Human samples and ethical statement

In this study, blood samples were collected from 20 acute myocardial infarction patients within 24 h of onset at Yantai Yuhuangding Hospital. Additionally, 20 healthy blood samples were used as a control group. Informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Yantai Yuhuangding Hospital.

Animal treatment

Male C57BL/6 mice, 8 weeks old, were obtained from Pengyue Laboratory Animal Breeding Co. Ltd (Jinan, China) and housed in an SPF-level laboratory animal room. A myocardial I/R model was constructed as described in the literature (Li et al. 2020). Briefly, mice were anesthetized with a small animal anesthesia machine (Shenzhen Ruiwode Lift Technology Co., Ltd. China, R5301E), and the left anterior descending coronary artery was ligated after thoracotomy. After 30 min, the ligature was loosened for 24 h of reperfusion. Mice in the sham operation group underwent the same procedure without ligation. The study included three groups of animals (n = 10 per group): I/R-treated mice (I/R), I/R-treated mice receiving intracardiac injection of recombinant human MANF protein (1.5 mg/kg, Novoprotein, Suzhou, China) (I/R + rMANF), and control group mice (Sham). All animal experiments and protocols were approved by the Animal Management and Use Committee of Yantai Yuhuangding Hospital.

Cell culture and construction of H/R

The mouse cardiomyocyte cell line (HL-1) was obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). HL-1 were cultured in MEM medium supplemented with 10% FBS, 100 IU/ml penicillin and 100ug/ml streptomycin in an incubator at 37 °C with 5% CO2. To mimic myocardial I/R injury in vitro, HL-1 were subjected to H/R treatment. Initially, HL-1 were allowed to grow in a three-gas cell culture chamber with 5% CO2, 1% O2, and 94% N2 at 37 °C for 20 h. Subsequently, HL-1 were returned to normal culture conditions (95% air and 5% CO2) for an additional 3 h. Control cells were cultured under normal conditions.

Construction and transfection of plasmids

Plasmids for Wt (wild-type MANF) and M1-M8 mutant (proline inserted between α1-α8 to disrupt α-helix) were constructed by subcloning the full-length cDNA into the pcDNA3.1(+) expression vector, with the addition of an HA tag at the 3′ end of MANF using PCR. The integrity of all plasmids was confirmed through DNA sequencing. Transfection of the plasmids was performed following the instructions of the Lipofectamine 3000 transfection reagent (Invitrogen, America). A mixture of plasmid and p3000 at a 1:2 ratio was prepared, followed by the addition of Lipofectamine 3000 (plasmid : lip3000 = 1:2). After mixing for 10–15 min, the resulting solution was added to the cells and cultured for 24 h, then transferred to an anoxic incubator for an additional 20 h.

Construction of shMANF

Small hairpin RNA targeting human MANF (shMANF) and negative control shRNA (shNC) were cloned into miRZip™ shRNA expression stimulation vectors (Systembio, Shanghai, China). The target sequences of MANF were 5′-TATCTTCCGGATATAGTCAG′ (sh1) and 5′- GGACCTCAAAGACAGAGATTT-3′ (sh2). A stable HL-1 cell line with knockout of MANF was established by lentiviral infection. Lentiviral particles were generated from HEK293T cells transfected with the packaging plasmid psPAX2, envelope plasmid pMD2.G and the target plasmid. Plasmids containing shNC and shMANF were introduced into HL-1 by transfection using Lipofectamine 3000 transfection reagent following the manufacturer’s instructions. After incubation of the cells for 48–72 h in the presence of lentivirus, different concentrations of puromycin were added to aid in the identification of cells with positive features.

Cell viability assay

Cell viability was determined using the CCK-8 assay. Specifically, cells were seeded at a density of 1 × 104 cells per well in 96-well plates and incubated for 24 h. CCK-8 reagent (Vazyme, China) was then added at a concentration of 1/10 of the medium volume, followed by a 2-hour incubation at 37 °C. Absorbance at 450 nm was measured using a multimode plate reader (VICTOR Nivo™, Finland) to obtain the results.

Western blot

Proteins were extracted from cardiomyocytes or cardiac tissues using RIPA lysate (Sparkjade, EA0002, China) and quantified with a BCA protein assay kit (Yeasen, 20201ES86, China). Subsequently, the protein samples underwent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. These membranes were blocked with 5% skimmed milk powder for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. After that, the membranes were exposed to secondary anti-rabbit or anti-mouse polyclonal antibodies (1:10000, D110087, D110058, Sangon, China) at room temperature for 1 h. Imaging and analysis were performed using a chemiluminescence imaging system (Clinx, ChemiScope 6000, China) and relative protein expression levels in different groups were determined using Image J software. GAPDH (1:10000, D110016, Sangon, China) and β-Tubulin (1:5000, 10094, Proteintech, China) served as internal references. The antibodies utilized in the study included MANF antibody (1:1000, ab67271, Abcam, America), Bcl-2 antibody (1:500, T40056, Abmart, China), Bax antibody (1:500, T40051, Abmart, China), Cleaved-Caspase3 antibody (1:1000, 9664, CST, America), HA-tag antibody (1:1000, 3724, CST, America), ATF6 antibody (1:1000, 65880, CST, America), p-IRE1α antibody (1:500, TD8322, Abmart, China), p-PERK antibody (1:500, TD8322, Abmart, China), BiP antibody (1:1000, 3177, CST, America), p-eIF2α antibody (1:500, 3398, CST, America), ATF4 antibody (1:500, T55873, Abmart, China), CHOP antibody (1:1000, 2895, CST, America), LC3B antibody (1:500, 381544, Zenbio, China), SQSTM1 antibody (1:500, YT7058, Immunoway, China), JAK1 antibody (1:500, YT2424, Immunoway, China), p-JAK1 antibody (1:500, YP0154, Immunoway, China), STAT1 antibody (1:1000, 14994, CST, America), p-STAT1 antibody (1:1000, 9167, CST, America), NF-κB antibody (1:1000, 8242, CST, America), and p-NF-κB antibody (1:1000, 3033, CST, America).

Real-time quantitative PCR (qRT-PCR)

Total RNA was extracted from cells or tissues samples using RNA extraction reagent (Vazume, China) according to the manufacturer’s instructions. RNA samples were reverse transcribed using the HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazume, America). qRT-PCR was performed on an Applied Biosystems 7500 (Thermo Fisher Scientific, America) using ChamQ Universal SYBR qPCR Master Mix (Vazume, China). The relative RNA level was analyzed by using 2−ΔΔct method, with GAPDH serving as the internal reference. The MANF primer pairs were custom-synthesized by Sangon: forward 5′-TCAATGAGGTGTCGAAGCCC-3′, reverse 5′-GTCCACTGTGCTCAGGTCAA 3′.

Immunofluorescence

Cell cultures or confocal petri dishes were fixed with 4% paraformaldehyde for 30 min at 4 °C. Subsequently, 0.5% Triton X-100 was added and allowed to incubate for 10 min at room temperature. Following blocking with 10% goat serum for 1 h at room temperature, the cells were incubated overnight at 4 °C with or without the primary antibody (used as a negative control). After rinsing with PBS, the cells were then exposed to the secondary antibody for 1 h at room temperature in the dark, followed by blocking with an anti-fluorescence quenching solution (containing DAPI) (Beyotime, China). Images were captured using a fully automated inverted fluorescence microscope (Axio Observer7, Carl Zeiss, Germany).

Immunohistochemistry

The hearts were sliced into 4 μm thick tissue sections, which were then dewaxed and underwent antigen retrieval. Subsequently, the tissue sections were treated with anti-MANF antibody (1:100, ab67271, Abcam, America) overnight at 4 °C, followed by incubation with HRP-coupled goat anti-rabbit IgG secondary antibody (1:10000, Sangon, China) for 1 h. After a 30-second hematoxylin re-staining, the sections were washed and stained with diaminobenzidine. The results were observed under a light microscope (Leica Microsystems, America) and captured in photographs.

Tunel staining

Cell apoptosis was assessed using the Tunel BrightGreen Apoptosis Detection Kit (A112, Vazyme, China). Cells were fixed in 4% paraformaldehyde for 25 min at 4 °C, permeabilized with 0.2% Triton X-100, and then incubated with 50 µL of Tunel reaction mixture for 1 h at 37 °C under light-avoidance conditions. Subsequently, cells were blocked with anti-fluorescence quenching blocking solution (including DAPI) (Beyotime, China). Images were captured using a fully automated inverted fluorescence microscope (Axio Observer7, Carl Zeiss, Germany).

Flow cytometry analysis

Samples were processed using the Annexin V-FITC/PI apoptosis detection kit following the provided instructions (A211, Vazyme, China). Negative controls were established and experimental groups were subjected to Annexin V-FITC and PI single staining for regulatory compensation. Flow cytometry analysis was performed on all samples using MoFlo XDP flow cytometer (Beckman) and FlowJo software (version 10.0).

Echocardiography assays

To assess cardiac function in mice, M-mode echocardiograms were performed using an animal echocardiography system (VINNO 6 LAB, VINNO, China). The procedure involved removing chest hair with a depilatory instrument, applying a medical ultrasound coupler to the mice, and placing the probe next to the sternum to capture at least six consecutive cardiac cycles. Data, including measurements from three consecutive heartbeats, are presented in Supplementary Table S1.

Evans Blue-2,3,5-triphenyltetrazolium chloride (TTC) staining

Before the end of reperfusion, use a syringe to inject 1–2 ml of 2% Evans Blue solution through the tail vein or the left ventricle of the heart. Heart samples were frozen and sliced into 1 mm thick sections. These sections were then incubated in TTC solution (Solarbio, G3005, China) at 37 °C for 15 min, followed by immediate termination of the reaction with 4% paraformaldehyde. The infarct area was identified through photography and analyzed using Image pro-plus (version 6.0) software. The percentage of infarct area was calculated by dividing the infarct area by the total area of the section.

Masson staining

Paraffin sections were deparaffinized in distilled water and stained with Weigert’s iron hematoxylin staining solution. Acidic ethanol differentiation solution was used to differentiate the sections, which were then washed and stained with Ponceau S acid fuchsin stain solution. Following a 30-second wash in weak acid working solution, the sections were treated with phosphomolybdic acid solution for 1–2 min. Aniline blue staining solution was applied for 1–2 min after another weak acid wash. The sections were dehydrated with anhydrous ethanol, transparentized with xylene, and sealed with neutral gum. After overnight drying at room temperature, the slides were examined and photographed under a light microscope (Leica Microsystems, America).

Enzyme-linked immunosorbent assay (ELISA)

Male mice were anesthetized and dissected 24 h after reperfusion. Blood was immediately collected from the heart, and then centrifuged at 1000 g for 20 min to obtain serum. The Lactate dehydrogenase (LDH) assay kit (A020-2-2) was acquired from Nanjing Jiancheng Bioengineering Research Institute. Additionally, mouse cardiac troponin I (cTnI) ELISA kit (JL11280), mouse creatine kinase MB isoform (CKMB) ELISA kit (JL12422), mouse interleukin 6 (IL-6) ELISA kit (JL20268), mouse interleukin 1Beta (IL-1β) ELISA kit (JL18442), and mouse tumor necrosis factor alpha (TNF α) ELISA kit (JL10484) were purchased from Shanghai Jianglai Biotechnology Co., Ltd. The indicators in mouse serum were detected following the instructions of the respective reagent kits, and the final optical density was measured at 450 nm.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 10 (GraphPad Software, America). Each experiment was replicated a minimum of three times, and results are presented as mean ± standard deviation. Student’s t-test was utilized for comparing two groups with normally distributed data, while One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed for comparing multiple groups. A significance level of P < 0.05 was used to determine statistical significance.