The authors have declared that no competing interests exist.
Conceived and designed the experiments: CZ LLM FJZ MY. Performed the experiments: CZ YX LLM FJK WNW CCW. Analyzed the data: CZ LBQ XBL JAW. Contributed reagents/materials/analysis tools: MY JAW. Wrote the manuscript: CZ LLM YX.
Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia. Therefore, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. In the present study, normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane for 5 min or 3 cycles of 10-s ischemia/10-s reperfusion. The hemodynamic parameters, including left ventricular developed pressure, left ventricular end-diastolic pressure and heart rate, were continuously monitored. The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. We found that both sevoflurane and ischemic postconditioning significantly improved heart pump function, reduced infarct size and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β in the healthy rats. In the hypercholesterolemic rats, neither sevoflurane nor ischemic postconditioning improved left ventricular hemodynamics, reduced infarct size and increased the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. In conclusions, hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.
Myocardial ischemia reperfusion injury can be reduced by multiple interventions, such as ischemic postconditioning [
In recent years, anesthetic postconditioning has mainly been documented in healthy subjects, and the effect of sevoflurane postconditioning on hypercholesterolemic rat heart remains unclear. A number of prospective clinical studies have shown that both coronary artery disease (CAD) and the risk factor for cardiac death after acute myocardial infarct (AMI) are directly related to hypercholesterolemia [
All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health). The Laboratory Animal Care Committee of Zhejiang University approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffering. The rats were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-hour light-dark cycle. Six-week-old male Sprague-Dawley rats, weighing 130-180 g, were used for all experiments.
To investigate whether sevoflurane-induced cardioprotection was maintained in hypercholesterolemic rats, the experiments were conducted as follows: 1) normocholesterolemic ischemia reperfusion group (IR): rats were fed standard pellet chow for 8 weeks and received no further treatment before myocardial ischemia; 2) normocholesterolemic sevoflurane postconditioning group (IR + SPO): rats were fed standard pellet chow for 8 weeks and then treated with 2.4% sevoflurane (Maruishi Pharmaceutical Co, Ltd, Osaka, Japan)
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 [
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 heart were harvested from 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 [
The IR surgery was performed according to the methods of Zhang et al [
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 left ventricle was separated, frozen, cut into transverse slices, and incubated in 1% triphenyltetrazolium chloride solution at 37°C for 10 min. The area of infarct (pale) and risk (red) was measured by planimetry using ImageJ 1.37 from the National Institutes of Health (Bethesda, MD, USA). The infarct size was expressed as a percentage of the area at risk. Samples with an area at risk <15% or >45% of the left ventricle were excluded [
Apoptosis was assessed using the TUNEL method. At the end of reperfusion, the hearts 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, IN, USA), following the manufacturer’s protocol. Ten microscopic fields (400×) from each section were assayed by counting brown nuclei. The percentage of TUNEL-positive nuclei (brown nuclei) was calculated.
Fifteen minutes following reperfusion, the samples were taken from ischemic zone. The expression of myocardial Akt, phosphorylated-Akt (Ser473) (p-Akt), ERK1/2 and phosphorylated-ERK1/2 (Thr202/Tyr204) (p-ERK1/2), GSK3β, phosphorylated-GSK-3β (Ser9) (p-GSK3β) (Cell Signaling Technology, Beverly, MA, USA) were determined by Western blotting as we described elsewhere [
Data are shown as mean ± SD. Lipid levels were analyzed using the unpaired Student’s
Seventy rats were used for myocardial infarction experiments (2 were excluded as a result of area at risk <15% and 4 died of refractory ventricular arrhythmias during the 30-min occlusion) and 79 animals were used for immunoblotting (7 died of refractory ventricular arrhythmias during the 30-min occlusion). An additional 53 rats were used for TUNEL staining (5 died of refractory ventricular arrhythmias during the 30-min occlusion). 10 rats were used for hematoxylin-eosin staining.
Levels of TC (136.5 ± 12.8 mg/dl), LDL-C (57.9 ± 7.2 mg/dl), and TG (128.2 ± 7.1 mg/dl) were increased in rats fed high-cholesterol chow compared with those (68.3 ± 6.6 mg/dl, 26.1 ± 4.8 mg/dl, and 59.4 ± 8.9 mg/dl) fed normal chow (
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 (
The arterial lumen is indicated by the arrow, n = 5 hearts/group.
As shown in
Group | Baseline | Ischemia | Reperfusion |
|||
---|---|---|---|---|---|---|
30 min | 60 min | 90 min | 120 min | |||
LVDP, mmHg | ||||||
IR | 127 ± 8 | 81 ± 5 | 108 ± 11 | 89 ± 9 | 84 ± 6 | 77 ± 10 |
IR + SPO | 130 ± 9 | 83 ± 5 | 111 ± 11 | 106 ± 5* | 101 ± 5* | 97± 6* |
IR + IPO | 123 ± 7 | 80 ± 7 | 115 ± 15 | 107 ± 11* | 102 ± 5* | 96 ± 7* |
HC + IR | 130 ± 12 | 85 ± 11 | 115 ±10 | 98 ± 9 | 88 ± 12 | 80 ± 13 |
HC + IR + SPO | 134 ± 8 | 80 ± 7 | 110 ± 7 | 94 ± 12 | 87 ± 13 | 83 ± 12 |
HC + IR + IPO | 127 ± 7 | 84 ± 5 | 107 ± 10 | 92 ± 7 | 86 ± 7 | 84 ± 8 |
LVEDP, mmHg | ||||||
IR | 4.1 ± 1.5 | 10.1 ± 1.6 | 5.3 ± 1.8 | 7.6 ± 1.8 | 7.9 ± 1.4 | 8.2 ± 1.5 |
IR + SPO | 3.9 ± 1.0 | 9.4 ± 1.7 | 4.5 ± 1.4 | 5.1 ± 1.2* | 5.9 ± 1.1* | 5.7 ± 1.5* |
IR + IPO | 4.0 ± 1.4 | 9.5 ± 2.1 | 5.0 ± 1.1 | 5.6 ± 1.2* | 5.5 ± 1.0* | 5.9 ± 1.1* |
HC + IR | 4.1 ± 1.0 | 9.9 ± 1.2 | 5.4 ± 1.0 | 6.9 ± 1.3 | 7.8 ± 1.0 | 8.4 ± 1.3 |
HC + IR + SPO | 4.5 ± 1.2 | 10.4 ± 1.1 | 6.3 ± 1.0 | 7.2 ± 1.2 | 7.4 ± 1.2 | 7.8 ± 1.5 |
HC + IR + IPO | 3.9 ± 1.4 | 10.0 ± 1.8 | 6.4 ± 1.6 | 7.0 ± 1.8 | 7.3 ± 1.5 | 7.7 ± 1.6 |
HR, beats/min | ||||||
IR | 345 ± 35 | 325 ± 41 | 312 ± 48 | 305 ± 39 | 315 ± 42 | 299 ± 41 |
IR + SPO | 332 ± 32 | 312 ± 41 | 315 ± 39 | 321 ± 35 | 306 ± 40 | 313 ± 39 |
IR + IPO | 350 ± 34 | 310 ± 34 | 318 ± 40 | 309 ± 37 | 310 ± 39 | 300 ± 41 |
HC + IR | 328 ± 31 | 308 ± 43 | 303 ± 39 | 300 ± 34 | 298 ± 41 | 288 ± 39 |
HC + IR + SPO | 340 ± 39 | 318 ± 39 | 300 ± 39 | 310 ± 30 | 313 ± 44 | 304 ± 41 |
HC + IR + IPO | 330 ± 38 | 320 ± 38 | 310 ± 32 | 308 ± 36 | 310 ± 33 | 300 ± 37 |
Effects of sevoflurane and ischemic postconditioning on left ventricular hemodynamic parameters in rat hearts exposed to ischemia-reperfusion. IR: ischemia reperfusion; SPO: sevoflurane posconditioning; IPO: ischemic posconditioning; HC: hypercholesterolemia. Data are mean ± SD, n = 8 hearts/group. *
The area at risk was not significantly different among all groups (data not shown). As shown in
IR: ischemia reperfusion; SPO: sevoflurane posconditioning; IPO: ischemic posconditioning; HC: hypercholesterolemia. Data are mean ± SD, n = 8 hearts/group. *
As shown in
TUNEL-positive nuclei (brown nuclei) was indicated by the arrow. a normocholesterolemic ischemia reperfusion group (IR), b normocholesterolemic sevoflurane postconditioning group (IR + SPO), c normocholesterolemic ischemic postconditioning group (IR + IPO), d hypercholesterolemic ischemia reperfusion group (HC + IR), e hypercholesterolemic sevoflurane postconditioning group (HC + IR + SPO), f hypercholesterolemic ischemic postconditioning group (HC + IR + IPO). IR: ischemia reperfusion; SPO: sevoflurane postconditioning; IPO: ischemic postconditioning; HC: hypercholesterolemia. Data are mean ± SD, n = 6 hearts/group. *
Neither sevoflurane nor hypercholesterolemia alters the expression of MG53 in the absence of IR, which indicates that sevoflurane doesn’t have a direct effect on MG53 expression (
Sevoflurane and ischemic postconditioning upregulated the expression of MG53 in healthy rats (
IR: ischemia reperfusion; SPO: sevoflurane postconditioning; IPO: ischemic postconditioning; HC: hypercholesterolemia. Data are mean ± SD, n = 6 hearts/group. *
The levels of total Akt were not significantly different among all groups. Therefore, the levels of p-Akt were expressed as the percentage of total protein. Sevoflurane and ischemic postconditioning significantly increased the expression of PI3K-p85 and p-Akt in healthy rats (
No significant differences were found in the expression of total ERK1/2 among any groups. Sevoflurane and ischemic postconditioning significantly increased the p-ERK1/2 in healthy rats (
Immunoblots of total GSK3β were not significantly different between groups. Sevoflurane and ischemic postconditioning significantly increased the p-GSK3β in healthy rats (
To investigate whether the significant inhibition of sevoflurane and ischemia-induced GSK3β phosphorylation in hypercholesterolemic rat hearts was due to the inactivation of Akt and ERK1/2, we treated healthy and hypercholesterolmic rats with GSK3β inhibitor SB216763.
While sevoflurane and ischemic postconditioning did not improve the hemodynamic parameters of hypercholesterolemic rat hearts, SB216763 significantly improved LVDP and LVEDP in the hypercholeslerolemic group as well as in the healthy group after 60 min of reperfusion (
Group | Baseline | Ischemia | Reperfusion |
|||
---|---|---|---|---|---|---|
30 min | 60 min | 90 min | 120 min | |||
LVDP, mmHg | ||||||
IR | 127 ± 8 | 81 ± 5 | 108 ± 11 | 89 ± 9 | 84 ± 6 | 77 ± 10 |
IR + SB | 125 ± 9 | 83 ± 9 | 110 ± 12 | 105 ± 8* | 102 ± 7* | 95 ± 7* |
HC + IR | 130 ± 12 | 85 ± 11 | 115 ±10 | 98 ± 9 | 88 ± 12 | 80 ± 13 |
HC + IR + SB | 127 ± 7 | 84 ± 8 | 117 ± 10 | 110 ± 7# | 100 ± 7# | 93 ± 8# |
LVEDP, mmHg | ||||||
IR | 4.1 ± 1.5 | 10.1 ± 1.6 | 5.3 ± 1.8 | 7.6 ± 1.8 | 7.9 ± 1.4 | 8.2 ± 1.5 |
IR + SB | 3.8 ± 1.1 | 9.8 ± 1.5 | 5.0 ± 1.4 | 5.3 ± 1.2* | 5.7 ± 1.3* | 5.9 ± 1.2* |
HC + IR | 4.1 ± 1.0 | 9.9 ± 1.2 | 5.4 ± 1.0 | 6.9 ± 1.3 | 7.8 ± 1.0 | 8.4 ± 1.3 |
HC + IR + SB | 3.9 ± 1.4 | 9.6 ± 1.4 | 4.9 ± 1.2 | 5.6 ± 1.1 | 5.8 ± 1.3# | 6.0 ± 1.4# |
HR, beats/min | ||||||
IR | 345 ± 35 | 325 ± 41 | 312 ± 48 | 305 ± 39 | 315 ± 42 | 299 ± 41 |
IR + SB | 322 ± 33 | 310 ± 36 | 320 ± 33 | 311 ± 39 | 315 ± 36 | 310 ± 33 |
HC + IR | 328 ± 31 | 308 ± 43 | 303 ± 39 | 300 ± 34 | 298 ± 41 | 288 ± 39 |
HC + IR + SB | 320 ± 38 | 310 ± 39 | 312 ± 30 | 318± 41 | 310 ± 37 | 302 ± 34 |
Effects of GSK3β inhibitor SB216763 on left ventricular hemodynamic parameters in rat hearts exposed to ischemia-reperfusion. IR: ischemia reperfusion; SB: SB216763; HC: hypercholesterolemia. Data are mean ± SD, n = 8 hearts/group. *
Interestingly, we found that SB216763 significantly reduced infarct size in both normal and hypercholesterolemic hearts (IR + SB, 29 ± 5%
IR: ischemia reperfusion; SB: SB216763; HC: hypercholesterolemia. Data are mean ± SD, n = 8 hearts/group. *
Moreover, SB216763 significantly decreased the number of TUNEL-positive nuclei in both healthy and hypercholesterolemic hearts (IR + SB, 14 ± 4%
a normocholesterolemic ischemia reperfusion group (IR), b normocholesterolemic SB216763-treated group (IR + SB), c hypercholesterolemic ischemia reperfusion group (HC + IR), d hypercholesterolemic SB216763-treated group (HC + IR + SB). IR: ischemia reperfusion; SB: SB216763; HC: hypercholesterolemia. Data are mean ± SD, n = 6 hearts/group. *
The current study suggested that sevoflurane and ischemic postconditioning-induced cardioprotection against IR was blunted in hypercholesterolemic rat hearts, with altered MG53/RISK signaling pathway that inhibit GSK3β. We found that inhibition of GSK3β may be a potential therapeutic intervention to reduce myocardial infarct and apoptosis in hypercholesterolemic subjects.
We used chronic treatment with a high-cholesterol diet for 8 weeks to induce hypercholesterolemia in rats [
Effects of hypercholesterolemia on myocardial IR have yielded controversial results in rodents. A number of studies have shown that hypercholesterolemia increases myocardial infarct size in the setting of IR [
Another principle finding of our current study was hypercholesterolemia alone increased myocardial apoptosis in the setting of IR. In the present study, the number of apoptotic nuclei was increased by 30% in hypercholesterolemic rat hearts than that in healthy ones. One reasonable explanation is increased free oxygen radicals and inflammation may prompt apoptotic signaling pathway [
The present study has shown that hypercholesterolemia induced by high-cholesterol diet abrogated sevoflurane and ischemic postconditioning-induced cardioprotection and modified cardioprotective signaling pathways. Here, we demonstrated that the myocardial p-Akt and p-ERK1/2 expression was upregulated by sevoflurane and ischemic postconditioning. Interestingly, such cardioprotection and upregulation of myocardial p-Akt and p-ERK1/2 expression were both lost in hypercholesterolemic rats. Furthermore, previous studies have demonstrated that both PI3k-Akt and MEK-ERK1/2 signals play a pivotal role in the cardioprotection of sevoflurane or ischemic postconditioning [
Our current study has shown that the cardioprotective effect of postconditioning was lost in hypercholesterolemic subjects, which suggests that hypercholesterolemic patients may not benefit from therapeutic application of sevoflurane in the clinical setting. Therefore, it is translationally important to explore novel cardioprotective strategies. We found that the GSK3β inhibitor SB216763 significantly improved heart pump function, reduced myocardial infarct size and apoptosis in hypercholesterolemic rats, which suggests that the downstream signal mechanisms of GSK3β in hypercholesterolemic myocardium is preserved. Together, these data indicate that inhibition of GSK3β would be a more promising therapeutic target to protect hypercholesterolemic hearts against IR injury.
Our findings are translationally important in that they determined whether the myocardial protection induced by sevoflurane occurs in hypercholesterolemic animals. However, there are several limitations in our current study. First, the loss of cardioprotection of sevoflurane postconditioning in hypercholesterolemic rats might also be due to changes in cardiovascular oxidative/nitrosative stress [
In summary, we report that sevoflurane-induced cardioprotection against IR injury was abrogated in hypercholesterolemic rats. Hypercholesterolemia blocked the ability of sevoflurane to phosphorylate components of RISK pathway and consequently, the phosphorylation of the downstream target GSK3β. These data indicate that direct inhibition of GSK3β before myocardial infarct may be a potential therapeutic approach to prevent IR injury in the presence of hypercholesterolemia.
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