Introduction:Cellular repressor of E1A-stimulated genes 1 (CREG1) is an evolutionarily conserved glycoprotein of 220 amino acids. It is localized in the endosomal-lysosomal system. In cultured cells, CREG1 inhibits proliferation and induces differentiation. Published literature suggests that CREG1 is required for early embryonic development.Methods:To elucidate the function of CREG1 in cardiac development, we generated Creg1conditional knockout mice using the CRISPR-Cas9 system. The UBC-Cre/ERT2 transgene was introduced into the Creg1fl/flbackground, allowing for tamoxifen-inducible Creg1knockout at a specific developmental timepoint. Creg1fl/flfemales were mated with Creg1fl/fl;Cremales. Timed pregnant mice were injected with tamoxifen on embryonic day 12.5 (E12.5) and sacrificed on E19.5, following cesarean delivery. Creg1komice were compared to within-litter Creg1fl/flcontrols.Results:Creg1komice died shortly after cesarean delivery with breathing difficulties and cyanosis. Microdissection revealed atrial congestion in Creg1kohearts but not Creg1fl/flcontrols. Hematoxylin and eosin staining demonstrated increased ventricular wall thickness in Creg1kohearts with increased trabecular-to-compact myocardium ratio. Myocardial tissue architecture in Creg1kowas disorganized and lacked cellular alignment. The nuclei of Creg1kocardiomyocytes were rounded and displayed euchromatin predominance, compared with the cigar-shaped nuclei and heterochromatin seen in control cardiomyocytes. Immunofluorescence staining for Ki67 demonstrated more actively proliferating cardiomyocytes in Creg1kohearts. Immunofluorescence staining for CREG1 in isolated cardiomyocytes displayed subcellular localization of CREG1 in vesicles adjacent to cell-cell contact sites of actively aggregating cells. Adenovirus-mediated overexpression of CREG1 in cardiomyocytes promoted cell-cell junction formation while knockout CREG1 inhibited cell-cell adhesion.Conclusions:These results suggest that CREG1 is required for the formation of cohesive myocardial structures. Ablation of CREG1 results in cardiac hypertrophy and compaction defects. Supported bythe AHA Founders AffiliateMedical Student Research Grant.