Untargeted Metabolomics of Heart Tissue

The detailed protocol for untargeted metabolomics analysis was described in our previous report [20 (link)]. Briefly, a mixture of 25 mg of heart tissue and 500 μL of extract solution (acetonitrile–methanol–water (2:2:1) with isotopically-labeled internal standard mixture) was homogenized and sonicated for 3 cycles and then kept at −40 °C for 1 h. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was transferred to a fresh glass vial for subsequent LC-MS/MS analyses, which were performed on a UHPLC system (Vanquish, Thermo Fisher Scientific, Cambridge, MA) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, Cambridge, MA, USA).