Pgc 1α

We analysed the expression of cardiac lipid metabolism proteins by the Western blot method as previously described in detail.²⁸ As severe LV hypertrophy is related to the impaired angiogenesis pathway, we also studied the expression of key proteins that regulate angiogenesis and signal hypoxia. Primary antibodies: HIF1α (ab463, Abcam; 1:500); fatty acid transporter (CD36; ab133625, Abcam; 1:1000); carnitine palmitoyltransferase 1 (CPT1; NBP1‐59576, Novus Biological; 1:1000); fatty acid‐binding protein (FABP3; ab133585, Abcam; 1:1000); acyl‐CoA dehydrogenase (ACADL; ab196655, Abcam; 1:3000); medium‐chain acyl‐CoA dehydrogenase (MCAD; ab110296, Abcam; 1:1000); 2,4‐dienoyl‐CoA reductase (DECR1; ab198848, Abcam; 1:2000). AMP‐activated protein kinase (AMPK; 2532 s, Cell Signaling; 1:1000); phosphorylated AMPK (Thr 172) (pAMPK; 2535 s, Cell Signaling; 1:500); SIRT1 (8469 s, Cell Signaling; 1:1000); PPARα (ab8934, Abcam; 1:1000); retinoid X receptor (RXRα; 3085 s, Cell; 1:1000); peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α; 20,658‐1‐AP, Proteintech; 1:1000). Targeted bands were normalized to the expression of cardiac GAPDH (sc‐32,233, Santa Cruz Biotechnology; 1:2000).

Proteins collected from myocardial tissues and H9C2 cells were lysed by RIPA lysate (Beijing Applygen Technolog Inc., China) according to the manufacturer's instruction. The protein content was quantified using the BCA method. After addition of loading buffer and boiling for 5 minutes at 99°C, protein samples (25 mg per sample) were separated by 10% Tris/Glycine SDS-PAGE and then electrotransferred to PVDF membranes at 300 mA for 1.5 h. Membranes were incubated in TBST containing 5% nonfat milk for 1 hours. Then the membranes were incubated with different primary antibodies including rabbit monoclonal antibodies against MFN1/2 (Proteintech), OPA1 (Proteintech), FIS1 (Proteintech), DRP1 (Proteintech), PGC-1α (Proteintech), NRF1(Proteintech), TFAM (Proteintech), and GAPDH (Proteintech) overnight at 4°C. The blots were washed with TBST three times and then incubated with anti-rabbit IgG (1 : 2000) for 4 h at 4°C. After washing three times with TBST, the immunolabeling was detected using Omni ECL reagent (EpiZyme, China) for 1 min at room temperature. The ultimate expression of every single protein was standardized by GAPDH. The band grayscale analysis was performed using Image J software.

The protein of liver tissues and HepG2 cells was lysed and extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beijing Beyotime, China), and the concentration was quantified using a BCA assay kit (Solarbio, PC0020, Beijing, China). The denatured protein samples (50 µg) from each group were loaded on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA, USA). Each PVDF membrane was blocked with 1×TBST containing 5% skimmed milk and incubated with primary antibodies at 4°C refrigerator overnight and incubated with secondary antibodies for 1 h in the dark. All the primary antibodies in the Western blot experiment are as follows: PGC1α (1:1000, Proteintech), PPARα (1:1000, Proteintech), PINK1 (1:1000, Proteintech), Parkin (1:1000, Affinity), ATG7 (1:1000, Affinity), SQSTM1/p62 (1:1000, Affinity), FAS (1:1000, ab128856), SREBP-1c (1:1000, A15586), LC3 I/II (1:1000, AB Clonal), and β-actin (1:10000, AB Clonal). The chemiluminescence intensity of all membranes was visualized with enhanced chemiluminescence system. The gray value of protein strips was analyzed using the Image J software.