form of 42/44 MAPK. Monoclonal mouse antibody to Akt1, which recognizes amino acids 345–480 of this kinase, was purchased form Santa Cruz Biotechnology. Monoclonal rabbit antibodies to AT1 and AT2 receptors, which recognize the NH2 terminus and COOH terminus of type 1 and type 2 angiotensin receptors in rats and humans were purchased from Santa Cruz Biotechnology. Equal amounts of protein (50 µg) from the hearts in each group were separated by 10% SDS-PAGE and transferred to nitrocellulose filters (Sigma). After incubating in blocking solution (5% nonfat milk, Sigma), membranes were incubated in a buffer containing 2.0 µg/ml of the antibodies to phosphorylated p42/44 MAPK AT1, AT2, and Akt1. Anti-mouse and anti-rabbit alkaline phosphatase-conjugated antibodies were used as a secondary antibody at 1:5,000 dilutions. Protein bands of interest were detected by chemiluminescence system, and relative intensities of protein bands were analyzed by MSF-300G Scanner, as described earlier (16).

Statistical Analysis

All data are expressed as means ± SD. Means of different groups were compared with ANOVA followed by Student’s t-test for paired and unpaired observations. A P value of <0.05 was considered significant. Morphological and immunohistochemical differences were assessed by three different individuals observing the same slides blindly.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
There was no significant difference in plasma glucose levels at the time of the experiment (nonfasting levels) among different groups of rats [285 ± 125 vs. 315 ± 148 vs. 305 ± 179 mg/dl for sham, control diet, and rosiglitazone-rich diet groups, respectively; P = nonsignificant (NS)]

Hemodynamic Findings

Baseline hemodynamic profiles (mean arterial pressure and ±dP/dt) were similar in the sham control, the control diet group exposed to ischemia-reperfusion, and the rosiglitazone-rich diet group of rats exposed to ischemia-reperfusion (Table 1).


View this table:
[in this window]
[in a new window]
  		Table 1. Hemodynamic data

 
Ischemia caused a decline in mean arterial pressure and ±dP/dt in the control-diet and rosiglitazone-rich diet groups of rats. The ischemia-induced reduction in cardiac function was similar in the rats fed the rosiglitazone-rich diet or the control-diet groups; however, the decline in cardiac hemodynamics during reperfusion was 75% less in the rats fed the rosiglitazone-rich diet (P < 0.01 vs. control-diet fed rats) (Table 1).

Infarct Size

The area of the myocardium at risk was determined by planimetry in five hearts in both the control-diet and rosiglitazone-rich diet groups. The mean areas at risk in both groups were similar (64 ± 4 vs. 61 ± 6% of left ventricle, P = 0.42).

After ischemia-reperfusion, the infarct size was 39 ± 6% of the area at risk in the control-diet group and 16 ± 9% in the rosiglitazone-rich diet group of rats, which reflects a 58% reduction in infarct size in the rosiglitazone-rich diet group compared with the control diet group (P = 0.002) (Fig. 1).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Infarct size in rats on regular chow (control diet) or rosiglitazone (Rosi)-rich diet. Note that the area at risk was similar in both groups. Data (means ± SD) are from 5 rats in each group.

 
Myocardial ANG II Receptor Expression

The expression of ANG II AT1 and AT2 receptors in the sham-control group was low with minimal staining for both receptors. Ischemia-reperfusion was associated with slight upregulation of AT1 receptors in the control-diet group of rats exposed to ischemia-reperfusion, and this upregulation was ameliorated in the rosiglitazone-rich diet group of rats (Fig. 2). More importantly, hearts from the rosiglitazone-rich diet group and exposed to ischemia-reperfusion showed an intense overexpression of myocardial AT2 receptors (vs. hearts from the control diet group) (Fig. 3). The immunohistochemical findings were subsequently verified with Western blotting, indicating suppression of AT1 protein expression with rosiglitazone supplementation in the diet. The AT1 protein expression in the control diet group exposed to ischemia-reperfusion did increase but did not reach a statistically significant different level compared with the sham group. The rosiglitazone-rich diet group also demonstrated a significant increase in AT2 protein expression (Fig. 4).


Figure 2
View larger version (82K):
[in this window]
[in a new window]
  		Fig. 2. Representative examples of AT1 receptor expression in the myocardium (supplied by the left coronary artery) by immunostaining. A: negative control with normal rabbit serum in the sham-control group. B: negative control in the regular-rat chow group. C: negative control in the rosiglitazone-rich diet group. D–F: staining with AT1 antibody in a heart from a sham-control group rat (D), from the ischemic-reperfused region of a rat from the control diet group (E), and from the ischemic-reperfused region from a rosiglitazone-rich group rat (F). Note the increased AT1 receptor expression in the myocardium of the rat given the control diet but not in the myocardium of the rat given rosiglitazone.

 

Figure 3
View larger version (80K):
[in this window]
[in a new window]
  		Fig. 3. Representative examples of AT2 receptor expression in the myocardium (supplied by the left coronary artery) by immunostaining. A: negative control with normal rabbit serum in the sham-control group. B: negative control in the regular-rat chow group. C: negative control in the rosiglitazone-rich diet group. D–F: staining with AT2 antibody in a heart from a sham-control group rat (D), from the ischemic-reperfused region of a rat from the control diet group (E), and from the ischemic-reperfused region from a rosiglitazone-rich group rat (F). Note that the AT2 receptor immunostaining is markedly increased in rosiglitazone-rich group.

 

Figure 4
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4. Myocardial AT2 (A) and AT1 (B) protein expression in the 3 experiment groups (sham control, control diet, and rosiglitazone-rich diet). *P < 0.05.

 
AT1 and AT2 receptor gene expression was determined by RT-PCR. Note that myocardial ischemia-reperfusion caused an eightfold upregulation of the AT1 mRNA in the control diet group of rats, and this effect was attenuated in the rats fed rosiglitazone-rich diet (Fig. 5). In contrast, AT2 receptor mRNA expression was ~100-fold higher in the hearts from rosiglitazone-rich diet group than in those from the control diet group (P = 0.02) (Fig. 6). These data support the observations on immunostaining and Western blotting for AT1 and AT2 receptors.


Figure 5
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 5. AT1 receptor mRNA as determined by real-time PCR. Note a marked increase in AT1 receptor RNA in control diet group of rats subjected to ischemia-reperfusion but not in the rosiglitazone-rich diet group of rats subjected to ischemia-reperfusion. Data based on 3 experiments in each group. The bar graph is summary of data from all 3 experiments.

 

Figure 6
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 6. AT2 receptor mRNA as determined by real-time PCR. Note the marked increase in AT2 receptor RNA in the rats given the rosiglitazone-rich diet and subjected to ischemia-reperfusion. This increase was not observed in the rats given the control diet. Data based on 3 experiments in each group. The bar graph is a summary (±SD) of data from all 3 experiments.

 
Myocardial p42/44 MAPK and Akt1 Expression

Ischemia-reperfusion induced a significant rise in p42/44 MAPK levels in the hearts from the control diet group. The ischemia-reperfusion-induced increase in p42/44 MAPK activity was significantly attenuated in the hearts from rats on the rosiglitazone-rich diet (P < 0.05) (Fig. 7).


Figure 7
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7. Phosphorylated p42/44 MAPK expression. Note the increase in p42/44 MAPK expression after ischemia-reperfusion in the rats fed the control diet but not in the rats given the rosiglitazone-rich diet. Data based on 4 experiments in each group. Top: representative gel. Bottom: the bar graph is a summary of data from all 4 experiments.

 
The Akt1 expression was similar in the hearts from the sham control, control diet, and rosiglitazone-rich diet groups, indicating no significant alterations with ischemia-reperfusion or type of diet (P > 0.5) (Fig. 8).


Figure 8
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. Akt1 expression determined by Western analysis. Note the expression was similar in the hearts from all 3 groups of animals. Data based on 4 experiments in each group. Top: representative gel. Bottom: bar graph is summary of data from all 4 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study confirms the results of previous studies showing the protective effect of PPAR-{gamma} ligand rosiglitazone against myocardial ischemia-reperfusion injury (40). The protective effect was evident by attenuation of decrease in ±dP/dt and reduction in infarct size in the rats given rosiglitazone-rich diet.

Activation of AT1 receptors has been implicated in the pathogenesis of ischemia-reperfusion injury (12, 38). In fact, previous studies have indicated that AT1 receptor antagonists are cardioprotective in models of ischemia-reperfusion injury (20, 24). Likewise, antisense directed at the AT1 receptor mRNA protects the hearts from the adverse effects of ischemia-reperfusion (38). It has been suggested that a part of the beneficial effect of AT1 receptor blockers may be mediated by upregulation of AT2 receptors (14, 35). Matsubara (18a) have reviewed the mechanisms of AT2 receptor-mediated cardioprotection, which involve stimulation of protein tyrosine or serine/threonine phosphatases in a Gi protein-dependent manner. Some studies have indicated that AT2 receptor overexpression may have deleterious effects on left ventricular systolic function and lead to heart failure (36), However, these studies were performed in a chronic setting on transgenic mice that were evaluated by echocardiography at 18 wk and did not involve acute ischemia-reperfusion injury. Yet multiple other studies have confirmed a cardioprotective role for AT2 receptor activation during ischemia-reperfusion and after acute myocardial infarction (3, 39). Diep et al. (7) showed that PPAR-{gamma} ligands are capable of modulating ANG II receptor expression in rats, and the resultant PPAR-{gamma}-mediated vasodilation is in part due to modulation of AT2 receptor expression. We wondered if rosiglitazone-mediated cardioprotection would involve alterations in ANG II receptor expression. Indeed, we observed that rosiglitazone-fed rats had >100-fold increase in AT2 receptor mRNA and a marked reduction in AT1 receptor mRNA. The results of alterations in mRNA expression were also evident on immunostaining and Western blot assays of the reperfused myocardium.

At the molecular level, the deleterious effects of ANG II have been shown to be mediated through AT1 receptors and subsequent downstream activation of stress-responsive MAPKs (23). MAPK activation has been shown to be a pivotal pathway in the setting of myocardial ischemia-reperfusion injury and can exert deleterious effects on myocardial contractile performance (25, 30). The upregulation of AT2 receptors in our study was associated with inhibition of p42/44 MAPK. p42/44 MAPKs are extracellular signal-regulated kinases that mediate a wide spectrum of intracellular signaling pathways, including those initiated by AT1 receptor activation (34). The inhibition of p42/44 MAPK through AT2 receptor can potentially antagonize the deleterious effects of ANG II and AT1 receptor activation on the myocardium during ischemia-reperfusion. This hypothesis is supported by the work of Nakajima and colleagues (23), who found that AT2 receptor overexpression by gene transfer prevented neointimal proliferation in balloon-injured rat carotid arteries. They were also able to demonstrate that the antiproliferative effects of AT2 overexpression were accompanied by a significant reduction of MAPK (23). Inhibition of p42/44 MAPK may relate to AT2 receptor-mediated activation of protein tyrosine phosphatase, which dephosphorylates the threonine 202 and tyrosine 204 residues of p42/44 MAPK (10).

It is noteworthy that we also observed that the AT1 receptor expression was attenuated in rosiglitazone-fed rats. It is unclear from these studies whether the cardioprotective effect of rosiglitazone is primarily due to inhibition of AT1 or upregulation of AT2 receptor expression. The 100-fold increase in AT2 receptor mRNA levels in the rosiglitazone-rich diet group of rats draws attention to the strong association between cardioprotective effects of PPAR-{gamma} ligands and upregulation of AT2 receptors. Yet this study cannot establish a direct causal relationship between upregulation of AT2 receptors and cardioprotection, and further experiments with genetically modified species expressing various levels of angiotensin receptors are needed in this regard. The precise basis of rosiglitazone-mediated upregulation of AT2 and downregulation of AT1 receptors is not known but may be related to a reduction in free radical generation during ischemia-reperfusion. In other studies, we have shown that rosiglitazone decreases P67 NADPH oxidase in ischemic-reperfused hearts (21). In recent studies, we have demonstrated that another PPAR-{gamma} ligand, pioglitazone, decreases superoxide anion generation in endothelial cells exposed to oxidative stress (19). Furthermore, pioglitazone also decreases adhesion of inflammatory cells to the activated endothelial cells, a common denominator in ischemia-reperfused-mediated tissue injury (19). AT1 receptor activation has been shown to activate NADPH oxidase in a variety of tissues (4). We propose that PPAR-{gamma} ligands break the positive feedback between AT1 receptor activation and reactive oxygen species generation. However, it should be noted that the cardioprotective effects of PPAR-{gamma} ligands may also stem from several other simultaneous modulations in signaling mechanisms, including NF-{kappa}B expression, which has been shown to be downregulated by PPAR-{gamma} ligands (21). Nonetheless, the most important information in this study is the dramatic increase in AT2 receptor expression at the transcriptional level in the rats on rosiglitazone-rich diet.

In addition to the modifications in AT1 and AT2 receptor expression, the cardioprotective effects of rosiglitazone in our study could be explained by its glucoregulatory effects. Although insulin sensitization and glucose-insulin infusion during acute myocardial infarction have been a time-honored strategy in myocardial preservation, it is unlikely that the cardioprotective effects of rosiglitazone in this study were related to improved glucose homeostasis. First, the animals in our study were nondiabetic, and there was no significant difference in plasma glucose levels among the sham-control, control diet, and rosiglitazone-rich diet groups. Second, the Akt expression was unaltered in all three groups of animals. Akt1 is an intracellular kinase that has been known to mediate insulin-sensitizing effects of rosiglitazone (13).

In summary, we show that the PPAR-{gamma} ligand rosiglitazone protects the myocardium against reperfusion injury and reduces infarct size in a nondiabetic setting. The cardioprotective effects of rosiglitazone are accompanied by downregulation of AT1 and overexpression of AT2 receptors. The upregulation of AT2 receptors was accompanied by reduced activity of p42/44 MAPK in the rosiglitazone-rich diet group. However, there was no significant change in insulin signaling as evidenced by similar levels of Akt1 in all three groups.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Molavi, Dept. of Internal Medicine, Div. of Cardiovascular Medicine, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., #532, Little Rock, AR 72205 (e-mail: molavibehzad{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adams M, Montague CT, Prins JB, Holder JC, Smith SA, Sanders L, Digby JE, Sewter CP, Lazar MA, Chatterjee VK, and O’Rahilly S. Activators of peroxisome proliferator-activated receptor-{gamma} have depot-specific effects on human preadipocyte differentiation. J Clin Invest 100: 3149–3153, 1997.[Web of Science][Medline]
  2. Bishop-Bailey D. Peroxisome proliferator-activated receptors in the cardiovascular system. Br J Pharmacol 129: 823–834, 2000.[CrossRef][Web of Science][Medline]
  3. Bove CM, Gilson WD, Scott CD, Epstein FH, Yang Z, Dimaria JM, Berr SS, French BA, Bishop SP, and Kramer CM. The angiotensin II type 2 receptor and improved adjacent region function post-MI. J Cardiovasc Magnet Res 7: 459–464, 2005.[CrossRef]
  4. Cai H, Li Z, Dikalov S, Holland SM, Hwang J, Jo H, Dudley SC Jr, and Harrison DG. NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II. J Biol Chem 277: 48311–48317, 2002.[Abstract/Free Full Text]
  5. Chen LY, Nichols WW, Hendricks J, and Mehta JL. Myocardial neutrophil infiltration, lipid peroxidation, and antioxidant activity after coronary artery thrombosis and thrombolysis. Am Heart J 129: 211–218, 1995.[CrossRef][Web of Science][Medline]
  6. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[Web of Science][Medline]
  7. Diep QN, El Mabrouk M, Cohn JS, Endemann D, Amiri F, Virdis A, Neves MF, and Schiffrin EL. Structure, endothelial function, cell growth, and inflammation in blood vessels of angiotensin II-infused rats: role of peroxisome proliferator-activated receptor-{gamma}. Circulation 105: 2296–2302, 2002.[Abstract/Free Full Text]
  8. Fang JM and Alderman MHM. Dissociation of hospitalization and mortality trends for myocardial infarction in the United States from 1988 to 1997. Am J Med 113: 208–214, 2002.[CrossRef][Web of Science][Medline]
  9. Folli F, Kahn CR, Hansen H, Bouchie JL, and Feener EP. Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels. A potential role for serine phosphorylation in insulin/angiotensin II crosstalk. J Clin Invest 100: 2158–2169, 1997.[Web of Science][Medline]
  10. Gallinat S, Busche S, Raizada MK, and Sumners C. The angiotensin II type 2 receptor: an enigma with multiple variations. Am J Physiol Endocrinol Metab 278: E357–E374, 2000.[Abstract/Free Full Text]
  11. Hiraoka M. A novel action of insulin on cardiac membrane. Circ Res 92: 707–709, 2003.[Free Full Text]
  12. Jalowy A, Schulz R, and Heusch G. AT1 receptor blockade in experimental myocardial ischemia/reperfusion. J Am Soc Nephrol 10, Suppl 11: S129–S136, 1999.[Web of Science]
  13. Jiang G, Dallas-Yang Q, Li Z, Szalkowski D, Liu F, Shen X, Wu M, Zhou G, Doebber T, Berger J, Moller DE, and Zhang BB. Potentiation of insulin signaling in tissues of Zucker obese rats after acute and long-term treatment with PPAR{gamma} agonists. Diabetes 51: 2412–2419, 2002.[Abstract/Free Full Text]
  14. Jugdutt BI and Balghith M. Enhanced regional AT2-receptor and PKC{epsilon} expression during cardioprotection induced by AT1-receptor blockade after reperfused myocardial infarction. J Renin-Angiotens-Aldosterone Syst 2: 134–140, 2001.
  15. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, and Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR {gamma}). J Biol Chem 270: 12953–12956, 1995.[Abstract/Free Full Text]
  16. Li DM, Saldeen TM, Romeo FM, and Mehta JLM. Oxidized LDL upregulates angiotensin ii type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-{kappa}B. Circulation 102: 1970–1976, 2000.[Abstract/Free Full Text]
  17. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, and Chambon P. The nuclear receptor superfamily: the second decade. Cell 83: 835–839, 1995.[CrossRef][Web of Science][Medline]
  18. Marrero MB, Fulton D, Stepp D, and Stern DM. Angiotensin II-induced insulin resistance and protein tyrosine phosphatases. Arterioscler Thromb Vasc Biol 24: 2009–2013, 2004.[Abstract/Free Full Text]
  19. Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res 83: 1182–1191, 1998.
  20. Mehta JL, Hu B, Chen J, and Li D. Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation. Arterioscler Thromb Vasc Biol 23: 2203–2208, 2003.[Abstract/Free Full Text]
  21. Miyoshi H, Takayama Y, Kitashiro S, Izuoka T, Saito D, Imuro Y, Mimura J, Yamamoto S, Tokioka M, and Iwasaka T. Influence of angiotensin II type 1-receptor antagonist CV11974 on infarct size and adjacent regional function after ischemia-reperfusion in dogs. Jpn J Pharmacol 89: 120–125, 2002.[CrossRef][Medline]
  22. Molavi B and CHLDMJL. Preservation of left ventricular function following ischemia-reperfusion by PPAR-{gamma} ligands. Circulation 106: 934, 2003.
  23. Moudgil R, Xu Y, Menon V, and Jugdutt BI. Effect of chronic AT1 receptor antagonism on post ischemic functional recovery and AT1/AT2 receptor proteins in isolated working rat hearts. J Cardiovasc Pharmacol Ther 6: 183–188, 2001.[Abstract/Free Full Text]
  24. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, and Dzau VJ. The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci USA 92: 10663–10667, 1995.[Abstract/Free Full Text]
  25. Preckel B, Schlack W, Gonzalez M, Obal D, Barthel H, and Thamer V. Influence of the angiotensin II AT1 receptor antagonist irbesartan on ischemia/reperfusion injury in the dog heart. Basic Res Cardiol 95: 404–412, 2000.[CrossRef][Web of Science][Medline]
  26. Ravingerova T, Barancik M, and Strniskova M. Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology. Mol Cell Biochem 247: 127–138, 2003.[CrossRef][Web of Science][Medline]
  27. Scheen AJ. Prevention of type 2 diabetes mellitus through inhibition of the Renin-Angiotensin system. Drugs 64: 2537–2565, 2004.[CrossRef][Web of Science][Medline]
  28. Scheen AJ. Renin-angiotensin system inhibition prevents type 2 diabetes mellitus. 1. A meta-analysis of randomised clinical trials. Diabetes Metab 30: 487–496, 2004.[Web of Science][Medline]
  29. Shimizu M, Wang QD, Sjoquist PO, and Ryden L. The angiotensin II AT1-receptor antagonist candesartan improves functional recovery and reduces the no-reflow area in reperfused ischemic rat hearts. J Cardiovasc Pharmacol 34: 78–81, 1999.[CrossRef][Web of Science][Medline]
  30. Spiegelman BM. PPAR-{gamma}: adipogenic regulator and thiazolidinedione receptor. Diabetes 47: 507–514, 1998.[Abstract]
  31. Talmor D, Applebaum A, Rudich A, Shapira Y, and Tirosh A. Activation of mitogen-activated protein kinases in human heart during cardiopulmonary bypass. Circ Res 86: 1004–1007, 2000.[Abstract/Free Full Text]
  32. Taniyama Y, Hitomi H, Shah A, Alexander RW, and Griendling KK. Mechanisms of reactive oxygen species-dependent downregulation of insulin receptor substrate-1 by angiotensin II. Arterioscler Thromb Vasc Biol 25: 1142–1147, 2005.[Abstract/Free Full Text]
  33. Vivaldi MT, Kloner RA, and Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol 121: 522–530, 1985.[Abstract]
  34. Wang QD and Sjoquist PO. Effects of the insurmountable angiotensin AT1 receptor antagonist candesartan and the surmountable antagonist losartan on ischemia/reperfusion injury in rat hearts. Eur J Pharmacol 380: 13–21, 1999.[CrossRef][Web of Science][Medline]
  35. Xie Z, Pimental DR, Lohan S, Vasertriger A, Pligavko C, Colucci WS, and Singh K. Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species. J Cell Physiol 188: 132–138, 2001.[CrossRef][Web of Science][Medline]
  36. Xu J, Carretero OA, Liu YH, Shesely EG, Yang F, Kapke A, and Yang XP. Role of AT2 receptors in the cardioprotective effect of AT1 antagonists in mice. Hypertension 40: 244–250, 2002.[Abstract/Free Full Text]
  37. Yan X, Price RL, Nakayama M, Ito K, Schuldt AJ, Manning WJ, Sanbe A, Borg TK, Robbins J, and Lorell BH. Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice. Am J Physiol Heart Circ Physiol 285: H2179–H2187, 2003.[Abstract/Free Full Text]
  38. Yang B, Li D, Phillips MI, Mehta P, and Mehta JL. Myocardial angiotensin II receptor expression and ischemia-reperfusion injury. Vasc Med 3: 121–130, 1998.[Abstract/Free Full Text]
  39. Yang BC, Phillips MI, Zhang YC, Kimura B, Shen LP, Mehta P, and Mehta JL. Critical role of AT1 receptor expression after ischemia/reperfusion in isolated rat hearts: beneficial effect of antisense oligodeoxynucleotides directed at AT1 receptor mRNA. Circ Res 83: 552–559, 1998.[Abstract/Free Full Text]
  40. Yang Z, Bove CM, French BA, Epstein FH, Berr SS, Dimaria JM, Gibson JJ, Carey RM, and Kramer CM. Angiotensin II type 2 receptor overexpression preserves left ventricular function after myocardial infarction. Circulation 106: 106–111, 2002.[Abstract/Free Full Text]
  41. Yue TlP, Chen JM, Bao WM, Narayanan PKP, Bril AP, Jiang WM, Lysko PGP, Gu JLM, Boyce RP, Zimmerman DMM, Hart TKP, Buckingham REP, and Ohlstein EHP. In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-{gamma} agonist rosiglitazone. Circulation 104: 2588–2594, 2001.[Abstract/Free Full Text]
  42. Yusuf SD, Ostergren JBM, Gerstein HCM, Pfeffer MAM, Swedberg KM, Granger CBM, Olofsson BP, Probstfield JM, McMurray JVM, and on behalf of the Candesartan in Heart Failure Assessment of Reduction in Mortality and Morbidity Program (C.HARM) Investigators. Effects of candesartan on the development of a new diagnosis of diabetes mellitus in patients with heart failure. Circulation 112: 48–53, 2005.[Abstract/Free Full Text]
  43. Zhu PM, Lu LM, Xu YM, and Schwartz GGM. Troglitazone improves recovery of left ventricular function after regional ischemia in pigs. Circulation 101: 1165–1171, 2000.[Abstract/Free Full Text]
  44. Zhu YZ, Chimon GN, Zhu YC, Li B, Hu HZ, Yap EH, Lee HS, and Wong PTH. Expression of angiotensin II AT2 receptor in the acute phase of stroke in rats. Neuroreport 11: 1191–1194, 2000.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 CirculationHome page
A. Jones, R. Deb, E. Torsney, F. Howe, M. Dunkley, Y. Gnaneswaran, D. Gaze, H. Nasr, I. M. Loftus, M. M. Thompson, et al.
Rosiglitazone Reduces the Development and Rupture of Experimental Aortic Aneurysms
Circulation, June 23, 2009; 119(24): 3125 - 3132.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
P. Lefebvre, B. Cariou, F. Lien, F. Kuipers, and B. Staels
Role of Bile Acids and Bile Acid Receptors in Metabolic Regulation
Physiol Rev, January 1, 2009; 89(1): 147 - 191.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
I. Kuipers, P. van der Harst, G. Navis, L. van Genne, F. Morello, W. H. van Gilst, D. J. van Veldhuisen, and R. A. de Boer
Nuclear Hormone Receptors as Regulators of the Renin-Angiotensin-Aldosterone System
Hypertension, June 1, 2008; 51(6): 1442 - 1448.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Q. Zhang, F. He, R. Kuruba, X. Gao, A. Wilson, J. Li, T. R. Billiar, B. R. Pitt, W. Xie, and S. Li
FXR-mediated regulation of angiotensin type 2 receptor expression in vascular smooth muscle cells
Cardiovasc Res, February 1, 2008; 77(3): 560 - 569.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
J. D. Brown and J. Plutzky
Peroxisome Proliferator Activated Receptors as Transcriptional Nodal Points and Therapeutic Targets
Circulation, January 30, 2007; 115(4): 518 - 533.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/H687    most recent
00926.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Molavi, B.
Right arrow Articles by Mehta, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Molavi, B.
Right arrow Articles by Mehta, J. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.