Abstract Body: Background: Cardiac transcription factors (CTFs) and coregulators drive the developmental transition from immature to mature cardiomyocytes (CMs). The nuclear receptor estrogen-related receptor (ERR), with coregulator PGC-1α and CTF GATA4, has been shown to serve as a critical role in CM maturation. Friend of GATA 2 (FOG2), a transcriptional coregulator, is crucial for normal cardiac development and exists in 2 isoforms: FOG2 and FOG2S, a shortened isoform lacking the N-terminal repressor domain and more prevalent in developing heart. Little is known about the role of FOG2S. We sought to investigate the mechanisms whereby FOG2S serves as a transcriptional regulator and assess its potential interaction with maturation CTFs.
Methods: FOG2S was overexpressed in 8-week-old male C57/BL6 mice and human iPSC-derived cardiomyocytes (iPSC-CMs) using cardiac-specific AAV (cTNT.AAV9). Hearts were processed for RNA-seq and electron microscopy. iPSC-CMs were also treated with 0.1 mM DMOG for 16 h to induce hypoxia, essential for metabolic flexibility, and assessed with Seahorse Bioanalyzer. Hearts and iPSC-CMs were also analyzed for oxidative phosphorylation genes by qRTPCR. Co-transfection studies using a 3XERR-RE upstream of a minimal promoter driving luciferase reporter was performed in HEK293 cells with 3xFLAG-ERRα, PGC-1α-Myc/His-, GATA4-HA-, FOG2-FLAG- and/or FOG2S-FLAG-tagged plasmids.
Results: FOG2S mice showed 678 differentially expressed genes (p<0.001, 424 genes up and 254 genes down). Gene set enrichment analysis identified oxidative phosphorylation as the top hallmark pathway, driven by decreased Nduf’s and Cox’s and increased Bnip3, a mitophagy gene. FOG2S hearts had smaller and rounder mitochondria with less cristae. In vitro, DMOG-treated FOG2S iPSC-CMs exhibited decreased basal and maximal respiration and reduction in mitochondrial network. There was a significant 40-80% decrease in mitochondrial gene expression, MFN1, COX10, TIMM21, TOMM22, NDUFA11, NDUFAB1. GSEA for transcription factor targets identified ERRα as the top hit. Luciferase reporter assay for 3XERR-RE activity showed FOG2S inhibited ERRα-PGC1α-GATA4 mediated activation in a dose-dependent manner. Inhibition was also mediated through GATA4 and independent of PGC1α.
Conclusion: FOG2S overexpression suppresses oxidative phosphorylation and mitochondrial metabolism by inhibiting GATA4 in the context of ERRα-PGC1α-GATA4 complex. These results suggest that FOG2S modulates CM metabolic maturation.