Bisoprolol protects myocardium cells against ischemia/reperfusion injury by attenuating unfolded protein response in rats

Bisoprolol (B) exerts potential cardioprotective effects against myocardial ischemia/reperfusion (I/R) injury. Unfolded protein response (UPR) attenuates I/R injury induced apoptosis by reducing oxidative damage and inflammation response. The current study investigated whether the protective effects of bisoprolol resulted from modulating UPR and anti-inflammatory during myocardial I/R condition and elucidated its potential mechanisms. Sprague-Dawley rats were treated with B in the absence or presence of the injected UPR activator dithiothreitol (DTT) and then subjected to myocardial I/R surgery. In vitro, cultured H9C2 cells were pretreated with B or DTT and then subjected to simulate ischemia reperfusion (SIR) operation. Bisoprolol conferred cardioprotective effects by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase levels, suppressing TNF-α and IL-6 secretion, inhibiting UPR signal pathways and downregulating caspase-12 and caspase-3 expressions. Consistently, B conferred similar antioxidative and anti-inflammatory effects against SIR injury in cultured H9C2 cardiomyocytes. Pretreatment with DTT or C/EBP homologous protein (CHOP) overexpression mediated by lentivirus administration both abolished these effects. In summary, our results demonstrate that Bisoprolol protects myocardium cells against ischemia/reperfusion injury partly by attenuating unfolded protein response.

injury activate all the three branches of UPR pathway is still largely unknown. Activated IRE1 pathway splices the mRNA of a transcription factor called X-box-binding protein-1 (XBP1), removing a 26-bp nucleotide intron from the full-length XBP1 mRNA that creates a translational frame shift leading to the expression of a spliced XBP1 (sXPB1) 15,16 . The molecular of sXBP1 is a highly active transcription factor for ER-resident enzymes and chaperones 15 . Activated PERK phosphorylates eukaryotic translation initiation factor 2 subunit α (eIF2α), which leads to inhibition of global protein synthesis 17 . However, phosphorylated eIF2α can also lead to an increase expression of ATF4 and CHOP 18 . When ER stress is excessive and/or prolonged, however, apoptotic signals are initiated by the UPR in the ER, including induction of CHOP, activation of Jun N-terminal kinase (JNK), and cleavage of caspase-12 [19][20][21] .
β-adrenoceptor antagonists (β blockers) are indicated for patients with mild to moderate heart failure (New York Heart Association (NYHA) class II and III) of ischaemic or non-ischaemic aetiology 22 . Treatment with β-blockers exerts a range of beneficial effects on cardiac function and, importantly, reduces HF-related morbidity and mortality 23,24 . Importantly, it has also been reported that bisoprolol significantly reduced long-term cardiac death and myocardial infarction in high-risk patients after successful major cardiac vascular surgery 25 . However, the underlying molecular mechanism(s) responsible for this effect remains unidentified.
In the article, Bisoprolol was used as an adjuvant to attenuate the UPR induced by myocardial I/R injury. In addition, the involvement of the UPR signaling pathway and apoptosis in bisoprolol's protective actions were also evaluated.

Results
Effect of exogenous bisoprolol and dithiothreitol (DTT) treatment on Sham operated rat heart. Firstly, in the in vivo experiment, we evaluated the effect of exogenous B or DTT treatment on the sham-operated rat hearts. Compared with the sham group, neither B nor DTT treatment in Sham operated rat significantly affected the cardiac function including Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) value (P > 0.05, Fig. 1A-C). In addition, neither the myocardial infarct size nor the myocardial apoptosis were markedly changed compared with the sham group (P > 0.05, Fig. 1D-F). Moreover, we detected the effects of B or DTT on the cellular UPR signaling pathway. As shown in Fig. 2, as a UPR activator, DTT treatment markedly actived the three branches pathway of UPR (P < 0.01), B treated alone had no Exogenous bisoprolol treatment significantly attenuated the myocardial I/R injury and UPR, where these effects can abolish by DTT co-treatment. In vivo experiment, we evaluated the effect of exogenous B or DTT treatment on the myocardial I/R-operated rat hearts. Compared with the myocardial I/R group, B treatment effectively improved the post-myocardial I/R cardiac function recovery as indicated by the increased LVEF and LVFS (P < 0.01, Fig. 3A-C). However, compared with the myocardial I/R + B group, these protective effects were blocked by DTT co-treatment (P < 0.01, Fig. 3A-C). Particularly, compared with the myocardial I/R group, treated with DTT alone aggravate the injury of heart ( Fig. 3A-C). Next, we evaluated the myocardial infarct sizes. As shown in Fig. 3D, the B-treated group showed a significantly decreased myocardial infarct size (compared with the I/R group, P < 0.01), while the DTT treatment significantly blocked this effect (compared with the I/R + B group, P < 0.01). We also find that the infarct size in I/R + DTT is bigger than the I/R groups (Fig. 3D). We also measured the levels of myocardial apoptosis. As shown in Fig. 3E and F, the myocardial apoptosis caused by the myocardial I/R injury was also markedly attenuated by B treatment, and this attenuation was also abolished by the DTT co-treatment. The B treatment significantly decreased the serum lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) levels, and these effects were also blocked by DTT treatment (P < 0.01, Fig. 3G).
To further understand the molecular mechanism of B's cardioprotective effect, we investigated the myocardial UPR signaling pathway. Figure 4 showed that B markedly down-regulated the GRP78, ATF6 and CHOP both in transcript and protein levels in the myocardial I/R-injured hearts (compared with the I/R group, P < 0.01). The splice effect of XBP in I/R and I/R + DTT group was activated which detected by RT-PCR (Fig. 4D). However, these effects were attenuated by the B co-treatment (compared with the I/R or I/R + DTT group, Fig. 4). Although DTT alone up-regulated the GRP78, ATF6 and CHOP levels, but treated with B together have a decrease effect of those molecular. (Fig. 4).
Exogenous bisoprolol treatment significantly attenuated the apoptosis and inflammation in myocardial cells caused by myocardial I/R injury. To further understand the molecular mechanism of B's cardioprotective action, we investigated the expression of caspase family molecules in myocardial. As shown in Fig. 5A-D, both in transcript and protein levels, the B treatment significantly decreased the expression of caspase-12 and caspase-3 compared with I/R group. In the I/R + B + DTT group, the B's reduce effect was abolished compared with I/R + B group. In addition, we measured the expression of IL-6 and TNFα in myocardial tissue homogenate. As shown in Fig. 5E and F, the myocardial I/R injury significantly up-regulated the expression, but this was markedly down-regulated by the B treatment. However, all the protective effects of B were abolished by the DTT co-treatment (compared with the I/R + B group, P < 0.01, Fig. 5E,F).

Bisoprolol significantly inhibited the UPR and inflammation response in the simulate ischemia reperfusion (SIR) injured H9C2 cells, where these effects can abolish by DTT co-treatment. The
in vitro experimental results were consistent with those obtained from the myocardial I/R-injured rat hearts. The H9C2 cells were treated with B at 0.2, 2, 20, or 200 mmol/L for 24 h, and then performed the cell viability by MTT assay. Compared with the control group, no significant cell viability changes were found in the 0.2, 2 and 20 mmol/L groups (P > 0.05, Fig. 6A). However, the 200 mmol/L B treatment markedly affected cell survival (P < 0.01, compared with the Con group, Fig. 6A). Therefore, the 20 mmol/L B dose was chosen for the present study. Firstly, we measured cardiomyocyte inflammation markers in the cultured H9C2 cells. As depicted in Fig. 6B and C, shown TNF-α and IL-6 levels were both markedly decreased by B treatment (P < 0.01, compared with the SIR group). As expected, DTT co-treatment blocked these effects by greatly upregulated TNF-α and IL-6 levels compared with SIR + B group. Then we investigated the cellular UPR signaling pathway in the B or DTT treatment H9C2 cells. Figure 7 showed that B markedly down-regulated the GRP78, ATF6 and CHOP in protein level in the SIR + B group (compared with the SIR group, P < 0.01). Although DTT alone up-regulated the GRP78, ATF6 and CHOP levels, but treated with B together have a decrease effect of those molecular. (Fig. 7A,C-E). The splice effect of XBP in SIR and SIR + DTT group was activated which detected by RT-PCR (Fig. 7B). However, these effects were attenuated by the B co-treatment (compared with the SIR or SIR + DTT group, Fig. 7B).
Bisoprolol protected the H9C2 cells from the SIR-induced apoptosis, but these effects were abolished by DTT co-treatment and CHOP overexpression. As the Fig. 8A shown, the H9C2 cells were treated LV CHOP or DTT, the cell viability was no significant changes compared with Con group. Second, we found that the B treatment effectively increased the cell viability following SIR by TUNEL assay (compared with SIR group, P < 0.01, Fig. 8B and C). However, the protective action of B was abolished by treatment with the

Discussion
In the present study, we utilized both in vivo and vitro models to investigate the protective effect of bisoprolol against myocardial I/R-induced cardiac damage. We found that bisoprolol markedly ameliorated the myocardial I/R injury by reducing the UPR signal pathway. Importantly, UPR signaling was proven to play a critical role in this process. This study could help to understand the pharmacology of bisoprolol in the treatment of ischemic heart disease and promote the development of an understanding mechanism of I/R injury.
The benefits of β blocker therapy in the treatment of heart failure are universally accepted. It is well understood that beta-blocker therapy can reduce the morbidity and mortality in heart failure patients 26,27 . With advances in the management of heart disease and the widespread recognition of their effects, beta-blockers are the abundant use of the current treatment for heart failure. However, the mechanism of the protective effects of the beta-blockers is rarely been studied.
In the current study, both in vivo and in vitro experiments showed that bisoprolol ameliorated myocardial I/R injury and proved that bisoprolol treatment induced a significant decrease of the molecules of UPR expression and reduced the inflammation response. The inhibit UPR signaling was also associated with a decrease of Caspase-12 and Caspase-3 expression. As expected, these effects were both abolished by UPR activator DTT and overexpression of CHOP expression. These results all suggest that the cardioprotection of bisoprolol treatment involves the inhibition of UPR signaling, and decrease the inflammation response, Caspase-12 and Caspase-3 expression. Interestingly, we found in bisoprolol treated animals or cells significantly reduced myocardial IL-6 accumulation and TNF-production, which indicated that bisoprolol also ameliorated cardiac inflammation.
Furthermore, the present study was carried out to elucidate the underlying mechanisms of bisoprolol's cardioprotective action by attenuate UPR signaling. It has been proven that excessive ER stress participates in ischemia/ reperfusion injury 28,29 . Of the many signaling pathways involved in ER stress, PERK/eIF2α/ATF4, one of the UPR signal pathway has been found to contribute strongly to programmed cell death during myocardial I/R injury 30 . Intriguingly, in the present study, we found that bisoprolol down-regulate all the three pathway of the UPR signal pathway, and treatment with DTT and overexpression CHOP can abolish the effect of bisoprolol. Furthermore, bisoprolol reduced the pro-inflammatory cytokine which induced by ER stress and significantly decreased the LDH and CK-MB levels both in vivo or vitro. There was study found that the ER stress pathway involving CHOP was activated and played a role in the pathogenesis of septic organ injury 31 . And in septic mice that CHOP inhibition effectively decreased the organ damage and increased the overall survival rate 32 . Thus, it is worthwhile to further investigate bisoprolol's potent therapeutic effect on myocardial I/R injury with a focus on CHOP. So far, several clinical investigations have established that there is a beneficial effect of bisoprolol treatment on the cardiovascular system in patients with high-risk patients after successful major cardiac vascular surgery 25,33,34 . Hence, bisoprolol was suggested to be a safe and effective option for patients under conditions of moderate cardiovascular risk. Based on our data, ameliorate the myocardial I/R injury by bisoprolol pretreatment is correlate with UPR signaling pathway in rats. The main novel finding of this study is to demonstrate a cardioprotective effect of treatment with bisoprolol via inhibition of UPR and the stress-associated intracellular factors. In addition, mitochondrial apoptosis pathway is another important target of cardioprotective signalling. There have many researches pointed out that myocardial hypoxia is closely associated with cardiac dysfunctions, in particular myocardial infarction (MI), ischemia reperfusion injury, and hypertrophy 35 . So the UPR is merely a part of participate in regulate the myocardial I/R injury. Further clinical studies might be needed before a conclusion can be reached on the utility of bisoprolol in this setting.
Taken together, we demonstrated that bisoprolol reduced myocardial I/R injury via inhibiting of the UPR signaling and inflammation response. These findings may provide new mechanistic insight into the cardioprotective effects of bisoprolol.

Materials and Methods
Antibodies and reagents. Evans blue dye, 2,3,5-triphenyltetrazolium chloride and Dithiothreitol (DTT) obtained from sigma Aldrich, USA. Antibodies for GRP78, CHOP, ATF6, XBP1 and GAPDH were obtained from Santa Cruz Biotechnology, USA; Antibody for Caspase-12 and Caspase-3 were obtained from Sigma Aldrich, USA. The secondary antibody HRP-labeled Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG were obtained from beyotime, China.

Creation of myocardial I/R rat model in vivo.
Adult male Sprague-Dawley (SD) rats (250-300 g) were procured from the Animal Center of Jinling Hospital (Nanjing, China). Animal maintenance and experimental procedures were conducted after approved by the Animal Care Committee of Nanjing University (Nanjing, China) and in accordance with the strict guidelines of the Institutional Animal Ethics Committee (IAEC). SD rats were treated with B (10 mg·kg −1 ·d −1 ) for 1 week, in the absence or presence of the injected UPR activator dithiothreitol (DTT, 100 umol·kg −1 ) before ischemia 10 minutes and then subjected to myocardial I/R surgery. The rats were anaesthetized using pentobarbital sodium (3%) via intraperitoneal injection. The surgical procedures were performed as previously described 36 . Briefly, after pericardiotomy, a 6-0 silk suture was placed under the left coronary artery (LCA), which was occluded by tightening the snare using a lightweight hemostatic clamp. After 30 min of myocardial ischemia, the suture was loosened and the myocardial was reperfused for 6 hr (for analysis transcript and protein levels and for quantification of myocardial apoptosis and infarct size), for 72 hr (for cardiac function determination). The sham-operated animals underwent the same surgical procedures except that the suture around the LCA was not fastened. Ischemia was confirmed by a transient decrease in blood pressure and cyanosis on the myocardial surface. Reperfusion was indicated by an epicardial hyperemic response and the rapid disappearance of cyanosis.
Identification of myocardial infarct size in rats with I/R. At the corresponding time of the experiment, the rats were anaesthetized and the LCA was reoccluded, injected with 2% Evans blue dye via the external jugular vein, then the area at risk (AAR) in the heart can be displayed. About 5 min later, the heart was rapidly excised and frozen at −80 °C approximately 10 min. After that, the frozen heart was cut into 5 transverse slices of equal thickness. Then the slices were incubated in phosphate-buffered 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 10 min at 37 °C. In the end, the slices fixed with 4% paraformaldehyde prepared in PBS (pH-7.4). The AAR means the area that was not stained with Evans blue dye. In the AAR area, where not stained by TTC were confirmed as the area of infarction (AI). The AAR, AI and ventricle size (VS) were assessed by using Image-J software 37 .

Measurement of LDH and CK activities in rats with I/R.
After the end of the reperfusion, plasma samples (1 mL) were obtained from carotid aorta using a heparinized syringe and immediately centrifuged. The activities of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) were assayed using spectrophotometrically (Beckman DU 640, Fullerton, CA, USA) in a blinded manner as described before 38 , which purchased from the Institute of Jiancheng Bioengineering (Nanjing, China).

Apoptotic Cell Assay. For detection of DNA fragmentation, terminal deoxynu-cleotidyl transferase (TdT)
-mediated dUTP nick end labeling (TUNEL) assay was performed as described previously 39 . Briefly, After in vivo I/R procedure, the hearts were fixed in 10% buffered formalin and subsequently embedded in paraffin to obtain 5 μm-thick sections. DeadEnd ™ Fluorometric TUNEL system (Promega) was used to stain for apoptotic nuclei. Detection of IL-6 and TNF-α Level. After reperfusion, the levels of IL-6, TNF-in cardiomyocytes supernatant, and myocardial tissue homogenate were detected in strict accordance with manufacturer's instructions 40,41 . BCA kit was used to detect the protein quantization.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR). For X-box binding protein (XBP1)
analysis, cDNA was synthesized as previous mentioned. PCR was performed using Geneamp PCR system (Singma) with Taq DNA polymerase (CWBIO, China). The primer specific for target sequences of XBP1: forward, GAGCAGCAAGTGGT GGAT; reverse, AGGCAACAGCGTCAGAAT. Amplification conditions included: 94 °C for 5 min; 30 cycles at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s; 72 °C for 5 min. When UPR is induced, XBP1mRNA is spliced and activated through activation of the ER stress sensor IRE1 42 . The primer sets for XBP1 were amplified the PCR products of 297 bp for the unspliced form and 271 bp for the spliced form. The amplified RT-PCR products were separated by 3% agarose gel electrophoresis and visualized with ethidium bromide staining under UV light.
Cell culture and cell viability measurement. H9C2 cells were cultured in high glucose Dulbecco's-modified Eagle's medium (DMEM; GIBCO, UK) containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS) (GIBICO, UK). Cultured H9C2 cardiomyocytes pretreated with B (2 μM, 48 h before simulate ischemia) or DTT (2 mM, 3 h before simulate ischemia), then subjected to SIR injury. The H9C2 cells, 1 × 10 4 per well, were seeded in 96-well culture plates. After different treatment, the SIR was performed. The cell viability was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously 43 . Briefly, after the cells were treated and washed with PBS, 10 μL of MTT dye was added to each well at a final concentration of 0.5 mg/mL. After 4 h of reperfusion, 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured using a microtiter plate reader (SpectraMax 190, Molecular Device, USA) at a wavelength of 570 nm. The cell viability was calculated by dividing the optical density of samples by the optical density of the control group.
Simulate ischemia reperfusion (SIR) treatment. The SIR treatment was performed using physiological concentrations of potassium, hydrogen, and lactate. The procedure was performed as described previously 44 . Briefly, the H9C2 cells were exposed to an ischemic buffer containing (in mmol/L) 137 NaCl, 12 KCl, 0.49 MgCl 2 , 0.9 CaCl 2 , and 4 HEPE S. This buffer was also supplemented with (in mmol/L) 10 deoxy-glucose, 0.75 sodium dithionate, and 20 lactate. The buffer pH was 6.5. Stimulated ischemia was the procedure that the cardiomyocytes were incubated for 2 h in a humidified cell culture incubator (1% O 2 , 5% CO 2 , 96% N 2 , 37 °C). Reperfusion was performed by returning the cells to normal culture medium for 4 h in a humidified cell culture incubator (21% O 2 , 5% CO 2 , 37 °C).
Lentivectors construction and lentivirus production. The primers used to over-express CHOP to generate pCDH-CMV-CHOP contained EcoRI and BamHI restriction sites, shown as forward: TGCTCTAGAATGGCAGCTGAGTCTCTG, reverse: CGGGATCCTCATGCTTGGTGCAGACTG. The CHOP gene was cloned into the over-expression lentivector pCDH-CMV-MCS-EF1-GreenPuro (CD513B-1) (SBI, Mountain View, CA, USA) to generate pCDH-CMV-CHOP. HEK-293T cells were transfected with the construct along with three other plasmids (pGag/Pol, pRev, pVSV-G) into HEK293T cells using TurboFect (Thermo scientific) according to the manufacturer's protocol. The overexpression experiment was performed as follows. H9C2 cells (4 × 10 6 /well) were seeded in a 6-well plate. After 24 h, 50% of the medium in each well was removed, and polybrene was added to a final concentration of 8 μg/ml. The CHOP overexpressing lentiviruses (MOI = 1) were added to the cells. After an overnight incubation at 37 °C, the medium was replaced with fresh medium and incubated for another 72 h. The cells were then used to evaluate the expression of CHOP or for further experiments. and blocked in 5% non-fat dry milk prepared in 1 × TBS. The membranes were incubated with the primary antibodies for 4 h at RT or overnight at 4 °C (dilutions 1:500-1:1,000). The following primary antibodies were used: GRP78, ATF6, CHOP, XBP1, Caspase-12, Caspase-3 and GAPDH. After washing the membranes were incubated with secondary antibodies (1:5000) for 2 h at RT. The secondary antibodies used were horseradish peroxidase (HRP) conjugated anti-IgG corresponding to the primary antibodies. The blots were developed by using ECL Plus detection system (Amersham Pharmacis). The relative band intensity was measured by Image J software. Statistical Analysis. Significant differences between sham and I/R groups were made using ANOVA followed by Newman-Keuls multiple comparison test. The statistical analyses were performed using a 2-tailed Student's t-test and represented as mean ± SEM. *P < 0.05 and **P < 0.01 were considered statistically significant.