Abstract 876: Chronic GPER1 Cardioprotection in H9c2 Myoblasts is Mediated by an MST1-YAP Pathway and Mitochondrial Function and Dynamics

Abstract

Introduction: We recently showed that cardiac tissue treated acutely with estrogen (17β-estradiol; E2), pre- and post-ischemia, exhibited reduced myocardial infarct size and better functional recovery after ischemia /reperfusion via the G protein-coupled estrogen receptor 1 (GPER1). These E2-GPER1 cardioprotective effects have been found to involve the preservation of mitochondrial structure and function. Here, we investigate the impact and mechanisms underlying chronic GPER1 activation in H9c2 cardiac myoblasts treated with a cytotoxic agent, H 2 O 2 . Methods: H9c2 cells were treated with 200 μM H 2 O 2 , for 24 h and reoxygenated in no-serum conditions for 4 and 8 days with the addition of E2 (80nM) and GPER1 agonist (G1; 100nM) and GPER1 antagonist (G15; 1μM). We measured cell death and proliferation, as well as gene expression, mitochondrial morphology and function using flow cytometry, cell assays, qRT-PCR, along with fluorescence and electron microscopy. We also analyzed cell lysates by Western blot to assess the mechanisms involved. Results: We found that chronic treatment of cardiac myoblasts with E2 and G1 results in reduced cell death. The surviving cells showed improved mitochondrial function as evidenced by better cristae morphology, increased ATP production and increased resistance to opening of the mitochondrial permeability transition pore. Particularly chronic GPER1 activation led to reduced cell cycle activity along with changes in mitochondrial dynamics of fusion and fission. Additionally, we found that chronic GPER1 activation signaling involves decreased phosphorylation of mammalian sterile-20-like kinase (MST1) and increased translocation of the transcription coactivator, yes-associated protein (YAP), to the nucleus for upregulation of pro-survival genes. These effects were mainly prevented by the G15, suggesting a GPER1-dominant mechanism. Conclusion: Chronic GPER1 activation in H9c2 cardiac myoblasts treated with oxidative stress results in reduced cell death by preserving mitochondrial structural integrity and function by increasing mitochondrial dynamics. This chronic GPER1 cardioprotection involves activation of MST1/YAP signaling pathways as well as upregulation of pro-survival gene.