Apela improves cardiac and renal function in mice with acute myocardial infarction

Abstract Apela was recently identified as a new ligand of the apelin peptide jejunum (APJ) receptor. The purpose of this study was to investigate the role of apela in post‐myocardial infarction (post‐MI) recovery from cardiorenal damage. A murine MI model was established, and apela was then infused subcutaneously for two weeks. Echocardiographs were performed before and after infarction at the indicated times. Renal function was evaluated by serum and urine biochemistry. Immunohistochemistry of heart and kidney tissue was performed by in situ terminal deoxynucleotidyl transferase‐mediated dUPT nick end‐labelling reaction. Compared to the control group (MI/vehicle), the average value of the left ventricular ejection fraction in apela‐treated mice increased by 32% and 39% at 2‐ and 4‐week post‐MI, respectively. The mean levels of serum blood urea nitrogen，creatinine, N‐terminal pro‐brain natriuretic peptide and 24‐hour urine protein were significantly decreased at 4‐week post‐MI in apela‐treated mice relative to that of control animals. At the cellular level, we found that apela treatment significantly reduced myocardial fibrosis and cellular apoptosis in heart and kidney tissue. These data suggest that apela improves cardiac and renal function in mice with acute MI. The peptide may be potential therapeutic agent for heart failure.


| INTRODUC TI ON
In recent years, research has found a non-coding RNA transcription gene called Apela (Elabela or Toddler); this gene can be transcribed and translated to produce a secreted hormone containing 55 amino acids, which becomes a mature short peptide of 32 amino acids after enzymatic cleavage. Apela and apelin can also act on an orphan G protein-coupled receptor called apelin peptide jejunum (APJ) receptor. 1,2 Previous studies showed that APJ receptors are widely expressed in human tissues and organs. 3 Exogenous apela can interact with APJ receptors in mammalian tissues and cells to exert corresponding biological effects. [4][5][6][7][8][9][10] Previous basic experiments have also shown that apelin can improve cardiac function in mice with myocardial infarction by inhibiting myocardial cell apoptosis, promoting angiogenesis and inhibiting myocardial fibrosis. 11 Sustained apela gene therapy can improve blood pressure in high-salt diet hypertension rats. 12 Exogenous apela can also reduce pulmonary arterial pressure in mice pulmonary hypertension models. 13 Recently, clinical studies have shown that endogenous plasma apela levels are higher in patients with acute myocardial infarction (MI) than in healthy controls; furthermore, apela levels are also negatively correlated with left ventricular ejection fraction (LVEF). 14 These basic experiments and clinical studies suggest that apela is valuable in the diagnosis and treatment of cardiovascular disease. In this study, we prepared a heart failure model of MI in mice to further investigate the effects of exogenous apela intervention on the heart and kidney function of mice with AMI as well as to explore the possible underlying mechanisms.

| Drug
The peptide of apela with a full length of 32-aa (QRPVNLTMRRK LRKHNCLQRRCMPLHSRVPFP) is synthesized by Hangzhou DGpeptides Co., Ltd with a purity of >98%. University. Eight-week-old male SPF-grade C57BL/6 mice were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. Mice had ad libitum access to food and water in the breeding environment, and the indoor temperature was controlled at 23°C ± 2°C. Mice were intraperitoneally injected with 10% chloral hydrate (0.02 mL/10 g), and the trachea was intubated with a ventilator to control ventilation frequency: 133 breaths/min, tidal volume (1-1.5 mL)]. The chest was opened to expose the heart, and then, a 7-0 suture was used to ligate the left anterior descending coronary artery about 2 mm below the left atrial appendage.

| Preparation of MI model
The sham-treated group underwent a similar procedure without ligation. Echocardiography was performed at 2 and 4 weeks after model preparation as previously described. 11 Briefly, mice were anesthetized with 2% isoflurane inhalation, and echocardiography was performed using a small animal ultrasound system (Vevo770, Canadian VISUALSONICS company) equipped with a 30-MHz probe. M-mode echocardiography was used to measure the left ventricular end-systolic and end-diastolic inner diameters, and the LVEF was calculated automatically.

| Grouping and drug administration
The experimental animals were divided three groups: sham operation procedure with saline treatment (sham + vehicle), MI with apela treatment (MI + apela), and MI with saline treatment (MI + vehicle).
Apela (1 mg/kg/d) or an equal volume of saline was continuously administered for 2 weeks by a micro-dosing osmotic pump (Alzet Model 1002, Alza Corp, Mountain View, CA, USA) implanted subcutaneously in the abdomen of mice.

| Blood and urine biochemistry
Serum endogenous apela (Eiaab, Wuhan, China) and NT-proBNP (Cloud-Clone Corp, Houston, TX, USA) was measured by ELISA according to the manufacturer's instructions. The serum urea nitrogen (BUN) and creatinine (Cr) were respectively detected using ELISA kits (Creative Diagnostics, USA) according to user's manual. The 24-h urine volume of mice was respectively collected before operation and 2 and 4 weeks after operation. The 24-h urine protein content was measured with a urine protein quantitative test box (Jiancheng bioengineering, Nanjing, China).

| Cardiac 2, 3, 5-triphenyltetrazolium chloride staining
Mice were euthanized by intraperitoneal injection of excessive pentobarbital. The heart was quickly removed and was rinsed with ice-cold saline to remove blood stains; it was then immediately fro-

| Heart tissue histology
Briefly, heart tissues were fixed with 4% formalin and embed-

| Immunohistochemical detection
Heart or kidney tissues were embedded in paraffin before staining. Then, 5-μm-thick sections were cut, deparaffinised and hydrated. After antigens were retrieved, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide. The tissue sections were incubated overnight at 4°C with primary antibody anti-CD31 (ab28364, Abcam, Cambridge, UK, 1:50) or MPO (ab208670, Abcam, Cambridge, UK, 1:1000). Samples were then incubated with a biotinylated secondary antibody and sequentially incubated in ABC-peroxidase solution. Finally, samples were stained with 3, 3'-diaminobenzidine, slightly counterstained with haematoxylin and fixed with neutral balata.

| TUNEL assay
Sections were detected by TUNEL assay using an In Situ Cell Death Detection Kit (KGA703, KeyGen Biotech, Nanjing, China). Briefly, heart or kidney tissue sections with paraffin-embedded were deparaffinised, rehydrated and antigen-retrieved. The following procedures were according to the instructions of the TUNEL assay kit.

| Statistical analysis
GraphPad Prism5 statistical software was used for data analysis. The data were expressed as mean value ± SD. The statistical differences were assessed using one-way ANOVA or Student's unpaired t test. A P value of <0.05 was considered significant.

| Apela improves heart function in mice after MI
There was no significant change in serum NT-proBNP or apela concentration in sham-treated mice before MI and at 2 and 4 weeks after MI, whereas the NT-proBNP or apela concentration in the control group (MI + vehicle) increased gradually after MI. The average NT-proBNP or apela concentration at 2 and 4 weeks after MI was significantly lower in the apela-treated group (MI + apela) than in the control group ( Figure 1A and B). Correspondingly, the mean value of LVEF at 2 and 4 weeks after MI was significantly higher in the apela-treated group than in the control group ( Figure 1C).
The average value of LVEF in apela-treated mice increased by 32% and 39% at 2-week and 4-week post-MI compared with control mice, respectively. Interestingly, we found a positive correlation between serum levels of apela and NT-proBNP in the control mice at 2 weeks after MI (r = 0.936, P < 0.05), whereas there was a negatively correlation between apela (r = −0.891, P < 0.05) or NT-proBNP (r = −0.976, P < 0.05) and LVEF in the control mice at 2-week post-MI (Table 1).

| Apela improves kidney function in mice after MI
Regarding serum BUN and Cr levels, compared to their baseline levels in mice before MI, their levels were higher at the 2 and 4 weeks after MI. The average BUN and Cr concentrations were lower in apela-treated mice than in control mice (MI + vehicle) at 2 and 4 weeks after MI (Figure 2A and B). Compared with control mice, the average value of 24-h urine volume significantly increased at 2 and 4 weeks after MI in apela-treated mice ( Figure 2C); furthermore, in apela-treated mice, the mean value of 24-h urine protein content was lower than that in control mice ( Figure 2D).

| Apela limits MI area and reduces myocardial fibrosis
Histopathological results showed that compared with the control group, the apela-treated group showed significantly decreased mean value of the area of myocardial infarction at 4 weeks after MI ( Figure 3A and B). Masson staining of the myocardial tissue in the control group showed obvious interstitial fibrosis at 4 weeks after MI, whereas the myocardial interstitial fibrosis was significantly reduced in apela-treated mice ( Figure 3C).

| Apela increases the proportion of CD31positive cells in the heart
To observe the myocardial neovascularization after infarction, we applied immunohistochemistry to detect the expression of neovascular endothelial cell marker CD31. 15 The results showed that the proportion of CD31-positive cells near the infarcted area of myocardial tissue was significantly higher in the apela-treated group than in the control group at 4 weeks after MI ( Figure 4A and B).

| Apela reduces the proportion of TUNELpositive cells in heart and kidney tissues
To observe the apoptosis of heart and kidney tissues after MI, we used TUNEL staining to evaluate the ratio of TUNEL-positive cells.
The results showed that at 4 weeks after MI, the percentage of TUNEL-positive cells in the non-infarcted area of the heart was significantly lower in the apela-treated group than in the control group ( Figure 5A). Similarly, the ratio of TUNEL-positive cells in kidney tissues was significantly lower in apela-treated mice than in control mice at 4 weeks after MI ( Figure 5B).

| Apela inhibits MPO expression in kidney tissue
Myeloperoxidase (MPO) is a haemoglobin protein that is rich in neutrophils and is widely used as a marker of inflammation. 16 Immunohistochemical tests showed that at 4 weeks after MI, the percentage of MPO-positive cells in kidney tissues of the control group was significantly higher than it was before MI, while the ratio of MPO-positive cells was significantly lower in the apela-treated group than in the control group ( Figure 6A and B).

| D ISCUSS I ON
The APJ receptor, also known as the orphan G protein-coupled receptor, was first isolated by O 'Dowd et al. in 1993. 17 In 1998, Tatemoto et al extracted and purified the first APJ receptor endogenous ligand from bovine gastric secretions and named it apelin. 18 After 2 decades of research, it has been found that apelin and APJ receptor are widely expressed in mammalian tissues and organs. The apelin/APJ receptor system exerts various biological effects through endocrine or para/autocrine manner, and it interacts with the digestive system, respiratory system, endocrine and reproductive system, central nervous system and cardiovascular system. In particular, apelin plays an important role in the occurrence and development of heart failure. 19,20 In recent years, two independent research teams from the United States and Singapore have discovered APJ receptor's second endogenous ligand named apela (Elabela/Toddler). The this suggests that apela and apelin have biological similarities. 1,2 Functional studies have also shown that apela and apelin can promote cardiomyocyte differentiation, induce angiogenesis and dilate aorta in vitro. [21][22][23][24] In vivo experiments have found that apelin and its structural homologs can alleviate myocardial ischaemia and reperfusion injury. 25,26 Sato et al. showed that apela can prevent pressure overload heart failure caused by transverse aortic constriction in mice. 27 Yurdaer et al found that the apela level was positively correlated with NT-proBNP (r = 0.586, P < 0.05), whereas the levels of apela ((r = −0.551, P < 0.05) or NT-proBNP (r = −0.684, P < 0.05) were negatively correlated with LVEF in patients with acute MI. 14 BNP or NT-proBNP is an important biomarker to evaluate the severity and prognosis of heart function. It is mainly secreted from the ventricle, and its synthesis and secretion are closely related with the ventricular load and wall tension. 28 As the BNP produced in decompensated heart failure is not enough to antagonize the activated rennin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS), so exogenous recombinant human BNP is widely used to treat decompensated heart failure. 29 In our study, we also observed that serum NT-proBNP increased significantly while LVEF declined in mice with AMI. After intervention with exogenous apela, NT-proBNP decreased significantly and LVEF increased significantly at 2 and 4 weeks after MI. These results suggest that apela can not only prevent heart failure caused by pressure overload but also improve heart failure caused by acute myocardial ischaemia. More interestingly, our results are partially similar to that of Yurdaer et al. We also found that endogenous apela or NT-proBNP was parallel regulated in mice post-MI, and the expression of apela was significantly correlated with LVEF at 2 weeks after MI. We speculate that endogenous apela is similar to BNP, which increases in decompensated heart failure, but the amount of endogenous apela is not enough to reverse decreased heart function. Therefore, exogenous apela is needed to improve cardiac function of heart failure. More interestingly, apela can improve not only the heart function in MI mice but also the kidney function. Coquerel et al. found that apela and apelin can improve renal function in septic shock rats and that apela has a stronger ability to inhibit plasma inflammatory cytokines than apelin. 32 Chen et al. made a mouse model of acute kidney insufficiency (AKI) by renal ischaemia/reperfusion and found that apela can inhibit renal cell apoptosis, DNA damage and inflammation and fibrosis of renal tissue in AKI mice. 33 In this study, we observed that apela increased 24-h urine volume and decreased urinary protein excretion significantly in mice after MI. Serum creatinine and urea nitrogen levels were also significantly lower in apela-treated F I G U R E 3 Photographs showing representative TTC staining and histology of heart. The slices were incubated with 2,3,5-triphenyltetrazolium-chloride (TTC) for 30 min. Non-infarcted myocardium was stained brick red, whereas infarcted tissue was unstained (A). The infarct size was measured and calculated as a percentage of the total area (B). Heart histology after Masson's trichrome staining of sections from each group (C). The data are presented as mean ± SD. *P < 0.05, vs. MI + vehicle group In recent years, cardiorenal syndrome (CRS) caused by the interconnection and influence of heart and kidney diseases has attracted the attention of cardiologists and nephrologists but mechanistic pathways have yet to be fully elucidated. 35 Cardiorenal syndrome is a clinical syndrome wherein acute or chronic dysfunction of the heart or kidney causes acute or chronic functional impairment of another organ. The American Heart Association classifies it into five types based on the incidence of heart and kidney disease. Acute renal failure secondary to acute heart failure is called type I cardiorenal syndrome, which is also a common type of cardiorenal syndrome in clinical practice. Once patients with acute heart failure are associated with renal insufficiency, the mortality and disability rates increase significantly.
Therefore, effectively preventing cardiorenal syndrome remains a challenge in clinical practice. 36,37 The data in our study suggest that apela improves cardiac and renal function in mice with acute MI. The most common cause of acute heart failure is AMI, and the abnormal haemodynamics as well as activation of the RAAS and SNS are the major factors that mediate acute heart failure secondary to AMI. 38 39,40 In the present study, the effects of apela on blood pressure, inflammation mediators or other major factors in mice after MI were not examined, which would be worthwhile to be studied in the future.

| CON CLUS IONS
In summary, our results reveal that apela can improve not only cardiac function after MI but also reserve renal function. We suggest that it is possible to develop apela as a clinical drug in the future that can not only treat heart failure but also prevent type I cardiorenal syndrome.

ACK N OWLED G EM ENTS
The work was supported from the National Natural Science

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

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.