Role of Cavβ subunits in cardiac differentiation

Abstract

Calcium entering the cell through voltage-gated calcium channels (VGCC) activates multiple signaling pathways controlling gene expression. By regulating both plasma membrane targeting and biophysical properties of VGCC, VGCC β-subunits actively regulate calcium influx and thus gene transcription. Emerging studies have reported alternative pathways through which Cavβ translocates to the nucleus and directly regulates gene expression. These studies have highlighted several partners of Cavβ which mediate its role in gene transcription. Several genes regulated by Cavβ have been previously shown to be involved in cell differentiation suggesting its role in this process. However, how this alternative feature of Cavβ impacts cell differentiation remains unclear and Cavβ partners and mechanisms involved are still to be discovered. Here, we investigate how nuclear Cavβ function regulates cardiomyocytes differentiation using iPS-derived cardiomyocyte model. Preliminary experiments show nuclear localization for Cavβ and expression of several Cavβ isoforms in iPS-derived cardiomyocytes. First part of the project will consist in identifying Cavβ partners involved in its nuclear targeting in iPS-derived cardiomyocytes and in its role in gene transcription regulation. This will be achieved using protein-protein interaction screening methods, such as immunoprecipitation of tagged-Cavβ and mass spectrometry analysis of co-immunoprecipitated proteins. Additionally, using ChIP sequencing, we aim at identifying what part of DNA interacts with those complexes and which gene expressions are regulated. Finally, we will decipher how this regulates cardiac differentiation through specific Cavb knockout approach, using CRISPR technology.