Male C57BL/6J wild type mice were purchased from Slac Laboratory Animals (Shanghai, China), and were studied at 10 weeks of age. Animals were housed at 22°C with a 12-h light/12-h dark cycle with free access to water and standard chow. The study conforms to the relevant guidelines and regulations of the Bio-X Institutes of Shanghai Jiao Tong University (Shanghai, China) and was performed after securing approval from the Animal Care and Use Committee of Shanghai Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China).
2.2 Experimental models
Ten weeks old C57BL/6J male mice (20-25g) were divided into two groups. In one group, mice were anesthetized with 80-100 mg/kg ketamine and 5-10 mg/kg xylazine intraperitoneally, and then the epididymal white adipose tissue (eWAT) and the main visceral adipose tissue (VAT) were removed. The other group served as normal controls with eWAT reserved. Both groups were then randomly divided into sham and heart failure (HF) groups. The HF mice were established by transverse aortic constriction (TAC). Briefly, mice were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally), and after opening the chest wall, the thoracic aorta was identified after blunt dissection through the intercostal muscles. Silk suture (7-0) was placed around the transverse aorta and tied around a 26-gauge blunt needle, which was subsequently removed. Sham-operated mice underwent a similar surgical procedure without aortic constriction. After 11 weeks, cardiac structure and function were determined by echocardiography. Mice were then euthanized under isoflurane anesthesia by cervical dislocation, and the hearts were weighed and collected for further analysis.
Echocardiography was performed 11 weeks after TAC under the guidance of M-shaped curves of two-dimensional ultrasonic echocardiogram. Every 3 continuous means of the cardiac cycle were selected as the test value. Some cardiac diameters such as fractional shortening (FS), diastolic left ventricular posterior wall (LVPWd), and diastolic left ventricular internal diameter (LVIDd) were quantified as previously described 
2.4 Processing of specimens
All mice were weighed before opening the chest wall. The heart was weighed after being taken out, leaving the left ventricle intact (including the inter-ventricular septum) for further weighing. Then, the weight index of the left ventricle (weight of the left ventricle/the body weight) was calculated. Mouse serum was isolated from whole blood, and blood samples were incubated for 60 min at room temperature to induce coagulation. Afterwards, samples were centrifuged at 5000 rpm for 5 min at 4˚C and the supernatant (serum) was collected and stored at -80℃ until further analysis.
2.5 Hematoxylin and eosin/Masson's staining
Heart tissue samples were formalin-fixed, paraffin-embedded and stained with Hematoxylin/Eosin (HE) and Masson. Cardiac myocyte cross sectional area was assessed using HE-stained and Masson-stained sections of myocardium. After staining, the myocytes were cut into transverse sections and the perimeter of the cell borders of 50 myocytes were measured using CellSens (Olympus). Myocytes were selected in up to ten 400x microscopic fields each separated into 20 equally sized squares. Only one transversally myocyte per square was measured to increase representativeness.
2.6 Quantitative real time PCR (qRT-PCR)
Total RNA was isolated from heart samples with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), purified with the RNEasy Kit (Qiagen, Hilden, Germany), and reverse transcribed using the oligo (dT) primers with the Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche diagnostics, Shanghai, china), as previously described . Samples were analyzed for mRNA levels using SYBR-green Master Mix (cat. no. 04887352001; Roche diagnostics), and amplification was monitored using the CF X96 Real-Time PCR system (Bio-Rad, Munich, Germany) with the following thermocycling conditions: 5 s at 95˚C followed by 30 s at 60˚C for 42 cycles. The reaction mixture was composed of 10 µl SYBR premix Ex Taq II, 0.2 µl primers, 8.8 µl H2O, and 1 µl DNA. Data were normalized to the expression levels of GAPDH (internal control) and were presented as arbitrary units normalized to wild type expression levels. Primer sequences in the present study are presented in Table 1.
2.7 Analysis of fatty acids
Mouse serum was isolated from whole blood. For FAs serum profiling, 100 μl of murine serum was hydrolyzed under alkaline-methanolic conditions, neutralized and diluted in methanol (1:10) containing internal FA standards. Analysis of fatty acids was performed using HPLC (Agilent 1200 HPLC system), coupled with an Agilent 6460 triple quad mass was performed, as previously described . Serum concentration of FFA was measured using a HR-NEFA kit (WAKO), as previously described . Triglycerides (TG) in serum were determined using a triglyceride assay kit (DiaSys GmbH), according to the manufacturer’s instructions.
2.8 Statistical analysis
Quantitative data were expressed as mean ± standard error of the mean (SEM). Comparison of mean values between groups was evaluated by the following tests: two-way ANOVA followed by post Bonferroni tests , two-way repeated measures ANOVA followed by post Bonferroni tests, one-way ANOVA followed by Tukey’s or Bonferroni multiple comparison test, or unpaired t tests, as appropriate, and data were analyzed with GraphPad Prism software. Exact value of number is provided for each type of experiments. Statistical significance was assumed at P< 0.05. Vertical lines in the histograms indicate standard error of the mean (SEM).