Abstract 170: Protein Kinase C regulates alternative splicing transitions during heart development
Introduction: Highly conserved genetic and molecular pathways coordinate gene expression during heart development. Alternative splicing (AS) regulates gene expression by generating distinct spliced isoforms of proteins such that embryonic variants are expressed only in fetus but not in adult. Importantly, mutations that affect splicing of embryonic genes such as Nkx2.5 and GATA4 can lead to congenital heart defect. Although AS is critical for proper gene expression in developing heart, the signals that regulate AS are not well known. We have previously shown that Protein Kinase C α/β (PKCα/β) phosphorylates splicing regulator CELF1 that controls AS in heart, suggesting that PKC signaling could be responsible for AS regulation during heart development.
Hypothesis: PKCα/β contributes to embryonic gene expression by regulating AS.
Methods: We used H9c2 rat embryonic cell differentiation into cardiomyocyte-like cells and rat heart tissues at different stages of development to determine whether PKC is responsible for AS transitions. The localization and levels of proteins were assessed by western blot and immunohistochemistry. Quantitative RT-PCR was performed to determine the splicing pattern of nine developmentally regulated genes using specific primers.
Results: The results indicate that AS of developmentally important genes transitioned during rat heart development and H9c2 differentiation. PKCα/β regulated embryonic to neonatal AS transitions by phosphorylating splicing regulators CELF1 and Rbfox2. Blocking PKCα/β activity by bisindolylmaleimide IX reduced both the phosphorylation and protein levels of CELF1 and Rbfox2 with concurrent changes in AS transitions. Depletion of PKCα was sufficient to alter a subset of AS events by down-regulating CELF1 and Rbfox2 proteins. Finally, our data suggest that nuclear localization of PKCα/β at embryonic stages could be the mechanism for AS transitions and increased steady state levels of splicing regulators.
Conclusions: Our results show that PKCα/β regulate splicing regulators and AS during heart development. Changes in PKCα/β subcellular localization is one of the determinants for embryonic to neonatal AS transitions in developing rat heart and during cardiomyocyte differentiation.
- © 2013 by American Heart Association, Inc.