Contributed equally
The protective effects of sevoflurane post-conditioning against myocardial ischemia/reperfusion (I/R) injury (MIRI) have been previously reported. However, the mechanisms responsible for these protective effects remain elusive. In this study, in order to investigate the molecular mechanisms responsible for the protective effects of sevoflurane post-conditioning on isolated rat hearts subjected to MIRI, Sprague-Dawley rat hearts were randomly divided into the following 6 groups: i) the sham-operated control; ii) 2.5% sevoflurane; iii) ischemia/reperfusion (I/R); iv) 2.5% sevoflurane post-conditioning plus I/R; v) 2.5% sevoflurane post-conditioning + NG-nitro-L-arginine methyl ester (L-NAME) plus I/R; and vi) L-NAME plus I/R. The infarct size was measured using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Additionally, the myocardial nitric oxide (NO), NO synthase (NOS) and nicotinamide adenine dinucleotide (NAD+) levels were determined. Autophagosomes and apoptosomes in the myocardium were detected by transmission electron microscopy. The levels of Bcl-2, cleaved caspase-3, Beclin-1, microtubule-associated protein light chain 3 (LC3)-I/II, Na+/H+ exchanger 1 (NHE1) and phosphorylated NHE1 protein were measured by western blot analysis. NHE1 mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction. Compared with the I/R group, 15 min of exposure to 2.5% sevoflurane during early reperfusion significantly decreased the myocardial infarct size, the autophagic vacuole numbers, the NHE1 mRNA and protein expression of cleaved caspase-3, Beclin-1 and LC3-I/II. Post-conditioning with 2.5% sevoflurane also increased the NO and NOS levels and Bcl-2 protein expression (P<0.05 or P<0.01). Notably, the cardioprotective effects of sevoflurane were partly abolished by the NOS inhibitor, L-NAME. The findings of the present study suggest that sevoflurane post-conditioning protects the myocardium against I/R injury and reduces the myocardial infarct size. The underlying protective mechanisms are associated with the inhibition of mitochondrial permeability transition pore opening, and with the attenuation of cardiomyoctye apoptosis and excessive autophagy. These effects are mediated through an increase in NOS and a decrease in phopshorylated NHE1 levels.
Myocardial ischemia/reperfusion (I/R) injury (MIRI) causes irreversible damage to myocardial cells, decreases cell viability and may have life-threatening consequences (
Previous studies have reported that ischemic post-conditioning, including pharmacological post-conditioning (
ROS play an important role in MIRI (
NHE1 has been demonstrated to be a key factor protecting against I/R injury in experimental animal models (
Even though a number of studies have demonstrated the cardioprotective effects of NHE1 inhibitors against MIRI, it is unknown whether NOS and/or phosphorylated (p-)NHE1 plays a role in mediating the cardioprotective effects of sevoflurane post-conditioning. In the present study, we investigated the underlying mechanisms through which sevoflurane post-conditioning reduces myocardial infarct size and mortality following MIRI. Using a rat heart model of I/R, we investigated new strategies for the basic and clinical treatment of MIRI.
Adult male Sprague-Dawley rats weighing 270–350 g (9–10 weeks old; n=144) were purchased from the Animal Center of Soochow University (Suzhou, China). Male rats were used to avoid any potentially confounding effects of female sex hormones (i.e., estrogen) on sevoflurane post-conditioning. All rats were kept under a 12-h light-dark cycle in a temperature-controlled environment, and were allowed free access to food and water for 1 week prior to euthanasia. All rats were randomly divided into the experimental groups described below following euthanasia (all animals were anaesthetized by single-dose intra-peritoneal injection of pentobarbital, 50 mg/kg body weight). All experimental protocols were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and the policies of Soochow University (protocol number: SZULL-20090309, approved on March 9, 2009).
Sevoflurane and 2,3,5-triphenyl tetrazolium chloride (TTC) were purchased from Abbott Laboratories S.A. (Shanghai, China). Sodium pentobarbital, p-NHE1 and total NHE1 were purchased from Sigma (St. Louis, MO, USA). Antibodies against Bcl-2 (Cat. no. 2876), cleaved caspase-3 (Cat. no. 9665), Beclin-1 (Cat. no. 3495) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), microtubule-associated protein light chain 3 (LC3-I/II; Cat. no. ab62721-100) was purchased from Abcam, Inc. (Cambridge, England), glyceralde-hyde-3-phosphate dehydrogenase (GAPDH; Cat. no. AG019) and the bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime Institute of Biotechnology (Nanjing, China). NO and NOS detection assay kits were purchased from Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China. NG-nitro-L-arginine methyl ester (L-NAME) was purchased from Beyotime Biotechnology Corp. (Shanghai, China). TRIzol and AMV First Strand cDNA Synthesis kits were obtained from Sangon Biotech Co., Ltd. (Shanghai, China).
The hearts were prepared according to the Langendorff heart model, as described in a previous study (
Following 30 min of equilibration, the isolated rat hearts were randomly divided into the following 6 groups: i) the sham-operated control group, continuously perfused with K-H buffer for 180 min; ii) the sevoflurane group, exposed to 2.5% sevoflurane for 15 min, as previously descrbied (
Myocardial infarct size was determined by TTC staining. Briefly, to visualize the unstained infarct area, the isolated hearts were removed from the Langendorff device following 2 h of reperfusion, cut into 5
Following reperfusion, the hearts were removed from the Langendorff device and homogenized in ice-cold 0.9% saline solution, and then centrifuged at 600 × g for 10 min. The NO and NOS levels were measured using a diagnostic assay kit (Nanjing Jiancheng Bioengineering Research Institute). The absorbance was determined using a DU-640 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA) at 530 nm and plotted as a percentage of the control according to the manufacturer's instructions.
The quantity of mitochondrial NAD+ in the heart tissue was determined as previously described (
After teh other experiments, the isolated heart tissues were fixed with 2.5% glutaraldehyde solution for 2 h, washed with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide. Following dehydration with 50–100% (v/v) ethanol and acetone, the sections were embedded in Epon 812 epoxy resin. Ultrathin sections were obtained at 50–70 nm and stained with 1% uranyl acetate and lead citrate. Apoptotic and autophagic cells were examined under a transmission electron microscope (HT7700; Hitachi, Ltd., Tokyo, Japan).
Western blot analysis was performed as previously described (
RT-qPCR was used to measure the NHE1 mRNA expression levels. Briefly, the hearts were harvested following reperfusion, total RNA was extracted from the left ventricular heart tissues using TRIzol reagent and the RNA purity was quantified spectrophotometrically at a ratio of 260 to 280 nm. cDNA was obtained from 1
All data are expressed as the means ± standard deviation (SD) and analyzed by one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. A value of P<0.05 (two-tailed) was considered to indicate a statistically significant difference. All statistical analyses were conducted using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA).
To evaluate the effects of sevoflurane post-conditioning on MIRI
We measured the NO and NOS levels in the heart tissues subjected to MIRI with or without sevoflurane post-conditioning. We observed that the NO and NOS levels were decreased in the heart tissues subjected to MIRI when compared with the levels in the sham-operated group (P<0.05, n=6 experiments;
Mitochondrial NAD+ levels may be used as a marker of MPTP opening in MIRI heart tissues and it has been suggested that NAD+ levels are significantly decreased in tissues subjected to MIRI (
Western blot analysis and quantitative analysis indicated that the Bcl-2 levels were significantly lower in the I/R group (29.92±3.75) when compared with the sham-operated group (61.54±7.89; P<0.05;
To examine whether the protective effects if sevoflurane post-conditioning in MIRI are mediated by the inhibition of autophagy
We investigated whether the NHE1 phosphorylation levels are altered in myocardial tissues subjected to I/R. We measured the protein expression levels of p-NHE1 and total (t-)NHE1 by western blot analysis. The ratio of p-NHE1 to NHE1 protein increased significantly in the I/R group when compared with the sham-operated group. p-NHE1 protein expression was significantly decreased following sevoflurane post-conditioning. However, treatment with L-NAME abolished the reducing effects of sevoflurane post-conditioning on p-NHE1 expression (
In the present study, we used a rat model of MIRI nvestigate a novel mechanism through which sevoflurane post-conditioning protects the heart against MIRI. Our results confirmed I/R induced apoptosis and excessive autophagy, eventually causing myocardial infarction. Notably, sevoflurane post-conditioning significantly attenuated MIRI and inhibited apoptosis and excessive autophagy. Furthermore, the NHE1 phosphorylation and mRNA expression levels were markedly downregulated by sevoflurane post-conditioning. These effects were all abolished by treatment with L-NAME.
To the best of our knowledge, the present study is the first to demonstrate that sevoflurane post-conditioning reduces MIRI
Apoptosis is one of the major mechanisms responsible for cell death in MIRI (
NO, which is generated from L-arginine by NOS1, NOS2 and NOS3, is a key factor which has been demonstrated to exert protective effects against cardiac injury (
The inhibition of MPTP opening is a potential cardioprotective mechanism. Thus, to investigate the role of MPTP opening in MIRI following sevoflurane post-conditioning, we measured the NAD+ levels, which indirectly represent MPTP opening (
MIRI has been demonstrated to be associated with a variety of significant intracellular and extracellular metabolic alterations, including elevated potassium, increased lactate and acidosis (
In conclusion, the findings of the present study confirm the cardioprotective effects of sevoflurane post-conditioning against MIRI through the inhibition of apoptosis and excessive autophagy. The protective effects of sevoflurane post-conditioning following I/R injury
The present study was supported by the National Natural Science Foundation of China (grant no. 81372024, to J.Z.), the Natural Science Foundation of Jiangsu Province, China (grant no. BK20141187, to C.W), and the Science and Technology Development Plan of Suzhou City, China (grant no. SYSD2012085, to J.C.)
Experimental protocol. After randomly dividing the rats into 6 groups (n=24), rats underwent a 30-min ischemia procedure, followed by 120 min of reperfusion. The sham-operated group underwent a procedure without ischemia, but it was time-matched. Sevoflurane (2.5%) was inhaled for 15 min immediately following ischemia. Sham, sham-operated group; SEVO, sevoflurane group (no I/R injury); I/R, ischemia/reperfusion injury group; SEVOP, sevoflurane post-conditioning group; L-NAME, group treated with NG-nitro-L-arginine methyl ester (NOS inhibitor).
Sevoflurane post-conditioning decreases infarct size which is increased by myocardial ischemia/reperfusion injury (MIRI) in rats. (A) Representative TTC-stained images and (B) quantification of the percentage of the injured area. The columns and error bars represent the means ± SD; n=6 experiments. A one-way ANOVA followed by Tukey's post-hoc test was used to determine statistical significance. *P<0.05 vs. I/R. Sham, sham-operated group; SEVO, sevoflurane group (no I/R injury); I/R, ischemia/reperfusion injury group; SEVOP, sevoflurane post-conditioning group; L-NAME, group treated with NG-nitro-L-arginine methyl ester (NOS inhibitor); TTC, triphenyltetrazolium chloride.
Sevoflurane post-conditioning increases nitric oxide (NO) and nitric oxide synthase (NOS) production following ischemia/reperfusion. (A) Sevoflurane post-conditioning increased NO production. (B) Sevoflurane post-conditioning increased NOS production. Sevoflurane post-conditioning-induced NO and NOS production was inhibited by L-NAME treatment. Data are presented as the means ± SD; n=6 experiments. A one-way ANOVA followed by Tukey's post-hoc test was used to determine statistical significance. *P<0.05 vs. sham. #P<0.05 vs. I/R. Sham, sham-operated group; SEVO, sevoflurane group (no I/R injury); I/R, ischemia/reperfusion injury group; SEVOP, sevoflurane post-conditioning group; L-NAME, group treated with NG-nitro-L-arginine methyl ester (NOS inhibitor).
Alterations in Bcl-2 expression and cleaved-caspase-3 activity following sevoflurane post-conditioning. Shown are myocardial western blots of Bcl-2 and cleaved caspase-3 expression. (A and B) Representative western blots of Bcl-2 and cleaved-caspase-3, respectively, in Sham, SEVO, I/R, SEVOP, SEVOP+L-NAME and L-NAME groups. (C and D) Representative transmission electron microscopy images from the I/R and SEVOP groups, respectively (original magnification, ×2000; scale bar, 2
Alterations in LC3-II/I and Beclin-1 expression following sevoflurane post-conditioning. Shown are myocardial western blots of LC3-II/I and Beclin-1 expression. (A and B) Representative western blots of Beclin-1 and LC3II/I, respectively, in the Sham, SEVO, I/R, SEVOP, SEVOP + L-NAME and L-NAME groups. Values are expressed as the means ± SD (n=5 experiments). A one-way ANOVA followed by Tukey's post-hoc test was used to determine statistical significance. *P<0.05 vs Sham. #P<0.05 vs I/R. (C and D) Representative transmission electron microscopy images from the I/R and SEVOP groups, respectively (original magnification, ×2,000; scale bar, 2
Sevoflurane post-conditioning inhibits p-NHE1 expression. (A and B) Shown are myocardial western blots of phosphorylated (p-)and total (t-)NHE1 expression. Values are expressed as the means ± SD. A one-way ANOVA followed by Tukey's post-hoc test was used to determine statistical significance. *P<0.05 vs. Sham. #P<0.05 vs. I/R. Sham, sham-operated group; SEVO, sevoflurane group (no I/R injury); I/R, ischemia/reperfusion injury group; SEVOP, sevoflurane post-conditioning group; L-NAME, group treated with NG-nitro-L-arginine methyl ester (NOS inhibitor).
Sevoflurane post-conditioning inhibits NHE1 mRNA expression. (A1) Representative amplification plot of GAPDH; 3 microwells/group, 6 groups; 3 different colors indicate 3 microwells. (A2) Representative amplification plot of GAPDH for 6 groups. Numbers 1, 2, 3, 4, 5 and 6, represent the amplification plot of GAPDH for the Sham, SEVO, I/R, SEVOP, SEVOP + L-NAME and L-NAME groups, respectively. (B1) Representative amplification plot of NHE1; 3 microwells/ group, 6 groups; 3 different colors indicate 3 microwells. (B2) Representative amplification plot of NHE1 for 6 groups. Numbers 1, 2, 3, 4, 5 and 6, represent the amplification plot of NHE1 for the Sham, SEVO, I/R, SEVOP, SEVOP + L-NAME and L-NAME groups, respectively. (C) mRNA expression of NHE1 relative to GAPDH, in the Sham, SEVO, I/R, SEVOP, SEVOP + L-NAME and L-NAME groups, respectively. Values are expressed as the means ± SD (n=3). A one-way ANOVA followed by Tukey's post-hoc test was used to determine statistical significance. ΔP<0.05 vs. Sham, *P<0.05 vs. I/R, #P<0.05 vs. SEVOP..
Proposed mechanism of sevoflurane post-conditioning cardioprotection against myocardial ischemia/reperfusion injury (MIRI). Black arrows indicate transition; red downward arrows indicate downregulation; red upward arrows indicate upregulation; and red bar-headed lines indicate inhibition.
Determination of NAD+ content.
Groups | Sham | SEVO | I/R | SEVOP | SEVOP + L-NAME | L-NAME |
---|---|---|---|---|---|---|
NAD+ (nmol/g) | 178±37 | 181±40 | 91±27 |
122±30a,b | 95±26 |
88±24 |
P<0.05 vs. SEVOP group;
P<0.05 vs. I/R group. Sham, sham-operated group; SEVO, sevoflurane group (no I/R injury); I/R, ischemia/reperfusion injury group; SEVOP, sevoflurane post-conditioning group; L-NAME, group treated with NG-nitro-L-arginine methyl ester.