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Corresponding author. Department of Cardiac Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.
The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro.
Methods
The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells.
Results
The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group.
Conclusion
Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).
Cardiovascular disease (CVD), which is caused by cardiovascular events and acute myocardial ischemic hypoxic cardiomyopathy, is a serious health problem in the world with growing morbidity and mortality [
]. In recent years, the clinical treatment of cardiovascular disease mainly includes reascularization, thrombolysis, percutaneous coronary interention (PCI) in a timely manner to restore blood supply and demand balance for the treatment of cardiovascular diseases [
]. To some extent, blood flow recovery can save the ischemic myocardial reperfusion, repair damaged tissue, but ischemia-reperfusion (I/R) may lead to the occurrence of ischemia-reperfusion injury [
]. I/R damage can lead to abnormal mitochondrial metabolism, endoplasmic reticulum stress (ERs), release of inflammatory cytokines, and ultimately accelerate cardiomyocyte apoptosis. Previous studies reported that sustained or severe ERs, which can be triggered by I/R damage, lead to apoptosis through increasing proapoptotic proteins (Bax) while decreasing antiapoptotic proteins (Bcl-2) [
]. The protein contains a CxxC zinc finger domain that identifies unmethylated CpG islands, as well as PHD, F-box, and leucine-rich repeat (LRR) domains. Some researchers reported FBXL10 exerted its H3K36 demethylase activity at different genome sites, and played an important role in anti-tumor activities by regulating cell proliferation and inhibiting apoptosis [
], accompanied by neural tube defects, suggesting that FBXL10 is involved in the regulation of proliferation and apoptosis in the early stages of neurogenesis. Although the protective role of FBXL10 in inhibiting apoptosis in cerebrovascular disease and tumors has been demonstrated, the potential protective effect in cardiovascular disease is still unclear. In the present study, we utilized adenoviral and plsmid transfection of FBXL10 into cardiomyocytes to explore and consummate underlying anti-apoptosis mechanism further in I/R or H/R model. Our data revealed that FBXL10 significantly alleviated ischemia reperfusion induced myocardial damage through inhibiting ERs induced apoptosis and inflammatory response in vivo and in vitro.
2. Materials and methods
2.1 Animals and groups
Healthy adult male Sprague-Dawley rats (n = 50, specific pathogen-free [SPF] Grade), weighing 220–260 g were obtained from Vitalriver company in Beijing. The rats were housed under a 12 h light/dark cycle at constant humidity (60%) and temperature (25 °C) in a controlled environment with free access to water and food. All animal procedures were performed in accordance with the Guidelines of the Chinese Society for Laboratory Animals Science. The rats were randomly allocated to experimental groups. The left anterior descending (LAD) coronary artery was ligated to reproduce myocardial I/R model, as described before [
For the experiments, we established the four groups which were as follows: (1) sham + AAV + GFP group (rats were only treated with threading without ligation, the single tail-vein injection of adeno-associated virus (AAV)-GFP); (2) I/R group, in which LAD was subjected to 30 min of MI followed by 24 h of reperfusion, the single tail-vein injection of AAV-GFP; (3) sham + AAV + FBXL10 group (rats were only treated with threading without ligation, the single tail-vein injection of AAV-FBXL10); (4) I/R + AAV + FBXL10 (After MI/R surgery, the single tail-vein injection of AAV-FBXL10). After treatment for 24 h, we sacrificed rats and the hearts were removed for following experiments. A single tail-vein injection of adeno-associated virus (AAV)-FBXL10 (ad-FBXL10) or AAV-GFP was given to the rats at 1011 particles/rat.
2.2 TTC staining
Animals were euthanized, hearts were removed, and 2 mm sections were made. Slices were immersed in a 2% solution of TTC (2,3,5-triphenyltetrazolium) in normal saline at 37 °C for 20 min. This salt accepts a proton from succinate dehydrogenase in the inner membrane of the mitochondria, which reduces it to its red insoluble form known as formazan [
]. Thus, an area with inactive enzymes and the infarction is not stained and appears pale. The unstained areas were measured by the Image J software and calculated as the percent of the white area.
2.3 H&E staining
Myocardial tissue samples were fixed in 4% paraformaldehyde solution and embedded in paraffin. Paraffin sections were cut at 4-μm thickness, mounted on glass slides and routinely stained with H&E for histopathological analysis.
2.4 Cell culture and H/R model
Rat embryonic cardiac myocytes H9c2 cells were obtained from ATCC. H9c2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 1% × 100 Mycillin at 37 °C in an atmosphere containing 5% CO2. To study the effects on H/R cells, H9c2 cells were then digested, seeded into 96-well plates (3 × 103 cells/well) and divided into four groups. The control group was incubated in normal culture medium. The H/R group was incubated at 37 °C in an atmosphere containing 5% CO2 and 1% O2 for 2 h, after which, cells were transferred into normal DMEM and were cultured for 6 h. For reoxygenation, plates were removed from the hypoxic chamber and were incubated under standard conditions at 37 °C for 24 h. Then H9c2 cells were collected for the following experiments.
2.5 Plsmid constructs and transfection
To upregulate FBXL10 expression, the recombinant sense expression vector plasmid Cytomegalovirus promoter DNA 3.1 for FBXL10 (pcDNA3.1-FBXL10) (INvitrogen) was constructed by subcloning the complementary deoxyribonucleic acid (cDNA) fragment of FBXL10 that contained the complete coding sequence between KpnI and BamHI. All cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Then in our experiment, the groups are as follows (1) control group, (2) con + pcDNA3.1-FBXL10, (3) H/R + pcDNA3.1, (4) H/R + pcDNA3.1-FBXL10.
2.6 Cell Counting Kit-8 (CCK-8) assay
Cell viability was assessed with a Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Japan). Briefly, H9c2 cells (2 × 103 cells/well) were seeded into a 96-well plate and allowed to attach for 24 h. After H/R treatment, CCK-8 reagents (10 μL/well, Beyotime) was added to each well followed by incubation for an additional 3 h at 37 °C. Then the absorbance (450 nm) was measured with a microplate reader (Bio-Rad, USA). Experiments were repeated at least three times.
2.7 Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining
To quantify apoptotic cardiomyocytes, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed following the manufactures’ instructions in paraffin sections. Briefly, paraffin-embedded sections were prepared from rat hearts, and each section was stained with TUNEL reagents after sequential deparaffinization. DAPI staining was used to count the total number of nuclei. The staining was analyzed by a Leica Scanning Microscope. Apoptotic cardiomyocytes were quantified and classified as a TUNEL-positive in a field.
2.8 Immunohistochemical stainig
Immunohistochemistry staining was performed as previously described [
]. The myocardial tissue was fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. About 4-μm paraffin sections were dewaxed and quenched in 3% H2O2 at room temperature for 10 min, then incubated at 4 °C overnight with CHOP (1:200) and GRP78 (1:200) antibodies. Slides were incubated with a biotinylated secondary antibody for 1.5 h, then develop with avidin peroxidase and DAB and counterstained with hematoxylin. Dehydrated slides were mounted with neutral resin. The positive cells were present as brown area. Three areas of each sample from the experiment were randomly captured using the camera (Leica, Germany) and analyzed using Image J.
2.9 Immunofluorescence staining
After different treatment, H9c2 cells were fixed with 4% paraformaldehyde for 30 min, washed 3 times by PBS, and then treated with 0.1% Triton X-100 (Solarbio, Beijing) at room temperature for 20 min. After being washed, cells were blocked in 5% BSA (Solarbio, Beijing) for 30 min at room temperature. Subsequently, cells were incubated with primary antibodies against CHOP (1:200) and GRP78 (1:200) overnight at 4 °C. After that, samples were incubated with anti-rabbit secondary antibody (1:500) at 37 °C for 1 h. And the cells were stained with DAPI for 5 min. Three areas of each sample from the experiment were randomly captured. Images were taken and measured by flurescence microscope (Olympus, Japan).
2.10 Flow cytometry
The apoptosis of H9c2 cells was assessed by flow cytometry using stained with Annexin V-FITC/propidium iodide (PI) kit (Sigma, USA).In brief, after washed with PBS and resuspended with the kit binding buffer, cells were incubated with 10 μL Annexin V-FITC and 5 μL PI for 10 min in the dark. The fluorescence was detected using a flow cytometer (BD, USA).
2.11 Western blotting assay
After treatment, the cells and the tissues were collected and lysed in ice-cold radio immunoprecipitation assay (RIPA) (Beyotime, China) buffer containing protease inhibitors phenylmethylsulfonyl fluoride (PMSF) (Beyotime, China) and protease inhibitor cocktail (Beyotime, China) and then centrifuged at 12,000 rpm at 4 °C for 15 min to obtain supernatants. Equal amount of protein lysates were loaded into a 5%-10–15% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies against Bax (1:1000, CST, USA), Bcl-2 (1:900, CST, USA), caspase-3 (1:200, Santa Cruz, USA), PERK (1:1000, Santa Cruz, USA), p-PERK (1:1000, Abcam, UK), FBXL10 (1:1000, Abcam, UK), and β-actin (1:1000, CST, USA). The membranes were washed three times with TBS containing Tween 20 (TBST). The membranes were subsequently incubated with fluorescent secondary antibody (1:15,000, Zsbio, China) for 1 h at room temperature. Then, the membranes were washed again with TBST for 3 times, 5 min of each time. The protein bands were detected with an Odyssey color infrared laser scan-imaging instrument (LI-COR, USA). The images were analyzed using Image J Software.
Quantitative Real-Time PCR analysis Total RNA was extracted from cells or tissues using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions, and 1 μg of total RNA was reversely transcribed into cDNA using a Reverse Transcription Kit (Takara, China). Primers were synthesized by Sangon Biotech (Shanghai, China). The expression was calculated via the comparative-threshold cycle method and normalized to β-actin mRNA levels. And the ratio of the relative expression levels was calculated using the 2 −ΔΔCT method. All experiments were in triplicate.
2.13 ELISA kit assay
ELISA kit was used to measure the concentration of IL-6 and TNF-α in myocardial tissues and H9c2 cells by determining the absorbance at 450 nm. Briefly, 10 mg myocardial tissue or 100 μL cultured H9c2 cells were homogenized according to the manufacturers’ protocols. The concentration of TNF-α and IL-6 were measured according to the standard curve.
2.14 Statistical analysis
Data were expressed as mean ± SEM of at least independent experiments. One-way ANOVA or a Student's t-test was performed to assess statistical differences between the groups using SPSS 22.0 software and Graphpad Prism 5.0. Differences were considered statistically significant if P < 0.05.
3. Results
3.1 FBXL10 was downregulated in the heart in I/R rat and in H/R H9c2 cells
To investigate whether FBXL10 participates in I/R injury, the expression of FBXL10 in I/R rats and H/R H9c2 cells were detected by Western blot and qRT-PCR analysis. As shown in Fig. 1, the mRNA expression and protein expression of FBXL10 in I/R group was significantly lower than those in sham group (Fig. 1 A, B). Similarly, the mRNA expression and protein expression of FBXL10 in H/R group was also lower than those in control group (Fig. 1 C, D). From our results, we found that FBXL10 was reduced in the heart of I/R rats and H/R H9c2 cells.
Fig. 1FBXL10 was downregulated in the heart in I/R rat and in H/R H9c2 cells. Western blot analysis and qRT-PCR were performed to investigate the expression of FBXL10 in the myocardial of I/R rat and H/R H9c2 cells. QRT-PCR was used to measured the mRNA expression of FBXL10 in the myocardial tissue (A) and H9c2 cells (C). Western blot analysis was used to measure the protein expression of FBXL10in the myocardial tissue (B) and H9c2 cells (D), equal protein loading was confirmed with the β-actin. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs sham or control, n = 6.
3.2 FBXL10 reduced inflammatory response in the rat model of myocardial ischemia reperfusion
Next we assessed the role of FBXL10 on inflammatory response in the rat model of myocardial ischemia reperfusion. As shown in Fig. 2, qRT-PCR and Western blot were performed to detect the mRNA and protein levels of FBXL10 in four groups (sham group, sham + AAV-FBXL10, I/R group and I/R + AAV-FBXL10) (Fig. 2 A, B). Compared with the sham or I/R + AAV-GFP group, the expression of FBXL10 was increased in the sham + AAV-FBXL10 or I/R + AAV-FBXL10 group. In order to investigate the effect of FBXL10 on the inflammation in I/R rats, the concentration of IL-6 and TNF-α in the myocardial tissue was evaluated using ELISA kits and qRT-PCR. Significant increases in the expression levels and mRNA level of TNF-α and IL-6 were detected in the I/R group, whereas these increased levels were reduced in the I/R + AAV-FBXL10 group (Fig. 2C and D).
Fig. 2FBXL10 reduced inflammatory response in the rat model of myocardial ischemia reperfusion. QRT-PCR analysis was used to measure the mRNA expression of FBXL10 in myocardial tissue (A). Western blot analysis was used to detect the expression of FBXL10 in the myocardial tissue of different groups (B), equal protein loading was confirmed with the β-actin. ELISA kits were used to determine the concentration of IL-6 and TNF-α in the myocardial tissue (C). QRT-PCR analysis was used to measure the mRNA of IL-6 and TNF-α in the myocardial tissue (D). Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs sham, n = 6; #P < 0.05, ##P < 0.01, ###P < 0.001, vs I/R + AAV-FBXL10, n = 6.
3.3 FBXL10 improved myocardial ischemia reperfusion injury in vivo
To determine the effect of FBXL10 on myocardial ischemia reperfusion injury in rats, TTC staining was performed to assess the effect of FBXL10 on the myocardial infarction. Our results showed that compared with the sham group, the infarct area of rats in I/R group was increased significantly. While the infarct area in I/R + AAV-FBXL10 group was reduced significantly (Fig. 3 A). H&E staining results showed that compared with the sham group, the infarct myocardium performed myocardial fiber deformation and disorganization, inflammatory cell infiltration and myocardial cell swelling in I/R rats. Whereas myocardial tissues from sham group were arranged regularly without necrosis (Fig. 3B). However, the intercellular space in myocardial tissue of rats in I/R + AAV-FBXL10 group was significantly reduced and the cell arrangement was improved.
Fig. 3FBXL10 improved myocardial ischemia reperfusion injury in vivo. TTC staining was used to detect the effect of FBXL10 on the myocardial infarction of rats (A). Effects of FBXL10 on myocardial tissue injury after I/R were evaluated by hematoxylin and eosin (H&E) staining (B). Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining was used to evaluate the effects of FBXL10 on myocardial tissue apoptosis after I/R (C). Western blot analysis was used to measure the protein expression of Bax, Bcl-2 and caspase-3 in the myocardial tissue of rats (D), equal protein loading was confirmed with the β-actin. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs sham; #P < 0.05, ##P < 0.01, ###P < 0.001, vs I/R + AAV-FBXL10, n = 3.
3.4 FBXL10 reduced inflammatory response in H/R H9c2 cells
In order to evaluate the effect of FBXL10 on inflammatory response in the H/R model, ELISA kits and qRT-PCR were performed to detect the concentration and the mRNA expression of IL-6 and TNF-α in H/R H9c2 cells. As shown in Fig. 4A and B, the expression of FBXL10 in H/R H9c2 cells was detected by qRT-PCR and Western blot. It showed that compared with the control or H/R group, the expression of FBXL10 was increased in the con + pcDNA3.1-FBXL10 or H/R+ pcDNA3.1-FBXL10 group significantly. ELISA kits results showed that the concentration of IL-6 and TNF-α in H/R H9c2 cells was higher than those in control or con + pcDNA3.1-FBXL10 group. In contrast, the concentration of IL-6 and TNF-α in H/R H9c2 cells was lower compared with that in H/R + pcDNA3.1-GFP group (Fig. 4C). QRT-PCR results showed that the mRNA levels of IL-6 and TNF-α in H/R H9c2 cells were increased significantly compared with those in control or con + pcDNA3.1-FBXL10 group. The mRNA levels of IL-6 and TNF-α in H9c2 cells were reduced significantly compared with those in control or con + pcDNA3.1-FBXL10 group (Fig. 4D). It indicated that FBXL10 reduced the inflammatory response in H/R H9c2 cells.
Fig. 4FBXL10 reduced inflammatory response in H/R H9c2 cells. QRT-PCR analysis was used to detect the mRNA expression of FBXL10 in H/R H9c2 cells (A). Western blot analysis was used to measure the expression of FBXL10 in H/R H9c2 cells (B), equal protein loading was confirmed with the β-actin. ELISA kits were used to determine the concentration of IL-6 and TNF-α in the H9c2 cells (C). QRT-PCR analysis was used to measure the mRNA of IL-6 and TNF-α in the H9c2 cells (D). Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs control, n = 6; #P < 0.05, ##P < 0.01, ###P < 0.001, vs H/R + AAV-FBXL10, n = 3.
Next we investigate the effect of FBXL10 on the apoptosis of H9c2 cells after H/R. Firstly, CCK-8 was performed to detect the cell viability of H9c2 cells. As shown in Fig. 5A, the cell viability of H/R group 72 h after 2 h hypoxia and 6 h reoxygenation was significantly lower than those of control and con + pcDNA3.1 groups. Compared with the H/R group, the viability of H/R + pcDNA3.1-FBXL10 72 h after 2 h hypoxia and 6 h reoxygenation was significantly higher. In addition, flow cytometry was used to detect the apoptosis in H/R H9c2 cells. The results showed that compared with the control or con + pcDNA3.1-FBXL10, the apoptotic rate was higher significantly with statistical difference. The apoptotic rate of H/R + pcDNA3.1-FBXL10 group was lower significantly compared with the H/R group (Fig. 5B and C). Later, we assessed the expression level of apoptosis-related proteins in H/R H9c2 cells via Western blot. As shown in Fig. 5 D, E, the expression of Bax and caspase-3 was increased significantly in H/R group compared with that in the control and con + pcDNA3.1-FBXL10 groups, whereas the expression of Bcl-2 was reduced. Conversely, the expression of Bax and caspase-3 in H/R + pcDNA3.1-FBXL10 group was reduced significantly compared with the H/R group, whereas, the expression of Bcl-2 was increased. The results indicated that FBXL10 could downregulate the expression level of apoptosis-related proteins in H/R H9c2 cells, reduce the apoptosis in H/R cells.
Fig. 5FBXL10 reduced cell apoptosis in H/R H9c2. Viability of H9c2 cells was determined using the CCK-8 assay (A). Apoptotic H9c2 cells were detected using flow cytometry after 2 h of Hypoxia and 6 h of reoxygenation (B). The apoptosis rate of H9c2 cells were quantified (C). The protein expression of Bax, Bcl-2 and caspase-3 in the H9c2 cells were determined by Western blot (D), equal protein loading was confirmed with the β-actin. The protein level of Bax, Bcl-2 and caspase-3 were quantified (E). Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs control, n = 3; #P < 0.05, ##P < 0.01, ###P < 0.001, vs H/R+ pcDNA3.1-FBXL10, n = 3.
3.6 FBXL10 inhibits endoplasmic reticulum stress induced by I/R in vivo
To demonstrate that FBXL10 could ameliorate endoplasmic reticulum stress (ER stress) during I/R, several ERs-associated markers were detected via immunohistochemical staining and western blotting assay. As shown in Fig. 6 A, B, immunohistochemical stainig results showed that compared with sham or sham + AAV-FBXL10 groups, the expression of CHOP and GRP78 was increased significantly in the I/R group. Whereas, the expression of CHOP and GRP78 was decreased in the I/R + AAV-FBXL10 compared with the I/R group. Furthermore, The expression of CHOP, GRP78, ATF4, PERK and p-PERK in the myocardial tissue was detected by Western blot. The results showed that the expression of CHOP, GRP78, ATF4, PERK and p-PERK was increased significantly in I/R group compared with sham or sham + AAV-FBXL10 groups. Whereas, compared with the I/R group, the expression of CHOP, GRP78, ATF4, PERK and p-PERK was reduced significantly in the I/R + AAV-FBXL10 (Fig. 6 C, D). From the above results, we found that FBXL10 could inhibit endoplasmic reticulum stress induced by I/R.
Fig. 6FBXL10 inhibits endoplasmic reticulum stress induced by I/R in vivo. Representative immunohistochemical images taken with the myocardial tissue were shown. The expression of CHOP (A) and GRP78 (B) in the myocardial tissues were detected by immunohistochemical staining. Representative immunohistochemical images were taken with the myocardial tissues in different groups. The expressions of CHOP and GRP78 were quantified. Western blot was used to detect the expression of ATF4, CHOP, GRP78, PERK and p-PERK in the myocardial tissues of different groups (C). The protein levels were quantified. Equal protein loading was confirmed with the β-actin. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs control, n = 3; #P < 0.05, ##P < 0.01, ###P < 0.001, vs H/R + AAV-FBXL10, n = 3.
3.7 FBXL10 inhibits endoplasmic reticulum stress in H/R H9c2 cells
To confirm that FBXL10 could inhibit endoplasmic reticulum stress (ER stress) during H/R, several ERs-associated markers were detected via immunofluorescence staining and western blotting assay. As shown in Fig. 7 A, B, immunofluorescence staining results showed that compared with control or con + pcDNA3.1-FBXL10 groups, the expression of CHOP and GRP78 was increased significantly in the H/R group. Whereas, the expression of CHOP and GRP78 was decreased in the H/R + pcDNA3.1-FBXL10 compared with the H/R group (Fig. 7C). Furthermore, The expression of CHOP, GRP78, ATF4, PERK and p-PERK in the H9c2 cells was detected by Western blot. The results showed that the expression of CHOP, GRP78, ATF4, PERK and p-PERK was increased significantly in H/R group compared with control or con + pcDNA3.1-FBXL10 groups. Whereas, compared with the H/R group, the expression of CHOP, GRP78, ATF4, PERK and p-PERK was reduced significantly in the H/R + pcDNA3.1-FBXL10 (Fig. 6 C, D). According the above results, we found that FBXL10 could inhibit endoplasmic reticulum stress in H/R H9c2 cells.
Fig. 7FBXL10 inhibits endoplasmic reticulum stress in H/R H9c2 cells. The expression of CHOP (A) and GRP78 (B) in the H9c2 cells were detected by immunofluorescence staining. Representative immunofluorescence images were taken with the H9c2 cells in different groups. The expressions of CHOP and GRP78 were quantified. Western blot was used to detect the expression of ATF4, CHOP, GRP78, PERK and p-PERK in the H9c2 cells of different groups (C). The protein levels were quantified. Equal protein loading was confirmed with the β-actin. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, vs control, n = 3; #P < 0.05, ##P < 0.01, ###P < 0.001, vs H/R+ pcDNA3.1-FBXL10, n = 3.
CHD is considered to be the leading cause of death around the world, with approximately 17.5 million people dying because of cardiovascular disease according to the estimates from the World Health Organization [
], which has greatly increased the economic burden of patients and society. Myocardial I/R is accompanied by sustained I/R injury and ERs induced apoptosis of cardiomyocytes [
GSK-3β inhibition confers cardioprotection associated with the restoration of mitochondrial function and suppression of endoplasmic reticulum stress in sevoflurane preconditioned rats following ischemia/reperfusion injury.
]. Our present study also confirmed that the expression of FBXL10 was down-regulated in both I/R and H/R models. A better understanding of the complex pathologic mechanisms of ERs reperfusion injury may contribute to the development of promising strategies for the treatment of cardiovascular disease.
Yin et al. found that overexpression of FBXL10 in the heart can prevent diabetic heart dysfunction, apoptosis of cardiomyocytes, inflammation and other pathological processes [
]. Furthermore, they found that overexpression of FBXL10 can significantly reduce the apoptosis of cardiomyocytes induced by high glucose. In particular, our results showed the expression levels of FBXL10 in both I/R and H/R models were significantly down-regulated. In addition, FBXL10 overexpression was sufficient to reduce the inflammation in I/R or H/R model, decreasing the production of the inflammatory cytokines TNF-α and IL-6. Then we confirmed that FBXL10 could inhibit cell apoptosis in H/R or I/R model, through downregulating the expression of Bax and upregualting the expression of Bcl-2. From the present study, we also found FBXL10 could improve cell viability in H/R model and attenuate the myocardial ischemia reperfusion injury in I/R model.
Apoptosis is a vital pathological process in myocardial I/R injury and the level of apoptosis determines the severity of myocardial I/R injury [
]. During myocardial ischemia reperfusion, Liu et al. confirmed that apoptosis of cardiomyocytes led to the loss of cardiomyocytes and directly affected cardiac function [
]. Our results also demonstrated a significant increase in myocardial apoptosis during myocardial ischemia-reperfusion. As the main terminal cleavage enzyme during apoptosis, caspase-3 is involved in DNA repair and basal integrity. Bcl-2 prevents apoptosis through inhibiting mitochondrial rupture, caspase-3 activation and Bax cytotoxicity. As shown in Fig. 5, we found that FBXL10 could downregulate the expression of Bax and caspase-3, and upregulate the expression of Bcl-2 in H/R H9c2 cells.
Endoplasmic reticulum (ER) stress has been reported to play an important role in inducing apoptosis of myocardial cells following myocardial infarction [
]. Prolonged ERs is associated with apoptosis of cardiomyocytes, resulting in myocardial ischemia injury and chronic heart failure. In addition, ERs-mediated apoptosis is also involved in the development of diabetic heart disease [
]. In the present study, overexpression of FBXL10 can reduce myocardial ischemia reperfusion injury and apoptosis induced by ERs, suggesting that inhibition of FBXL10 may regulate ERs and play a protective role in I/R injury.
Cardiomyocytes alleviate ERs through the unfolded protein response (UPR), which is regulated by ER chaperones and folding enzymes, such as GRP78 and CHOP. The GRP78, the major ER-resident chaperone, is well known as a biomarker of ERs in the course of disease [
]. If sustained ERs persisted, inflammatory and apoptotic pathways are activated, including the induction of CHOP, PERK, caspase-3, caspase-12 and NF-κB [
Hepatoprotective effect of quercetin on endoplasmic reticulum stress and inflammation after intense exercise in mice through phosphoinositide 3-kinase and nuclear factor-kappa B.
]. Based on the considerations, we used GRP78 as an ERs marker and CHOP as an ERs-mediated apoptosis marker, and detected the expression levels to evaluate the effect of FBXL10 on ERs in I/R and H/R model.
The present study also supported that ERs response induced apoptosis is involved in the pathological process of myocardial ischemia reperfusion injury. GRP78 and CHOP showed enhanced expression in I/R or H/R group, which paralleled an increase of apoptotic cells. To further demonstrate which UPR components were involved in ER stress induced cardiac damage in vivo and in vitro, we determined the ERs related proteins expression levels, and ATF4 and p-PERK were found to have increased protein amounts in I/R or H/R group. The findings manifest that increased ERs contributed to cardiomyocytes apoptosis following myocardial I/R injury. Cullinan et al. [
]has demonstrated that ERs may be associated with the apoptosis of cardiomyocytes during myocardial ischemia reperfusion injury. Furthermore, Wang et al. [
] suggested that the inhibition of ERs played a potential therapeutic approach for attenuating myocardial I/R injury. Nevertheless we believed that overexpression of FBXL10 inhibited ERs induced apoptosis to play a cardioprotective role in the I/R or H/R groups. The expression of GRP78 and CHOP in rat heart tissue and H9c2 cells were significantly reduced by the expression of FBXL10. In addition, we observed that ATF4 and p-PERK levels were decreased by the overexpression of FBXL10 in vivo and in vitro. Furthermore, our results showed that FBXL10 could reduce the apoptosis in H/R H9c2 cells.
In conclusion, our present study indicated that FBXL10 could attenuate myocardial ischemia reperfusion injury by inhibiting ERs-induced apoptosis and inflammatory response. Our findings provide a reliable theoretical basis for FBXL10 in the prevention and treatment of myocardial ischemia-reperfusion injury. Therefore FBXL10 may be a potential target to alleviate myocardial I/R injury.
Declaration of competing interest
All authors declare that there is no any conflict of interest.
Acknowledgements
We are grateful to all participates for their contributions for the present study. The study was supported by the funded project of 1351 talent training plan of Beijing chaoyang hospital affiliated to capital medical university (No. CYMY-2017-18).
Appendix A. Supplementary data
The following is the Supplementary data to this article:
GSK-3β inhibition confers cardioprotection associated with the restoration of mitochondrial function and suppression of endoplasmic reticulum stress in sevoflurane preconditioned rats following ischemia/reperfusion injury.
Hepatoprotective effect of quercetin on endoplasmic reticulum stress and inflammation after intense exercise in mice through phosphoinositide 3-kinase and nuclear factor-kappa B.
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