Requirements for Cyp26 enzymes in second heart field addition and ventricular maintenance

Doctor's Name: 
Ariel Rydeen
Children’s Hospital, Cincinnati

Collaboratively awarded through the CHF and AHA Congenital Heart Defect Research Awards
(Total Request = $28,080;  CHF portion = $14,040)

Congenital heart defects (CHDs) are the most common type of birth defect with one third of CHDs being outflow tract (OFT) defects. Understanding the mechanisms underlying proper heart and OFT development is necessary for improving and designing novel therapies aimed at the prevention and treatment of CHDs. Normal heart development requires proper regulation of retinoic acid (RA) signaling. Vitaminosis A or excess RA in mothers during development can lead to RA embryopathies, which often include OFT defects. Cyp26 enzymes limit embryonic RA signaling through metabolizing RA into easily degraded derivatives, suggesting their loss could lead to congenital defects similar to excess RA. Moreover, alterations in Cyp26 enzyme function have been implicated in several human diseases, including DiGeorge syndrome, Klippel-Feil anomaly and Antley-Bixler syndrome, which also incur OFT defects. Although previous studies show Cyp26 deficient embryos have cardiovascular defects, the precise nature of the heart defects have not been addressed. Our preliminary data using zebrafish indicates that depletion of Cyp26 enzymes first leads to a disruption of second heart field (SHF) addition to the OFT and then a previously unrecognized progressive loss of differentiated ventricular cardiomyocytes (VCs), which is due to cells migrating away. Additionally, we observe an ectopic population of cardiac-like cells in the pharyngeal region of Cyp26 deficient embryos, suggesting increased RA signaling leads to ectopic differentiation and migration of cardiac fated cells. In Specific Aim 1, we will use Kaede-based lineage tracing to determine the origin of the ectopic cardiac-like cell population in Cyp26 deficient embryos. In order to identify the mechanisms underlying the progressive loss of VCs, we performed RNAseq and identified MMP9 as a candidate effector of excess RA signaling in Cyp26 deficient embryos. Importantly, treatment with pan-MMP inhibitor can restore SHF addition. In Specific Aim 2, we will use loss-of-function experiments to specifically address if increased MMP9 activity in Cyp26 deficient embryos promotes SHF addition to the OFT. Ultimately, the information obtained from our studies will help to broaden our understanding of the signaling networks and mechanisms driving heart development and cardiomyocyte maintenance, which will allow for generation of new strategies aimed at preventing OFT defects and improving the quality of life for individuals living with CHDs.

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