Exercise‐induced peptide EIP‐22 protect myocardial from ischaemia/reperfusion injury via activating JAK2/STAT3 signalling pathway

Abstract Recent studies have revealed that exercise has myocardial protective effects, but the exact mechanism remains unclear. Studies have increasingly found that peptides play a protective role in myocardial ischaemia‐reperfusion (I/R) injury. However, little is known about the role of exercise‐induced peptides in myocardial I/R injury. To elucidate the effect of exercise‐induced peptide EIP‐22 in myocardial I/R injury, we first determined the effect of EIP‐22 on hypoxia/reperfusion (H/R)‐ or H2O2‐induced injury via assessing cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) was assessed by fluorescence microscope. Meanwhile, Western blot and TUNEL methods were used to detect apoptosis level. Then, we conducted mice I/R injury model and verified the effect of EIP‐22 by measuring cardiac function, evaluating heart pathology and detecting serum LDH, CK‐MB and cTnI level. Finally, the main signalling pathway was analysed by RNA‐seq. In vitro, EIP‐22 treatment significantly improved cells viabilities and MMP and attenuated the LDH, ROS and apoptosis level. In vivo, EIP‐22 distinctly improved cardiac function, ameliorated myocardial infarction area and fibrosis and decreased serum LDH, CK‐MB and cTnI level. Mechanistically, JAK/STAT signalling pathway was focussed by RNA‐seq and we confirmed that EIP‐22 up‐regulated the expression of p‐JAK2 and p‐STAT3. Moreover, AG490, a selective inhibitor of JAK2/STAT3, eliminated the protective roles of EIP‐22. The results uncovered that exercise‐induced peptide EIP‐22 protected cardiomyocytes from myocardial I/R injury via activating JAK2/STAT3 signalling pathway and might be a new candidate molecule for the treatment of myocardial I/R injury.


| INTRODUC TI ON
Ischaemic heart disease is a type of cardiovascular disease with high morbidity and high fatality rate. The annual death toll is nearly 17.8 million, which has long occupied the top of various lethal factors. 1 Although interventional surgery has relieved clinical symptoms to a certain extent and improved the survival rate of patients with ischaemic heart disease, the myocardial injury caused by ischaemiareperfusion (I/R) is a difficult and cutting-edge problem in the field of cardiovascular disease prevention and treatment, 2 which needs to be solved urgently. Numerous studies have shown that cardiomyocytes apoptosis and reactive oxygen species (ROS) accumulation are strongly associated with myocardial ischaemia-reperfusion injury. [3][4][5] However, there is no effective method to protect myocardial from ischaemia-reperfusion induced injury.
Accumulating evidence suggested that moderate exercise can prevent and improve cardiovascular disease. 6 Exercise training can reduce long-term complications and improve the quality of survival after myocardial infarction. 7,8 Moderate exercise can alleviate myocardial ischaemia-reperfusion injury and has myocardial protective effects. 9,10 Recent studies have shown that some beneficial bioactive substances, such as irisin, are secreted in response to exercise stimuli, mediating the protective effects of exercise through antiapoptotic, antioxidant and anti-inflammatory effects. 11,12 However, the exact mechanism of the cardioprotective effect of exercise remains unclear. Further study of the specific mechanisms by which exercise protects the heart will hopefully lead to the development of new cardioprotective drugs.
Various peptides have been shown to be involved in cardiovascular-related pathophysiological processes and have been found to be promising drugs for the treatment of cardiovascular diseases, such as brain natriuretic peptide (BNP), 13 Apelin-13 14 and glucagon-like peptide-1 (GLP-1). 15 In recent years, peptide drugs, which have the advantages of small molecular weight, low toxicity, low immune response and high biological activity, have been widely used in clinical disease treatment. 16 These reports indicate that peptides may be effective molecules in the treatment of cardiovascular disease. In a recent study, differentially expressed peptides were identified in the peripheral plasma of four healthy male volunteers in the last minute of exercise and 1, 2 and 5 hours after exercise. 17 A total of 425 peptides were significantly regulated during or in the immediate hours after exercise (P < .05; ANOVA with permutationbased correction), of which a peptide, we named as EIP-22 (exerciseinduced peptide, 22 amino acids), that was up-regulated during exercise and had a high bioactivity prediction score (Peptide Ranker score is 0.80, greater than 0.5 indicate potential bioactivity) caught our attention.
Janus kinase (JAK)-signal transducer and activator of transcription (STAT) has been confirmed to be involved in the various pathophysiological response and play a key role in the development of cardiovascular diseases. 18,19 Janus kinase 2-signal transducer and activator of transcription 3 (JAK2/STAT3) is the most deeply studied signal pathway in JAK/STAT family. Growing evidence suggests that the JAK2/STAT3 signalling pathway plays a key role in mediating protection of myocardium from I/R injury. 20 Therefore, targeting the JAK2/STAT3 pathway will potentially hold a certain promise for the treatment of myocardial I/R injury.
Our study aims to evaluate the role and mechanism of a novel exercise-induced peptide EIP-22 in myocardial I/R injury. In this study, we proved that EIP-22 has cardioprotective effects against myocardial I/R injury in vitro and vivo through increasing cell viabilities, reducing apoptosis and ROS accumulation, improving cardiac function and ameliorating myocardial infarction area and fibrosis. In addition, we provided evidence that EIP-22 up-regulated the expression of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3

| Cell culture, hypoxic stimulation and experimental design in vitro
The cardiomyocytes were extracted from 1 to 3 days SD rats. After ventricular tissues were collected, cut and washed under aseptic conditions, 0.1% trypsin was added for digestion at 37℃ for 20 minutes, repeated 10 times. The supernatant was collected and transferred to a centrifuge tube containing 15 mL of 10% foetal bovine serum (FBS) for termination of pancreatin digestion. Next, the supernatant was centrifuged at 1000 r/min for 5 minutes at room temperature and resuspended with the appropriate DMEM medium. The collected cells were cultured in 37℃ and 5% CO 2 incubator for 2 hours, and the myofibroblasts were removed by differential attachment. The differential attachment of myofibroblast means to remove myofibroblasts by using the different time of adhesion between cardiomyocytes and myofibroblasts. After 2 hours, myofibroblasts were almost completely adherent, but cardiomyocytes were not adherent.
The cardiomyocytes in suspension were seeded on gelatin-coated (Sigma-Aldrich) culture plates, and then, cells were cultured on gel plates for 24-48 hours before further experiments. The cardiomyocytes were cultured in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Wisent, Canada) in an incubator with 95% air and 5% CO 2 at 37℃. For the H/R model, cardiomyocyte culture medium was converted into H/R buffer (4 mM HEPES, 12 mM KCl, 117 mM NaCl, 0.49 mM MgCl 2 , 0.9 mM CaCl 2 , 20 mM sodium lactate, 5.6 mM 2-deoxy-glucose, pH 6.2) and placed in a hypoxia chamber (95% N2/5% CO 2 , Billups-Rothenberg) at 37℃ for 30 minutes. The chamber was closed, and normal oxygen supply was given for an additional 4 hours, followed by reperfusion with DMEM medium supplemented with 10% FBS. 21 The peptides were added to the cell culture medium during reperfusion.

| Detection of cell viability
CCK-8 kit was used to detect cell viability according to the following steps. The cardiomyocytes (5 × 10 3 /well) were seeded in 96well plates, and upon reaching the adherence stage, the cells were treated with drugs as described above. 10 µL CCK-8 reagent was added to each well and cultured away from light in 37℃ incubators.
After 2 hours, a microplate reader was used to measure the intensity of the light absorption at 450 nm wavelength. In brief, we first calculated the number of inoculated cells in each group to ensure that the number of cells in each group is basically equal and then measured the OD 450nm value of each group after drugs treatment. The OD 450nm value was normalized by the number of inoculated cells. Cell viability was calculated with the normalized OD ratio of experimental and control well (Cell viability = OD 450nm experiment / OD 450nm control).

| Detection of LDH
Levels of LDH released were detected in the cell supernatant or mice serum using an LDH release assay kit (Nanjing Jian) according to the manufacturer's instruction. The cardiomyocytes (5 × 10 3 /well) were calculated and seeded in 96-well plates and were treated with drugs as described above. The cell supernatant or serum (120 µL/well) were collected and mixed with reaction solution (60 µL/well), and then, the mixtures were added into 96 well plates. The plate was maintained away from light for 30 minutes at room temperature on the shaker.
Finally, a microplate reader was used to measure the intensity of the absorbance at OD490nm wavelength. The OD 490nm value was normalized by the number of inoculated cells in each group.

| Detection of ROS, MDA, SOD and GSH-Px
The cardiomyocytes were plated in 6-well plates at a density of 5 × 10 5 per well and treated as described above. The levels of intracellular ROS were determined using a ROS assay kit following the manufacturer's instruction. The cells were incubated in serum-free DMEM medium including 0.1% DCFH-DA at 37°C for 20 minutes and then washed with serum-free DMEM three times. A fluorescence microscope (BX61; Olympus Corporation) was used to photograph. The ROS fluorescence was quantitated by Image J software 1.26. MDA, SOD and GSH-Px assay kits were used to detect intracellular MDA, SOD and GSH-Px levels according to the manufacturer's protocol.

| Determination of mitochondrial membrane potential
The mitochondrial membrane potential was measured by a JC-1 assay kit according to the manufacturer's instructions. The cardiomyocytes were plated in 6-well plates at a density of 5 × 10 5 per well and treated as described above. After treatment, the cardiomyocytes were cultured with serum-free DMEM medium including (1×) JC-1 staining working fluid at 37°C for 20 minutes. Then, the cells were washed twice with JC-1 buffer, and then 2 mL DMEM medium was added. The cardiomyocytes were photographed by a fluorescence microscope (BX61; Olympus Corporation) and quantitated by Image J software 1.26. JC-1 aggregates glow red fluorescence and monomers glow green.
After H/R treatment, the cardiomyocytes were washed twice with PBS and fixed with 4% paraformaldehyde. TUNEL staining (Promega) was used to visualize apoptotic cells following the manufacturer's instructions. The percentage of positive cells was calculated by TUNEL fluorescence density/DAPI fluorescence density, and the density was analysed using ImageJ software 1.26.

| Western blot analysis
The protein in the cardiomyocytes was extracted with lysis buffer (including RIPA and 1% PMSF) and quantified by a BCA assay kit (23229; Thermo Fisher Scientific). 1 × SDS loading buffer was added the protein samples and denatured by boiling at 95℃ for 5 minutes. After cooling on ice, the equivalent amounts of protein samples (20 mg) were separated by 8%-10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skimmed milk at room temperature and then incubated with the corresponding specific antibody overnight at 4°C.
The membranes were washed three times with TBST buffer. After that, the membranes were incubated with horseradish peroxidasecombined secondary antibodies (anti-rabbit/mouse) for 1 hour at room temperature. Protein expression was quantitatively analysed by Image Lab software (Bio-Rad). Western blot results were normalized by β-actin.

| Mice and I/R model
The male C57BL/6 mice, 8 week-old, were purchased from Shanghai Slake Experimental Animal Co., Ltd., and raised at the SPF Laboratory Animal Center of Nanjing Medical University.
The mice were fed adaptively for one week. All mice experiments were conducted according to the Guide for the Care and Then, the chest was opened and the heart was exposed to determine the position of the left anterior descending (LAD) coronary artery. The LAD was ligated with 6-0 silk thread for myocardial ischaemia. After 45 minutes of ischaemia, the ligation was released and the heart was reperfused for 1 week. The whole procedure was used in the sham operation without LAD ligation and release. The experimental mice were randomly allocated to four groups: the sham with Scr peptide group, the sham with EIP-22 peptide group, the I/R with Scr peptide group and the I/R with EIP-22 peptide group, and 12 mice in each group. The peptide EIP-22 or Scr were dissolved in normal saline and injected into the tail vein before reperfusion. The dosage of peptide (EIP-22 or Scr) was 10 mg/kg.

| Echocardiography
After a week reperfusion, all mice were anaesthetized lightly with 1.5% isoflurane and allowed to breathe spontaneously, and then, mice cardiac function was detected using high-resolution small

| Evans blue-TTC double staining
After a week reperfusion, the LAD coronary artery was religated, and 2 mL 2% Evans blue was injected intravenously to delineate the area at risk (AAR). Then, the heart was flushed with normal saline and frozen. After 30 minutes of cold storage at -20℃, it was cut into 1-2 mm thick myocardial sections along the long axis of the heart. The sections were incubated in 1% of 2,3,5-Triphenyltetrazolium chloride (TTC) at 37℃ for 30 minutes to delineate the myocardial infarct area (MI). The area of AAR and MI were measured using NIH Image J software.

| Masson staining
After the ventricular tissue was harvested, immediately fixed in 4% paraformaldehyde for 48 hours, the tissue samples were dehydrated, paraffin embedded and sectioned into 5 µm thick slices using a sliding microtome (Leica, Nussloch). Then, the tissue sections were dewaxed, rehydrated and stained with Masson's trichrome. Lastly, the microscope was used to observe the degree of myocardial fibrosis. Masson staining was quantified using ImageJ software.

| Detection of CK-MB and c-TnI
After reperfusion 2 hours, about 2 mL blood was taken from abdominal aorta and centrifuged at 4℃ for 10 minutes at 4000 r/min. After centrifugation, the serum was collected and stored in the ultralow temperature refrigerator for the detection of CK-MB and cTnI level. The detection instrument was enzyme-labelled instrument.
All operations were carried out strictly according to the instructions provided by the kit (Nanjing Jiancheng Biocompany).

| Statistical analysis
The experimental data and statistical graphs were analysed using GraphPad Prism 8 software. The data are presented as the means ± standard deviation (SD). Statistical differences were measured with an unpaired 2-sided Student t test or two-way ANOVA with Bonferroni correction for multiple comparisons.
When the P-value was < .05, the difference was considered significant.

| EIP-22 increased cell viability and reduced LDH release in cardiomyocytes exposed to H/R and H 2 O 2
To study the function of EIP-22, we constructed the model of  Figure 1F,H).
Next, we demonstrated that 20 µM EIP-22 could reduce LDH release in H 2 O 2 and H/R models ( Figure 1J,L), whereas the scramble peptide had no effect ( Figure 1I

| EIP-22 ameliorates myocardial I/R injury
To study the effect of EIP-22 in vivo, we established an myocardial I/R injury model by ligating the left anterior descending (LAD) coronary artery for 45 minutes, followed by reperfusion for 1 week.
The EIP-22 peptide was given through tail vein before reperfusion ( Figure 4A). The injection of EIP-22 peptide protected the heart from I/R injury, as demonstrated by improved cardiac function assessed by echocardiography analysis (Figure 4B,C). The myocardial infarction area was evaluated through the Evans Blue-TTC double staining. As shown in Figure 4D

| EIP-22 activates JAK2/STAT3 signalling pathway
To elucidate the potential mechanism of EIP-22 in cardioprotection, the RNA-seq approach was used to map the transcriptome after EIP-22 treatment in H/R model. 23 A total of 353 genes showed significant differences (P < .05, fold change > 2), of which 189 genes were up-regulated and 164 genes were down-regulated compared to H/R group ( Figure 5A,B). KEGG pathway analysis was shown in

| AG490 eliminates the cardioprotection of EIP-22
To further confirm that EIP-22 peptide protected cardiomyocytes from H/R-and H 2 O 2 -induced injury through activating JAK2/STAT3 signalling pathway, AG490, a selective inhibitor of JAK2/STAT3 signalling pathway, was used for rescue experiments. 25 As shown in Figure 6, the protein levels of p-JAK2 and p-STAT3 were inhibited whereas cells were treated with AG490 ( Figure 6A-D). Then, we Exercise has been shown to play an important role in various physiological process. Studies have shown the beneficial effects of exercise on the obesity and cardiovascular disease. 26,27 However, the specific mechanism of exercise in such process was still unknown.
Previous studies have shown that exercise-induced exosomal miR-342-5p can protect heart against myocardial infarction. 28 Ghrelin was a hormone which was induced by exercise and possessed an anti-apoptosis effect. 20 Similar, proteomics analysis also showed that protein was significantly changed during exercise. 29 Thus, we wondered to explore more components during exercise, which provide a better understanding for the beneficial effects of exercise.
It was well known that oxidative stress and apoptosis were the main events of myocardial injury. 30 33 In addition, cyclosporine A attenuated oxidative stress induced cardiomyocytes apoptosis through alleviating the production of ROS and up-regulating heat shock protein 70. 34 Therefore, from the perspective of anti-oxidative stress and anti-apoptosis, we may find the effective therapeutic target of myocardial injury caused by ischaemia-reperfusion.
The JAK/STAT signalling pathway is mainly composed of the JAKs protein family and the STATs protein family, among which JAK is the upstream kinase of STAT. The JAK/STAT signalling pathway is widely involved in a variety of biological effects such as cell stress, differentiation, proliferation and apoptosis. 35 Although we identified a novel exercise-induced peptide, which we named as EIP-22, protect cardiomyocytes from I/R injury and oxidative stress, there were still some limitations in our study. For example, whether different modification methods could influence the function of EIP-22 remains to be verified. In addition, the effect of EIP-22 peptide on other kinds of cardiomyocytes needs to be clarified in the future, such as H9c2 cells and AC16 cells. Therefore, in future work, we will evaluate the effect of EIP-22 on other types of cardiomyocytes and make different modifications to clarify its cardioprotective function.

| CON CLUS ION
In summary, this is the first study that we verified the function and mechanism of EIP-22 in myocardial I/R injury. Our study provided a new insight into treatment strategy of myocardial I/R injury.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.