Calhex231 ameliorates myocardial fibrosis post myocardial infarction in rats through the autophagy‐NLRP3 inflammasome pathway in macrophages

Abstract The calcium‐sensing receptor (CaSR) is involved in the pathophysiology of many cardiovascular diseases, including myocardial infarction (MI) and hypertension. The role of Calhex231, a specific inhibitor of CaSR, in myocardial fibrosis following MI is still unclear. Using Wistar rats, we investigated whether Calhex231 ameliorates myocardial fibrosis through the autophagy‐NLRP3 inflammasome pathway in macrophages post myocardial infarction (MI). The rats were randomly divided into sham, MI and MI + Calhex231 groups. Compared with the sham rats, the MI rats consistently developed severe cardiac function, myocardial fibrosis and infiltration of inflammatory cells including macrophages. Moreover, inflammatory pathway including activation of NLRP3 inflammasome, IL‐1β and autophagy was significantly up‐regulated in myocardial tissue, infiltrated cardiac macrophages and peritoneal macrophages of the MI rats. These impacts were reversed by Calhex231. In vitro, studies revealed that calindol and rapamycin exacerbated MI‐induced autophagy and NLRP3 inflammasome activation in peritoneal macrophages. Calhex231 and 3‐Methyladenine (a specific inhibitor of autophagy) attenuated both autophagy and NLRP3 inflammasome activation; however, the caspase‐1 inhibitor Z‐YVAD‐FMK did not. Our study indicated that Calhex231 improved cardiac function and ameliorated myocardial fibrosis post MI, likely via the inhibition of autophagy‐mediated NLRP3 inflammasome activation; this provides a new therapeutic target for ventricular remodelling‐related cardiovascular diseases.


| INTRODUC TI ON
Myocardial infarction (MI), a serious cardiovascular event, is the main cause of cardiac death. 1 The inflammatory response is critical in cardiac healing after MI; however, excessive inflammation can lead to adverse pathological remodelling such as cardiac enlargement, myocardial fibrosis and then cardiac dysfunction. 2 Macrophages, as the main response cells after MI, participate in the regulation of wound healing post MI, 3 in several ways including clearing necrotic cardiomyocytes, secreting cytokines such as interleukin 1 beta (IL-1β) and chemokines. 4 IL-1β promotes ventricular remodelling by inducing myocardial inflammation following MI. 5,6 The NLRP3 inflammasome promotes the activation and secretion of inflammatory cytokines IL-1β and IL-18, 7 and contributes to inflammation and infarct size post MI. 8 Autophagy is a conservative process of intracellular component recovery and degradation. 9 Studies have shown that autophagy is activated in cardiac hypertrophy and ischaemia-reperfusion injury. 10,11 Autophagy is related to the innate and adaptive immune systems, 12,13 and it is also closely associated with the NLRP3 inflammasome. 14 Evidence suggests that autophagy can regulate macrophage activity and responses to stimuli, [15][16][17] while the role of macrophage autophagy in MI is still unknown.
Calcium sensing receptor (CaSR), one of the seven-transmembrane (7TM) receptors, 18 works by sensing extracellular calcium concentration. CaSR is functionally expressed in the parathyroid, kidney and immune cells, including macrophages. 19 Calhex231 is a specific inhibitor of CaSR 20 which blocks Ca 2+ by competitive binding to the 7TM domains. 21 Calhex231 has been reported to exert a protective effect in cardiac hypertrophy 22 and diabetic cardiomyopathy. 23 Our previous study reported that CaSR activates the NLRP3 inflammasome by PLC-IP3 in macrophages and amplifies inflammation and ventricular remodelling post MI 24 ; however, the role and mechanism of Calhex231 in ventricular remodelling after MI remain unclear.
In this study, MI rats and peritoneal macrophages were treated with Calhex231, CaSR agonist, autophagy agonist, and antagonist or the inhibitor of NLRP3 inflammasome in order to elucidate the role and molecular mechanism of Calhex231 in myocardial fibrosis post MI.

| Rat MI model
Male Wistar rats (200 ± 20 g) were provided by the Animal Research Institute of Harbin Medical University. The experimental procedure was approved by the Institutional Animal Care and Use Committee of Harbin Medical University. All rats were raised in a 12h light/dark cycle and fed standard chow and water. After 1 week of adaptation, rats received a permanent ligation of the left anterior descending (LAD) artery. 25 Briefly, rats were intubated and ventilated after being anaesthetized with 10% chloral hydrate (0.3 mL/100 g). Then, the heart was visualized via left thoracotomy, and the LAD was exposed and ligated under the left atrium by using the 4-0 silk suture after separating the pericardium. Rats that died during anaesthesia resuscitation were not included in the analysis. After the operation, the rats were randomly divided into groups. The sham group: rats underwent the same procedure excluding LAD ligation (

| Isolation of rat peritoneal macrophages and groups
To identify the relationship between CaSR, autophagy, and the NLRP3 inflammasome in macrophages after MI, peritoneal macrophages were collected from the abdominal cavity of rats in each group by cold PBS aseptic lavage after anaesthesia. To detect the expression of the proteins, the cells were collected and prepared for Western blotting. Some peritoneal macrophages from MI 7 d rats were plated into 60 mm culture dishes containing 3 mL RPMI 1640.
To detect the relationship between CaSR and autophagy, cells

| Echocardiography
To evaluate the cardiac morphometry and function of rats in each group, a Vivid 7 Dimension echocardiographic system (GE Healthcare, Waukesha, WI, USA) was used to conduct echocardiography at predetermined intervals after anaesthesia (10% chloral hydrate, 0.3 mL/100 g). Heart rate (HR), left ventricular end-systolic dimension (LVESD), left ventricular posterior wall diameter (LVPWD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured and analysed.

| HE and Masson staining
Following immersion in 4% paraformaldehyde for 24 h, the myocardial specimens were embedded in paraffin and cut into 4 μm sections. Then, the cardiac slices were stained with haematoxylin, eosin and Masson's trichrome reagent. These stained slices were viewed by light microscopy (Olympus BX60, Beijing, China). The collagen area was calculated by randomly selecting 10 visual fields (×400 magnification) from each cardiac section. The area of blue-dyed collagen fibres, indicating fibrosis, was calculated using Image-pro Plus software. The degree of fibrosis was determined by the ratio of the fibrotic area to the myocardial area.

| Immunofluorescence
Immunofluorescence was used to detect the expression of CaSR, autophagy, NLRP3 inflammasome and IL-1β in myocardial infiltrating macrophages post MI. After antigen retrieval, antibodies against

| Western blotting
After treatment, peritoneal macrophages were collected and pro-

| IL-1β concentration measurement
The concentration of IL-1β in cell supernatant was detected using ELISA kit according to the manufacturer's instructions.

| Statistical analysis
The data were collected from at least three independent experiments. Data are presented as the mean ± standard error of the mean (SEM). For normally distributed data, t tests were used to test the difference between two groups, and one-way analysis of variance (ANOVA) was used between multiple groups, followed by Tukey's test. For non-normally distributed data, the differences between two groups were evaluated by Mann-Whitney U test, and Kruskal-Wallis test was used between multiple groups. A P-value of < 0.05 was considered to be statistically significant.  ing; Calhex231 ameliorated these changes (Figure 1 A, Figures S1 and S2). Moreover, Masson staining of the myocardium revealed that the ratio of fibrotic area to myocardial area was increased in slices of the myocardium in MI rats compared with similar parts in the sham group at each interval, and this effect was attenuated by Calhex231 (Figure 1 B). Proteins related to the myocardial fibrosis

| Calhex231 inhibited infiltration of inflammatory cells and activation of NLRP3 inflammasome post MI in rats
Histochemical findings showed that, compared with the levels in the sham rats, infiltration of macrophages (CD68 + cells) and IL-1β  Figures S2 and S3 A, B).
Moreover, compared with that of the sham group rats at the same point in time, immunofluorescence analysis of myocardial tissue of the MI rats showed that the colocalization of CD68 + CaSR + , CD68 + NLRP3 + , CD68 + ASC + , CD68 + pro-/Casp-1 + and CD68 + IL-

| Calhex231 alleviated activation of autophagy and the NLRP3 inflammasome in peritoneal macrophages post MI in vitro
Calindol, an agonist of CaSR, was used to detect the relationship  Meanwhile, the ELISA detection of IL-1β verified the results of Western blot ( Figure 7J).

| D ISCUSS I ON
The reperfusion injury in rats. 32 Guo et al also reported that CaSR expression was positively correlated with the sensitivity of cardiomyocytes to MI in rats. 33 In the present study, we successfully established a rat MI model as shown by significantly enlarged LVEDD and LVESD, dramatic decreases in LVEF and LVFS, obvious myocardial fibrosis, inflammatory cell accumulation and scar tissue formation compared to sham rats, which was consistent with the previous study. 25 We also found that Calhex231 improved LV Monocytes are closely related to infarction size and LVEF in MI patients, 39 and macrophages affect inflammation and heart failure post MI. 40,41 We reported that CaSR in macrophages increases ventricular remodelling by promoting the activation of the NLRP3 inflammasome. 24 The NLRP3 inflammasome, which consists of NLRP3, ASC and pro-Casp-1, promotes pro-Casp-1 conversion into its activated form, Casp-1, 8 and triggers the activation of proinflammatory cytokines such as pro-IL-1β to IL-1β; this results in initiation and amplification of inflammation-mediated fibrotic processes. 42 The NLRP3 inflammasome was examined in both myocardium-infil-  47 as well as promoting angiotensin II-induced inflammation and cardiac fibrosis. 48 Our study also showed that Rapa, an agonist of autophagy, increased MI-induced autophagy, NLRP3 activation and IL-1β secretion; however, specific antago-

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