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ATORVASTATIN POST-CONDITIONING ATTENUATES MYOCARDIAL ISCHEMIA REPERFUSION INJURY VIA INHIBITING ENDOPLASMIC RETICULUM STRESSYRELATED APOPTOSIS Jing-gang Xia,*† Fei-fei Xu,‡ Yang Qu,§ Dan-dan Song,‡ Hong Shen,|| and Xiu-hua Liu‡ *Medical School of Chinese People’s Liberation Army, Chinese PLA General Hospital; † Department of Cardiology, Xuanwu Hospital, Capital Medical University; ‡ Department of Pathophysiology, Chinese PLA General Hospital; §Department of Pathology, Beijing Chest Hospital, Capital Medical University; and ||Department of Emergency, Chinese PLA General Hospital, Beijing, China Received 19 Mar 2014; first review completed 8 Apr 2014; accepted in final form 11 Jun 2014 ABSTRACT—The present study examined whether atorvastatin, when used for pharmacological postconditioning, attenuated myocardial ischemia-reperfusion (I/R) injury in a manner similar to ischemic postconditioning (I-PostC), that is, by inhibition of endoplasmic reticulum (ER) stressYrelated apoptosis. In the present study, markers for myocardial injury, infarct area, and hemodynamics, and indicators of ER stress and apoptosis were compared in ischemic and atorvastatin-induced postconditioning as a means of evaluating the protective effect of atorvastatin postconditioning in I/R injury and whether, as in I-PostC, inhibition of ER stress is involved. Both ischemic and atorvastatin-mediated postconditioning significantly decreased indications of cardiac damage and reduced serum concentrations of markers for myocardial injury, reduced the infarct area seen at the end of reperfusion, and improved left ventricular systolic function. We found that high-dose atorvastatin- and I-PostC significantly downregulated expression of glucose-regulating protein 78 and calreticulin (CRT; ER stress markers), expression of C/EBP homologous protein (CHOP), and caspase 12 (markers for ER stressYrelated apoptosis), and Bax (downstream molecule of CHOP), in the myocardial area at risk. Atorvastatin and I-PostC have similar cardioprotective effects in I/R injury and inhibit the ER stressYrelated apoptotic pathway. KEYWORDS—Atorvastatin, postconditioning, ER stress, ischemia-reperfusion, myocytes apoptosis

INTRODUCTION

examination of PPC use in humans is mostly confined to smallscale clinical trials (10). Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) exert pleiotropic effects that are independent of their lipid-lowering effect (11) and have favorable effectiveness and safety in PPC compared with other drugs (12). And although it is known that PPC with statins attenuates myocardial I/R injury, the mechanisms underlying this cardioprotection are not completely understood. One mechanism through which I/R causes injury is the induction of excessive myocardial endoplasmic reticulum (ER) stress. Moderate ER stress increases the release of chaperone proteins, such as glucose-regulating protein (GRP) 78 and calreticulin, which are involved in recognition and degradation of misfolded proteins, and is protective. However, prolonged or severe stress, such as can occur in I/R injury, will cause apoptosis through increased production of C/EBP homologous protein (CHOP), an imbalance of proapoptotic (Bax) and antiapoptotic (Bcl-2) proteins, and activation of caspases (13). Therefore, inhibition of ER stressYrelated apoptosis has become a target in treatment of myocardial I/R injury (14). As shown in our previous study, I-PostC inhibits ER stressYrelated apoptosis (15). In the present study, we examined whether atorvastatin, when used for PPC, attenuated myocardial I/R injury in a manner similar to I-PostC, that is, by inhibition of ER stressYrelated apoptosis. In the present study, markers for myocardial injury, infarct area, hemodynamics, and indicators of ER stress and apoptosis were compared in ischemic and atorvastatin-induced postconditioning as a means of evaluating the protective effect of atorvastatin postconditioning in I/R injury and whether, as in I-PostC, inhibition of ER stress is involved.

Early thrombolysis, interventional therapy, and coronary artery bypass grafting are common strategies used for the treatment of acute ST-segment elevation myocardial infarction (1, 2). However, the sudden increase in blood flow that results from successful treatment causes reperfusion injury, and ischemia-reperfusion (I/R) injury is a major cause of death in acute myocardial infarction patients after reperfusion. Ischemic preconditioning and ischemic postconditioning (I-PostC) induce endogenous protective mechanisms that attenuate reperfusion injury (3). But the repeated transient I/R cycles used in preconditioning and postconditioning may cause injury (4), a drawback that limits clinical use. Recently, attempts have been made to substitute drug administration for the transient ischemia/perfusion cycles used in I-PostC. The resulting pharmacological postconditioning (PPC) has shown potential in both animal studies and clinical practice (4). It avoids the potential injury induced by I-PostC, is easier to perform, and therefore has better clinical potential than I-PostC. Commonly used drugs in PPC include anesthetics (5, 6), adenosine (7, 8), levosimendan (9), and statins. However, currently,

Address reprint requests to Hong Shen, PhD, Department of Emergency, Chinese PLA General Hospital, Beijing 100853, China. E-mail: [email protected]. Co-correspondence: Xiu-hua Liu, PhD, Department of Pathophysiology, Chinese PLA General Hospital, Beijing 100853, China. E-mail: [email protected]. J.-G.X. and F.-F.X. contributed equally to this work. This work was supported by a grant from the National Natural Science Foundation of China (no. 81170140 and 81070130) Conflicts of interest and source of funding: none. DOI: 10.1097/SHK.0000000000000224 Copyright Ó 2014 by the Shock Society

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SHOCK VOL. 42, NO. 4 MATERIALS AND METHODS

Preparation of animal model and grouping The current study followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996). The research was approved by the Animal Research Councils and the Ethical Council, Health Center, Peking University, China. Pathogen-free, healthy male Sprague Dawley rats (280 T 20 g) were purchased from the Experimental Animal Center of Military Medical Sciences. Animals were fasted with ad libitum access to water for 12 h prior to surgery. Rats were anesthetized intraperitoneally with 2% pentobarbital sodium solution (4.6 mg/100 g body weight), and then, after tracheal intubation, mechanical ventilation was performed (50Y60 breaths/min, tidal volume of 4Y6 mL) using a small animal ventilator (Medical Instrument of Zhejiang University). An SMUP-PC1 biological signal processing system and MFL Lab200 ECG software (Department of Physiology, Medical School, Fudan University) were used for measurement of ECG. After thoracotomy, the pericardium was opened, the left atrial appendage exposed, and a 6-0 suture placed between the pulmonary cone and left atrial appendage and beneath the left anterior descending coronary artery, 2 mm downward from the left appendage. A small latex pad was placed between the vessel and suture, followed by ligation to induce myocardial ischemia. The ischemic myocardium became white, the distal myocardium remained dark red, and ECG monitoring showed ST-segment elevation or bundle-branch block, indicating successful ligation. Different treatments were then administered before reperfusion. Animals developing ventricular fibrillation, pneumothorax, excessive blood loss, and anesthesia accident or that died before the end of the study were not used for further analysis. Animals were divided into five groups (n = 12 per group): (1) sham group: the suture was placed, but no ligation was performed; (2) I/R group: the left anterior descending coronary artery was ligated for 45 min to induce ischemia, followed by 24-h reperfusion; (3) ischemia postconditioning group (I-PostC): after ischemia for 45 min, three cycles of 10-s reperfusion and 10-s ischemia were performed, followed by 24-h reperfusion; (4) low-dose atorvastatin (Pfizer, New York, NY) postconditioning group (PPC 0.5 mg): after ischemia for 45 min, 0.5 mg/kg i.p. atorvastatin (in 10% dimethyl sulfoxide; Sigma, St Louis, Mo) was injected 5 min before 24-h reperfusion; (5) high-dose atorvastatin postconditioning group (PPC 2 mg): after ischemia for 45 min, 2 mg/kg i.p. atorvastatin (in 10% dimethyl sulfoxide) was injected 5 min before 24-h reperfusion. The doses of atorvastatin were chosen according to those used in previous studies (16, 17) and correlate with those used in humans, At the end of reperfusion, six rats were randomly selected from each group for detection of markers for myocardial injury and average myocardial perfusion (n = 5), and the remaining six rats were used for detection of hemodynamics and myocardial infarction by 2,3,5-triphenyltetrazolium chloride (TTC; Shanghai Chemical Reagent Company, Shanghai, China) staining (n = 5). Three of the six were also used for detection of ER stressYrelated molecules by Western blot assay, and five of the six were also used for detection of myocardial apoptosis by TUNEL (TdT-mediated, dUTP nick-end labeling) staining.

Detection of myocardial perfusion A PeriCam PSI bloodstream video monitoring system (PERIMED) (PeriCam PSI NR; PSIN-01037) was used to monitor the epicardial perfusion in the portion of the left ventricular anterior wall supplied by the left anterior descending coronary artery, using laser speckle contrast analysis. PIM Soft flow imaging system software was used to evaluate mean blood flow at 1 min before ligation, 1 min after ligation, and 1 min after the initiation of the 24-h reperfusion.

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and infarcted areas) that was infarcted (AN, infarcted area), that is, AN/AAR, was used to express the extent of the infarction. The proportion of AAR to left ventricle (LV) area (AAR/LV) was used to express the extent of the AAR. Image-Pro Plus image analysis software (version 4.1; Media Cybernetics, Rockville, Md) was used to measure AAR and AN.

Measurement of hemodynamics After the end of reperfusion, a tube was inserted, using sterile procedure, into the right common carotid artery and connected to a fluid-filled pressure transducer and SMUP-PC1 biological signal processing system. Mean arterial pressure (MAP) tracings were digitized with an analog-to-digital converter and stored in a computer for later analysis. The tube was then passed retrogradely into the LV, and LV pressure tracings were also digitized and stored. Maximum rate of systolic pressure rise (Tdp/dtmax) and 1eft ventricular end-diastolic pressure (LVEDP) were analyzed in a blinded fashion with dedicated software. The MFL Lab200 system was used to record the heart rate (HR), MAP, maximum rate of systolic pressure rise (Tdp/dtmax), and LVEDP.

Western blot assay of CRT, GRP78, caspase 12, Bax, and Bcl-2 At the end of reperfusion, thoracotomy was performed, the AAR was collected, and the total protein extracted as described previously (16). The extracted protein (200 2g) was subjected to sodium dodecyl sulfateYpolyacrylamide gel electrophoresis (8% separating gel) and then transferred onto a nitrocellulose membrane. Following blocking, the membrane was incubated at room temperature for 4 h with one of the following polyclonal antibodies: calreticulin (1:1,000) (CRT; Stressgen, Victoria, British Columbia, Canada), GRP78 (1:1,000) (Stressgen), CHOP (1:200) (Santa Cruz, Santa Cruz, Calif), caspase 12 (1:200) (Biovision, Exton, Pa), Bax (1:200) (Upstate, Lake Placid, NY), or Bcl-2 [1:200] (Santa Cruz), and then with the corresponding secondary antibody at room temperature for 1 h. In the control group, GAPDH (1:200) monoclonal antibody (Santa Cruz) was used for the above procedures. ImageProPlus image analysis software was used to detect the integrated optical density (IOD): IOD = mean optical density  area. The IOD of target protein was normalized to that of GAPDH as the relative expression of target protein.

Detection of myocyte apoptosis by TUNEL staining At the end of reperfusion, the myocardium in the AAR was harvested and fixed in 4% formaldehyde (pH7.4) for 48 h at 4-C, then in 75% ethanol, and embedded in paraffin. Four-micrometer transverse sections were obtained, dried, and stored at room temperature until use. Five sections underwent TUNEL (Promega, Madison, Wis) to label the nuclei of apoptotic cells. Cells were counterstained with DAPI. Sections were observed under a microscope, five fields randomly selected, and 100 TUNEL-positive cells counted in each field. The apoptosis index (AI) was calculated as follows: AI = (apoptotic cells / total cells)  100%.

Statistical analysis Continuous variables are presented as mean and SD and are compared between groups by one-way analysis of variance (ANOVA). Categorical variables are expressed by count and are compared between groups by Fisher exact test. When a significant difference between groups was apparent, multiple comparisons were performed using the Bonferroni procedure with type I error adjustment. P G 0.05 was considered statistically significant. Statistical analysis was done using SAS software version 9.2 (SAS Institute Inc, Cary, NC).

RESULTS Blood perfusion

Measurement of plasma troponin and lactate dehydrogenase At the end of reperfusion, arterial blood was collected, and the plasma separated. A rat Tn-I (troponin I) enzyme-linked immunosorbent assay kit (Wuhang Yilai Ruite Biotech Co, Ltd, Wuhang City, China) and a lactate dehydrogenase (LDH) detection kit (Nanjing Jiancheng Biotech Co, Ltd, Nanjing City, China) were used to measure Tn-I and LDH, according to the manufacturers_ instructions.

Detection of myocardial ischemia and infarct area At the end of reperfusion, the heart was harvested, the left anterior descending coronary artery ligated, and retrograde perfusion performed with 2 to 3 mL of 1% Evans blue. The atria were removed, and the ventricles stored at j20-C for 20 min. Next, 2-mm sections along the longitudinal axis of the ventricle were placed in 2% TTC buffered with phosphate-buffered saline (pH7.4), incubated at 37-C for 10 min, and fixed in 10% formaldehyde at 4-C for 24 h. The sections were mounted and scanned into a computer. The blue region indicated normal myocardium; the light red region, ischemic myocardium; and the white region, infarcted myocardium. The proportion of the area at risk (AAR, sum of ischemic

Blood perfusion in the I/R and I/R + treatment groups is shown in Table 1. All groups showed a decrease in perfusion at the onset of ischemia and an increase in perfusion at the onset of reperfusion. There were no significant differences between the groups (P90.05). Myocardial blood perfusion in the I/R group before ischemia, during ischemia, and after reperfusion is shown in Figure 1. Troponin and LDH

Ischemia-reperfusion significantly increased plasma levels of Tn-I and LDH (P G 0.001 compared with sham surgery). All three postconditioning treatments (I-PostC, PPC 0.5 mg, and PPC 2 mg) decreased the I/R-induced raised plasma levels of these markers of tissue damage (all P G 0.001, Table 2).

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TABLE 1. Blood perfusion and blood perfusion ratio among the five groups

Before the onset of ischemia (PU) The onset of ischemia (PU) Ratio of before to the onset of ischemic The onset of perfusion (PU) Ratio of the onset of perfusion to before the onset of ischemia

I/R (n = 5)

I-PostC (n = 5)

1,310.9 T 56.0

1,293.6 T 71.9

1,183.1 T 107.8

1,185.1 T 128.9

0.090

868.3 T 64.1

825.0 T 220.5

888.0 T 210.5

0.763

0.74 T 0.10

0.367

776.9 T 169.6 0.60 T 0.15 959.2 T 173.2 0.74 T 0.15

0.67 T 0.03 1,116.4 T 106.8 0.87 T 0.13

PPC 0.5 mg (n = 5) PPC 2 mg (n = 5)

0.70 T 0.17 1,014.9 T 193.5

1,080.6 T 189.1

0.87 T 0.20

0.91 T 0.08

P

0.487 0.302

Continuous variables are presented as mean and SD, which were compared between survival and death by one-way ANOVA.

Comparison of hemodynamics among groups

The Tdp/dtmax in the I/R group was significantly lower than that in the sham group (both P G 0.001). All postconditioning groups increased the low value seen in the I/R group (all P e 0.003), but there was no significant difference between the three postconditioning groups. There was no significant difference among the five groups in HR. Mean arterial pressure in the I/R group was significantly lower than that in the sham group (P G 0.001). Of the three treatment groups, only the high-dose atorvastatin group had significantly higher MAP than the I/R group (P=0.001). The LVEDP of the I/R group was significantly higher than that of the sham group (P G 0.001). Although LVEDP was lower in the I-PostC, PPC 0.5 mg, and PPC 2 mg compared with the I/R group, this difference did not reach statistical significance (Table 3).

was significantly higher than that of the sham group (P G 0.001). High-dose atorvastatin postconditioning increased this value still further (P = 0.013 vs. the I/R group; Fig. 3D). Bax protein expression in the I/R group was significantly higher than that of the sham group (P G 0.001). Ischemic and high-dose atorvastatin postconditioning significantly decreased this value (both P G 0.001 vs. I/R; Fig. 3E). Representative Western blot images are shown in Figure 3F. Apoptosis

Figure 4 shows apoptotic (TUNEL-positive) myocytes in the different groups. Apoptotic cells had yellow or brown granules in the nuclei, and viable cells had blue nuclei. Ischemiareperfusion significantly increased the apoptosis rate (P G 0.001 vs. sham), and all postconditioning treatments significantly lowered this rate (all P G 0.001 vs. I/R; Figure 4B).

Apoptosis-related proteins

Both GRP78 and calreticulin protein expression in the I/R group were significantly higher than that of the sham group (both P G 0.001; Fig. 2). All three postconditioning treatments significantly lowered this I/R-induced increase in the two indicators of ER stress (all Pe0.003; Fig. 2). Representative Western blot images are shown in Figure 2C. CHOP protein expression in the I/R group was also significantly higher than that of the sham group (P G 0.001), and this increase was significantly lessened by all three postconditioning treatments (all P e 0.006; Fig. 3A). There was no significant difference between sham, I/R, or postconditioning groups in proYcaspase 12 protein expression (Fig. 3B). Expression of the active caspase 12 protein itself was significantly higher in the I/R than in the sham group (P G 0.001), and all postconditioning treatments significantly decreased this value (all P G 0.001; Fig. 3C). Bcl-2 protein expression in the I/R group

AAR and infarct area among groups

All three postconditioning treatments also lessened the percentage of the AAR that became infarcted; the AN/AAR ratios of the I-PostC, PPC 0.5 mg, and PPC 2 mg groups were all significantly lower than that of the I/R group (all P e 0.011; Fig. 5A). However, the AAR itself was similar in the I/R and the three treatment groups (AAR/LV; Fig. 5B). TTC staining of infarct volume in the four groups is shown in Figure 5C. DISCUSSION In the current study, both ischemic and atorvastatin-mediated postconditioning significantly decreased indications of cardiac damage and reduced serum concentrations of markers for myocardial injury, reduced the infarct area, and improved left ventricular systolic (+dp/dtmax) function. The cardioprotection of atorvastatin postconditioning was comparable to that of I-PostC

FIG. 1. Myocardial blood perfusion in the I/R group. Red: myocardial blood perfusion in end-diastolic phase. A, Blood perfusion before ischemia. B, Blood perfusion during ischemia. C, Blood perfusion after reperfusion.

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TABLE 2. Troponiin and LDH among the five groups Sham (n = 5)

I/R (n = 5)

I-PostC (n = 5)

PPC 0.5 mg (n = 5)

†

†

PPC 2 mg (n = 5) †

P

Tn-I (2g/L)

1.75 T 0.26

4.06 T 0.68*

2.49 T 0.31

2.74 T 0.51

2.39 T 0.32

G0.001

LDH (U/L)

197.9 T 35.8

472.0 T 58.3*

263.3 T 23.1†

258.3 T 26.2†

254.7 T 27.9†

G0.001

Continuous variables are presented as mean and SD, which were compared between survival and death by one-way ANOVA. *Significant difference when compared with sham. † Significant difference when compared with I/R.

and consistent with the cardioprotective effect of statins reported by others (12, 16, 18). And the findings on the cardioprotective effect of I-PostC are also consistent with those of previous reports (19). Atorvastatin and I-PostC also significantly downregulated expression of GRP78 and CRT (ER stress markers), CHOP and caspase 12 expression (markers for ER stressYrelated apoptosis), and Bax (downstream molecule of CHOP), in the myocardial AAR. Ischemic postconditioning and low-dose atorvastatin had no effect on expression of the antiapoptotic protein, Bcl-2, but high-dose atorvastatin did increase the expression of this protein, and all three treatments decreased the number of apoptotic myocytes. These results show that the myocardial protection of atorvastatin postconditioning, like that of I-PostC, is accompanied by inhibition of the ER-related apoptotic pathway. High-dose atorvastatin postconditioning may be more effective than low-dose atorvastatin postconditioning in decreasing myocytes apoptosis. Low-dose atorvastatin and high-dose atorvastatin both inhibit ER stress and significantly downregulate the expression of GRP78 and CRT, CHOP, caspase 12, and Bax. However, postconditioning with high-dose atorvastatin dramatically alters the ratio of Bcl-2 to Bax compared with postconditioning with low-dose atorvastatin. This difference between the two doses suggests that the protective effect of high-dose atorvastatin on myocyte apoptosis may be more pronounced than that of the low dose. The current I-PostC results on inhibition of the ER stress pathway are similar to those of our previous study (15). The atorvastatin results on inhibition of this pathway are also similar to previously reported results. For example, Song et al. (20) reported that atorvastatin downregulated the expression of caspase 12 and CHOP in myocytes after myocardial infarction; Jia et al. (21) reported that statins inhibited ER stress to protect against vascular injury secondary to hyperhomocystinemiainduced atherosclerosis; Chen et al. (22) found that fluvastatin markedly increased the expression of GRP78 in RAW267.4 macrophages, an action that might be able to protect macrophages against hypoxia-induced death, and Urban et al. (23)

reported that simvastatin attenuated forebrain I/R and increased mRNA expression of GRP78 during ischemia and early reperfusion, actions suggesting that the neuroprotection of simvastatin is associated with the inhibition of excessive ER stress. A number of studies have reported the PI3K/Akt/endothelial nitric oxide synthase (eNOS) pathway to be involved in ischemic and atorvastatin-induced postconditioning (12, 16, 18, 24Y26). Atorvastatin has also been reported to have an antiinflammatory action during myocardial I/R by inhibiting tumor necrosis factor ! mRNA and protein (27), to act through Tolllike receptor 4 in liver I/R (28) and to increase endotheliumdependent relaxation through increasing eNOS (29). An innovation in our experimental protocol was the use of laser speckle contrast imaging to observe blood flow in microvessels in the AAR (30Y32). Laser speckle contrast imaging records the interference pattern produced by light scattered from different parts of the substance being imaged. When a component of the substance, such as a red blood cell, moves, the intensity of the speckle it produces changes, and the magnitude of the change is related to the velocity of the movement. With the imaging connected to a Laser Speckle Analysis software system, blood flow can be monitored in video form in a real-time manner (32). With this method, transient changes in microcirculatory perfusion can be visualized with high resolution and high sampling frequency. A real-time analysis of dynamic response and spatial resolution can be carried out, and a real-time curve and video record of regions and time intervals of interest be provided. The clinical benefits of statins have been shown as a reduction in perioperative mortality in interventional therapy in patients with acute coronary syndrome and a decrease in major adverse cardiac events within 30 days after surgery (33) and in significant improvement of fractional flow in myocardial infarction patients after thrombolysis in myocardial infarction trial (34). Basic studies have also shown that high-dose atorvastatin exerts protective effects on the heart, liver, and kidney during myocardial infarction (35). An advantage of the use of atorvastatin over other drugs in postconditioning is that it is a wellknown drug with a good safety record. Many cardiovascular

TABLE 3. Hemodynamics among the five groups PPC 0.5 mg (n = 6)

PPC 2 mg (n = 6)

+dp/dtmax (mmHg/s)

3,700.3 T 237.8

Sham (n = 5)

2,208.3 T 286.2*

2,955.3 T 393.3†

2,982.7 T 369.3†

3,012.3 T 341.5†

G0.001

jdp/dtmax (mmHg/s)

3,357.6 T 307.1

2,022.4 T 381.8*

2,746.9 T 391.2†

2,732.8 T 396.8†

2,889.0 T 331.4†

G0.001

448.4 T 22.6

453.3 T 56.8

427.3 T 61.8

486.8 T 44.2

463.0 T 54.4

0.383

†

HR (beats/min) MAP (mmHg) LVEDP (mmHg)

I/R (n = 6)

I-PostC (n = 6)

P

139.6 T 9.3

113.1 T 8.8*

125.7 T 9.9

119.3 T 8.4

131.5 T 7.9

G0.001

9.2 T 1.2

16.8 T 3.1*

13.4 T 2.9

13.7 T 2.8

13.5 T 2.2

0.002

Continuous variables are presented as mean and SD, which were compared between survival and death by one-way ANOVA. *Significant difference when compared with sham. †Significant difference when compared with I/R.

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FIG. 2. Protein expression of GRP78 and calreticulin in the five groups. Comparisons between the five groups in (A) GRP78 and (B) calreticulin expression. n = 3 per group. Variables are presented as mean and SD and compared by one-way ANOVA. *Significant difference compared with sham. †Significant difference when compared with I/R. C, Representative images of protein expression of GRP78, calreticulin, and GAPDH. Note: GAPDH is a reference. 1. Sham group; 2. I/R group; 3. I-PostC group; 4. PPC 0.5 mg group; 5. PPC 2 mg group.

patients are taking atorvastatin clinically, and chronic use of atorvastatin has been reported to interfere with the cardioprotective effects of I-PostC (24, 25, 36). Statins activate the p13K/Akt/eNOS pathway, and activation of this pathway is thought to mediate both the ischemic preconditioning and I-PostC effect on infarct size. However, during chronic statin use, negative feedback from activation of the p13K/Akt/eNOS pathway causes PTEN, an inhibitor of the pathway, to increase. The increase in PTEN may be the cause of the interference of chronic statin with I-PostC (25, 26). Other differences between acute and chronic statin are that chronic statin has been shown to block the increase in eNOS caused by postconditioning (25) and that chronic statin lacks the acute statin-induced increase in phosphor-p42, a mediator of the prosurvival p42/p44MAPK/ ERK pathway (36). Two studies suggest that chronic atorvastatin will not interfere with the beneficial effects of acute atorvastatin on I/R injury. Mensah et al. (26) have reported that the addition of acute atorvastatin to chronic atorvastatin causes a significant decrease

in I/R-induced infarct size. In addition, Di Sciascio et al. (37) reported that addition of acute atorvastatin to patients on chronic atorvastatin who underwent percutaneous coronary intervention significantly decreased the incidence of adverse coronary events, the major adverse event being perioperative myocardial infarction. Further studies of the effect of an acute atorvastatin dose superimposed on chronic atorvastatin need to be performed to determine the biochemistry involved in any interaction. Limitations of the present study

The mechanism of I/R injury is complex and multifaceted, and the mechanism of statin-induced cardioprotection of the myocardium is also multifaceted and is mediated by many intracellular pathways. So although this study focused on inhibition of ER stressYmediated apoptosis as a mechanism for cardioprotection and the involvement of this mechanism was supported by some of the conclusions, inhibition of ER stressYrelated apoptosis is only one of the pathways involved in cardioprotection from I/R injury. In addition, in the present

FIG. 3. Protein expression of apoptosis-related proteins in the five groups. Comparisons between the five groups in (A) CHOP, (B) ProYcaspase 12, (C) caspase 12, (D) Bcl-2, and (E) Bax expression. n = 3 per group. Variables are presented as mean and SD and compared by one-way ANOVA. *Significant difference compared with sham. †Significant difference compared with I/R. F, Representative images of protein expression of CHOP, ProYcaspase 12, caspase 12, Bcl-2, and Bax. 1. Sham group; 2. I/R group; 3. I-PostC group; 4. PPC 0.5 mg group; 5. PPC 2 mg group.

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FIG. 4. Comparisons of apoptosis rates of the five groups. n = 5 per group. Representative images of TUNEL staining. B, Histogram of TUNEL assay. Variables are presented as mean and SD and compared by one-way ANOVA. *Significant difference compared with sham. †Significant difference compared with I/R.

FIG. 5. Comparisons between the five groups in (A) AN/AAR and (B) AAR/LV. n = 5 per group. Variables are presented as mean and SD and compared by one-way ANOVA. *Significant difference compared with the I/R group. C, TTC staining of myocardium: from right to left: heart base to apex. Blue: nonischemic region; gray: infarcted region; red: ischemic but noninfarcted region. AN, infarcted region; LV, area of LV.

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