Signaling Scaffolds in Cardiovascular Development and Disease
- Author(s): Spindler, Matthew
- Advisor(s): Conklin, Bruce R.
- et al.
Background: G-protein signaling pathways regulate many aspects of cardiovascular development and disease. A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate these signaling events into specific physiological processes. One family member, AKAP13, integrates Gs, Gq/11, and G12/13 signals through binding of protein kinase A, C (PKC), and D (PKD) and encoding an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. AKAP13 is required for mouse development as null embryos die by E10.5 and exhibit cardiovascular defects. Additionally, the Rho-GEF and PKC-PKD binding domains mediate cardiomyocyte hypertrophy in isolated cardiomyocytes. However, the specific developmental processes regulated by AKAP13 and the requirements for the Rho-GEF and PKC-PKD binding domains during development and cardiac hypertrophy are unknown.
Methodology/Principal Findings: We used an RNAi, loss-of-function approach, to determine if AKAP13 mediates differentiation of mouse embryonic stem (ES) cells or angiogenic processes in human umbilical vein endothelial cells (HUVECs). We found that AKAP13 knockdown did not decrease ES cell differentiation into Nkx2.5-GFP-positive cardiac mesodermal cells or functionally beating cardiomyocytes. Similarly, the expression of endodermal, endothelial, or cardiac structural marker genes was not affected. We also found that AKAP13-deficient HUVECs had normal tube formation, VEGF-induced wound healing, and VEGF-induced PKD phosphorylation. We then generated AKAP13-truncation mutant mice to determine if the Rho-GEF and PKC-PKD binding domains are required for development. Surprisingly, homozygous mutant mice were born at normal Mendelian ratios and had normal survival, fertility, and cardiac structure. Finally, to determine if these domains mediate β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size increased normally in mice lacking the Rho-GEF and PKC-PKD binding domains. However, these hearts failed to increase cardiac ejection fraction or fractional shortening.
Conclusions: These results indicate that AKAP13 is not required for differentiation into cardiovascular tissue, the formation of functional cardiomyocytes, or angiogenic processes. They also indicate that the AKAP13 Rho-GEF and PKC-PKD binding domains are not required for mouse development or the cardiac structural response to β-adrenergic-induced hypertrophy. However, these domains are required for the proper cardiac functional response to β-adrenergic stimulation.