Male Sprague-Dawley rats (n=44; weight, 220–260 g; age, 7 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were fed standard chow and water and raised under specific pathogen-free conditions at 22°C and 50% relative humidity with an artificial 12-h light/dark cycle. The present study was approved by the Animal Ethics Committee of Qingdao Municipal Hospital (approval no. 2020-L-054).

After one week of adjustment, rats were divided into Control (n=10), Sham (n=10) and MI/RI (n=24) groups. Rats without any treatment were defined as the Control group. The Sham group was subjected to all procedures except ligation. The MI/RI model was induced via ligation of the left anterior descending coronary artery (LAD) as previously described, with some modifications (30). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg) via intraperitoneal injection. Subsequently, the LAD was ligated with a 6.0 thread and the ligation line was released after 45 min, followed by reperfusion for 24 h. Thus, an MI/RI model was established. In order to determine the effect of HRIM on MI/RI rats, MI/RI rats were divided into the following groups: MI/RI (MI/RI rats without treatment), MI/RI + small interfering RNA (siRNA) negative control (si-NC; MI/RI rats injected with 200 µg/kg/d si-NC via the tail vein following surgery) and MI/RI + HRIM siRNA (si-HRIM; MI/RI rats injected with 200 µg/kg/d si-HRIM via the tail vein following surgery) (n=8/group). Both si-NC (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′) and si-HRIM (forward, 5′-GCGCCAUUGCUGCAAAUUATT-3′ and reverse, 5′-UAAUUUGCAGCAAUGGCGCTT-3′) were purchased from Shanghai GenePharma Co., Ltd.

TTE was performed using a Vevo770 scanner (VisualSonics, Inc.). The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVEDD), ventricular end-systolic diameter (LVESD), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum velocity of LV contraction (+dp/dt_max) and maximum velocity of LV diastolic function (-dp/dt_max) were measured via TTE in rats.

At one week after establishment of the MI/RI model, rats were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg) and sacrificed by neck dislocation. Myocardial tissue was isolated, sectioned (thickness, 3 mm), stained with 1% TTC (cat. no. A610558; Sangon Biotech Co., Ltd.) for 30 min at 37°C, and fixed in 10% formalin for 10 min at 37°C. After washing with water, infarct size was quantified using the Motic Med 6.0 Digital Medical Image Analysis system (Motic Instruments). Infarcted myocardium was stained white, whereas surviving myocardium was stained brick red. The infarct size was calculated as follows: Infarct size (%)=(weight of white sections/weight of whole cardiac) ×100%.

Myocardial tissue was collected, fixed in 4% paraformaldehyde at 37°C for 24 h, dehydrated with an ethanol gradient (70% ethanol for 2 h, 80% ethanol overnight, 90% ethanol for 2 h, 100% I ethanol for 1 h and 100% II ethanol for 1 h) vitrified with xylene, waxed, embedded in paraffin and cut into sections (thickness, 4 µm). The sections were then stained with 0.5% hematoxylin for 5 min at 37°C, followed by 0.5% eosin for 1 min at 37°C. The ratio of inflammatory cells/field (5 random fields) in myocardial tissues was calculated by light microscopy (magnification, ×400). Stained cells were analyzed using Image-ProPlus (version 6.0; Media Cybernetics, Inc.)

MI/RI was mimicked in vitro by establishing an OGD/R model using myocardial cells (H9C2 cells). H9C2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS at 37°C and 5% CO₂. In order to establish an OGD/R model, serum/glucose-free DMEM was used. H9C2 cells were placed in an anaerobic chamber (95% N₂ and 5% CO₂) at 37°C for 3 h. The medium was then replaced with normal culture medium and incubated for 0, 4 and 8 h to reoxygenate the H9C2 cells. The OGD3h/R0h, OGD3h/R4h and OGD3h/R8h groups received reoxygenation at 3 h post-OGD for 0, 4 and 8 h, respectively. In addition, H9C2 cells without treatment were regarded as the Normal group.

For transfection experiments, H9C2 cells at >80% confluency were harvested and divided into three groups: OGD/R (H9C2 cells with reoxygenation at 3 h post-OGD for 8 h), OGD/R + si-NC (cells transfected with 50 nmol si-NC for 48 h) and OGD/R + si-HRIM (cells transfected with 50 nmol si-HRIM for 48 h). Asatone (an activator of the NF-κB pathway; cat. no. HY-N6826; MedChem Express) (32) was used to further investigate the association between HRIM and the NF-κB pathway. Thus, the OGD/R + si-HRIM + Asatone group comprised cells transfected with 50 nmol si-HRIM and treated with 20 µmol/l asatone at 37°C for 48 h. Cell transfection was performed using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 25°C.

Total RNA was extracted from myocardial tissue or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and subsequently reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. Subsequently, qPCR was performed using the MiScript SYBR Green PCR kit (Qiagen, Inc.) and a ABI 7500HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 3 min and 40 cycles of 95°C for 15 sec and 60°C for 30 sec, and a final extension step at 72°C for 10 min. Relative expression levels were calculated via the 2^−ΔΔCq method (33). HRIM expression levels were normalized to those of GAPDH. The primer sequences are presented in Table I.

A Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology) was used to extract nuclear and cytoplasmic proteins according to the manufacturer's protocol. Myocardial tissue from Sprague-Dawley rats or H9C2 cells were lysed using lysis buffer on ice to extract the total protein. Protein concentration was determined using a bicinchoninic acid protein concentration assay kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (20 µg) were separated via 10–12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline, containing 0.01% Tween-20 at 4°C overnight. The membranes were then incubated with the following primary antibodies (all 1:1,000; all Cell Signaling Technology, Inc.) overnight at 4°C: Anti-NF-κB p65 (cat. no. 8242), anti-phosphorylated (p)-NF-κB p65 (cat. no. 3033), anti-IκBα (cat. no. 4812) and anti-p-IκBα (cat. no. 4812), followed by incubation with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:5,000; cat. no. ab205718; Abcam) for 1 h at 37°C. The protein bands were visualized using enhanced chemiluminescence exposure solution (Pierce; Thermo Fisher Scientific, Inc.) and quantified with Quantity One 1-D analysis software (version 4.62; Bio-Rad Laboratories, Inc.). β-actin (1:1,000; cat. no. sc-517582; Cell Signaling Technology, Inc.) was used as the internal reference.

Cell Counting Kit-8 (CCK-8) reagent (BD Biosciences) was used to evaluate the proliferation of H9C2 cells according to the manufacturer's instructions. H9C2 cells (5×10³ cells/well) were seeded onto a 96-well plate and subjected to OGD/R. CCK-8 solution (10 µl/well) was then added and incubated for 2 h at 37°C. The optical density at 450 nm (OD₄₅₀) was measured using a microplate reader (BioTek Instruments Inc.). Wells containing only culture medium and CCK-8 reagent were used as the blank group.

After 48 h transfection in 24-well plates (3×10⁵ cells/well), cells were collected in 10 ml centrifuge tubes and centrifuged twice at 1,000 × g for 5 min at 4°C. Subsequently, the supernatant was collected and resuspended in binding buffer (140 mM NaCl, 2.5 mM CaCl₂, 10 mM HEPES/NaOH, pH 7.4) to adjust the cell density to 1–5×10⁶ cells/ml. Next, 100 µl cell suspension was cultured with 5 µl V-FITC and 10 µl PI for 15 min in the dark at 25°C. An additional 400 µl 1X binding buffer was added to each tube. The apoptotic rate was then detected using a FACSArray BD Bioanalyzer flow cytometer (BD Biosciences), and data were analyzed via CytoDiff CXP software (version 2.0; Beckman Coulter, Inc.).

Following cell transfection, cells from each group were centrifuged at 3,000 × g at 4°C for 10 min and the resulting supernatant was collected. The supernatant was collected and levels of TNF-α, IL-1β, IL-6, lactate dehydrogenase (LDH) and creatine kinase (CK) were measured via ELISA kits (TNF-α, cat. no. KRC3011; IL-1β, cat. no. BMS627; IL-6, cat. no. BMS625; all Thermo Fisher Scientific, Inc.; LDH, cat. no. ab183367; CK, cat. no. ab264617; both Abcam). The absorbance of each pore was measured at a wavelength of 450 nm via an enzyme mark sensing instrument (Molecular Devices LLC).

Statistical analysis was performed via SPSS software 23.0 (IBM Corp.). Data are presented as the mean ± SD of three independent repeats. Comparisons between groups were performed via ANOVA followed by post hoc Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Once the MI/RI model was established, hemodynamic indices were evaluated via TTE. Compared with the Control and Sham groups, LVEF, LVFS, LVSP, +dp/dt_max, and -dp/dt_max significantly decreased while LVEDD, LVESD, and LVEDP were significantly increased in the MI/RI group (P<0.01 and P<0.01; Fig. 1A). TTC staining results demonstrated that myocardial infarct size was significantly higher in the MI/RI group compared with the Sham group (P<0.001; Fig. 1B). HE staining revealed that the ratio of inflammatory cells/field in myocardial tissue was significantly increased in the MI/RI group compared with the Sham group (P<0.001; Fig. 1C). Additionally, HRIM expression levels were significantly upregulated in the MI/RI group compared with the Sham group (P<0.001; Fig. 1D).

H9C2 cells were subjected to OGD/R to mimic MI/RI conditions in vitro. Following reoxygenation at 3 h post-OGD for 0, 4 and 8 h, the survival rate of H9C2 cells significantly decreased compared with normal cells (P<0.01 and P<0.001; Fig. 2A). RT-qPCR results demonstrated that the expression levels of HRIM in H9C2 cells significantly increased in the OGD3h/R0h, OGD3h/R4h and OGD3h/R8h groups compared with the Normal group (P<0.001; Fig. 2B). The OGD3h/R8h group was selected for subsequent experiments due to the relative high HRIM expression.

In order to examine the role of HRIM, effective HRIM knockdown was performed in H9C2 cells via si-HRIM transfection (P<0.001; Fig. 2C). The CCK-8 assay results demonstrated that the OD₄₅₀ value of the OGD/R group was lower compared with the Normal group at 48 and 72 h post-culturing (P<0.01), and that silencing of HRIM significantly increased the OD₄₅₀ value of H9C2 cells compared with the OGD/R + si-NC group at 48 and 72 h post-culturing (P<0.01; Fig. 2D). Additionally, apoptosis of H9C2 cells significantly increased following OGD/R compared with the Normal group (P<0.001), whereas HRIM knockdown decreased H9C2 cell apoptosis compared with the OGD/R + si-NC group (Fig. 2E).

In order to investigate the effect of HRIM knockdown on inflammation in H9C2 cells, levels of IL-1β, TNF-α and IL-6 were determined via ELISA. Compared with the Normal group, the levels of IL-1β, TNF-α and IL-6 in H9C2 cells were significantly increased in the OGD/R group (P<0.001); however, HRIM knockdown significantly attenuated these effects (P<0.001; Fig. 3A). Moreover, compared with the Normal group, the levels of LDH and CK in H9C2 cells were significantly elevated in the OGD/R group (P<0.001), and HRIM inhibition significantly decreased these levels in the OGD/R + si-HRIM group (P<0.001; Fig. 3B and C).

In order to evaluate the effect of HRIM inhibition on the NF-κB signaling pathway in H9C2 cells, the expression levels of NF-κB-associated proteins were measured via western blotting. The results demonstrated that the levels of p-NF-κB p65/NF-κB p65 and p-IkBα/IkBα were significantly increased in the OGD/R group compared with the Normal group (P<0.001). However, compared with the OGD/R + si-NC group, knockdown of HRIM significantly decreased the levels of p-NF-κB p65 and p-IkBα (P<0.01; Fig. 4A). Furthermore, compared with the Normal group, the levels of cytoplasmic NF-κB p65 significantly increased (P<0.001), but those of nuclear NF-κB p65 significantly decreased in the OGD/R group (P<0.001); however, HRIM knockdown attenuated these effects (P<0.01; Fig. 4B).

In order to investigate the association between HRIM and NF-κB signaling, H9C2 cells were treated with the NF-κB signaling pathway activator asatone. CCK-8 assay and flow cytometry results demonstrated that HRIM knockdown significantly increased the OD₄₅₀ value at 48 and 72 h (P<0.001) post-culturing and significantly decreased the apoptosis of H9C2 cells (P<0.001; Fig. 5A and B). Moreover, HRIM knockdown significantly decreased the levels of TNF-α, IL-1β, IL-6, CK and LDH in H9C2 cells (P<0.001; Fig. 5C-E). However, the inhibitory effects of HRIM knockdown on H9C2 cells were reversed by treatment with asatone (P<0.01).

The biological function of HRIM in MI/RI rats was investigated. TTE analysis demonstrated that silencing of HRIM significantly increased the LVEF, LVFS, LVSP, +dp/dt_max and -dp/dt_max (P<0.05), but significantly decreased the LVEDD, LVESD and LVEDP in MI/RI rats compared with the MI/RI + si-NC group (P<0.001; Fig. 6A). Moreover, the ratio of inflammatory cells/field in myocardial tissue was significantly decreased in the MI/RI + si-HRIM group compared with that in the MI/RI + si-NC group (P<0.001; Fig. 6B). HRIM knockdown significantly decreased the expression levels of HRIM in MI/RI rats (P<0.001) compared with the MI/RI + si-NC group (Fig. 6C). Additionally, knockdown of HRIM significantly decreased the levels of p-NF-κB p65/NF-κB p65, p-IkBα/IkBα and cytoplasmic NF-κB p65 and significantly increased nuclear NF-κB p65 levels in MI/RI rats compared with the MI/RI + si-NC group (P<0.01; Fig. 6D and E).