Reagents

Zr17-2 was purchased from AOBIOUS INC (Cat No: AOB33334, USA). Fetal bovine serum (FBS), DMEM medium, streptomycin, and penicillin were obtained from GIBICO (USA) for cell culture.

Ethical approval

Male Sprague-Dawley (SD) rats (6 weeks, weighing from 180 to 220g) were purchased from HFK Biotechnology Company (Beijing, China). All the animal experiment protocols were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University and in accordance with ARRIVE gudelines. During the experiment, rats were housed in a temperature-regulated room where the ambient temperature was 22°C and the humidity was 50%. Rats were provided with food and water ad libitum in a 12-h:12-h light/dark cycle. The rats were placed in a bell jar at the end of the experiment, and they were deeply anesthetized with isoflurane for euthanasia.

Myocardial Infarction model

Rats were randomized to sham, MI+saline, and MI+zr17-2 groups. Rats were anesthetized with pentobarbital sodium (40mg/kg, IP) and ventilated by endotracheal intubation using a Zoovent ventilator. MI was induced by ligating the left anterior descending coronary artery (LAD) as previously described [10]. Successful occlusion of the artery was confirmed by the ST-segment elevation in the electrocardiogram (ECG). The MI+zr17-2 group was treated with zr17-2 (20nmol/kg, i.p.) once every other day 3 times before the operation. The sham group underwent the same procedure as MI-operated rats except for the ligation of LAD.

Echocardiography

Echocardiography was performed using a Vevo2100 imaging system (Visual Sonics Inc., Toronto, Canada). Rats were anesthetized with pentobarbital sodium (40mg/kg), and transthoracic echocardiography was performed by an experienced operator to assess cardiac function. The left ventricular (LV) ejection fraction (LVEF), LV internal diameters in end-diastole (LVIDd), LV fractional shortening (LVFS), LV end-diastolic volume (LVEDV), LV end-systolic volume(LVESV), LV internal diameter at end-systole (LVIDs) and interventricular septum diameter at end-diastole (IVSD) were detected by M-mode tracing system.

Histological analysis

Rats were euthanized and hearts were isolated and immersed in buffered formalin for 24 hours.LV tissues were cut into a series of 5-μm-thick slices and stained with hematoxylin-eosin (H&E) and Sirius red to evaluate collagen deposition, or stained with CD68 antibody followed by FITC conjugated-secondary antibody to evaluate macrophage infiltration. Nuclei were counterstained with DAPI and sections were examined with a fluorescent microscope. The percentage of LV circumference was measured to evaluate infarction size at 7 days post-infarction.

Quantitative real-time PCR

Total RNA was obtained using the Trizol reagent. 5 μg RNA was used to synthesize complementary DNA. qRT-PCR was performed using an Applied Biosystems 7500 Fast Dx Real-time PCR instrument (Thermo Fisher). The primer sequences were shown in Table 1. β-action was used as an internal reference and the fold alterations of each target mRNA level relative to β-actin under different conditions were detected based on the threshold cycle (CT) as r=2-Δ(ΔCT), where ΔCT=CT (target)-CT (β-action) and Δ(ΔCT) = ΔCT (experimental)- ΔCT (control).

Table 1 The primer sequences for qRT-PCREnzyme-linked immunosorbent assay (ELISA)

Serum interleukin-6 (IL-6) level was detected using rat commercially available ELISA assay kit (R&D SYSTEM), relying on the product description.

Cell culture and treatment

H9C2 cells (obtained from the Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were cultured using DMEM containing high glucose (4.5 g/L) ,10% heat-inactivated FBS, penicillin(100 units/ml), and streptomycin(100 µg/ml) at 37℃in a 5% CO2-humidified incubator. Cells were pretreated with zr17-2 (10µmol/L) for 2 days before the stimulation with H2O2(600µM) for 24 h. Subsequently, cells were subjected to the assay of CCK8 and western blot study.

siRNA transfection

All chemical CIRP-targeting siRNA (siCIRP) and nonsense siRNA (negative control siRNA, siCtrl), described in this study were synthesized by Genscript (Shanghai, China). After cells were plated, siRNA was mixed with lipofectamine RNAiMAX (catalog number:13,778,075, Invitrogen) and then added into cell culture for 48 h according to the manufacturer’s instructions. The final concentration for the nonsense siRNA or CIRP siRNA was 10 nM. The silencing efficiency of the siRNA was verified by the protein level of the targets.

Western blot analysis

Total proteins were extracted from the left ventricular tissues. The protein concentration was determined using the Pierce BCA protein Assay kit (Pierce) and 50 µg protein was collected for SDS-PAGE (Invitrogen) and transferred to a polyvinylidene fluoride membrane (Millipore). Proteins were then incubated with several primary antibodies [NQO-1 (ab2346), IL-1β (ab9722), CIRP (ab106230), collagen III (ab7778), and collagen 1(ab6308) obtained from Abcam; ICAM-1 (sc-107), Bax (sc-7480), VCAM-1(sc-13,160), Bcl2(sc-7382), Nrf2 (sc-81,342), and HO-1 (sc-136,960), GAPDH (sc-47,724), obtained from Santa Cruz; cleaved-caspase 3 (#9661) and TGF-β1 (#3711), purchased from Cell Signaling; collagen I (PA1-26204), purchased from Invitrogen] overnight at 4℃ and then incubated with HRP-conjugated secondary antibodies at room temperature for another 2-hour and the blots were visualized with ECL (Bio-Rad) reagent. The protein expression was normalized to GAPDH.

Statistical analysis

Data are represented as the mean ± S.D. Data were analyzed by one-way ANOVA, followed by Tukey’s Multiple Comparison Test. All statistical analyses were performed in GraphPad Pro 5.0. Statistical significance was set at P < 0.05 (*p < 0.05, **p < 0.001, ***p < 0.001 and ****p < 0.0001).