Reduced β2-glycoproteinI Alleviated Rat Myocardial Ischemia/Reperfusion Injury via Mitochondrial Protection and Inhibition of Apoptosis

Background Myocardial ischemia/reperfusion (I/R) injury is one of the most important reasons for death of coronary heart disease after vascular recanalization. New evidences have shown that β2-glycoprotein I (β2GPI) plays a protective role in cardiovascular diseases. This study aims to evaluate the effects of reduced β2GPI (R-β2GPI), one form of β2GPI, on myocardial I/R injury, and to explore related mechanisms. Methods The in vivo myocardial I/R models of Sprague Dawley rats and in vitro hypoxia/reoxygenation(H/R) models of H9c2 cells were established. The myocardial infarction and morphological changes in SD rats were measured by the TTC staining and HE staining. Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels in plasma were detected by ELISA Assay kit. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method and caspase-3 colorimetric assay kit were used to determine myocardial apoptosis. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential of H9c2 cells were measured by uorescent probe DCFH-DA and JC-1 uorescent staining respectively. To evaluate cell damage, cell viability was assessed by determining the release of lactate dehydrogenase (LDH). The ratio of Bcl-2/Bax at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis was used to detect the expression levels of total Akt and phosphorylated Akt as well as the expression levels of total GSK-3βand phosphorylated GSK-3β in H9c2 cells. indicates JC-1 in JC-1


Abstract Background
Myocardial ischemia/reperfusion (I/R) injury is one of the most important reasons for death of coronary heart disease after vascular recanalization. New evidences have shown that β2-glycoprotein I (β2GPI) plays a protective role in cardiovascular diseases. This study aims to evaluate the effects of reduced β2GPI (R-β2GPI), one form of β2GPI, on myocardial I/R injury, and to explore related mechanisms.

Methods
The in vivo myocardial I/R models of Sprague Dawley rats and in vitro hypoxia/reoxygenation(H/R) models of H9c2 cells were established. The myocardial infarction and morphological changes in SD rats were measured by the TTC staining and HE staining. Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels in plasma were detected by ELISA Assay kit. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method and caspase-3 colorimetric assay kit were used to determine myocardial apoptosis. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential of H9c2 cells were measured by uorescent probe DCFH-DA and JC-1 uorescent staining respectively. To evaluate cell damage, cell viability was assessed by determining the release of lactate dehydrogenase (LDH). The ratio of Bcl-2/Bax at mRNA level was detected by reverse transcriptionpolymerase chain reaction (RT-PCR). Western blot analysis was used to detect the expression levels of total Akt and phosphorylated Akt as well as the expression levels of total GSK-3βand phosphorylated GSK-3β in H9c2 cells.

Results
Our results suggested that R-β2GPI improved I/R model rats' heart function, decreased infarct size, reduced serum CK-MB, cTnI levels, cell apoptosis and caspase3 activity. In vitro, R-β2GPI decreased LDH leakage, reduced ROS generation, maintained mitochondrial membrane potential and increased bcl-2/bax mRNA ratio; increased phosphorylation of Akt and GSK-3β in H9c2 cells following Hypoxia/Reoxygenation (H/R) jnjury.
Conclusion R-β2GPI alleviated myocardial I/R (or H/R) injury by reducing oxidative stress and inhibiting mitochondrial apoptotic pathway via increasing the phosphorylation of Akt/GSK-3β.

Background
Coronary heart disease (CHD) is a common and frequently-occurring disease in cardiovascular system and the main cause for disability and death worldwide [1][2][3]. Myocardial ischemia/reperfusion (I/R) injury is not only one important reason for the death after coronary artery revascularization surgery, but also a hot and di cult problem studied by many people [4,5]. The pathological mechanism of myocardial I/R injury is very complex. Previous studies have demonstrated that the lack of ATP production is the basis for myocardial I/R injury, and the sharp increase of reactive oxygen species (ROS) along with the intracellular calcium overload are the central parts of I/R injury. In addition, I/R injury involves in many other factors such as apoptosis, mitochondrial permeability transition, in ammation, and so on [6][7][8].
β2-glycoprotein I (β2GPI) is the main auto-antigen for anti-phospholipid syndrome, and its molecular weight is about 50KD, with a circulating concentration of about 4 µmol/L in human plasma [9]. β2GPI is composed of ve complement control protein modules, which named from domain I to domain V, and domain V contains the binding site for negatively charged phospholipids [10]. In the crystal structure of β2GPI, a disul de bond is formed between Cys288 and Cys326 of domain V, exposing on the surface of this protein [10,11]. This disul de bond could be opened by thioredoxin-1 (TRX-1), resulting in the formation of two free thiols, and this form of β2GPI is called reduced β2GPI (R-β2GPI) [10,12].
Two forms of β2GPI both exist in vivo. Studies have found that β2GPI is involved in atheromatous plaque formation, oxidative stress, apoptosis and many other biological or pathological processes [13][14][15][16][17][18]. However, the role of R-β2GPI in cardiovascular diseases remains unknown. We speculated that, R-β2GPI may play a role in myocardial I/R injury through affecting oxidative stress, apoptosis and related signaling pathways. This study aims to explore the effect of R-β2GPI on myocardial I/R injury and related mechanisms through experiments in animal and cellular levels. Establishment of rat myocardial I/R model Adult male SD rats were fasted overnight, and then anesthetized via intraperitoneal (IP) administration of 0.3 g/kg chloral hydrate. A poly ethylene 50 (PE50) catheter was inserted into the left ventricle through the right carotid artery to measure the left ventricular pressure. Then a left thoracic incision was made to explore the heart, and myocardial ischemia model was produced by ligature the left anterior descending coronary artery (LAD) approximately 2-3 mm from its origin using a 6 − 0 silk slipknot. The change of ST segment on electrocardiogram was used to prove and monitor myocardial ischemia and reperfusion.

Materials
After 30 minutes ischemia, the slipknot was released to recover the bloodstream. After 2 hours' reperfusion, the rats were executed for next experiments. MedLab-U/4C501H Biological Signal Acquisition and Processing System was used to record the biological signals, such as blood pressure, heart rate, left ventricular systolic pressure, left ventricular end-diastolic pressure, ±LVdP/dtmax, and so on.
Rats were randomly divided into four groups with the following treatments (n = 8): 1) Sham group, received vehicle IP injection and operative procedures without coronary ligature; 2) I/R group, received vehicle IP injection 30 minutes prior to coronary I/R; 3) I/R + β2GPI group, received 1 mg/kg body weight β2GPI IP injection;and 4) I/R + R-β2GPI group, received 1 mg/kg body weight R-β2GPI IP injection.

TTC staining and HE staining
After 2 hours reperfusion, rats were executed and the hearts were taken off rapidly. The atriums and right ventricles were removed, then the left ventricles were frozen at -20℃ for about 20 minutes and cut into 1 mm thick slices perpendicular to the base-apex. Slices were incubated in 1% TTC dissolved in phosphate buffer at 37℃ for 10 minutes (pH 7.4). Pictures were taken and measurements of infarct area were performed by image analysis software Image-Pro Plus. Myocardial infarct size was expressed as percentage of infarct area over total area. On the other hand, some cardiac tissues were made into para n-embedded sections and stained with Haematoxylin-Eosin (HE).
Determination of CK-MB and cTnI using ELISA Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels in plasma were used to evaluate the degree of myocardial cellular damage and necrosis. After 2 hours reperfusion, 1 ml blood samples were drawn from aorta and centrifuged at 3000 rpm for 15 min. Then the upper serum was collected into another tube and stored at -20℃. Enzyme Linked Immunosorbent assay (ELISA) was used to detect the serum CK-MB and cTnI levels in rats. The detailed experimental procedure was performed per the manufacturer's instruction of CK-MB ELISA Assay kit and cTnI ELISA Assay kit.

Determination of myocardial apoptosis
Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method was used to determine myocardial apoptosis. After 2 hours reperfusion, the left ventricles were made into para nembedded sections. Then the sections were stained per the manufacturer's instruction of TUNEL assay kit and analyzed by optical microscope. TUNEL-positive cardiomyocytes in ischemic myocardium were counted in double-blinded method. The percentage of TUNEL-positive cells was determined by dividing the number of positive-staining nuclei by the total number of nuclei in a given eld of view (at 200 microscopic magni cations).
Caspase-3 activity in cardiac tissue was also used to determine myocardial apoptosis. The cardiac tissue from left ventricles was made into homogenate and caspase-3 activity was detected by caspase-3 colorimetric assay kit. In brief, myocardial tissue homogenate was centrifuged at 4℃ and supernatants were collected into new tubes. Protein concentration was measured by Bradford method. To each well of a 96-well plate, supernatant containing 200 mg of protein was loaded and incubated with 25 mg caspase-3 substrate N-acetyl-Asp-Glu-Val-Asp p-nitroanilide at 37℃ for 2 hours. The optical density was measured at 405 nm with a microplate spectrophotometer. Caspase-3 activity was calculated using a standard curve and expressed as fold increase over the mean value of sham group.

H9c2 cell Hypoxia/Reoxygenation (H/R) injury model
H9c2 cells were cultured in DMEM with 4500 mg/L glucose supplemented with 10% (v/v) fetal bovine serum at 37℃ in a humidi ed atmosphere with 5% CO2. Cells were routinely subcultured when grown to subcon uency (> 90% by visual estimate). Cells H/R injury model was established as previously described [23]. In brief, H9c2 cells were harvested and seeded onto 6-well plates, placed into a hypoxic trigas incubator (5% CO2/0%O2/95% N2) at 37℃ for 21 hours, and then re-oxygenated in a standard incubator for 6 hours with new medium.
Cells were randomly divided into four groups with the following treatments (n = 8) : 1) Control group; 2) H/R group,; 3) H/R + β2GPI group,; 4) H/R + R-β2GPI group. Cells incubated with β2GPI or R-β2GPI (100ug/ml) in hypoxic tri-gas incubator for 21 hours, and then reoxygenated in a standard incubator for 6hours with new medium.
Determination of intracellular reactive oxygen species (ROS) Intracellular ROS generation was measured by uorescent probe DCFH-DA. Cell-permeable nonuorescent DCFH-DA oxidizes to the highly uorescent 2,7-dichloro uorescin in ROS presence. H9c2 cells were plated upon a 6-well plate, and incubated with medium containing β2GPI or R-β2GPI (100ug/ml) in hypoxic tri-gas incubator for 21 hours, and then re-oxygenated in a standard incubator for 6 hours with new medium. Then cells were harvested and washed by PBS. 10 mM DCFH-DA was added for 20 minutes at 37℃ in the dark. Fluorescence intensity was measured by ow cytometry at excitation wavelength 488 nm, and emission wavelength 525 nm.

Measurement of mitochondrial membrane potential
Mitochondrial transmembrane potential was assessed using a sensitive uorescent dye, a lipophilic cationic probe JC-1. H9c2 cells were collected, incubated with 5 mM JC-1 dye (Molecular Probes) at 37 °C for 15 min, and washed with PBS for three times and analyzed immediately with Flow Cytometry. Some cells were grown on cover slips, stained with JC-1 dye and then analyzed with uorescent microscope. Red emission indicates membrane potential-dependent JC-1 aggregates in mitochondria. Green uorescence re ects the monomeric form of JC-1 appearing in cytoplasm after mitochondrial membrane depolarization. The green/red uorescence intensity rate was used to evaluate the change of mitochondrial membrane potential.

Western blot analysis
Whole cell extracts were prepared as follows: Cultured H9c2 cells were washed twice with cold PBS and immersed in lysis buffer (composition: 50 mM HEPES, pH 7.4, 0.1% Chaps, 5 mM DTT, 0.1 mM EDTA, and 0.1% Triton X-100). Cell lysates were centrifuged. Protein concentrations in the supernatants were determined by BCA method. Equal samples were loaded onto and separated by 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes by electrophoretic transfer system (Bio-Rad). Membranes were blocked in 5% skim milk for 2 hours at room temperature and incubated with primary antibody (rabbit anti-Akt, rabbit anti-GSK-3β) overnight at 4℃, followed by secondary antibody conjugated to horseradish peroxidase for 2hours. Immunoblot was visualized with DAB method and analyzed with Image-Pro Plus software.

Statistical analysis
All data are expressed as means ± SEM unless indicated otherwise. Differences among groups were determined by ANOVA. Differences between groups were determined by Student's t-test with P < 0.05 considered statistically signi cant.

R-β2GPI improved cardiac function and reduced myocardial infarct in I/R injury rats
Firstly, we investigated whether R-β2GPI could protect cardiac from I/R injury in vivo. we used LVSP and LVEDP to evaluate myocardial contractile function and ventricular compliance (Fig. 1A). In addition, ± LVdP/dtmax was used to evaluate the systolic and diastolic function. The results showed that LVSP, + LVdP/dtmax and -LVdP/dtmax were signi cantly lower in I/R model group than those in sham group; whereas LVEDP was signi cantly increased in I/R model group than that in sham group. R-β2GPI reduced the change of these indicators to some extent (Fig. 1A). we used TTC staining to detect ischemic infarction in myocardial tissue. Results showed that the myocardial infarction area rate of sham group was 0, I/R model group (6.50 ± 0.7)%, I/R + β2GPI group (5.0 ± 0.6)% and I/R + R-β2GPI group (4.8 ± 0.8)%. This indicated that β2GPI and R-β2GPI both reduced ischemic infarction area of myocardial tissue, and R-β2GPI has more obvious effect on myocardial ischemic infarction than β2GPI (Fig. 1B). We next assessed the biochemical marker of myocardial infarction of CK-MB level and cTnI level in serum. The results showed that the serum CK-MB level and cTnI level were signi cantly reduced in R-β2GPI group compared with I/R group (Fig. 1C and 1D).

R-β2GPI reduced cardiomyocytes apoptosis after I/R injury in vivo and in vitro
We next investigated whether R-β2GPI would affect cardiomyocytes apoptosis after I/R injury. We used TUNEL staining to evaluate cardiomyocytes apoptosis in vivo. Unsurprisingly, the number of apoptotic cells in R-β2GPI group was signi cantly decreased compared with β2GPI group and other groups(p < 0.05). (Fig. 2A). Caspase3 activity is another indicator re ecting myocardial apoptosis. In our results, the caspase3 activity of R-β2GPI group decreased signi cantly compared with I/R group (Fig. 2B). H9c2 cell, a myocardial cell line, was used to investigate the effect of R-β2GPI on cardiomyocytes apoptosis after H/R injury. Bcl-2/bax mRNA ratio was an important indicator re ecting the level of apoptosis. H/R treatment decreased bcl-2 expression, and increased bax expression, thus decreased the bcl-2/bax ratio. Pretreating H9c2 cells with R-β2GPI prior to H/R promoted bcl-2 expression and inhibited bax expression, ultimately increased the bcl-2/bax ratio (Fig. 2C). LDH content in cell culture medium represented the level of cell damage. In control group, the LDH content was about 1000U/mL. After H/R injury, this indicator increased more than 50%, indicating that cell damage signi cantly increased (p < 0.01), but incubated with R-β2GPI, the LDH level decreased by about 1/4(p < 0.05) (Fig. 2D).
The change of ROS and mitochondrial membrane potential (MMP) after H/R injury in vitro ROS generation and the change of MMP are important causes of myocardial cell injury and apoptosis.
Then we observed the change of ROS and MMP in H9c2 cells treated with R-β2GPI. Fluorescent results showed, compared with H/R group, R-β2GPI decreased ROS generation in H9c2 cells (Fig. 3A). Fluorescent probe and Flow cytometry were used to detect the change of MPP in H9c2 cells. Compared with H/R group, FITC/PE ratio decreased by about 1/3 and 1/2 in β2GPI and R-β2GPI group respectively (Fig. 3B).
R-β2GPI increased phosphorylation of Akt and GSK-3β in H9c2 cells PI3K/Akt and GSK-3β signaling play an important role in maintaining cardiomyocyte function. In addition, PI3K/Akt and GSK-3β are vital signaling pathways of R-β2GPI. We next explored the effect of R-β2GPI on PI3K/Akt and GSK-3β in H9c2 cells. Consistent with previous reports, I/R treatment increased phosphorylation of Akt and GSK-3β; Pretreatment with R-β2GPI signi cantly increased phosphorylation of Akt and GSK-3β, and consequently increased p-Akt/Akt and p-GSK-3β/GSK-3β ratios (Fig. 4A, 4B) (P < 0.05).

Discussion
In this study, we investigated the effect of R-β2GPI on I/R injury and its underlying mechanism using a myocardial I/R injury model. Firstly, our results revealed the salutary effect of R-β2GPI on cardiac function, which mainly re ected in the improvement of LVSP LVEDP and ± LVdP/dtmax. TTC staining, HE staining, serum CK-MB and cTnI level all showed coincident results, indicating that R-β2GPI could reduce myocardial infarction. We also observed that R-β2GPI could signi cantly decrease cardiomyocyte apoptosis after I/R injury in rats by TUNEL experiment and caspase 3 activity measurements.
It is generally known that I/R injury is associated with mitochondrial dysfunction and cell death via apoptosis [19,20]. Mitochondria are vulnerable to attack from ROS, which are considered to be key activators during I/R. In early reperfusion, the burst of ROS is generated from electronic respiratory chain due to leakage of electrons during the period of ischemia, which generally leads to the swelling and rupture of mitochondria [21]. It in turn affects the functions of cardiomyocytes; resulting in the depletion of ATP that contributes to myocardial infract. However, mitochondria are not only major sites of energy metabolism, but also regulate cell apoptosis in the high energy-consuming organ [22]. There is growing evidence that indicates that mitochondria dysfunction is closely related to cardiomyocyte apoptosis induced by I/R injury [23,24].With stimulation to mitochondria of ROS may lead to apoptosis. Mitochondria-related apoptotic pathways can be activated after the release of pro-apoptotic proteins. In the present study, we showed that myocardial cell apoptosis was reduced in rats with pretreatment of R-β2GPI. These results supported the hypothesis that the effect of cardio-protection of R-β2GPI is associated with mitochondrial protection.
Mitochondria not only play a major role in generation of ROS, but also are vulnerable to oxidative stress injury from ROS [25]. In particular, under conditions of excess ROS during reperfusion, the structure and function of mitochondria are susceptible to severe damage, which can lead to energy metabolism disorder and trigger apoptosis [25]. In this study, we rst demonstrated reduction of the accumulation of ROS when treated with β2GPI, which might explain the improvement of mitochondrial structure and energy metabolism status.
β2GPI could act on many receptor molecules on cell surface, such as annexin A2, Toll-like receptor 4 (TLR 4), Toll-like receptor 2 (TLR 2), VEGFR-2 and calreticulin [16,26]. β2GPI also played a role in a variety of intracellular signaling pathways, including p38-MAPK, PI3K/Akt, ERK1/2 [17,[26][27][28]. R-β2GPI is another form of β2GPI. Studies have showed that most β2GPI existed in reduced form in the blood of healthy human, which is thought to play a physiological role [16]. The Akt signaling pathway plays a central role in modulating cell proliferation, survival, and motility. A variety of reports have demonstrated that the constitutive activation of Akt signaling is su cient to block cell death induced by a range of apoptotic stimulus in various cell types [29,30]. Previous studies also have showed that the activation of AKT signaling pathways is closely related to the stabilization of mitochondrial membrane potential [31]. Hence, we next investigated whether β2GPI in uences mitochondrial function and apoptosis through AKT signaling pathways. Our results showed that R-β2GPI activated key intracellular signaling molecules Akt, and thus phosphorylated its downstream target GSK-3β. Therefore, we considered that R-β2GPI may act on TLR2 or TLR4, activate the Akt signaling pathway and then protect mitochondrial function and inhibit cell apoptosis.

Conclusion
We concluded that R-β2GPI has protective effects against myocardial Ischemia/ reperfusion injury via regulating PI3K/Akt and GSK-3β signaling pathway. This study provides good basis for the future study on the speci c mechanism of R-β2GPI in cardiovascular disease.

Consent for publication
Not applicable.

Availability of data and materials
The Additional le used and analysed during the current study are available from the corresponding author on reasonable request.