Enhancer-mediated metabolic pre-patterning defines trabecular cardiomyocyte identity prior to morphogenesis

Cardiac trabeculae are essential structures of the vertebrate heart critical for ventricular wall integrity and contractility. Despite their importance, the transcriptional regulatory program defining trabecular cardiomyocyte identity and driving their development remains poorly understood. We identify a novel enhancer, -6.8got2b, that drives gene expression specifically in the trabecular myocardium of the zebrafish heart. Functional analyses revealed that - 6.8got2b is required for trabeculae formation, likely acting through its target gene, got2b, which encodes a mitochondrial enzyme. We delineated a 166bp core region responsible for restricting gene expression to trabecular cardiomyocytes, harboring binding motifs for multiple transcription factors (TFs), including Tbx20, whose motif deletion abolished trabeculae-specific expression. A human genomic region sharing partial sequence similarity and TF motifs with -6.8got2b was found near GOT2P4 - a pseudogene related to the human GOT2 gene. Strikingly, the human sequence drove expression in the zebrafish trabeculae and ventricular myocardium, indicating partial but functionally relevant conservation. Using - 6.8got2b as a molecular marker, we transcriptionally profiled trabecular cardiomyocytes and show that, compared to compact layer cardiomyocytes, they exhibit distinct gene signatures indicative of transcriptional priming for elevated energy metabolism prior to the formation of overt trabecular structure. Our findings reveal a conserved -6.8got2b-got2b regulatory axis driving trabecular cardiomyocyte development downstream of Tbx20, establishing a new layer of transcriptional regulation in heart morphogenesis.