Upstream open reading frame inactivation augments GATA4 translation and cardiomyocyte hypertrophy in mice

Upstream open reading frames (uORFs) are short peptide-encoding sequences residing in the 5' untranslated region (5' UTR) of mRNAs enabling translational repression of main (m)ORFs. Despite their prevalence, with ~50% of mRNAs harboring at least one uORF in humans, our comprehension of the biological function of these elements remains limited. This study aims to elucidate the role of the uORF in the 5' UTR of the Gata4 (GATA binding protein 4) gene in the context of cardiac biology through inactivation of its start codon (?uORF) in the mouse genome. Our investigation reveals that mice with Gata4 uORF inactivation manifest spontaneous cardiac hypertrophy without apparent fibrosis during aging. Leveraging single-nucleus RNA sequencing (snRNA-seq), we unveiled significant transcriptional variations between wild-type (WT) and ?ORF mice. Notably, mRNAs linked to sarcomeric and contractile functions exhibit heightened expression levels, mirroring the hypertrophic phenotype. Functional assessments with isolated primary adult cardiomyocytes validated enhanced hypertrophy and contractility in ?ORF mice. Furthermore, we employed single-nucleus transposase-accessible chromatin (snATAC)-seq to probe alterations in chromatin accessibility. Our results revealed augmented accessibility within specific transcription-regulatory elements associated with amplified gene transcription. These putative cis-regulatory elements (pCREs) are significantly enriched in MEF2 (myocyte enhancer factor 2) binding motifs. In vitro luciferase reporter assays further validated the regulatory potential of three of these pCREs, emphasizing their role in the transcriptional enhancement of three GATA4 target genes that are bound by MEF2 and GATA4. These findings shed light on the role of uORF in regulating GATA4 protein expression and cardiomyocyte hypertrophy. Overall design: We performed Single-nucleus ATAC-seq (snATAC-seq) and RNA-seq on wildtype and ?ORF mice. Whole hearts were harvested from both mice at an age of 2 month (n = 3), and were fast frozen in liquid nitrogen. Nuclei were isolated from the frozen mouse heart tissue using the 10x Genomics single nuclei isolation kit, adhering to the manufacturer's instructions.