Animals

The PKCβ knockout mice utilized in this research were obtained from Cyagen Biosciences (Jiangsu, China). PKCβ-/- mice were generated through the targeted disruption of exon 3, one of the 17 exons of the transcribed PKCβ gene located on mouse chromosome 7, utilizing CRISPR/Cas9 gene editing technology. PKCβ-/- mice were generated by combining the Cas9 protein and four gRNAs targeting exon 3 flanking introns (gRNA-A1: GGA AGT GGA GCG TCC CCA GCT GG and gRNA-A2: CTA GAT AAC TTT CCT TAG ATA GG; gRNA-B1: GCA GGT GAG GAT GTT TCA TCT GG and gRNA-B2: GAT GAT TAT TAT TAT CTG G and gRNA-A1: GAT GAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAG G). (GAT GAT TAT TGC AAG CAG TCA GG) were co-injected into fertilized mouse eggs. The injected embryos were transferred into female recipient mice to obtain F0 mice. F0 mice were genotyped and tested using F1/R1 (F1: 5’-AAT GTA AGG CCG TTC AAT GAA AG-3’, R1: 5’-AAT ACT GAG CCA AGA AGT GGA GAA G-3’) primers. Genotyped and sequenced F0 mice were mated with wild-type (WT) mice to obtain genetically stable F1 heterozygous mice. Genotypically correct F1 heterozygotes were mated with each other to obtain PKCβ-/- mice.

The db/db mice, aged 6–8 weeks, utilized in this study were procured from Junke Biological Co. (Jiangsu, China) and were characterized as DM mice. Prior to the commencement of the experiment, all mice underwent a one-week acclimatization period and were provided with standard laboratory housing conditions at 25 ± 2 °C with a relative humidity of 50 ± 15% and a normal photoperiod of 12 h of darkness and 12 h of light. The experimental procedures were ethically approved by the Ethics Committee of Guangxi Medical University and adhered to the ARRIVE guidelines for the ethical use of experimental animals.

RNA synthesis

Y4 RNA (native and modified forms) was custom synthesized by IBSBIO (Shanghai, China). As reported previously [15, 19], the 56-nucleotide sequence for Y4 RNA is as follows: 5’-GGC UGG UCC GAU GGU AGU GGG UUA UCA GAA CUU AUU AAC AUU AGU GUC ACU AAA GU-3’.

Mouse myocardial I/R injury model

To induce myocardial I/R injury, the mice were subjected to 40 min of myocardial ischemia followed by 24 h of reperfusion. The mice were anesthetized via intraperitoneal injection of tribromoethanol (0.02 mL/g) and positioned in the supine position for intubation and connection to a small ventilator (model 845, Harvard Apparatus, Germany). The edges of the pectoralis major muscle were obliquely cut, and the pectoralis minor muscle was bluntly separated to expose the heart through the left fourth intercostal space. Subsequent to the ligation of the left anterior descending artery (LAD) using a No. 7 − 0 silk suture, a noticeable alteration in color from red to white was observed in the left anterior myocardium, accompanied by a significant reduction or cessation of ventricular wall motion. After 40 min, the ligature was released, and coronary blood flow was restored. Y4 RNA was injected into the myocardium of db/db mice. Specifically, the aorta was clamped using an aortic cross-clamp, and 100 µl of Y4 RNA was injected into the left ventricular cavity for 20 s. Y4 RNA was incubated with Dharmafect transfection reagent (Dharmacon, USA) in IMDM basal medium (Thermo Scientific, USA) at a concentration of 0.15 µg/g for 10 min at room temperature and then resuspended in 100 µl of IMDM for injection. In contrast, the sham-operated db/db mice and the I/R control mice were injected with equal amounts of Y4 RNA-free transfection reagent (Vehicle) in the left ventricular cavity. Twenty-four hours after reperfusion, all mice were evaluated for cardiac function and then euthanized, and their hearts were harvested for further analysis.

HE staining and immunofluorescence staining

The excised hearts were fixed in 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, and sectioned into 5 μm slices. These sections were dehydrated using an ethanol gradient and cleared with xylene prior to staining with hematoxylin-eosin (HE). The myocardial samples were subjected to immunostaining with specific antibodies against F4/80 (1:200, Cell Signaling Technology, USA), CD86 (1:200, Cell Signaling Technology, USA), and CD206 (1:100, Abcam, UK) on paraffin sections. DAPI was used to stain the nucleus. The quantification of macrophages was performed through image acquisition using Zeiss confocal microscopy and subsequent analysis with ImageJ software.

Treadmill

Following a 24-hour reperfusion period, the mice from each experimental group underwent exercise treadmill testing. The initial stage consisted of a speed range of 0–10 m/min for 1200 s, followed by a second stage with a speed range of 10–25 m/min for 1800 s. Subsequently, locomotor ability was assessed after acclimatization training, with continuous electrical stimulation of the running table throughout the acclimatization period. Human-induced repulsive stimulation was administered as needed during the testing process. When the mice reached a state of immobility after remaining in the rear third of the running platform for more than six instances, they were classified as exhausted. The distance covered by each mouse at the point of exhaustion was documented.

Echocardiographic assessment of cardiac function

Mice were anesthetized using tribromoethanol (0.02 mL/g) by intraperitoneal injection, and cardiac function was assessed by an ultrasound machine Vevo2100 (Visualsonics, Toronto, Ontario, Canada) as described previously. The ejection fraction (EF) was determined automatically by the machine, and fractional shortening (FS) was calculated according to the following equation: ((LVIDd-LVIDs)/LVIDd) × 100.

Infarct area assessment

The infarct area was determined using Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Following a 24-hour reperfusion period, the mice were ventilated with a ventilator, and the left anterior descending artery (LAD) was religated. A 2% solution of Evans blue (Sigma Aldrich, USA) was slowly administered through the right ventricle, followed by clamping of the ascending aorta. Subsequently, the hearts were swiftly excised, stained blue, and frozen at -20 °C for 20 min. The hearts were then sectioned into 4–5 segments located 2 mm below the ligature. Each segment, approximately 1–2 mm in thickness, was dissected along the longitudinal axis of the left ventricle. Normal myocardial tissue appears blue, while ischemic regions exhibit a pink hue. A 1% solution of 2,3,5-triphenyltetrazolium chloride from Sigma‒Aldrich, USA, was applied at 37 °C for 10 min. The extent of infarction is quantified as a percentage of the area at risk (AAR).

Cell culture

Bone marrow-derived macrophages (BMDMs) and neonatal murine ventricular myocytes (NMVMs) were isolated and cultured as previously reported [17, 18]. Y4 RNA (50 nM) was introduced into BMDMs via transfection with Dharmafect 4 reagent. Following centrifugation and cell counting at a 1:8 ratio using a cell counter, the transfected BMDMs were cocultured in Transwell cell culture dishes with NVNMs. The BMDMs were positioned in the upper layer of the Transwell cell culture dishes, while the NVNMs were located in the lower layer. Following a 24-hour coculture period, the Transwell cell culture dishes were transferred to an anoxic chamber and exposed to high-purity N2 gas in low-sugar, serum-free IMMM for 30 min to induce hypoxia and simulate an ischemic environment for an additional 24 h. Subsequently, the cells were incubated in oxygen-saturated, high-sucrose DMEM supplemented with 10% fetal bovine serum to mimic reperfusion for 48 h before harvesting both the cells and their supernatants.

High-glucose BMDMs were infected with an adenovirus overexpressing PKCβ obtained from Hanbio (Shanghai, China), followed by transfection of Y4 RNA into the PKCβ-overexpressing BMDMs. NMVMs were exposed to UV light at a wavelength of 520 nm for 7 min and then incubated at 37 °C in a 5% CO2 environment. Necrotic cell suspensions were subsequently added to macrophage culture flasks transfected with Y4 RNA after a 2-hour interval. After an overnight incubation period of 24 h, the cells were harvested for RNA and protein extraction as well as flow cytometry analysis.

Western blot analysis

Total protein was extracted from the hearts of db/db mice and BMDMs through a series of procedures, including determination of protein concentration using a bicinchoninic acid assay kit (Solarbio, China). Subsequently, 30 µg of total protein was separated via SDS‒PAGE and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed by stimulating the membrane with specific antibodies against PKCβ (1:1000, Cell Signaling Technology, USA), p-Ser661-PKCβ(1:1000, Mlbio, China), JNK (1:1000, Abcam, UK), p-JNK (1:1000, Abcam, UK), ERK (1:1000, Abcam, UK), p-ERK (1:1000, Abcam, UK), P38 (1:1000, Abcam, UK), p-P38 (1:1000, Abcam, UK) and β-actin (1:10000, Abcam, UK).

qRT‒PCR

Total RNA was extracted from cardiac tissues and cells. Subsequently, 1 µg of RNA was reverse transcribed into cDNA using a T100 Thermal Cycler (Bio-Rad; USA), and quantitative RT‒PCR was carried out with a CFX96 Total (Bio-Rad; USA) following the provided protocol. All the results were normalized against β-actin expression.

ELISA

To evaluate the levels of creatine kinase MB (CK-MB) and inflammatory factors in the serum and cell supernatants, enzyme-linked immunosorbent assay (ELISA) assays were conducted using a kit from Mlbio (Shanghai, China). Following a 24-hour reperfusion period, the mice were intubated, and blood samples were obtained from the right ventricle postchest cavity opening. The blood samples were centrifuged at 3000 × g for 15 min to collect the supernatant. Cell supernatants were obtained according to established protocols. Subsequent ELISA analysis was carried out in accordance with the provided instructions.

Flow cytometry

Macrophages were dissociated and isolated using prewarmed Accutase at 37 °C to generate single-cell suspensions. The isolated cells were subsequently incubated with a FITC-conjugated anti-F4-80 antibody, an APC-conjugated anti-CD86 antibody, and a PE-conjugated anti-CD206 antibody (BioLegend, USA) on ice for 20–30 min for phenotypic sorting. Following the incubation period, the cell samples were subjected to two consecutive washes. The samples were subsequently examined utilizing an Attune NxT flow cytometry apparatus (Thermo Fisher Scientific, USA). The macrophage phenotypes were classified as either proinflammatory (F4-80+/CD86+) or anti-inflammatory (F4-80+/CD206+). Subsequently, calibration and compensation of experiments involving unstained and single fluorescent controls were performed manually or automatically.

Statistics

All quantitative data are presented as the mean ± standard error of the mean (SEM). The results were analyzed using Prism 9.0 software (GraphPad, CA). Normal distribution and homogeneity of variance were tested using the Shapiro–Wilk test and Bartlett’s test, respectively. For data that passed both normality and equality of variance tests, comparisons between two groups were performed using the unpaired 2-tailed Student’s t test, and multiple comparisons were analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc test. Otherwise, the nonparametric Kruskal–Wallis test was applied, followed by Dunn’s test. P < 0.05 was considered to indicate statistical significance.