Abstract 24010: Muscle-specific A-Kinase Anchoring Protein Polymorphisms Pre-dispose Humans to Cardiovascular Diseases by Affecting cyclic AMP/PKA Signaling

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In-depth understanding of key cardiac signaling pathways is crucial in finding new targets for cardiovascular diseases (CVDs), the no. 1 cause of deaths globally. One of such pathways is cAMP-dependent PKA signaling which is modulated by scaffold proteins, A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) regulates expression of hypertrophic factors partly by controlling cardiac cAMP levels. Furthermore, published literature revealed a strong correlation between single nucleotide polymorphisms (SNPs) in proteins and risk of developing CVDs by varying cAMP/PKA signaling. This aspect of AKAPs has been largely unexplored. Hence, we hypothesized that mAKAP SNPs alter cAMP/PKA signaling making individuals susceptible to CVDs. We analyzed selected mAKAP SNPs found often in human patients with CVDs through multiple online tools that predict functional effects of SNPs to finalize two SNPs, Ser(S)1653Arg(R) in PDE4D3 binding domain and Glu(E)2124Gly(G) in 3’-PKA binding domain of mAKAP. After making both mAKAP mutant plasmids from WT using site-directed mutagenesis, we studied them in HEK 293T cells. Four separate samples were used for each experiment. In immunoprecipitation studies, S1653R mutant showed increased binding to PDE4D3 at baseline but significantly reduced binding after stimulation with 1 μM isoproterenol as compared to WT. Similarly, E2124G mutant exhibited significantly lower PKA binding at baseline and higher binding after stimulation. cAMP levels and PKA activity were significantly lower at baseline but higher after stimulation in S1653R mutant cells. Also, E2124G expressed cells showed no significant change in cAMP levels when compared to WT but PKA activity was significantly lower at baseline followed by abrupt increase after stimulation. PDE activity assay was in congruent with cAMP changes in S1653R mutant cells. Fluorometric assay showed higher intracellular calcium in E2124G mutant cells after stimulation. Lastly, immunoblotting data showed altered phosphorylation of hypertrophic markers in both mutants. To conclude, human mAKAP SNPs may pre-dispose humans to the risk of developing CVDs by affecting cAMP/PKA signaling and thus confirming the clinical significance of PKA-mAKAP-PDE4D3 interaction.

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