Cardiovascular Research

Cardiovascular Research 2004 62(1):135-144; doi:10.1016/j.cardiores.2003.12.027
© 2004 by European Society of Cardiology

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Copyright © 2004, European Society of Cardiology

Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion

Hui-Rong Liu, Ling Tao, Erhe Gao, Bernard L Lopez, Theodore A Christopher, Robert N Willette¹, Eliot H Ohlstein¹, Tian-Li Yue^*,1 and Xin-Liang Ma^*

Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building, Room 241, Philadelphia, PA 19107, USA

^* Corresponding authors. Tel.: +1-215-955-4994; fax: +1-215-503-4458. or: Tel.: +1-610-270-5313; fax: +1-610-270-5080. Email address: tian-li.yue{at}gsk.com xin.ma{at}jefferson.edu

Received 1 October 2003; revised 30 November 2003; accepted 29 December 2003

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objectives: This study examined the effects of diet-induced hypercholesterolemia on the extent of postischemic myocyte apoptosis and elucidated the potential mechanisms involved. Furthermore, the effects of rosiglitazone (RSG) on postischemic myocardial apoptosis in HC rabbits were investigated. Methods: Male New Zealand rabbits were fed normal or high cholesterol diets for 8 weeks. Three weeks after being fed a high cholesterol diet, HC rabbits were randomized to receive vehicle or RSG during the remaining 5 weeks. Rabbits were then subjected to 60 min of coronary occlusion followed by 4 h of reperfusion. Results: Compared with rabbits fed with a normal diet, HC rabbits had increased caspase-3 activity, apoptotic cell death and retarded contractile function recovery after reperfusion. HC increased iNOS expression, total NOx and nitrotyrosine contents, indicating that an increased nitrative stress occurred in HC myocardial tissue. Activity of a hypertrophic/anti-apoptotic mitogen-activated protein kinase (MAPK), ERK1/2, was significantly decreased while activation of a pro-apoptotic MAPK, p38, was increased. Treatment with RSG in HC rabbits attenuated postischemic myocardial nitrative stress, restored a beneficial balance between pro- and anti-apoptotic MAPK signaling, reduced postischemic myocardial apoptosis, and improved cardiac functional recovery. Conclusions: Our results demonstrated that HC increased postischemic myocardial apoptosis likely by increasing the production of pro-apoptotic molecules, activating pro-apoptotic signaling pathways and inhibiting anti-apoptotic signaling. In addition, our results suggest that peroxisome proliferator-activated receptor (PPAR) agonists may not only attenuate the formation of atherosclerosis associated with hypercholesterolemia as previously reported, but may also protect the heart from subsequent ischemic/reperfusion-induced myocardial apoptosis.

KEYWORDS Hypercholesterolemia; Reperfusion injury; Nitric oxide

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Considerable evidence indicates that apoptosis is a significant contributor to myocardial cell death following ischemia and reperfusion [1,2]. However, to date, the vast majority of research on postischemic myocardial apoptosis has utilized healthy animals, and whether the extent of postischemic cardiomyocyte apoptosis varies in different pathophysiological settings remains largely unknown. Hypercholesterolemia and ensuing atherosclerosis have been implicated in the pathophysiology of coronary artery disease and myocardial ischemia (MI) [3]. Elevated cholesterol concentrations may play a role in not only initiating ischemia, but also in propagating ischemic damage. In a recent study, Wang et al. [4] reported for the first time that postischemic myocardial apoptosis is markedly increased in rabbit fed with a 10% cholesterol diet for 8 weeks, suggesting that hypercholesterolemia may aggravate myocardial apoptotic death and thus contribute to increased cardiac vulnerability to ischemia–reperfusion in hypercholesterolemia.

Reactive oxygen species (ROS) have long been known to cause oxidative protein modification and to act as the major mediators of postischemic myocardial injury [5]. However, increased susceptibility of the hypercholesterolemic heart to ischemia and reperfusion injury cannot be exclusively explained by ROS production [6], and many fundamental questions remain unresolved. Recent studies have identified nitric oxide (NO)-derived reactive nitrogen intermediates (collectively known as reactive nitrogen species, RNS) as critical contributors of protein modification and cell injury [7], providing potential targets for therapeutic interventions.

Peroxisome proliferator-activated receptors (PPARs, including PPAR, and ) are transcription factors belonging to the nuclear receptor super family [8]. Emerging evidence indicates that the PPAR signaling pathway plays a critical role in the regulation of a variety of biological processes within the cardiovascular system [9]. Treatment with PPAR agonists such as rosiglitazone (RSG) has been shown to improve cardiovascular function in diabetic patients and diabetic animal models [10]. Moreover, several recent studies have demonstrated that treatment with PPAR agonists, such as RSG, reduces myocardial infarct size in normal animals subjected to regional ischemia and reperfusion [11–14]. However, whether PPAR agonists may still exert cardiac protection in a more clinically relevant model that exhibits more severe heart injury, such as myocardial ischemia and reperfusion in hypercholesterolemic animals, has not been previously studied.

Therefore, the purposes of the present study were to (1) determine whether hypercholesterolemia may exacerbate cardiomyocyte apoptosis in response to ischemia/reperfusion; (2) and if so, to elucidate the contribution of nitrative stress and disturbed pro- and anti-apoptotic signaling to increased vulnerability of myocardial injury to ischemic–reperfusion insult in hypercholesterolemia; and (3) to investigate whether treatment with RSG (a PPAR- agonist) may attenuate the cardiomyocyte apoptosis induced by ischemia/reperfusion in a hypercholesterolemic rabbit model.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Animals and dietary protocol
Adult male New Zealand white rabbits weighing 2.0–2.5 kg were provided with food and water ad libitum. Blood was drawn from the central ear artery of each rabbit, and baseline plasma lipids were determined by GlaxoSmithKline Clinical Laboratories. On day zero of the experiment, rabbits were assigned to either a normal (control, n=10) or high cholesterol (HC, normal rabbit diet supplemented with 1% cholesterol, n=20) diet for 8 weeks. Three weeks after being fed an HC diet, the rabbits' blood was redrawn and plasma lipids were determined. Hypercholesterolemic rabbits were then randomized to receive either vehicle (n=10) or RSG (3 mg/kg/day, oral gavage) during the remaining 5 weeks. All rabbit diets were prepared by and purchased from Zeigler Bros. (Gardners, PA). The experiments were performed in adherence to NIH Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University Committee on Animal Care.

2.2. Experimental preparation
At the end of 8 weeks of an HC diet, rabbits were anaesthetized with sodium pentobarbital (30 mg/kg, iv) and ventilated with a Harvard small animal respirator. A polyethylene catheter was inserted into the right external jugular vein for supplemental pentobarbital injection. The arterial blood pressure was measured via a polyethylene catheter cannulated to the right femoral artery, and the left ventricular pressure (LVP) was measured via a Millar Mikro-Tip catheter pressure transducer inserted into the left ventricular cavity through the left carotid artery. After midline thoracotomy, a 4–0 silk ligature was placed around the major marginal branch of the left circumflex coronary 10–12 mm from its origin. After a 10-min period of stabilization, myocardial ischemia was initiated by complete ligation of the marginal coronary artery. After 60 min of ischemia, the ligature was untied and the ischemic myocardium was reperfused (R) for 4 h. Sham myocardial ischemia and reperfusion rabbits were subjected to the same surgical procedures performed on MI/R rabbits, except that coronary artery was left untied. The heart was quickly excised and ischemic–reperfused tissue (area-at-risk, AAR) was isolated for apoptotic, biochemical assays and Western blots.

2.3. Myocardial functional injury
MI/R-induced cardiac dysfunction was continuously monitored during the entire MI/R period. The arterial blood pressure and LVP were digitally processed via a hemodynamic analyzing system (Po-Ne-Mah Physiology Platform P3 Plus, Gould Instrument Systems). Mean arterial blood pressure (MABP), heart rate (HR), maximal positive and negative values of the instantaneous first derivative of LVP (+dP/dt_max and –dP/dt_max) and cardiac contractile index (CI=+dP/dt_max/p) were derived by computer algorithms.

2.4. Myocardial apoptosis
Myocardial apoptosis was qualitatively analyzed by detection of DNA fragmentation (DNA ladders) and quantitatively analyzed by TUNEL assay as described previously [15] and caspase-3 activity assay. Myocardial tissue caspase-3 activity was performed by using a caspase-3 colorimetric assay kit (Chemicon International, Temecula, CA) following manufacturer's instructions. The activity of caspase-3 in tissue samples was calculated using a standard curve (7.81–500 µM of pNA), and expressed as µmol pNA/mg protein.

2.5. Measurement of ERK1/2 activity and p38 mitogen-activated protein kinase (MAPK) phosphorylation
ERK1/2 (a hypertrophic/anti-apoptotic member of MAPKs) was measured using a non-radioactive MAP kinase activity assay kit (Chemicon International) according to the manufacturer's instruction. Results were expressed as a fold change over the sham MI/R group. p38 MAPK activation (a pro-apoptotic MAPK member) was determined using a phosphorylated p38 MAPK assay kit (Assay Design) according to the manufacturer's instruction. Results were expressed as fold change over sham MI/R group.

2.6. Determination of total NOx contents in cardiac tissue
Cardiac tissue samples from AAR and area-not-at-risk (ANAR) were rinsed, homogenized in deionized water (1:10, wt/vol) and centrifuged at 14,000 x g for 10 min. The tissue NO and its in vivo metabolic products (NO₂ and NO₃) in the supernatant, collectively known as NOx, were determined using a chemiluminescent NO detector (SIEVER 280i NO Analyzer) as described in our previous study [16].

2.7. Quantitation of tissue nitrotyrosine content
Nitrotyrosine content, a footprint of in vivo peroxynitrite (ONOO^–) formation, was determined using an ELISA method described in our recent publication [17]. The results were presented as nanomoles of nitrotyrosine per gram protein.

2.8. Immunohistochemical detection of cardiomyocyte iNOS expression and nitrotyrosine formation
Myocardial tissue was removed and stored in 4% formalin for less than 48 h. Fixed myocardial tissues were dehydrated and embedded in paraffin, and sections were cut at 5 µm and mounted onto glass slides. Immunohistochemical detections of iNOS and nitrotyrosine were performed as described previously [17].

2.9. Statistical analysis
All values in the text, tables and figures are presented as means±S.E.M. of n independent experiments. All data were subjected to ANOVA followed by Bonferroni correction for post hoc t test. Probabilities of 0.05 or less were considered to be statistically significant.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. RSG had no effects on plasma lipid profiles after a high cholesterol diet
There were no significant differences between groups in plasma lipid profiles before the onset of the study (time 0) or after 3 weeks of a high cholesterol diet (before RSG treatment) (Table 1). After an additional 5 weeks of a high cholesterol diet during which the rabbits were treated with either vehicle or RSG, plasma cholesterol (CHOL), triglyceride (TRIG), HDL and LDL levels further increased. However, there was no significant difference between the vehicle and RSG-treated groups. These results indicate that RSG failed to improve plasma lipid profiles in this diet-induced hypercholesterolemic model, and therefore, any anti-apoptotic effects observed could not be attributed to this mechanism.

View this table: [in this window] [in a new window]   Table 1 Lipid profiles in vehicle-treated and RSG-treated groups

 
3.2. Hypercholesterolemia increases postischemic myocardial injury and worsens cardiac functional recovery
It is now well accepted that DNA ladder formation is highly specific for apoptotic cell death, but lacks sensitivity and is difficult to quantify. In contrast, TUNEL staining of nuclei is extremely sensitive but is less specific for apoptosis as some necrotic cells may stain positive. These two methods were thus used in combination to improve accuracy and reliability of our results. In myocardial tissue from sham MI hearts, no DNA ladder was detected. In contrast, the formation of DNA nucleosome ladders was clearly detected in myocardial tissues obtained from ischemic reperfused hearts. Most importantly, in myocardial tissue from HC rabbits, the ladder formation was markedly intensified (Fig. 1A).

View larger version (80K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 DNA ladder formation (A) and TUNEL positive staining (B) in myocardial tissues from sham MI/R, rabbits with a normal diet subjected to MI/R, rabbits with a high cholesterol diet subjected to MI/R (HC+MI/R) and rabbits with a high cholesterol diet treated with RSG subjected to MI/R (HC+MI/R+RSG). MM in panel A=molecule marker. Cardiac myocytes are identified by anti--actinin antibody (red), total nuclei were labeled with DAPI (blue) and apoptotic nuclei were detected by TUNEL staining (green).

 
Consistent with the absence of DNA ladders, few cells stained positive in tissue from sham MI hearts. In contrast, TUNEL-positive nuclei were prevalent in tissues from ischemic–reperfused hearts (area-at-risk), and high cholesterol feeding further increased postischemic myocardial apoptosis in ischemic–reperfused myocardial tissues (Figs. 1B and 2A). The TUNEL positive cells in the non-ischemic/reperfused area was slightly higher in rabbits fed with a high cholesterol diet (1.2±0.3%) than that with a normal diet (0.7±0.2%). However, this difference was not statistically significant. This result indicates that although hypercholesterolemia markedly increased myocardial apoptosis after ischemia and reperfusion, this acute diet-induced hypercholesterolemia itself failed to increase myocardial apoptosis under physiologic conditions in the adult rabbit.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Summary of TUNEL positive myocytes (A) and caspase-3 activity (B). Rabbits were subjected to 4.5 h of sham ischemia and reperfusion (Sham MI) or 30 min of myocardial ischemia followed by 4 h of reperfusion (MI/R). At the end of experiment, the heart was removed, ischemic/reperfused myocardial tissue was identified, and TUNEL positive staining and caspase-3 activity were determined. HC=high cholesterol diet; V=vehicle. *P<0.05, **P<0.01 vs. normal diet group; ##P<0.01 vs. HC group treated with vehicle.

 
To provide more evidence in a quantitative and specific manner that hypercholesterolemia enhanced postischemic myocardial apoptosis, myocardial caspase-3 activation, a final common pathway in caspase-dependent apoptosis, was determined. As summarized in Fig. 2B, a fourfold increase in caspase-3 activity was observed in HC rabbits compared with that in normal diet rabbits.

Myocardial infarction represents the total myocardial injury caused by necrosis and apoptosis. Because of the chronic model used in this study and the limited number of animals in each group, direct measurement of infarct size was not possible (the study required an entire heart for TTC staining; this tissue subsequently could not be used for any other measurements). To further ensure that hypercholesterolemia not only increases myocardial apoptosis but also increases myocardial necrosis, thus enlarging the myocardial infarction, plasma creatine kinase (CK) activity was determined. There was no difference in plasma CK activity between the normal diet and the cholesterol diet groups before coronary occlusion (3.46±0.22 vs. 4.93±0.51 U/g protein, P>0.05). However, at the end of 4 h reperfusion, plasma CK activity was significantly higher in the cholesterol diet group than that seen in the normal diet group (21±1.28 vs. 15±0.8 U/g protein, P<0.01), indicating that hypercholesterolemia increased myocardial necrotic injury following ischemia and reperfusion.

To determine whether increased postischemic myocardial injury in hypercholesterolemic rabbits may aggravate cardiac functional injury after ischemia and reperfusion, left ventricular pressure was measured, and dP/dt_max and a contractile index (CI=dP/dt_max/p) were obtained. Myocardial ischemia and reperfusion significantly decreased HR in all three groups. An HC diet had no significant effect on MABP and HR before, during or after ischemia (Fig. 3). However, an HC diet markedly aggravated myocardial contractile function as evidenced by decreased dP/dt_max and CI (Fig. 4).

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Mean arterial blood pressure (MABP, panel A), and heart rate (HR, panel B) in three experimental groups subjected to myocardial ischemia and reperfusion.

 

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Left ventricular dP/dtmax (A) and contractile index (CI, panel B) in three experimental groups subjected to myocardial ischemia and reperfusion. **P<0.01 vs. normal diet group; ##P<0.01 vs. HC group treated with vehicle.

 
3.3. Hypercholesterolemia increased nitrative stress and disturbed a beneficial balance between anti-/pro-apoptotic MAPK signaling
Recent experimental results have demonstrated that besides oxidative stress, nitrative stress also plays a critical role in tissue injury under a variety of pathologic conditions. Excessive NO produced from iNOS and more importantly, the reaction product between NO and superoxide, peroxynitrite, have been demonstrated to activate apoptotic pathways and result in apoptotic cell death. To determine whether hypercholesterolemia may cause myocardial nitrative stress and thus contribute to increased myocardial apoptosis after ischemia and reperfusion, we determined iNOS expression, myocardial total NOx content, and nitrotyrosine formation (a footprint of in vivo peroxynitrite formation). As illustrated in Figs. 5 and 6, hypercholesterolemia markedly upregulated iNOS expression, increased myocardial total NOx content, and increased nitrotyrosine formation. These results demonstrated that the pro-apoptotic reactive nitrogen species are significantly increased in the hypercholesterolemic heart.

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Myocardial iNOS expression (A) and total NOx contents (B). Rabbits were subjected to 4.5 h of sham ischemia and reperfusion (Sham MI) or 30 min of myocardial ischemia followed by 4 h of reperfusion (MI/R). At the end of experiment, the heart was removed, ischemic/reperfused myocardial tissue was identified, and iNOS expression and total NOx were determined. **P<0.01 vs. normal diet group; ##P<0.01 vs. HC group treated with vehicle.

 

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Myocardial nitrotyrosine staining (A) and content (B). Rabbits were subjected to 4.5 h of sham ischemia and reperfusion (Sham MI) or 30 min of myocardial ischemia followed by 4 h of reperfusion (MI/R). At the end of experiment, the heart was removed, ischemic/reperfused myocardial tissue was identified, and nitrotyrosine localization and content were determined. **P<0.01 vs. normal diet group; ##P<0.01 vs. HC group treated with vehicle.

 
To further elucidate the mechanisms by which hypercholesterolemia may increase postischemic myocardial apoptosis, we investigated the balance between an anti- and a pro-apoptotic MAPK in ischemic/reperfused myocardial tissue. Interestingly, hypercholesterolemia differentially regulated ERK1/2 and p38 MAPK activation. Compared with myocardial tissues from rabbits fed with a normal diet, the hypertrophic/anti-apoptotic ERK1/2 activity was significantly inhibited in myocardial tissue from rabbits fed with a high cholesterol diet. In contrast, activation of the pro-apoptotic p38 MAPK was markedly enhanced in myocardial tissue from hypercholesterolemic rabbits (Fig. 7). These results demonstrated that hypercholesterolemia disturbs a beneficial anti-/pro-apoptotic balance and renders the cardiomyocyte more vulnerable to ischemic/reperfusion injury.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Myocardial ERK1/2 activity (A) and p38 MAPK activation (B). Rabbits were subjected to 4.5 h of sham ischemia and reperfusion (Sham MI) or 30 min of myocardial ischemia followed by 4 h of reperfusion (MI/R). At the end of experiment, the heart was removed, ischemic/reperfused myocardial tissue was identified, and MAPK activities were determined. **P<0.01 vs. normal diet group; ##P<0.01 vs. HC group treated with vehicle.

 
3.4. Treatment with RSG attenuated nitrative stress, restored anti-/pro-apoptotic MAPK signaling, reduced postischemic myocardial apoptosis and necrosis, and improved cardiac function
Previous studies have demonstrated that PPAR agonists, such as RSG, exert beneficial cardiovascular effects in diabetic patients. In our recent study, we have demonstrated that treatment with RSG significantly reduced vascular NADPH oxidase activity and superoxide generation, and improved endothelial function in hypercholesterolemic rabbits [18]. To determine whether the PPAR signaling pathway may inhibit apoptosis by attenuating nitrative stress in a non-diabetic model, 10 rabbits fed with a high cholesterol diet were treated with RSG for 5 weeks. As summarized in Figs. 1 through 4, treatment with RSG in hypercholesterolemic rabbits exerted significant protective effects as evidenced by reduced DNA ladder formation, decreased TUNEL positive myocytes, and reduced caspase-3 activity. RSG treatment tended to increase both dP/dt_max and CI before ischemia, although this difference was not statistically significant. However, treatment with RSG markedly attenuated cardiac contractile dysfunction after ischemia (CI) and reperfusion (dP/dt_max and CI). In addition, RSG treatment also significantly reduced plasma CK activity (14±0.5 U/g protein, P<0.01), a parameter that primarily reflects necrotic injury in vivo. To further determine the potential mechanisms by which RSG may exert its anti-apoptotic properties, the effect of RSG treatment on nitrative stress and anti-/pro-apoptotic MAPK balance was determined. As illustrated in Figs. 5–7, treatment with RSG in hypercholesterolemia significantly inhibited iNOS expression, reduced myocardial NOx content, decreased toxic peroxynitrite formation and restored a beneficial anti-/pro-apoptotic MAPK balance.

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
We have made several novel observations in our present experiment. First, we demonstrated that hypercholesterolemia significantly exacerbated cardiac reperfusion injury as evidenced by increased caspase-3 activation and cardiomyocyte apoptosis, and decreased cardiac function recovery after reperfusion. Second, we documented for the first time that increased nitrative stress in hypercholesterolemia and the disturbance of anti- and pro-apoptotic MAPK signaling may contribute to the increased vulnerability of myocyte apoptosis to ischemia and reperfusion. Finally, we have provided direct evidence that the PPAR signaling pathway may play a significant role in regulating nitrative stress and subsequent tissue injury. To our knowledge, this novel phenomenon has not been previously reported and suggests that PPAR agonists such as RSG may have a broad spectrum of cardiovascular protective effects in addition to its reported beneficial effects in diabetes.

Hypercholesterolemia has long been recognized as a major risk factor for coronary artery disease and acute myocardial infarction. In a recent study, Wang et al. [4] reported that experimental hyperlipidemia induced by a high cholesterol diet (10%) increased infarct size and apoptotic cell death. Our present experimental results confirmed their report and demonstrated that a moderate hypercholesterolemia (1% instead of 10% cholesterol diet for 8 weeks) also increased postischemic myocardial apoptosis. Furthermore, we have demonstrated that postischemic cardiac function recovery was markedly blunted in rabbits with hypercholesterolemia.

Although apoptosis has been observed in cultured cardiomyocytes, in isolated perfused hearts, and in in vivo animal models as well as in clinical observations after ischemia/reperfusion [19–25], its relevance to the pathogenesis of myocardial reperfusion injury and cardiac dysfunction remains controversial. However, growing evidence strongly suggests that apoptosis may contribute to myocardial ischemia/reperfusion injury by several mechanisms. Loss of cardiomyocytes leads to loss of "cardiac mass" and hence "diminished pump power". Loss of cardiomyocytes may also result in electrical conduction inhomogeneity that may lead to arrhythmias. Finally, apoptosis may lead to cardiac remodeling due to realignment of neighboring cardiomyocytes. This latter mechanism is unique to the heart, where function is extremely dependent on optimal geometrical and structural alignment. Thus, apoptosis, even if limited in scope, may result in widespread mechanical and electrical disturbances. These possibilities are further supported by studies in which inhibition of apoptosis by a variety of pharmacological and genetic approaches results in smaller infarction [26–32], improved cardiac function [33,34] and attenuated ischemia–cardiac remodeling [35,36].

A novel finding in our study is that in hypercholesterolemic rabbits, myocardial nitrative stress is markedly increased (iNOS expression, pathological levels of NOx, and more importantly, peroxynitrite formation). High concentrations of NO have been reported to induce apoptotic cell death in several cell types including macrophages, thymocytes, pancreatic islets, certain neurons and tumor cells [37]. More importantly, peroxynitrite has been reported to increase apoptotic cell death in a variety of cell types. In contrast to NO, which has both anti- and pro-apoptotic properties, no anti-apoptotic effect of ONOO^– has been reported to date. The pro-apoptotic mechanisms of ONOO^– include protein and DNA oxidation [38], lipid peroxidation [39], protein nitration [40] and endoplasmic reticulum stress with the subsequent release of caspase 12 [41].

In addition, our results demonstrated for the first time that the anti- and pro-apoptotic MAPK signaling pathway is differentially regulated in hypercholesterolemic myocardial tissue. Previous studies by other investigators as well as our group have demonstrated that both ERK1/2 and p38 MAPK, two of the three members of the MAPK superfamily, are activated following myocardial ischemia and reperfusion [42,43]. However, activation of ERK1/2 (anti-apoptotic) and p38 MAPK (pro-apoptotic) exert opposite effects on postischemic myocardial apoptosis. Since the activation of ERK1/2 after ischemia/reperfusion is markedly reduced, whereas activation of p38 MAPK is significantly increased in hypercholesterolemic hearts, this disturbed anti-/pro-apoptotic MAPK signaling may provide another likely explanation for increased postischemic myocardial apoptosis in hypercholesterolemic animals.

The mechanisms responsible for the differential regulation of ERK1/2 and p38 MAPK activation in hypercholesterolemic hearts are unknown. Using cultured Rat-1 cells, Furuchi and Anderson [44] demonstrated that cholesterol depletion causes hyperactivation of ERK1/2. One can speculate that hypercholesterolemia may exert opposite effects and inhibit ERK activation. A likely mechanism explaining enhanced p38 MAPK activation is the increased nitrosative stress in hypercholesterolemic hearts. Schieke et al. recently reported that ONOO^– markedly activates p38 MAPK, but only slightly activates ERK1/2 in cultured rat liver epithelial cells [45]. Moreover, Oh-Hashi et al. [46] demonstrated that treatment with 3-morpholinosydonimine (SIN-1), a peroxynitrite generator, induced the expression of three different growth arrest and DNA damage-inducible mRNA's in a p38 MAPK-dependent manner.

Another important finding of the present study is that treatment with RSG markedly reduced myocardial injury as evidenced by decreased apoptosis as well as improved cardiac function. There are several potential mechanisms by which a PPAR agonist may exert its anti-apoptotic effect in hypercholesterolemia. However, the present experiment demonstrated that a PPAR agonist most likely achieves its myocardial protection via inhibition of nitrative stress and subsequent pro-apoptotic MAPK activation. This conclusion is supported by reduced iNOS expression, total NO production, nitrotyrosine formation, and p38 MAPK activation in RSG-treated hypercholesterolemic animals. Therefore, our results suggest that RSG may not only attenuate the formation of atherosclerosis as previously reported, but also protect postischemic myocardial injury by inhibiting toxic peroxynitrite production.

In summary, our present study demonstrated that HC increased postischemic myocardial apoptosis likely by increasing the production of pro-apoptotic molecules (such as peroxynitrite), activating pro-apoptotic signaling pathways (such as p38 MAPK) and inhibiting anti-apoptotic signaling (such as ERK1/2). In addition, our results demonstrate that RSG markedly reduced postischemic myocardial apoptosis in a non-diabetic, hypercholesterolemic model, suggesting that PPAR agonists may have broad applications in treatment of cardiovascular diseases, in addition to their current application in treatment of type 2 diabetes.

4.1. Limitations
A major limitation of the present study is that a conclusive cause–effect relationship between pro-/anti-apoptotic signaling and increased postischemic myocardial apoptosis could not be established in this large animal model. Future studies utilizing a gene manipulated mice model (such as iNOS and p38 MAPK knockout) may provide more complete answers, and these experiments are being planned. It is now recognized that apoptosis is regulated by multiple pathways at multiple levels (e.g., transcriptional and posttranscriptional). Our present study demonstrated that hypercholesterolemia resulted in disturbance of ERK1/2 and p38 MAPK pathway. However, whether other pro- and anti-apoptotic molecules may also be altered under this pathological condition remains unknown, and this should be directly addressed in future studies.

It should also be indicated that ERK1/2 and p38 MAPK activity was determined 4 h after reperfusion in the present experiments. In previous studies reported by other investigators as well as our group, maximal ERK1/2 and p38 MAPK activation occurred within 20 min after reperfusion in isolated rat hearts subjected to global ischemia and reperfusion [34,47]. The time course of ERK1/2 and p38 MAPK activation after myocardial ischemia and reperfusion in vivo in a large animal model has not been previously studied. Whether or not hypercholesterolemia and RSG may exert similar regulatory effect on their activation at earlier time points was not directly addressed in the present study.

	Acknowledgements

 
This research was supported in part by an NIH grant HL-63828 and EMF established Investigator Award (X.L. Ma).

	Notes

 
¹ GlaxoSmithKline Pharmaceuticals, King of Prussia, PA.

Time for primary review 24 days

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Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

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