Animals model of myocardium infarct (MI) and leptin treatment

All animal experiments conformed to the guidelines of the Animal Use Committee of the Guizhou Provincial People’s Hospital. After anesthesia by intraperitoneal injection of sterile pentobarbital sodium (50 mg/kg), the 8–12-weeks old male C57BL/6 J mice (20–25 g; Animal Experiment Center of Hangzhou Medical College) were performed to build the HF model by ligation of the left anterior descending coronary artery, as described previously (Yang et al. 2018). Suture without ligation served as a sham operation. Immediately after the surgery, the recombinant mouse leptin (1 mg/kg) was intraperitoneally injected twice daily for 14 days. An equal volume of PBS served as control. 56 days after surgery, the mice were sacrificed by cervical dislocation, and the heart tissues were harvested for the subsequent experiments.

Cells culture and transfection

The cardiomyocytes H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 15% fetal bovine serum (FBS) under 95% air/5% CO2, and were pretreated with leptin (50 ng/ml) for 24 h. The leptin concentration was determined according to previous studies (Yang et al. 2018; Luo et al. 2021). The solvent of leptin served as the control medium. To establish the HF model in vitro, the cells were subjected to 20 μM ISO for an additional 24 h (Cheng, et al. 2023; Wang et al. 2021).

Cell transfection: ①shOPA1 transfection: The H9c2 cells were transfected with lentiviruses containing shOPA1 or empty vectors (NC) at a multiplicity of infection (MOI) of 10 for 24 h. Then, the cells were replaced with fresh medium for an additional 24 h. The sequences of shOPA1 are shown in Table S1. ②Flag-OPA1 transfection: On the whole, the 2 μg of plasmid encoding Flag-OPA1 was used to transfect H9c2 cells via Lipofectamine 3000 transfection reagent (Invitrogen, USA) for 24 h, and the fresh medium was replaced to culture the cells for another 24 h. The overexpression efficiency of OPA1 by western blot analysis in H9c2 cells (Fig. S2B). Similarly, H9c2 cells were transfected with HA-HDAC5 plasmid (2 μg) in the same way.

LMK-235 inhibition assay: After being transfected by lentiviruses containing shOPA1 or vector for 24 h, the cells were replaced with fresh medium for an additional 24 h. Then, H9c2 cells were pretreated with leptin for 24 h. Next, H9c2 cells were exposed to ISO with LMK-235 (100 nM; MedChemExpress, USA) for another 24 h. Then the luciferase reporter assay was performed.

co-Immunoprecipitation (Co-IP)

After transfected with 2 μg of Flag-OPA1 and/or HA-HDAC5 plasmid, the HEK293T cells (1.5 × 106 cells) were harvested in immunoprecipitation (IP) lysis buffer (containing 25 Mm Tris-Base-PH 8.0, 300 Mm NaCl, 10% glycerin, 1% NP-40 and protease inhibitors). Then, 10% cell lysates were used for input, and the supernatant was incubated in IP washing buffer (containing 25 Mm Tris-Base-PH 8.0, 300 Mm NaCl, and 0.2% NP-40) containing Flag-Beads (Smart-Lifesciences Biotechnology, China) in a gently agitated manner at 4℃ overnight. The following day, the Flag-Beads were washed and detected through western blotting.

OCR assay

According to the manufacturer’s instructions for the extracellular O2 consumption reagent kit (Abcam, USA), H9c2 cells (5 × 104 cells per sample in 96-well-plate) were prepared to be added to the extracellular O2 consumption reagent after being replaced with fresh medium, accompanied by closed with warm mineral oil. The O2 consumption of H9c2 cells was measured at Ex/Em = 380/650 nm by Tecan (Tecan Austria GmbH, Switzerland) every 1.5 min for 90 min. Equal volume fresh culture medium served as blank control. The OCR was calculated as a percentage relative to the control medium.

ECAR assay

According to the manufacturer’s instructions for the ECAR reagent kit (Abcam, USA), H9c2 cells (4 × 104 cells per sample in a 96-well plate) were prepared and incubated with CO2-free for 2 h. After washing with PBS, 150 μL ECAR buffer was added into each well, followed by an additional 10 μL glycolysis assay reagent. Subsequently, the H9c2 cells were incubated at a temperature of 37 °C prior to being analyzed using a Cayman assay kit (Cayman Chemical, USA). The absorbance was measured at wavelengths of 380 nm and 615 nm at intervals of 1.5 min over a period of 2 h. Equal volume fresh culture medium was served as blank control. The ECAR was calculated as a percentage relative to the control medium.

Transmission electron microscopy (TEM)

The mitochondrial network ultrastructure of H9c2 cells was observed via TEM as previously described (Yang et al. 2018). In brief, the specimens of H9c2 cells were fixed in 2.5% glutaraldehyde for 6 h and post-fixed with 1% osmium tetroxide for 2 h. After being dehydrated by an ethanol gradient, the specimens were infiltrated overnight. Next, the H9c2 cells were embedded in Spurr resin, followed by sectioned (85 nm) by Leica EM UC7 Manufacturer (Leica, Germany). After being stained with uranyl acetate and alkaline lead citrate, the images of mitochondria were obtained by Talos L120C 120 kV (Thermo, USA). 5 cells were randomly detected in each group, and 30 mitochondria were randomly measured in each cell. The number, length, and area of mitochondria were measured using Image J.

Luciferase gene reporter assay

This assay was performed according to the manufacturer's instructions for the Luciferase Reporter Assay kit (Biovison, USA). The sequence of the luciferase gene reporter is shown in Table S2. The MEF2 binding domain was included in the promoter region, according to a previous study (Santalucia et al. 2001). H9c2 cells were transfected with the pGL4.14 vector containing the MEF2 luciferase reporter gene by Lipofectamine 3000 for 12 h. The pGL4.14 vector without the MEF2 binding domain served as control. Then, H9c2 cells were harvested into cell lysis buffer, and luciferin Substrates A and B were added. The fluorescence was detected by Tecan.

Echocardiography

Transthoracic echocardiography was performed to evaluate cardiac function at baseline and at 3, 14, 28, and 56 days after coronary ligation. The mice were anesthetized with inhalation of 1.5% isoflurane gas. Two-dimensional and M-mode images were acquired and cardiac function was analyzed using a D6VET system (Vinno, Jiangsu, China). The images were acquired through a parasternal long-axis view using a 23 MHz transducer. The long-axis left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were detected for at least three interval cardiac cycles. The measurements of LVESD and LVEDD are shown in Table S3.

Additional detailed information on cell survival assay, TUNEL, Annexin V/PI staining, Sirius Red staining, ATP measurements, Glycogen staining assay, Glucose oxidase method, ELISA, Western blots, qPCR and antibodies are acquired in Supporting Information (SI). The primer sequences are shown in Table S4.

Statistical analysis

All data were expressed as mean ± SEM. Comparison of two sets of data was measured using Student's t-test, and comparison of more than three sets of data was made using One-way ANOVA analysis of variance followed by Tukey's post-test. The Kaplan–Meier curve and log-rank test were used to analyze the survival rate. The p-value less than 0.05 was considered statistical significance. Repeated experiments were performed at least three times. The statistics were exhibited by GraphPad Prism 5.