Role of ERRα in regulating ATP synthesis in hiPSCs-derived cardiomyocytes / 解放军医学杂志

ABSTRACT

Objective: To investigate the role of estrogen-related receptor alpha (ERRα) in regulating the adenosine triphosphate (ATP) synthesis in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods: Undifferentiated human induced pluripotent stem cells (hiPSCs) were induced to differentiate into cardiomyocytes by sequential transient activation/inhibition of Wnt signaling pathway. Immunofluorescence was used to detect the expression of pluripotency markers sex determining region Y-box 2 (SOX2), stage specific embryonic antigen 4 (SSEA4) and tumor resistance antigen 1-60 (TRA-1-60) in hiPSCs and the expression of cardiac specific markers cardiac troponin T (cTnT) and connexin 43 (Cx43) in hiPSCCMs, respectively; RT-qPCR was used to detect the mRNA expression levels of cardiac troponin T2 (TNNT2) and myosin heavy chain 6 (MYH6) in hiPSCs and hiPSCs-CMs; Western blotting was performed to detect the protein expression levels of ERRα, cytochrome C (CytC) and mitochondrial pyruvate carrier 1 (MPC1) in hiPSCs-CMs. Additionally, the ERRα-specific inhibitor XCT790 was used to treat the hiPSC-CMs, and then the protein expressions of ERRα, CytC and MPC1 were detected by Western blotting, and the changes of cell viability, intracellular ATP content and mitochondrial membrane potential were measured by assay kits. Results: Immunofluorescence results showed that hiPSCs expressed SOX2, SSEA4 and TRA-1-60, while hiPSC-CMs expressed cTnT and Cx43; compared with hiPSCs, the mRNA levels of TNNT2 and MYH6 in hiPSC-CMs increased significantly (82.820 ± 2.005 vs. 1.001 ± 0.029, 90982.000 ± 1968.000 vs. 1.003 ± 0.053, respectively, P<0.05), and intracellular ATP content and protein expression levels of ERRα, CytC and MPC1 also increased significantly [(9.905 ± 1.286) nmol/mg protein vs. (4.582 ± 0.141) nmol/mg protein, 5.392 ± 0.313 vs. 1.050 ± 0.076, 8.954 ± 0.293 vs. 1.071 ± 0.067, 2.605 ± 0.088 vs. 1.031 ± 0.091, respectively] with significant differences (P<0.05). Furthermore, compared with the control group, 10 μmol/L XCT790 could effectively inhibit the protein activity of ERRα in hiPSC-CMs without cytotoxicity, and reduced intracellular ATP content and mitochondrial membrane potential [(4.903 ± 1.158) nmol/mg protein vs. (9.310 ± 0.980) nmol/mg protein, 1.407 ± 0.022 vs. 1.977 ± 0.093, respectively], meanwhile down-regulated the protein expression levels of MPC1 and CytC in hiPSC-CMs (0.705 ± 0.019 vs. 0.897 ± 0.011, 0.594 ± 0.021 vs. 0.797 ± 0.025, respectively, P<0.05). Conclusions: The increase of ATP content after differentiation of hiPSCs into cardiomyocytes is related to the increase of ERRα expression. In hiPSC-CMs, ERRα may regulate the ATP synthesis though regulating the mitochondrial membrane potential and the protein expression of CytC and MPC1.