Knockdown of circ_0060745 alleviates acute myocardial infarction by suppressing NF‐κB activation

Abstract It has been shown that circRNAs are involved in the development of heart diseases. However, few studies explored the role of circRNAs in acute myocardial infarction (AMI). The present study aims to investigate the role of circ_0060745 in the pathogenesis of AMI. We found that the expression of circ_0060745 was significantly increased in the myocardium of AMI mice and was mainly expressed in myocardial fibroblasts. The knockdown of circ_0060745 decreased myocardial infarct size and improved systolic cardiac functions after AMI. The knockdown of circ_0060745 in cardiac fibroblasts inhibited the migration of peritoneal macrophage, the apoptosis of cardiomyocytes and the expressions of IL‐6, IL‐12, IL‐1β, TNF‐α and NF‐κB under hypoxia. Overexpression of circ_0060745 caused an increase in infarct size and worsened cardiac functions after AMI. In summary, our findings showed that knockdown of circ_0060745 mitigates AMI by suppressing cardiomyocyte apoptosis and inflammation. These protective effects could be attributed to inhibition of NF‐κB activation.

low-and middle-income countries. In China, AMI has become a major cause of hospitalization and mortality. 3 In light of this, further understanding of the molecular mechanisms of AMI pathogenesis may provide a basis for the development of new treatment methods.
Circular RNAs are involved in the development of human cardiovascular diseases. 4 Deep RNA-sequencing identified a total of 15 318 and 3017 circRNA in human and mouse hearts. 5 Microarray analysis showed that knockdown of circCHFR could effectively suppress the proliferation and migration of oxidatively modified low-density lipoprotein-induced vascular smooth muscle cells. 6 Peripheral blood hsa_circ_0124644 was found to be a potential biomarker for coronary artery disease diagnosis. 7 The expression of circDNAJC6, circTMEM56 and circMBOAT2 was significantly lower in patients with hypertrophic cardiomyopathy. 8 For AMI, the down-regulation of circNifx could improve myocardial function and prognosis in AMI mice. 9 It has been known that NF-κB regulates cell growth, apoptosis and immune-inflammatory responses. 10 Suppression of NF-κB signalling pathway impeded the cardiac rupture and ventricular remodelling in AMI mice. 11 However, the interaction of circRNAs and NF-κB in the development of MI are still poorly understood.
In this study, we used an AMI mouse model to investigate the role of circ_0060745 in the pathogenesis of AMI in an attempt to find a new therapeutic target for AMI.

| Animals and AMI model
Male C57BL/6J mice, 8-10 weeks of age, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were kept at a 12-h light-dark cycle with ad libitum access to food and water. All experimental procedures were approved by the Ethics Committee of The Affiliated Hospital of Jiaxing University. Efforts were made to minimize the suffering of the animals. The AMI mouse model was established by permanent left anterior descending coronary artery (LAD) occlusion as previously described. 12 The mice were anaesthetized by intraperitoneal injection of 1% phenobarbital sodium (30 mg/kg), intubated and connected to a rodent ventilator. The left thoracic cavity was exposed by thoracotomy at the fourth intercostal space. The LAD coronary artery was ligated with a 6-0 suture at the lower edge of the left atrial appendage. The success of establishing the AMI model was confirmed if there was an immediate colour change on the heart surface after LAD ligation. Sham animals underwent the same surgical procedures, except the LAD was not occluded.

| Echocardiography and haemodynamics
Echocardiography was performed to assess the cardiac functions using the Vevo 2100 Ultrasound system (VisualSonics Inc, Toronto, Ontario, Canada) 3 days after surgery. Haemodynamics was measured using a Millar SPR-1000 catheter. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), diastolic (LVID,d) and systolic left ventricular internal diameters (LVID,s), left ventricular (LV) maximum pressure and dP/dt maximum rate were measured. 13 Subsequently, the mice's hearts were harvested and fixed in 4% paraformaldehyde overnight, and the infarcted myocardial tissues were collected for subsequent experiments.

| Isolation of adult mouse cardiomyocytes and cardiac fibroblasts
The cardiomyocytes and cardiac fibroblasts of adult mice were isolated as previously described. 14

| Isolation of neonatal mouse cardiomyocytes and cardiac fibroblasts
Procedures were outlined in a previous study. 15 Briefly, the hearts of 1-day-old mice were harvested, washed and minced into 1 mm 3 pieces. Heart tissues were detached with collagenase type II (240 U/mL) in HBSS at 37°C for 10 minutes. The supernatant was collected, filtered and then centrifuged at 100 g for 5 minutes. The pellet was re-suspended in DMEM/F12 (1:1) medium with 10% foetal bovine serum and 1% penicillin and incubated with 5% carbon dioxide at 37°C for 2 hours. Then, the non-adherent cardiomyocytes were collected while the adherent cells were mainly myocardial fibroblasts. Cardiomyocytes and fibroblasts were cultured for 48 hours and then used for subsequent experiments. The hypoxic condition was created in a tri-gas incubator containing 1% O 2 , 5% CO 2 and 94% N 2 .
Adenovirus (1 × 10 8 PFU, 50 μL) was injected intramyocardially into the apex with a 30-G needle 7 days before the surgery. Another group of mice injected with vectors (50 μL) was regarded as the control group. The cells were transfected using the Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA).

| Migration assay
Peritoneal macrophages were harvested from C57BL/6 mice (8-10 weeks old) as described. 16 Cells were seeded at a density of 1 × 10 6 to the upper chamber of the Transwell chambers (8 μm; Corning Inc., Corning, New York, USA). The lower chamber contained medium collected from the fibroblasts cultured under hypoxia. After incubation at 37°C for 16 hours, we removed the cells on the upper side of the membrane, fixed cells on the underside with 4% paraformaldehyde and stained them with crystal violet. Three randomly selected fields were viewed under a microscope.

| Measurement of the area at risk (AAS) and infarct size (IS)
Three days after ligation, Evans blue (Sigma-Aldrich, St. Louis, Missouri, USA) was injected into the apex of AMI mice to determine non-ischaemic tissues. The heart was detached, rapidly frozen at −20°C and cut into 2-mm slices. The slices were incubated in 2, 3, 5-triphenyl tetrazolium chloride (TTC, Sigma-Aldrich) at 37°C for 30 minutes and then fixed in 4% formaldehyde for 2 hours.
Finally, the slices were photographed, and AAR and IS were measured using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).

| RNA extraction and qRT-PCR
Total RNA of cells and heart tissues were extracted using TRIzol

| TUNEL staining
The cell apoptosis in the infarcted myocardium was assessed using the TUNEL staining kit (Roche, Basel, Switzerland). Heart tissue slices were fixed in 4% paraformaldehyde for one h, permeabilized with 0.1% Triton X-100 for 2 minutes and incubated with the TUNEL mixture for 1 hour. Nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Apoptotic cells were counted in five random fields under a fluorescence microscope. The per cent of TUNEL-positive cells in total myocardial cells was calculated.

| Western blot analysis
The total protein of mouse cardiac tissues and cells was extracted with the Pierce IP Lysis Buffer (Thermo Fisher Scientific Inc.). The concentration of proteins was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of extracted protein (60 μg) were separated by 10% SDS-PAGE and were transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 hours and incubated with primary antibodies: Bcl-2 antibody, Bax antibody and NF-κB antibody (Abcam, Cambridge, MA, USA) at 4°C overnight and incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. GAPDH antibody was used as the internal control. The bands were analysed using the Enhanced Chemiluminescence Kit (GE Healthcare, Chicago, IL, USA). SPSS 21.0 (SPSS, IBM, USA) was used to analyse the data. Data are described by means and standard deviations (SD). Differences between groups were tested using a two-tailed Student's t test.

| Relative circ_0060745 expression was upregulated in the myocardium of AMI mice
RT-PCR was employed to monitor the trend of circ_0060745 expression in the infarcted myocardium of AMI mice. The expression of circ_0060745 was significantly increased at 2 hours after AMI and continued to increase within 24 hours ( Figure 1A). To locate the circ_0060745 in the myocardium, we isolated the cardiomyocytes and cardiac fibroblasts and tested the expression of circ_0060745 separately. No significant difference in circ_0060745 expression was found in the cardiomyocytes between the AMI and the sham group ( Figure 1B). The level of circ_0060745 in myocardial fibroblasts was approximately 10 times higher than that of the cardiomyocytes and was significantly increased in the AMI group ( Figure 1B).
We then examined the effects of circ_0060745 by knockdown and overexpression. We found that circ_0060745 was successfully knocked down by shRNA against circ_0060745 in both AMI and sham group ( Figure 1C). Using Evens blue and TTC staining, we found that the infarct size (IS) of the sh-Circ group was significantly lower than that of the sh-NC group. We observed no significant difference in areas at risk (AAR) between the two groups ( Figure 1D). Besides, the expression of circ_0060745 was higher after transfection with circ_0060745 overexpression plasmid, which was related to an increase in infarct size in AMI mice ( Figure 1E,F). These observations indicated that circ_0060745 had a role in AMI in vivo.

| Circ_0060745 modulated the cardiac function after AMI
We tested whether knockdown or overexpression of circ_0060745 may influence the recovery of cardiac dysfunction post-AMI. was not observed in the sham group (Figure 2A-F). In addition, the trends of these cardiac function indicators were strengthened by circ_0060745 overexpression (Figure 2G-L). These results suggested that the systolic cardiac function of AMI mice was impaired and was modulated by circ_0060745.

| Circ_0060745 regulated apoptosis after AMI
We then used TUNEL staining to assess the apoptosis after AMI in mice. As shown in Figure 3A,B, the cell apoptosis rate in the infarcted areas of the sh-Circ group was significantly lower than that of the sh-NC group. Using Western blot, we found a significantly higher expression of Bcl-2 but a lower expression of Bax in the sh-Circ group than that of the sh-NC group ( Figure 3C-E). Conversely, overexpression of circ_0060745 enhanced the apoptosis of cells after AMI ( Figure 4A,B). Overexpression of circ_0060745 decreased the expression of Bcl-2 and increased the expression of Bax after AMI ( Figure 4C-E). These observations indicated that circ_0060745 regulated cell apoptosis after AMI.

| Knockdown of circ_0060745 in cardiac fibroblasts inhibited the migration of peritoneal macrophage under hypoxia
To further elucidate the mechanism of circ_0060745 on the cardiac function in mice, cardiac fibroblasts were isolated from newborn C57 mice (nCFs). Using RT-PCR, we found that the relative circ_0060745 expression was significantly increased at 2 hours under the hypoxic condition and peaked at 12 hours ( Figure 5A). Therefore, cardiac fibroblasts treated with 12-hour hypoxia were

| Knockdown of circ_0060745 in cardiac fibroblasts inhibited the apoptosis of cardiomyocytes under hypoxia
To examine the effects of circ_0060745 knockdown on cardiomyocytes, we co-cultured neonatal cardiomyocytes with nCFs transfected with H + sh-Circ or H + sh-NC under normal or hypoxic conditions. Flow cytometry showed that the apoptosis rate of neonatal mouse cardiomyocytes was significantly increased in the H + sh-Circ group than that of the H + sh-NC group ( Figure 6A,B).
The H + sh-Circ group also had a higher expression of Bcl-2 and a lower expression of Bax than the sh-NC group ( Figure 6C-E). These results indicated that circ_0060745 knockdown in nCFs inhibited the apoptosis of cardiomyocytes under hypoxia.

| Knockdown of circ_0060745 in cardiac fibroblasts inhibits the activation of NF-κB under hypoxia
Western blot was used to determine the expression of p65, a subunit of NF-κB, in the nuclear extract of nCFs. The results demonstrated that the NF-κBp65 was significantly increased in nCFs cultured under hypoxia compared with the normoxic group. The hypoxia-induced protein overexpression of NF-κBp65 was restored by shRNA against circ_0060745 ( Figure 7A,B). Moreover, knockdown of circ_0060745 decreased the p-IκBα/IκBα ratio in nCFs under hypoxia ( Figure 7C).
These data indicated that knockdown of circ_0060745 suppressed the activation of NF-κB.

| D ISCUSS I ON
It has been established that circular RNAs play a crucial role in the development of a variety of human diseases, 17  improving the myocardial function and prognosis of MI. 9 It has been revealed that circ-Ttc3 reduced myocardial ATP depletion and apoptosis by sponging miR-15b. 18 Both in vivo and in vitro experiments showed that overexpression of circFndc3b reduced the apoptosis of cardiomyocyte and endothelial and improved myocardial function by binding to the FUS protein instead of being a miRNA sponge. 19 It was found that the expression of circRNA MICRA in the group of cardiac dysfunction was higher than that in the control group, suggesting that MICRA can be used for the risk classification of AMI and early judgment of cardiac prognosis. 20 circRNA_081881 was significantly reduced in the plasma of MI patients and associated with the expression of miR-548 and PPARγ synthesis. 21 Our study explored the role of circ_0060745 in the development of AMI. We found that circ_0060745 was significantly elevated in the myocardium of AMI mice. Furthermore, knockdown Prior studies have noted the importance of cardiac fibroblasts in the reparative response during heart remodelling post-myocardial infarction. 22,23 After the injury, the fibroblasts proliferate and become the majority of cells in the infarct area. In the early stages of infarct healing, cardiac fibroblasts are crucially involved in the inflammatory response and production of cytokines, chemokines and proteases. 24 Our study discovered that circ_0060745 was mainly expressed in cardiac fibroblasts, which is in accord with previous studies that The apoptosis of cardiomyocytes plays a crucial role in the process of AMI. 25 Myocardial apoptosis was strongly associated with unfavourable left ventricular remodelling and early symptomatic post-infarction heart failure. 26 Apoptosis was inhibited by the down-regulation of miR-200a in AMI models. 27 In the present study, the degree of apoptosis was less severe in mice treated with shRNA against circ_0060745, while apoptosis was enhanced by overexpression of circ_0060745. The apoptosis of cardiomyocytes was also inhibited when they were co-cultured with nCFs that were treated with shRNA against circ_0060745 and hypoxia. Based on these results, we hypothesized that circ_0060745 regulates the infiltration or migration of inflammatory cells and the apoptosis of cardiomyocytes by regulating the production of inflammatory cytokines in cardiac fibroblasts.
The nuclear factor kappa B (NF-κB) plays a vital role in a variety of biological processes such as inflammation, immunity, cell growth, development and survival. 10 Activation of the NF-κB signalling pathway increases the production of proinflammatory cytokines, including IL-6, IL-12, IL-1β and TNF-α. 28 Inhibition of the expression of MIRT1 suppressed the activation of the NF-κB signalling pathway, alleviating myocardial fibrosis, myocardial cell apoptosis, oxidative stress and inflammatory damage. 29 The silencing of IRAK3 inactivated the NF-κB signalling pathway, thereby attenuating the progression of AMI in mice. 11 The silencing of ATP2B1-AS1 had a protective effect post-MI by blocking the NF-κB pathway. 30 In our study, knockdown of circ_0060745 suppressed the expressions of IL-6, IL-12, IL-1β, TNF-α and NF-κBp65 under hypoxia. These data suggest that circ_0060745 may exert its effects in AMI by modulating the activation of NF-κB, thereby influencing the production of proinflammatory cytokines. However, the mechanisms involved in the NF-κB inhibition mediated by circ_0060745 knockdown require further studies.
In summary, our study found that the expression of circ_0060745 was increased in the myocardium post-AMI. Circ_0060745 regulated myocardial infarct size and improved left ventricular cardiac functions after AMI. Moreover, knockdown of circ_0060745 inhibited the expressions of IL-6, IL-12, IL-1β, TNF-α and NF-κB pathway.
These findings provide insights into the roles of circRNAs in the development of AMI.

ACK N OWLED G M ENTS
None.

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

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