In Aim 1, I proposed to characterise the initiation and progression of CVC phenotypes in vivo by dissecting the role of specific genes and cell types, as well as the functional impact of tissue degradation. To achieve this, we have been establishing models to study CVC in both developing and adult zebrafish. Notably, we identified a mutant line exhibiting calcification of the outflow tract (OFT) and cardiac valves, from 3 and 6 months of age, respectively. This model is now being used for single-cell transcriptomics to identify potential cell populations specific to CVC. In parallel we have performed single-cell transcriptomic analysis of wild-type cardiac valves (and surrounding tissues), and were able to identify all main cell clusters using specific markers. This central dataset will significantly enhance future analyses of samples derived from our CVC models. Additionally, we identified a second mutant model displaying OFT calcification at juvenile stages, starting from 30 days post fertilization, and we are actively working to identify CVC at earlier stages. Once the model and the earliest time points showing CVC are established, we will perform single-cell analysis of these samples, to later combine with the data from adult animals. This will allow us to uncover not only the factors driving CVC but also how these factors differ with aging. Furthermore, we are developing and analyzing transgenic zebrafish lines overexpressing pro-osteogenic factors in specific cardiovascular cell populations to determine the potential of each cell lineage to contribute to CVC. Using these mutant and transgenic models, we will also use live microscopy to characterise the cellular dynamics and functional impact of CVC in vivo.
In Aim 2, I proposed to identify new local and systemic therapeutic strategies to block/reverse CVC, leveraging the zebrafish’s amenability for genetic manipulation and suitability for high-throughput screening. We have already used a model of generalized ectopic calcification to establish a protocol for testing small compounds as potential calcification inhibitors. Using known inhibitory compounds as proof of concept, we successfully defined the conditions and measurements of our experimental setup.
Additionally, our host institution has purchased the Vertebrate Automated Screening Technology (VAST) platform, which we are currently optimizing for large-scale screening of small-compound libraries. From this screening, we will select a shortlist of small molecules for further testing in adult zebrafish and in cardiovascular cells derived from hiPSCs of CVC patients.
