The genes heg, san/ccm1, and vtn/ccm2 are key molecules in a novel genetic pathway regulating zebrafish cardiac development. Their importance extends to the mammalian cardiovascular system where these genes are essential for development of the heart and for maintenance of vascular integrity. The link to cardiovascular patterning is reinforced by studies showing that patients with mutations in the human versions of the san (CCM1) and vtn (CCM2) genes develop the vascular anomaly Cerebral Cavernous Malformations (CCM). One of the CCM pathway genes, CCM2, has been linked directly to MAPK signaling, suggesting that investigation of this second pathway may provide insights into disease pathogenesis. We have demonstrated that we are able to rescue the defects in the zebrafish mutants by circumventing upstream regulation of this pathway and directly activating ERK. This proposal aims to determine how these two pathways intersect to pattern growth of heart muscle and regulate the maintenance of blood vessel integrity. We will examine how the CCM pathway regulates MAPK signaling to promote proper cardiovascular patterning and early myocardial development and whether this is mediated through release of a secreted endothelial/endocardial factor. We propose two aims to establish the role of the Heg-CCM pathway in cardiovascular development. In the first aim, we will examine how disruption of the CCM pathway and the associated phenotype could be mediated by MEK1 signaling in CCM mutants and morphants (embryos injected with morpholinos to interfere with RNA function and knockdown protein levels to mimic a mutant). In the second aim, we will perform a systematic analysis of alterations in secreted factors when CCM1 and CCM2/CCM2L levels are downregulated in tissue culture cells, with particular focus on secreted factors. We will further determine the effects of knockdown of CCM2 and CCM2L gene expression levels in comparison to wild-type cells to define whether candidate secreted factor expression is altered. Rather than restricting our analysis to a select group of genes, we will use RNAseq to provide an unbiased analysis of changes in any transcripts normally expressed in these cells. Positive factors will be examined using inducible or myocardial expression in the zebrafish model as described above. Determination of how the CCM and MAPK pathways intersect could lead to important insights into how CCM disease occurs and progresses. Since the MAPK genes are popular drug targets, there are numerous small molecules available for testing in our studies. This work could ultimately suggest new approaches for therapy by targeting the MAPK signaling components instead of directly trying to manipulate members of the CCM pathway itself.
Our proposal will identify key elements essential to the growth and function of the heart and vasculature. The genetic pathways characterized through this proposal, and their interdependencies, will highlight approaches to manipulate them for treatments of cardiovascular diseases such as Cerebral Cavernous Malformations (CCM). The results of our studies will also provide important insights into how heart muscle develops and may also lead to new approaches for heart regeneration.
