Acute myocardial infarct (AMI) remains a leading cause of mortality throughout the world. Timely reperfusion is the most effective strategy to salvage cardiac cell viability (1). Nonetheless, reperfusion itself may produce additional damage, leading to cell apoptosis and necrosis (2). Remote ischemic preconditioning (RIPC) is an attractive new strategy, whereby transient ischemia and reperfusion of an organ or tissue remote from the heart confers protection against subsequent lethal injury (3, 4). Activation of reperfusion injury salvage kinase (RISK) pathway (mainly PI3K-Akt-GSK3β axis and ERK1/2) may contribute to RIPC-induced myocardial protection (5–8). Multiple prosurvival signaling pathways, including MG53-PI3K-Akt and ERK1/2, converge on glycogen synthase kinase 3β (GSK3β). And the phosphorylation of GSK3β inhibits the opening of mitochondrial permeability transition pore (MPTP) and reduces mitochondria-dependent apoptosis and necrosis (9). Recent studies have demonstrated that MG53, a newly identified tripartite motif-containing (TRIM) family protein, elicits cardioprotection through the activation of PI3K-Ak-GSK3β axis and ERK1/2 pathway in response to IR (8); however, whether MG53 is related to the cardiac RIPC remains elusive.
In recent years, RIPC has been investigated extensively in healthy subjects, RIPC induced by three cycles of 5 min ischemia and 5 min reperfusion in the left upper arm provided perioperative myocardial protection and improved the prognosis of patients undergoing elective CABG surgery (3). However, the effect of RIPC on hypercholesterolemic hearts remains unclear. Hypercholesterolemia is prevalent in patients suffering from myocardial infarct, which is not only detrimental for coronary artery disease progression but also a risk factor for cardiac death in patients after AMI (10). Hypercholesterolemia is associated with increased myocardial oxidative stress indicators and pro-inflammatory proteins, attenuation of prosurvival signals, leading to myocardial infarct expansion in the setting of AMI (11). Furthermore, hypercholesterolemia has been shown to abrogate IPC-induced myocardial infarct-sparing effect by inhibiting nitric oxide synthase expression (12, 13). These studies indicate that hypercholesterolemia may adversely affect RIPC-induced cardioprotection. Therefore, we tested the hypothesis that hypercholesterolemia may attenuate remote ischemic preconditioning-induced cardioprotection by alteration of reperfusion injury salvage kinase signals.
MATERIALS AND METHODS
Male Sprague–Dawley rats weighing 130 g to 180 g were supplied by the Shanghai Laboratory Animal Center. All the protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (the 8th Edition, NRC 2011), and approved by the Institutional Review Board of Zhongshan Hospital at Fudan University.
In the first protocol, we assessed the effect of RIPC on myocardial infarct size and apoptosis in normocholesterolemic and hypercholesterolemic (HC) rats. The experiments were conducted as follows: normocholesterolemic ischemia reperfusion group (IR): rats were fed with standard pellet chow for 8 weeks and received no further treatment before myocardial ischemia; normocholesterolemic remote ischemic preconditioning group (IR + RIPC): rats were fed with standard pellet chow for 8 weeks and then treated with three episodes of 5 min femoral artery ischemia followed by 5 min reperfusion immediately prior to myocardial ischemia; hypercholesterolemic ischemia reperfusion group (HC + IR): rats were fed with 2% cholesterol pellet chow for 8 weeks and received no further treatment before myocardial ischemia; hypercholesterolemic remote ischemic preconditioning group (HC + IR + RIPC): rats were fed with 2% cholesterol pellet chow for 8 weeks and then treated with three episodes of 5 min femoral artery ischemia followed by 5 min reperfusion immediately prior to myocardial ischemia. To determine whether both PI3K-Akt and MEK-ERK1/2 signals are essential for the myocardial protection induced by RIPC, the selective PI3K-Akt inhibitor wortmannin (10 μg/kg, i.v., 45 min before ischemia) and the specific MEK-ERK1/2 blocker PD98059 (1 mg/kg, i.v., 45 min before ischemia) were administered to normocholesterolemic and hypercholesterolemic rats (14, 15)
In the second protocol, we assessed the role of GSK3β in RIPC-induced cardioprotection in both normocholesterolemic and hypercholesterolemic rats. GSK3β inhibitor SB216763 (0.6 mg/kg, i.v., 10 min before ischemia) was administrated to normocholesterolemic and hypercholesterolemic rats exposed to IR (16, 17).
Models of hypercholesterolemia
Male Sprague–Dawley rats were fed with 2% cholesterol diet for 8 weeks to produce hypercholesterolemia (18). A control group was fed with normal chow for 8 weeks. At the end of the 8 weeks, the animals fed with cholesterol diet developed a moderate and steady increase in serum cholesterol and were used for left anterior descending artery ligation experiments.
Serum lipid assay
Blood was harvested from the caudal vein and centrifuged (3000 rpm, 10 min, 4°C) to obtain serum. Serum lipid levels were measured by spectrophotometry using commercial assay kits for total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) (Beijing BHKT Clinical Reagent Co, Ltd, Beijing, China) according to the manuals and as described by Ballantyne et al. (19).
To determine that chronic treatment with a high-cholesterol diet for 8 weeks does not result in the development of coronary atherosclerosis in rats, hematoxylin-eosin staining for thoracic aorta, and coronary artery was conducted. The small segments of thoracic aorta and the hearts were harvested from five rats fed with either high cholesterol or normal chow for 8 weeks for histologic examination. The samples were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin-eosin staining was conducted as Iliodromitis et al. (20) have described elsewhere.
The IR surgery was performed according to the methods of Zhang et al. (16) with modifications. Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), plus additional doses (25 mg/kg, i.p.) every 60 min to maintain effective anesthesia (21), and were mechanically ventilated with room air to maintain arterial pH, PCO2 and PO2 within the normal physiological range. The pericardium was opened and a 6-0 silk suture was placed around the left anterior descending coronary artery through a small polytetrafluoroethylene tube. The tube was pulled to occlude the coronary artery for 30 min and the occlusion was confirmed by epicardial cyanosis in the area at risk, while successful reperfusion (for 120 min) was verified by epicardial hyperemia.
Remote ischemic preconditioning protocol
Remote ischemic preconditioning was induced by occluding the femoral artery with a microvessel clip for 5 min followed by 5 min reperfusion in 14-week male Sprague–Dawley rats after anesthetized with sodium pentobarbital (50 mg/kg, i.p.) (22). Occlusion was confirmed by a change in the color of skin and a reduction in the temperature of the paw. Hyperemia and restoration of normal color and temperature confirmed reperfusion. Three episodes of 5 min ischemia followed by 5 min reperfusion were used.
Determination of infarct size
At the end of reperfusion, the coronary artery was reoccluded and perfused with 1% Evans blue dye to identify the unstained area as the area at risk. The heart was quickly excised, frozen, cut into transverse slices, and incubated in 1% triphenyltetrazolium chloride solution at 37°C for 10 min. The areas of infarct (pale) and risk (red) were measured by planimetry using ImageJ 1.37 from the NIH (Bethesda, Md). The infarct size was expressed as a percentage of the area at risk. The area at risk was expressed as a percentage of the left ventricular. Samples with an area at risk of less than 15% or more than 45% of the left ventricle were excluded (23). Eight samples were used in each group.
Detection of myocardial apoptosis
Apoptosis was assessed using the TUNEL method. At the end of reperfusion, six hearts taken from each group were fixed in 4% paraformaldehyde and embedded in paraffin for TUNEL staining. The heart tissue sections were stained using an in situ cell death detection kit (POD; Roche Diagnostics Corp, Indianapolis, Ind), following the manufacturer's protocol. Ten microscopic fields (×400) from each section were assayed by counting TUNEL-positive cells. The percentage of TUNEL-positive nuclei (green nuclei) was calculated.
Five minutes following reperfusion, six hearts were taken from each group. The samples taken from ischemic zone were used for immunoblotting study. The expression of myocardial Akt, phosphorylated-Akt (Ser473) (p-Akt), ERK1/2, and phosphorylated-ERK1/2 (Thr202/Tyr204) (p-ERK1/2), GSK3β, phosphorylated-GSK3β (Ser9) (p-GSK3β) (Cell Signaling Technology, Beverly, Mass), phosphorylated-GSK3β (Tyr216) (Abcam, Cambridge, Mass) were determined by Western blotting as we described elsewhere (24–26). At the end of reperfusion, the expression of MG53, PI3K-p85 (Gene Tex, San Antonio, Tex) and cleaved Caspase-3 (Abcam, Cambridge, Mass) were determined. The quantitative protein band density was assayed by ImageJ 1.37.
Data are shown as mean ± SD. Lipid levels were analyzed using the unpaired Student t test. All other data were analyzed by 1-way ANOVA following Tukey post hoc test. A value of P <0.05 was considered to be statistically significant. All statistical analyses were performed using GraphPad Prism Version 6.0 (GraphPad Prism Software, San Diego, Calif).
High-cholesterol diet induced a moderate and steady increase in serum cholesterol without substantial development of coronary atherosclerosis in rats
Levels of total cholesterol (TC) (136. 8 ± 16.1 mg/dL), low-density lipoprotein cholesterol (LDL-C) (60.1 ± 9.1 mg/dL), and triglyceride (TG) (129.7 ± 10.8 mg/dL) were increased in rats fed with high-cholesterol chow compared with those (69.7 ± 7.1 mg/dL, 22.9 ± 4.8 mg/dL, and 65.3 ± 8.9 mg/dL) fed with normal chow (P <0.05). The level of high-density lipoprotein cholesterol (HDL-C) (31.5 ± 5.1 vs. 28.7 ± 4.6) was not significantly different between normocholesterolemic and hypercholesterolemic rats. Atherogenesis was detected in the form of subintimal accumulation of lipids and foamy macrophages. There was no deposition of lipids and foamy macrophages in the subintimal area in the cross-section of the aortic wall (Fig. 1A) and the myocardial branch of coronary artery from rats fed with either high cholesterol or normal chow for 8 weeks (Fig. 1B).
Hypercholesterolemia abrogates the myocardial infarct-sparing effect of RIPC
The area at risk was not significantly different among all groups (data not shown). As shown in Figure 2, the myocardial infarct size was larger by 18% in hypercholesterolemic rats than normocholesterolemic ones (HC + IR vs. IR; P <0.05), indicating increased myocardial necrosis in hypercholesterolemic rats exposed IR. RIPC significantly reduced the infarct size in normocholesterolemic rats (IR + RIPC vs. IR, P <0.05), a phenomenon was completely blocked by PI3K inhibitor wortmannin but not MEK-ERK1/2 inhibitor PD98059. The reduced myocardial infarct conferred by RIPC was not found in hypercholesterolemic rats exposed to IR. Neither wortmannin nor PD98059 further exacerbated the myocardial infarct in hypercholesterolemic rats received RIPC compared with the HC + IR and HC + IR + RIPC groups.
Hypercholesterolemia inhibits the myocardial anti-apoptotic effect of RIPC
As shown in Figure 3, RIPC significantly decreased the number of TUNEL-positive nuclei expressed as a percentage of total nuclei in healthy rats but was less effective to reduce the number of TUNEL-positive nuclei in hypercholesterolemic ones. Interestingly, the number of TUNEL-positive nuclei was significant higher in hypercholesterolemic hearts compared with normocholesterolemic ones (HC + IR vs. IR; P <0.05), indicating increased myocardial apoptosis in hypercholesterolemic rats exposed to IR.
Hypercholesterolemia abrogates the upregulation of MG53 expression induced by RIPC
RIPC significantly upregulated the expression of MG53 in healthy rats (P <0.05), a phenomenon was completely blocked by hypercholesterolemia (Fig. 4A). The expression of MG53 was not significantly different between IR and HC + IR groups.
Hypercholesterolemia abrogates the upregulation of PI3K-p85 and p-Akt expression induced by RIPC
The levels of total Akt were not significantly different among all groups. Therefore, the levels of p-Akt (Ser473) were expressed as the percentage of total protein. RIPC significantly increased the expression of PI3K-p85 and p-Akt in healthy rat hearts (P < 0.05); however, this did not occur in hypercholesterolemic ones subjected to RIPC. The expression of PI3K-p85 and p-Akt did not significantly differ between IR and HC + IR groups (Fig. 4, B and C).
The expression of p-ERK1/2 was not altered by neither RIPC nor hypercholesterolemia
No significant differences were found in the expression of total ERK1/2 among any groups. Importantly, the expression of p-ERK1/2 (Thr202/Tyr204) was not altered by either RIPC or hypercholesterolemia in the setting of IR (Fig. 4D), confirming the role of the MEK-ERK1/2 pathway in the cardioprotective effect of RIPC.
Hypercholesterolemia abrogates the upregulation of p-GSK3β expression induced by RIPC
Immunoblots of total GSK3β were not significantly different between groups. RIPC significantly increased the p-GSK3β (Ser9) in healthy rats (P <0.05); however, RIPC was less effective to enhance the phosphorylation levels of GSK3β (Ser9) in hypercholesterolemic rat hearts. The expression of p-GSK3β (Ser9) did not significantly differ between IR and HC + IR groups (Fig. 4E). The expression of p-GSK3β (Tyr216) was not altered by either RIPC or hypercholesterolemia in the setting of IR (Supplementary figure 1, http://links.lww.com/SHK/A466).
Hypercholesterolemia abrogates the downregulation of cleaved Caspase-3 expression induced by RIPC
As shown in Figure 4F, RIPC significantly decreased the expression of cleaved Caspase-3 in normal hearts exposed to IR, a phenomenon was blocked by hypercholesterolemia. Importantly, the expression of cleaved Caspase-3 was significantly higher in hypercholesterolemic hearts compared with normocholesterolemic ones (P <0.05), indicating increased myocardial apoptosis in hypercholesterolemic hearts subjected to acute coronary occlusion.
Pretreatment with GSK inhibitor exerted cardioprotection in hypercholesterolemic hearts exposed to IR
The area at risk was not significantly different among all groups (data not shown). As shown in Figure 5, RIPC significantly reduced the infarct size in normocholesterolemic rats but not in hypercholesterolemic ones. Interestingly, we found pretreatment with SB216763 before myocardial ischemia significantly reduced infarct size in both normal and hypercholesterolemic hearts exposed to IR (IR + SB vs. IR, P < 0.05; HC + IR + SB vs. HC + IR, P < 0.05); however, no additional cardioprotective effect was achieved when we combined RIPC with SB216763 pretreatment in hypercholesterolemic hearts exposed to IR (HC + IR + RIPC + SB vs. HC + IR + SB; P >0.05).
Although remote ischemic preconditioning confers cardioprotection against IR injury, few studies have investigated the myocardial effect of RIPC on subjects with hypercholesterolemia. We found that the myocardial infarct size and apoptosis was significantly increased in hypercholesterolemic rat hearts than that in healthy ones in the setting of IR. Furthermore, RIPC induced cardioprotection against reperfusion injury, in terms of decreased infarct size and apoptosis, in normocholesterolemic rats, which was lost in hypercholesterolemic rat hearts with altered MG53-RISK signaling pathway that inhibit GSK3β.
We used chronic treatment with a high-cholesterol diet for 8 weeks to induce hypercholesterolemia in rats (18, 25, 27). We found that this diet induced a moderate and steady increase in serum cholesterol without substantial development of coronary atherosclerosis (Fig. 1), which is consistent with previous studies and suggests that the factor influencing ischemic conditioning in our study was hypercholesterolemia itself, and not subsequent atherosclerosis or other hypercholesterolemic complications (28, 29). Therefore, we used this hypercholesterolemic model to study the direct effects of hypercholesterolemia on remote ischemic preconditioning.
Effects of hypercholesterolemia on myocardial IR have yielded controversial results in rabbits and rodents. Recent studies have shown that hypercholesterolemia increase myocardial necrosis in the setting of IR (18, 27, 30), but the underlying mechanism is poorly understood. Increased inflammation and oxidative stress may contribute to increased myocardial infarct size in the setting of hypercholesterolemia during myocardial IR (11). In our study, the myocardial infarct size was increased by 18% in hypercholesterolemic rat hearts than in healthy ones. The finding that hypercholesterolemic rat hearts have larger infarct size than normocholesterolemic ones was consistent with the results of several previous studies (18, 27, 30). In contrast, infarct size did not significantly differ between normocholesterolemic and hypercholesterolemic rat hearts exposed to IR (31, 32). The reasons for these controversial results from different study groups are largely unclear, but may be attributable to the different IR models (including the period of IR, in vivo or in vitro), the different high-cholesterol diet components (1% cholesterol diet vs. 2% cholesterol diet) and treatment periods (2 weeks vs. 8 weeks) used by different research groups.
Another principle finding of our current study was hypercholesterolemia alone increased myocardial apoptosis in the setting of IR. One reasonable explanation is increased free oxygen radicals and inflammation may prompt apoptotic signaling pathway (33). Furthermore, hypercholesterolemia is associated with decreased expression of anti-apoptotic Bcl-2 (11), which plays a pivotal role in preventing apoptosis by inhibiting the activation of executioner Caspases-3 and the release of mitochondrial Cytochrome C (34, 35). In the present study, we also noted that the expression of cleaved Casepase-3 was significantly increased in hypercholesterolemic hearts compared with that in normal ones in the setting of IR. All the above may contribute to the increased myocardial apoptosis in the setting of hypercholesterolemia during IR.
RIPC is an inexpensive technique that is used to reduce myocardial infarct for patients suffering from AMI. Hypercholesterolemia has been known for some time to increase mortality in patients suffering from AMI (10, 36). However, effect of hypercholesterolemia on myocardial response to RIPC is still unclear. The present study has shown that hypercholesterolemia induced by high-cholesterol diet impaired myocardial response to RIPC and modified cardioprotective signaling pathways. Studies using healthy animals have demonstrated that PI3K-Akt-GSK3β signal plays a major role in reducing myocardial infarct-induced by RIPC (5, 7). In contrast, the role of ERK 1/2 as part of the signaling pathway and the phosphorylation of this kinase during RIPC stimulus are more debatable and has led to conflicting conclusions. In the present study, we did not detect any significant alteration in the phosphorylation status of ERK 1/2 in the normal myocardium directly after RIPC, which is consistent with previous studies investigating ERK1/2 activation in the myocardium after RIPC in rat in vivo(5, 6, 7). Our further investigation showed that MEK-ERK1/2 inhibitor PD98059 did not abolish RIPC-induced myocardial infarct-sparing effect in normal myocardium exposed to IR. These results indicate that the activation of ERK1/2 in the myocardium directly after RIPC is not necessarily an essential event for myocardial protection in our current study. In contrast, a recent study showed RIPC upregulated the expression of ERK1/2 and prompted the translocation of PKC in the rabbit heart in vivo(37). And, Jin et al. (38) observed increased phosphorylation of ERK1/2 in rat hippocampus after limb ischemic preconditioning. The reasons for these conflicting results are largely unknown, but may be related to animal species difference or tissue-specific differences.
The signaling mechanisms upstream of Akt responding for RIPC-induced cardioprotection is still poorly understood. MG53, a TRIM-family protein, forms a functional complex with the p85 subunit of PI3K and contributes to acute membrane repair in cardiomyocytes (8, 39). In addition, the myocardial infarct size was larger in MG53-deficient mice exposed to IR and overexpression of MG53 attenuates hypoxia- and oxidative-induced cell death by enhancing the phosphorylation of Akt (8, 39). These results indicate that MG53 plays a pivotal role in reducing cell death and is the upstream signal of PI3K-Akt pathway. The importance of enhanced phosphorylation of Akt and GSK3β has been indicated in cardioprotection afforded by RIPC and phosphorylation of GSK3β activated by Akt has been shown to inhibit mitochondrial-dependent apoptosis and necrosis by preventing the opening of MPTP (9). In healthy rat hearts, RIPC induced up-regulated MG53 and PI3K-p85, phosphorylated Akt, and GSK3β, however, which did not occur in hypercholesterolemic ones. We also noted that hypercholesterolemia did not alter the expression of MG53 and PI3K-p85, phosphorylated Akt and GSK3β compared with untreated healthy rats. These results imply that the dysfunction of MG53, the signaling mechanism upstream of Akt, is responsible for impaired Akt activation. Furthermore, the inactivation of GSK3β may in part be attributed to the observed deficient in Akt phosphorylation. If, as mentioned above, up-regulation of phosphorylated Akt and GSK3β plays a pivotal role in the cardioprotective effect of RIPC; we reasoned that the loss of benefit of RIPC in hypercholesterolemic myocardium, at least in part, be attributed to the dysfunction of Akt and GSK3β phosphorylation.
During myocardial IR, the phosphorylation of GSK3β inhibits the opening of MPTP and reduces mitochondria-dependent apoptosis and necrosis. Regarding its pivotal role in reperfusion injury, the GSK3β has become an obvious target for ischemic/pharmacological preconditioning-induced cardioprotection (9). Indeed, we found direct inhibition of GSK3β with SB216763 conferred myocardial cardioprotective effect in normal and hypercholesterolemic hearts, but had no additional cardioprotective effect was achieved in hypercholesterolemic ones received RIPC. These findings indicate that GSK3β is involved in RIPC-induced cardioprotection and direct inhibition of GSK3β would be a more promising therapeutic target to protect hypercholesterolemic hearts against IR injury.
In summary, we report that RIPC-induced cardioprotection against IR injury was abrogated by alteration of MG53-mediated PI3K-Akt-GSK3β pathway in hypercholesterolemic rats, whereas GSK3β inhibitor SB216763 maintained its cardioprotective effect in both normal and hypercholesterolemic rats. These data suggest that direct inhibition of GSK3β before myocardial ischemia would be a potential therapeutic approach to prevent IR injury in the presence of hypercholesterolemia.
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