Abstract Thu067: ZO-1,2 knockout in cardiac myocytes results in embryonic lethality

Introduction: Zona occludens (ZO) are scaffolding proteins in cardiomyocyte (CM) junctions. Their function in the mature CM has begun to be explored, but their role in cardiac development is poorly understood. We found CM loss of ZO-1 protein from mature CM led to post-natal conduction system defects, but no functional impairment as ZO-2 was upregulated in working CM. We hypothesized that combined loss of ZO-1 and ZO-2 would impact embryonic cardiac development. Objective: This study evaluates the function of ZO-1 and ZO-2 in cardiac development. Methods: ZO-1/ZO-2 floxed mice were crossed to Xenopus laevis myosin light-chain 2 Cre mice to produce CM-specific excision of ZO-1 and ZO-2 (XcZO-1/ZO-2 KO). Pregnant females were evaluated at embryonic stages (E) 9.5 to 14.5. Histological and immunofluorescence analyses characterized phenotypic and molecular alterations due to XcZO-1/ZO-2 KO. Results: Timed pregnancies showed at E12.5 the XcZO-1/ZO-2 KO embryos displayed 31% of those expected by Mendelian ratios (n=115, 9 observed, 28.75 expected), with only 14% of those expected by E13.5 (n=55, 2 observed, 13.75 expected), with no surviving XcZO-1/ZO-2 KO embryos by E14.5 (n=51, 0 observed, 12.75 expected). Histological assessment showed a thinned compact layer 27.9% at E10.5 (±7.8%, p = 0.0072) and 41.6% by E11.5 (±9.9%, p = 0.0029) in XcZO-1/ZO-2 KO as compared to Cre negative control embryos. Trabeculation in the XcZO-1/ZO-2 KO were also less developed and disordered, suggesting arrest of normal cardiogenesis. EdU labeling of E11.5 embryos (n=5) indicated a 33% reduction in CM proliferation (±9.7%, p = 0.027) in XcZO-1/ZO-2 KO vs. Cre negative control embryos. Immunofluorescence microscopy revealed reduced expression of the ZO-1 binding protein, coxsackie virus and adenovirus receptor (CAR), and altered localization of Connexin 45 localization to immature myocyte junctions. Conclusion: Our study highlights a previously unappreciated role for zona occludens proteins in de novo cardiomyocyte junction development, and for normal embryonic development. Continued investigation is being pursued to ascertain a detailed mechanistic role of these proteins in myocardial development.