Animals

38 male C57BL/6J mice, aged 6–8 weeks, were purchased from Beijing Sipeifu Biotechnology Company. The mice were acclimatized and fed for one week, and then randomly divided into control (n = 8) and diabetic groups (n = 30). The control group was fed with normal chow (#D12492, Sipeifu Biotechnology Company, Beijing, China), while the diabetic group was fed with a high-fat chow (#SPF-F02-001, Sipeifu Biotechnology Company, Beijing, China) for 8 weeks. Then the diabetic groups randomly divided into Vehicle + T2DM group (n = 10) and FG-4592 (#HY-13426, MCE, Shanghai, China) pretreated group (n = 20). The FG-4592 pretreated group mice were continuous gavaged for 9 days with two dosage: 10 mg/kg and 25 mg/kg, which were defined as FG10 + T2DM group (n = 10) and FG25 + T2DM group (n = 10), respectively. And the mice in the Vehicle + T2DM group were treated with an equal volume of double-distilled water by gavage for 9 days. At the end of the gavage, mice in these three groups were injected intraperitoneally with streptozotocin (40 mg/kg, once a day)(#18883-66-4, SIGMA, USA) for 2–5 days to destroy the pancreatic islets to accelerate diabetes modeling. Diabetic mice were confirmed by blood glucose (fasting blood glucose > 11.1 mmol/L) and body weight, then euthanized immediately. All animals were housed in standard, plastic rodent cages, under controlled conditions, with a constant temperature of 24 ± 1 ◦C and a 12-h light/12-h dark cycle (lights on at 07:00 h) and ad libitum access to food and water.

Body weight, blood pressure, and blood glucose measurements in mice

Body weight measurement: Ensure the scale (#ZK-DST2, Zhike, Henan) is clean and calibrated. Then, gently place the mouse on the scale. Allow the mouse to settle and stabilize on the scale. Once the mouse is stationary, immediately record the body weight displayed on the scale. Blood pressure measurement: Carefully place the mouse on the Softron sphygmomanometer (#BP-98 A, Softron Bio Tech Co. Ltd., Beijing, China) and position the blood pressure sensor as close as possible to the anterior end of the mouse’s tail. Allow the mouse to relax and its heart rate to stabilize. Once the heart rate is stable, record the blood pressure reading displayed on the sphygmomanometer. Blood Glucose Measurement: Carefully restrain the mouse and stabilize its body. Gently snip the end of the mouse’s tail to initiate blood flow. Wipe away the first drop of blood with a paper towel. Collect the second drop of blood and carefully place it on the Roche Glucose Meter (Accu-chek, Roche, Irish). Record the blood glucose value displayed on the meter. Body weight, blood glucose and blood pressure were recorded every fortnight.

Myocardial tissue specimen collection and processing

After all the experiments were completed, mice were anaesthetized by intraperitoneal injection of 1% pentobarbital (45 mg/kg)(#P11011, Merck, USA), then blood was collected from the eyeballs to collect the serum of the mice, and the thoracic cavities were dissected under a dissecting microscope rapidly to expose the heart to a good range of operation, and the heart was irrigated with physiological saline to exclude the blood that might be in the heart and removed immediately after irrigation. The mouse heart was removed immediately after the perfusion was completed. After 1 mm3 of myocardial tissue was fixed in 2.5% glutaraldehyde (#111-30-8, Merck, USA) for 24 h, images were collected by transmission electron microscopy(#H-7650, Hitachi Limited, Japan), half of the remaining cardiac tissue was fixed in 4% paraformaldehyde(#BL593A, Biosharp, China) for subsequent morphological experiments, and the rest was stored in a refrigerator (#702, Thermo, USA) at -80 ◦C for subsequent molecular biology experiments.

Echocardiographic measurements

Left ventricular (LV) function was assessed by a fijifilm visualsonic 3100LT echo-cardiographic system (Fujifilm Visual Sonics, China) at a probe frequency of 10 MHz with M-mode recording. Mice were anesthetized with avertin and depilated with depilating cream on the chest. The limbs of mice were attached to heating pads to maintain body temperature at 37 ◦C. Medical ultrasound gel (Tianjin Yajie Medical Material Co. Ltd., Tianjin, China) was used as a coupling agent between the ultrasound scan-head and the skin. Left ventricular parameters such as Left Ventricular Ejection Fraction and Left Ventricular Fraction Shortening were measured by M-mode recording. The data are presented as the average of measurements of three consecutive beats.

Transmission electron microscopy (TEM)

Myocardial tissues were collected and fixed by 2.5% glutaraldehyde (#111-30-8,Merck, USA). After being washed in 0.1 M sodium cacodylate buffer (#18839, Merck, USA), tissues were postfixed with 1% buffered osmium (20816-12-0, Merck, USA). The tissues were dehydrated through the graded alcohol (#64-17-5, Merck, USA) and embedded in resin (#JM3852, HPBIO, Shanghai, China). After dehydration through graded alcohol and embedding in resin, tissues were incubated in a 60 ◦C oven for 3 days. Subsequently examined by a transmission electron microscope (#H-7650, Hitachi Limited, Japan).

Brain natriuretic peptide (BNP) detection assay

Brain Natriuretic Peptide (BNP) was measured in serum from mice treated with different treatments using a Brain Natriuretic Peptide assay kit (#E-EL-M0204c, Elabscience, Wuhan, China) according to the manufacturer’s instructions and quantified with absorbance at 450 nm on a microplate reader (#A51119700DPC, Thermo Fisher, USA).

Cell culture and treatment

The rat heart-derived cardiac cells H9c2 (H9c2 cells) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. H9c2 cells were cultured in DMEM (#SH30022.01, HyClone, Shanghai, China) medium with 10% FBS (#164210-50, Procell, Wuhan, China), 1% penicillin and streptomycin antibiotics (#PB180120, Procell, Wuhan, China). Cells were incubated in an atmosphere of 5% CO2 at 37 ˚C. When the cells reached 50% confluency, H9c2 cells were pretreated with FG-4592 (10 mM) for 24 h and then stimulated with glucose (45 mM)(#ab147494, Procell, Wuhan, China) for additional 24 h. After these treatments, cells were harvested for appropriate analyses.

Western blot assay

Briefly, protein was extracted from tissue samples using RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitors (#ST506-2, Beyotime, Shanghai, China). The protein concentrations in the resulting lysates were determined using a BCA kit (#23225, Thermo Fisher Scientific). For traditional western blot procedure, the following primary antibodies were used: anti-HIF-1α (1:500, #36169S), anti-NRF2 (1:500, #D1Z9C), anti-UCP2 (1:500, #D1O5V), anti-HO-1 antibody (1:500, #70081S), anti-COX4 (1:1,000, #4850S) (all from Cell Signaling Technology, Danvers, Massachusetts, USA), anti-β-actin (1:1,000, #20536-1-AP, Proteintech, Wuhan, China), anti-SOD2 (1:500, #WL02506), anti-AKT (1:500, #WL0003b), anti-P-AKT (1:500, #WLP001a), anti-PI3K (1:500, #WL03380), anti-Bax (1:500,#WL01637) (all from Wanlei Technology, Shenyang, China), anti-P-PI3K (1:500, #AF3242, Affinity, Biosciences, Jiangshu, China), anti-GADPH (1:5,000, #ab9484, Abcam, United States). The membranes were then incubated with appropriate secondary horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin G (IgG) antibodies at a 1:5000 dilution for 45 min at 37◦C. Blots were visualized using an enhanced chemoluminescence (ECL) reagent (#P0018AS, Beyotime, China) and quantified by scanning densitometry (Tanon-5200T, Tanon, Shanghai, China).

MDA, GSH, SOD detection

Malondialdehyde (MDA) levels in myocardial tissues under different conditions were measured by the thiobarbituric acid method (#BC0025, Solarbio, Beijing, China). GSH levels (#BC1175, Solarbio, Beijing, China) were measured in tissue samples using a GSH/GSSG assay kit. SOD levels (#BC0175, Solarbio, Beijing, China) were also measured in tissue samples using a SOD assay kit.

Mitochondrial complexes I and IV and ATP content detection

The activities of the mitochondrial complex I and IV were determined by the Micro Mitochondrial Respiratory Chain Complex I and IV Activity Assay Kit (#BC0515, #BC0945, Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, mitochondrial homogenates were added into the respective reaction buffer. The reaction mixture was transferred to a prewarmed (30 °C) quartz cuvette and immediately put into a spectrophotometer. The absorbance of reaction mixture was measured at 340 nm for Complex I, or 550 nm for Complex IV, respectively. Mitochondrial complex activity was expressed as nmol/min/mg protein. ATP was quantified using an ATP assay kit (#S0026, Beyotime, Shanghai, China) according to the manufacturer’s protocols on a microplate reader.

Cell counting Kit-8 (CCK-8) assay

The H9c2 cell were seeded in 96-well plates, approximately 3,000 cells were seeded per well. After culturing for 24 h at 37 ◦C in 5% CO2, glucose at different concentrations were added to the cells, each group had at least 3 repetitions. After 24 h, 10uL of CCK-8 reagent (#C0037, Beyotime, Shanghai, China) was added to each well and incubated for 1 h under the above conditions. The absorbance at 450 nm was measured by a microplate reader (PerkinElmer, Wellesley, Massachusetts, USA).

OCR measurements

Experiments were performed using 300,000 H9c2 cells per chamber. And mitochondrial function was determined using high-resolution respirometry (Oxygraph 2k, Oroboros, Austria). The analysis was blinded. Briefly, respirometry was performed in respiration medium containing EGTA (0.5 mM), MgCl2 (3 mM), K-lactobionate (60 mM), taurine (20 mM), KH2PO4 (10 mM), HEPES (20 mM), sucrose (110 mM), and fatty-acid-free BSA (1 g/L). OCR was determined by adding oligomycin (#O4876, Oroboros, Austria), proximity compound carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)(#C2759, Oroboros, Austria), and antimycin A (#A8674, Oroboros, Austria) treatments. OCR (pmol/min) was calculated automatically via Oxygraph 2k.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using RNAiso Reagent (#9109, TaKaRa Bio, Tokyo, Japan) and reverse-transcribed into cDNA using PrimeScript RT Master Mix (#RR047A, TaKaRa Bio). qRT-PCR was performed using TB Green qPCR Master Mix (#RR420A, TaKaRa Bio). The sequences of the primers used were, HIF-1α: 5′-ACCGCCACCACCACTGATG-3′ and 5′-GTACCACTGTATGCTGATGCCTTAG-3′;GAPDH:5′-GACATGCCGCCTGGAGAAAC-3′ and 5′-AGCCCAGGATGCCCTTTAGT-3′; UCP2: 5′-CTGGCGGTGGTCGGAGATAC-3′ and 5′-TGGCATTTCGGGCAACATTGG-3′.

Synthesis and transfection of overexpression plasmids and siRNAs

The siRNA of UCP2, HIF-1α and a negative control was designed by Huatuobio (Shenyang, China). DMEM (#SH30022.01, HyClone, Shanghai, China) was used to dilute transfection reagent (#KC112-1.5, Huatuobio, Shenyang, China) and siRNA, respectively. After 5 min, the two were mixed, and the mixture was added to serum-free cell culture medium 20 minutes later. The sequences of siRNAs for rat are in Supplementary Table 1.

Mitosox red staining

After 24 hours’ exposure to FG-4592 and 45 mM glucose levels in normoxia, H9c2 cells were stained with MitoSOX™Red Mitochondrial Superoxide Indicator (#M36008, Thermo Fisher Scientific, Germany). A working concentration of 500nM was used, and cells were incubated at 37 ◦C for 30 minutes protected from light. After washing off excess dye, the cell crawls were placed under a confocal microscope (Nikon AXR, Japan) for observation and photography.

Immunohistochemical (IHC) staining

For IHC studies, paraffin-embedded cardio sections were deparaffi-nized and hydrated using slide warmers and alcohol. After antigen re-trieval, the sections were permeabilized with 3% H2O2 and blocked with 5% bovine serum albumin (BSA). Then, the sections were individually incubated with the following antibodies at appropriate concentrations: antibody against HIF-1α. Then, the sections were incubated with secondary antibodies and reacted with diaminobenzidine (DAB) in accordance with the manufacturer’s instructions.

Bioinformatics analysis of RNA-seq

Fastp software (https://github.com/OpenGene/fastp) were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases with default parameter. Then sequence quality was also verified using fastp. We used HISAT2 (https://ccb.jhu.edu/software/hisat2) to map reads to the reference genome of RAT. The mapped reads of each sample were assembled using String Tie(https://ccb.jhu.edu/software/stringtie) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare(https://github.com/gpertea/gffcompare/). After the final transcriptome was generated, StringTie and was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads (millions) × exon_length(kB) ] ). The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and with parametric F-test comparing nested linear models (pvalue < 0.05) by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html).

Statistical analysis

All experiments were conducted as biological replicates, with sample size determined by statistical power requirements. Data are expressed as mean ± standard deviation. All analyses were carried out using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Comparisons between groups were made using a t-test (two-sided). One-way ANOVA test were used for comparison of data from multiple groups, respectively. p < 0.05 was considered statistically significant.