Abstract 934: Physical and Genetic Interaction Analyses in Human Pluripotent Stem Cell-Derived Cardiomyocytes to Study Protein Quality Control Disease in the Heart

Abstract

Cardiomyocytes must maintain constant contractile function of the heart throughout a human lifetime. To achieve this, a complex network of chaperones and other proteins maintains the homeostasis of the proteins in the cell. Protein quality control pathways are increasingly recognized for their importance in both inherited and sporadic cardiac disease, and as potential therapeutic targets. The co-chaperone BAG3 is particularly interesting, because genetic evidence suggests that variants of BAG3 can be both pathologic and protective. However, understanding the molecular mechanism underlying most of these disease-genetic variant associations remains a major challenge. Unbiased analysis of physical and genetic interaction networks holds the potential to provide critical information to fill in this knowledge gap. Using genome engineering tools, we have generated a series of isogenic human induced pluripotent (iPS) cell lines bearing different variants on the BAG3 gene, along with an epitope tag fusion. Using mass spectrometry methods, we have been able to identify interacting partners for the BAG3 protein expressed at endogenous levels in iPS-derived cardiomyocytes. Our results show a surprisingly high number of cardiac-specific interactions. In addition, some BAG3 protein variants present distinct gain or loss of specific interactions, allowing us to narrow down the list of candidate hits. Using microscopy image analysis, we performed a targeted knockdown screening on BAG3 interaction partners to dissect pathways of BAG3 interactors that are involved in the development disease phenotype.We hope the information obtained from this study will improve our understanding of the heart proteostasis network, enable the identification of potential therapeutic targets, and provide clues towards a broader understanding of the role of genetic variation in complex disease.