Abstract 13757: Suppression of Ischemia-Hypoxia Reperfusion Injury in Human iPS Cell-Derived Cardiomyocytes Through Downregulation of EGR1 Mediated by miR-124-3p

Abstract

Background: Ischemic heart diseases are one of the leading causes of death globally. Although timely revascularization is performed, some cases may still result in heart failure due to ischemia-hypoxia reperfusion (IH/R) injury. Early growth response 1 (EGR1) was reported to involved in reperfusion injury, but its specific role in IH/R-induced apoptosis in human cardiomyocytes (CMs) remains unexplored. Hypothesis: EGR1 is involved in the exacerbation of the IH/R-mediated apoptosis in human CMs, and miR-124-3p acts as an antagonist of EGR1. Methods: Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured in serum-free low-glucose medium with a hypoxic chamber. EGR1 expression and the extent of apoptosis were evaluated by qRT-PCR, Western blotting (WB), and immunofluorescent assay (IFA). RNA-seq was conducted to identify specific pathways. SiRNA targeting EGR1 and miR-124-3p mimic were used to downregulate EGR1 under IH/R. Results: qRT-PCR (Control vs IH/R: 1.000 ± 0.000 vs 5.749 ± 2.947; P<0.01) and WB (0.037 ±0.038 vs 0.953 ± 0.306; P<0.0001) analyses revealed higher expression of EGR1 in IH/R group. IFA demonstrated a higher ratio of cleaved Caspase-3-positive apoptotic cells in IH/R group (0.202 ±0.206 vs 0.377 ± 0.252; P<0.001). RNA-seq analysis indicated significant downregulation of pathways related to cell survival in IH/R. The siRNA downregulated EGR1 successfully, as confirmed by qRT-PCR, WB, and IFA (IH/R Water vs IH/R siRNA: 0.612 ±0.307 vs 0.257 ±0.122; P<0.01), which led to the suppression of apoptosis (0.288 ±0.143 vs 0.142 ±0.049; P<0.05). miR-124-3p mimic downregulated EGR1 under IH/R conditions, as shown by PCR (IH/R Water vs IH/R miRNA mimic: 3.731 ±1.126 vs 2.568 ±0.959; P<0.0001) and WB (0.947 ±0.438 vs 0.478 ±0.277; P<0.05). IFA proved the suppression of EGR1 by miR-124-3p mimic (0.414 ±0.101 vs 0.062 ±0.064; P<0.0001), which subsequently reduced CM apoptosis (0.234 ±0.127 vs 0.092 ±0.047; P<0.001). Conclusion: EGR1 participate in exacerbating apoptosis mediated by IH/R in human CMs. The downregulation of EGR1 suppresses IH/R-induced apoptosis. Treatment with miR-124-3p targeting EGR1 may potentially serve as a novel therapeutic approach for cardioprotection in the future.