Chronic GPER1 Activation Prevents H9c2 Cardiac Myoblast Death after Oxidative Stress by Protecting Mitochondrial Structure and Function Via a Non‐Canonical MST1/YAP Pathway

Abstract

IntroductionEstrogen (17β‐estradiol; E2) is known to be cardioprotective against the damaging effects of ischemia/reperfusion (I/R) injury. We found that acute pre‐ischemic E2 treatment reduces cell death in cardiac tissue via direct effects on mitochondria by the G protein‐coupled estrogen receptor 1 (GPER1). We recently showed that acute application of E2 at the onset of reperfusion results in similar cardioprotection against I/R injury via GPER1, a non‐genomic receptor that mediates rapid E2 actions. Here, we investigated the impact and mechanism underlying chronic E2 protective actions in H9c2 cardiac myoblasts subjected to treatment with the cytotoxic agent, H2O2.MethodsUsing the H9c2 cardiac myoblasts, we subjected cells to H2O2 oxidative stress with no serum, followed by reoxygenation with E2 for 2 and 4 days. Cells were then collected and analyzed for cellular, mitochondrial and genetic function. We measured cell death, gene upregulation, mitochondrial structure, morphology and function using flow cytometry, cell assays, qRT‐PCR, along with fluorescence and electron microscopy. We also collected cell lysates and analyzed them by Western blot to assess the mechanism involved.ResultsWe found that chronic treatment of cells with E2 after oxidative stress results in decreased cell death but remarkably does not affect cell cycle activity. Surviving cells showed improved mitochondrial dynamics as evidenced by better cristae morphology and increased resistance to opening of the mitochondrial permeability transition pore, but with little change to mitochondrial fusion/fission. Additionally, we found that this chronic E2 action predominantly occurs through mitochondria to reduce cytochrome c release and decrease phosphorylation of mammalian sterile‐20‐like kinase (MST1) and increase yes‐associated protein (YAP) translocation to the nucleus for upregulation of TGF‐β and PGC‐1α. All these E2‐observed effects were prevented by G15, a GPER1 antagonist, suggesting a GPER1‐dependent mechanism.ConclusionWe have found that chronic E2 treatment to H9c2 cardiac myoblasts after stress leads to rescue from cell death by reducing apoptotic signaling and protecting mitochondrial morphology and function. We also found that this process occurs via GPER1 acting directly on mitochondria in a non‐canonical MST1/YAP mechanism that leads to genetic upregulation of cell growth.Support or Funding InformationNIH RO1 Grant/Award number: HL138093;American Heart Association, Grant/Award Numbers:18PRE34030307, 17SDG33100000This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.