
viously. [10] [11] [12] In brief, ligation of the LAD coronary artery was performed in penbarbital-anesthetized rats, which were orally intubated and mechanically ventilated. Left thoracotomy was performed by opening the fourth intercostal space. The heart was exposed and the pericardium was cut. The LAD was ligated with a 6.0 silk suture between the left atrium and the pulmonary artery outflow tract. Myocardial ischemia was verified by the appearance of epicardial cyanosis and ischemic ST-segment changes in the electrocardiogram. Then, the thorax was de-aired and closed. Surviving rats were maintained in stand rat chow and water ad libitum. To further verify the successful LAD ligation, acute infarct size was determined 12 h after coronary artery ligation using 1% 2,3,5-triphenyltetrazolium chloride (TTC) staining method (nϭ5), and a mean infarct size of 33Ϯ5% was obtained (Table 1) . Separate experiment was performed to evaluate the hemodynamic changes caused by chronic infarction. To do so, five rats of both post-infarct and sham-operated groups were sacrificed 6 weeks after surgery, five agematched rats without previous surgery served as healthy control. Sham-operated animals underwent the same surgical procedure except that the suture was passed under LAD without ligation. Baseline cardiac function of these postinfarcted, sham-operated and healthy control hearts on the Langendorff apparatus was compared.

Langendorff Heart Preparation and Hemodynamic Recordings Six weeks after LAD ligation, post-infarct rats were anesthetized by intraperitoneal injection of sodium pentobarbital 40 mg/kg and heparinized (1000 IU/kg). Once surgical anesthesia was achieved (confirmed by no response to tail clamping), hearts were rapidly excised, placed in ice-cold Krebs-Henseleit (K-H) buffer, and mounted on a modified non-circulating Langendorff apparatus via aorta cannulation. Then, hearts were retrogradely perfused at a constant pressure of 80 mmHg with K-H buffer (pH 7.40Ϯ0.05, 37.0Ϯ 0.5°C), which was continuously gassed with 95%O 2 ϩ 5%CO 2 . The composition of the K-H buffer was (in mmol/l): NaCl 118.0, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, CaCl 2 1.8, NaHCO 3 25.0, glucose 11.1 and ethylenediaminetetraacetic acid (EDTA) 0.5. A small latex balloon connected to a pressure transducer was inserted through the left atrium and pushed through the mitral valve into the left ventricle. The balloon was filled with saline to achieve an end-diastolic pressure of 0-10 mmHg. Then, the balloon volume remained unchanged during the following experiment. All analog signals were digitized with Biopac 5100 (Biopac Systems Inc., CA, U.S.A.) and recorded at 1000 Hz with AcqKnowledge version 4.0 software (Biopac Systems Inc., CA, U.S.A.) on a personal computer. Characteristic data derived from left ventricular pressure measurement were left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal and minimal first derivatives of LVP (Ϯdp/dt) as indices of contractility and relaxation, and heart rate (HR). All variables were collected when hearts beat in sinus rhythm and averaged over a sampling period of 2 s. Coronary flow (CF) was measured by timed collection of coronary effluent.

Experimental Protocol Spontaneously beating hearts were equilibrated for 20 min. After that, hearts were assigned randomly into one of the following groups, as displayed in Fig. 1 . Hearts in time control (TC) group were constantly perfused with K-H buffer for 90 min. Hearts in ischemiareperfusion (ISCH) group were subjected to 30 min of ischemia followed by 60 min of reperfusion alone (Ischemia was achieved by interruption of buffer perfusion to the hearts). After 30 min of ischemia, hearts in sevoflurane post- 5, 6) In order to show whether DMSO affect the results of the experiments, ISCHϩDMSO group with 0.02% DMSO given alone for the first 15 min after ischemia was performed. For each experimental group, ten hearts were prepared, and functional parameters were recorded (nϭ10). LY, PD and DMSO were all purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.).
Determination of Myocardial Infarction and Injury
Myocardial infarct size (IS) was determined by TTC staining. [10] [11] [12] At the end of the experiments, hearts were frozen at Ϫ20°C for 2 h and subsequently cut into five cross-section slices. Heart slices were incubated in a 1% TTC in 0.1 M phosphate buffer solution (pH 7.4) at 37°C for 20 min, and fixed overnight in 10% formaldehyde. Then, infarcted myocardial areas (pale color) can be differentiated from viable ones (brick-red color). Slices were photographed by digital camera, and computerized planimetry using Image J 1.37 software was performed. IS was determined by dividing the total necrotic area of the left ventricle by the total left ventricle slice area. For IS determination, five hearts (nϭ5) were assessed in each experimental group. As additional markers of myocardial injury, lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were determined from collected effluent by an automatic biochemistry analyzer (Hitachi 7600, Tokyo, Japan) using commercial LDH and CK-MB assay kits (Roche Diagnostics, Mannheim, Germany). For these purposes, coronary effluent (1 ml) was spot collected from hearts in the three groups (nϭ10) during equilibration and at the end of experiments, respectively. All coronary effluent samples were coded, and the laboratory investigator was blinded to the treatment regimen.
Determination of Myocardial Infarction and Injury
Western Blotting Analysis To determine the effects of sevoflurane exposure on phosphorylation of PKB/Akt and ERK 1/2, five hearts of each experimental group were prepared (nϭ5). At 15 min of reperfusion, left ventricular tissue samples were collected, immediately snap frozen in liquid nitrogen, and stored at Ϫ80°C till analysis. Samples were then powdered under liquid nitrogen and homogenized in lysis buffer (20 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton, 3% SDS, 1 mmol/l phenylmethylsulfonyl fluoride, and 5 mg/ml protease inhibitor cocktail). The homogenates were vortexed for 15 s and then centrifuged at 14000 g for 10 min at 4°C. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard. Equal amounts of protein were electrophoresed on a 12.5% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with polyclonal primary antibodies specific for total PKB/Akt, phosphorylated PKB/Akt , total ERK 1/2 and phosphorylated ERK 1/2
Determination of Myocardial Infarction and Injury
Thr-202/Tyr-204 (Santa Cruz Biotechnology Inc., CA, U.S.A.). Coomassie blue and Ponceau red were used to verify adequate transfer of proteins from the gel to the membrane. b-actin was used to ensure equal protein loading. The membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology Inc., CA, U.S.A.). Proteins were detected using chemiluminescence, bands were visualized by exposure to photographic film (Fuji Protein, Tokyo, Japan). Quantitive analysis of the band densities was performed using NIH Image 1.63 software, phosphorylated PKB/Akt and ERK 1/2 densities were normalized against those of total PKB/Akt and ERK 1/2, respectively. mPTP Opening Determination mPTP is an important end-effector in MIRI. [13] [14] [15] mPTP remains closed during ischemia and opens in the first few minutes of reperfusion. [16] [17] [18] Pharmacologically inhibiting the opening of mPTP upon reperfusion is cardioprotective. 19, 20) Mitochondria possess more than 90% of the total tissue content of nicotinamide adenine dinucleotide (NAD ϩ ). 21, 22) During reperfusion, NAD ϩ released from inactive and dysfunctional mitochondria upon opening of the mPTP, was washed out by coronary effluent. Therefore, low NAD ϩ content in cardiac tissue is an indicator of mPTP opening. 21, 22) After 15 min of reperfusion, NAD ϩ was extracted from left ventricular tissue at risk using the Klingenberg method 23) (nϭ5). To do so, 30 mg freeze-clamped tissue was powdered in a mortar and thoroughly mixed with 14% perchloric acid. The mixture was then homogenized and centrifuged at 0°C at 1970 g for 10 min. The centrifuged sediment was dissolved in 0.1 mol/l NaOH solution and the protein concentration was measured with Protein Concentration Measurement Kit (Applygen, Beijing, China) using bicinchoninic acid (BCA) method. In an ice bath, 0.2 ml of 1.0 mol/l K 2 HPO 4 solution was added to the supernatant and then 3.0 mol/l KOH was added until the pH was neutralized. After the KClO 4 was precipitated, the supernatant fluid was removed and NAD ϩ concentrations were determined fluorometrically using alcohol dehydrogenase (ADH) at a wavelength of 340 nm (DU 640, Beckman Coulter, Fullerton, CA, U.S.A.).
Determination of Myocardial Infarction and Injury
Statistical Analysis Data are presented as meanϮS.D. For hemodynamic data, repeated-measures analysis of variance was used to evaluate differences over time between groups. An unpaired t test was used to compare groups at identical time points, and a paired t test was used to compare within groups over time. For all other data, one-way analysis of variance (ANOVA) with post hoc Tukey test for multiple comparisons was used, and p value Ͻ0.05 was considered statistically signficant. All statistical analysis was performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, U.S.A.).
RESULTS

Characterization of Post-infarct Hearts
In accordance with previous studies, [10] [11] [12] our study demonstrated that LAD ligation induced significant infarction (approximately 33% of left ventricular area) (Table 1) , which then initiated myocardial remolding in post-infarct healing process. Compared with sham-operated and healthy control hearts, the heart weight and the ratio of heart weight over body weight were significantly increased 6 weeks after LAD ligation in chronically-infarcted hearts, suggesting compensatory hypertrophy (Table 1) . Consistent with loss of viable myocardium and cardiac hypertrophy, impaired cardiac functions were observed. The baseline values of LVDP, inotrophy and lusitropy were significantly lower in the post-infarcted hearts when compared with sham-operated and healthy control ones (Table 1) .
Characterization of Post-infarct Hearts
Protective Effects of SpostC on Cardiac Function Baseline hemodynamics among all experimental groups of post-infarct hearts (pϾ0.05), data were summarized in Table  2 . Hemodynamic variables remained constant during the experiment in the TC hearts. In contrast to that, the ISCH hearts showed significantly deteriorated myocardial function, as manifested by decreased LVDP, ϩdp/dt, Ϫdp/dt, HR, CF and increased LVEDP (pϽ0.05). All hemodynamic variables were better in the SpostC hearts, when compared with the ISCH ones ( pϽ0.05). Cardioprotection of sevoflurane was completely abolished by coadministration of LY or PD (pϽ0.05). Administration of LY or PD alone did not further deteriorate postischemic recovery (pϾ0.05).
Protective Effects of SpostC on Myocardial Infarction and Injury
The infarct size of hearts in the ISCH group averaged 32Ϯ4%, which was significantly reduced in the SpostC group (20Ϯ3%, pϽ0.05) (Fig. 2) 
Fig. 2. Infarct Size Determination
Infarct size was determined using 1% tripphenyltetrazolium chloride (TTC) staining. In each group, five hearts were prepared (nϭ5). Infarct size was expressed as percent of necrotic area over left ventricular area. Filled triangles represent data points of each group, and respective meanϮS.D. value is presented on the right side. Time control (TC) group was not included in the figure because no significant necrotic area was identified. ISCHϭischemia; LYϭLY294002; PDϭPD98059; DMSOϭdimethyl sulphoxide; SpostCϭsevoflurane postconditioning. * pϽ0.05 vs. ISCH. influence IS (ISCHϩDMSO: 34Ϯ6%, pϾ0.05) (Fig. 2) .
Fig. 2. Infarct Size Determination
Effects on LDH and CK-MB Release LDH and CK-MB in coronary effluent during the baseline and after 60 min of reperfusion in the eight experimental groups are shown in Fig. 3 Effects on PKB/Akt and ERK 1/2 Activation Western blotting study showed that, ischemia-reperfusion enhanced both PKB/Akt and ERK 1/2 phosphorylation in the ISCH hearts when compared with the TC ones ( pϽ0.05) (Fig. 4) . Administration of 3% sevoflurane for 15 min further increased phosphorylation of both PKB/Akt and ERK 1/2, when compared with the ISCH hearts ( pϽ0.05). Both LY and PD, administered alone or together with sevoflurane, abolished PKB/Akt and ERK 1/2 phosphorylation, respectively (pϽ0.05). Either LY or PD, has no influence over ischemia-enhanced ERK 1/2 or PKB/Akt phosphorylation, respectively ( pϾ0.05). DMSO did not influence either PKB/Akt or ERK 1/2 phosphorylation when compared with the ISCH hearts (pϾ0.05).
Fig. 2. Infarct Size Determination
Effects on mPTP Opening TC hearts have the highest myocardial NAD ϩ content among all groups ( pϽ0.05). While, sevoflurane postconditioned hearts retained higher contents of NAD ϩ than other hearts exposed to ischemiareperfusion, which suggested that sevoflurane prevented NAD ϩ release upon reperfusion by inhibiting mPTP opening. LY, PD or DMSO, has no effect on mPTP opening when given alone ( pϾ0.05). However, either LY or PD abolished the inhibitory effect of SpostC on mPTP (pϽ0.05) (Fig. 5) . These results suggest that both PI3K and ERK 1/2 mediate SpostC-induced cardioprotection by inhibiting mPTP opening.
DISCUSSION
Several findings of the current study extended our understanding regarding anesthetic postconditioning. Firstly, our study showed that anesthetic postconditioning by sevoflurane retains its protection in chronically infarcted rat hearts, as manifested by significantly improved functional recovery, reduced infarct size and decreased release of LDH and CK-MB when compared to unprotected hearts. Till now, most studies investigating the phenomenon of anesthetic preconditioning and postconditioning are performed in healthy and juvenile animals. This is different from real clinical situations where those who need anti-MIRI interventions most are elderly patients with a variety of pathological conditions (e.g. coronary heart disease, heart failure). Those pathological conditions trigger short-term adaptive and long-term maladaptive process which leads to structural and metabolic changes, which make the viable myocardium more vulnerable to further ischemic damage and less receptive to anti-MIRI interventions. For example, Miki and colleagues 8, 9) found that infarcted hearts of rabbit were refractory to ischemic preconditioning. Hyperglycemia and hypercholesterolemia were reported to inhibit ischemic preconditioning, too. [24] [25] [26] Trabecula from heart failure patients also has impaired response to ischemic preconditioning. 7) However, in accordance with our findings in the current study, evidence from clinical studies shows that sevoflurane has superior cardioprotective effects than propofol in elderly patients undertaking coronary artery bypass surgery. [27] [28] [29] [30] [31] Similarly, experimental studies have demonstrated that anesthetic preconditioning and postconditioning induced by isoflurane, another volatile anesthetic commonly used in clinical anesthesia, retain anti-MIRI protection in infarct-remodeled rat hearts. 10, 12) Taken together, it is highly possible that cardioprotection elicited by volatile anesthetics is more effective than ischemic conditioning in diseased myocardium. To further confirm this, more studies are warranted to investigate the effects of disease-related alteration in signaling on cardioprotection induced by different stimuli.
DISCUSSION
Secondly, we found that inhibiting the activation of either PKB/Akt or ERK 1/2 at the time of reperfusion eliminated cardioprotection conferred by sevoflurane. Evidence indicates that the RISK pathway, comprised of PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades, could potentially become a common target of myocardial protection against MIRI. 4) However, the relative contribution of these two cascades to the cardioprotection induced by different stimuli remains controversial. Some studies suggested a predominant role for PI3K-PKB/Akt, 11, 32) some supported MEK 1/2-ERK 1/2 as the major mediator, 33) the other argued for equally importance for these two cascades. [34] [35] [36] [37] It is difficult to explain the discrepancy of these findings, possible reasons might include the difference in ischemia-reperfusion models, species and the stimuli (e.g. anesthetics) used. To be more specific to sevoflurane, one research group recently demonstrated in separate studies that the activation of both PKB/Akt and ERK 1/2 were essential in sevoflurane-induced cardioprotection in healthy rat hearts in vitro. 5, 6) Whether these protective signaling cascades are abolished or differentially regulated in diseased myocardium is unclear. Here, we demonstrated in chronically-infarcted hearts that inhibiting either PKB/Akt or ERK 1/2 at the time of reperfusion eliminated sevoflurane elicited cardioprotection, indicating that SpostC-induced cardioprotection requires the activation of both PI3K and ERK 1/2. Consistent with our findings, Hausenloy et al. 34) reported that inhibition of PKB/Akt and ERK 1/2 during the first 15 min of reperfusion after index ischemia completely abolished protection induced by ischemic preconditioning in isolated rat hearts. Schwartz and Lagranha. 35) found ischemic postconditioning also activated both PKB/Akt and ERK 1/2 in pigs subjected to regional ischemia in vivo. Similarly, another research group demonstrated in separate studies that both PKB/Akt and ERK 1/2 play an important role in isoflurane-induced cardioprotection. 36, 37) Convergence on the common downstream targets, such as p70S6K and BAD may explain, at least partially, the requirement for both PKB/Akt and ERK 1/2 to be activated to mediate protection. 38) The activation of p70S6K requires the phosphorylation at both Thr389 (the site phosphorylated by PKB/Akt) and Thr421, Ser424 (the sites phosphorylated by ERK 1/2), which would necessitate the activation of both the PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades to mediate cardioprotection. 39, 40) Studies have also demonstrated that signaling through PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades can converge on another distal target, the pro-apoptotic protein, BAD, the phosphorylation and inactivation of which would mediate cellular survival by an anti-apoptotic mechanism. [41] [42] [43] BAD may be phosphorylated and inactivated by PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades via the recruitment of p70S6K. 44) Thirdly, we found that ERK 1/2 activation was resistant to PI3K inhibitor LY294002, and that PKB/Akt activation was also resistant to MEK 1/2 inhibitor PD98059, indicating PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades may be independent upon each other. In contrast to that, previous studies suggested there exists cross-talk between PI3K-PKB/Akt and MEK 1/2-ERK 1/2 cascades, such that inhibiting PI3K activates ERK 1/2 and vice versa. For example, Hausenloy et al. 38) reported in ischemic preconditioning that inhibiting PI3K-PKB/Akt with LY 294002 at the time of reperfusion resulted in the activation of the MEK 1/2-ERK 1/2 cascade, and the inhibition of MEK 1/2-ERK 1/2 with PD98059 at the time of reperfusion resulted in the activation of the PI3K-PKB/Akt. The disparity in the findings between Hausenloy's investigation and ours may rest with the different anti-MIRI stimulus (ischemic preconditioning vs. sevoflurane postconditioning). Additionally, we can't rule out the possible existence of a complex time course of PI3K-PKB/Akt and MEK 1/2-ERK 1/2 interaction, by determining kinase activation at single time point. Actually, previous study has shown that PKB/Akt and ERK 1/2 manifest a biphasic change in response to ischemic preconditioning. 34) Therefore, additional experiments will be required to further delineate the roles of PI3K and ERK 1/2 and the time course of their interaction in sevoflurane postconditioning mediated cardioprotection.
DISCUSSION
Fourthly, we demonstrated that both PKB/Akt and ERK 1/2 activation mediated SpostC-induced cardioprotection by inhibiting mPTP opening, as shown by increased myocardial NAD ϩ contents compared to unprotected hearts. mPTP is the most important end-effector in the signaling network in MIRI, with numerous modulating pathways all finally converging on it.
DISCUSSION
13,14,16,19,45-47) At present, the exact mechanism through which PKB/Akt and ERK 1/2 manipulate mPTP is unclear. GSK-3b, a PI3K-PKB/Akt downstream target, has been reported to mediate convergence of myocyte protection signaling through inhibition of mPTP opening. 48) Other studies also have suggested similar manipulative effects over mPTP imposed by PI3K-PKB/Akt. 13, 47) Using murine myocytes over-expressing PKC-e, Baines and colleagues have demonstrated that ERK 1/2 could form signaling modules with PKC-e at the level of mitochondria, 49) and PKC-e has been shown to associate with components of mPTP and inhibit its opening. 50) Through p90RSK, ERK 1/2 might phosphorylate and inhibit GSK-3b, 51) then inhibit mPTP opening upon reperfusion. 48) Fifthly, it is worth to mention that, although the activation of both PI3K-PKB/Akt and MEK 1/2-ERK 1/2 has been reported by us and other investigators who examined the phenomenon of anesthetic preconditioning and postconditioning, the mechanism through which anesthetics activate these two key kinases is still unknown and rarely explorated. [52] [53] [54] Actually, our group has been conducting research in investigating the relationship between sevoflurane-induced reactive oxygen species (ROS) production and ERK 1/2 activation in healthy rat heart in vitro, and we found a stimulative role of ROS in ERK 1/2 activation during sevoflurane postconditioning (Yao et al.) . 55) Therefore, it would be interesting and crucial to further ascertain the exact mechanism of how these two key pro-survival kinases are activated by sevoflurane in future investigation.
DISCUSSION
In summary, our findings in this study showed that 3% sevoflurane postconditioning provides significant cardioprotection against MIRI in isolated post-infarct rat hearts. We also demonstrated that the two key players of RISK pathway, namely PKB/Akt and ERK 1/2, are both essential mediators in SpostC-induced cardioprotective mechanisms. Furthermore, our results indicated that sevoflurane postconditioning exerted its cardioprotection by inhibiting mPTP opening through the recruitment of these pro-survival kinases. Although the species of rat and human share very high degree of similarity in genome, our findings generated from rats in vitro should be cautiously extrapolated to human.

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