Mechanistic Role of Human mAKAP Polymorphisms in Cardiovascular Diseases



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Cardiovascular diseases (CVDs) are the no. 1 cause of deaths in the world. CVDs can affect any individual irrespective of his/her age and gender. Despite the continuous improvement in the clinical research, there is no reduction in a number of deaths and/or hospitalizations from CVDs. We believe that comprehensive understanding of basic cardiac signaling is crucial in finding new therapeutic targets for CVDs. One of such vital cardiac signaling pathways is cAMP-dependent PKA signal transduction which is tempered by a family of scaffolding proteins called A-kinase anchoring proteins. By anchoring key enzymes along with their upstream and downstream signaling partners, AKAPs create order in intracellular signal transduction. Heart-specific muscle AKAP (mAKAP, AKAP6, AKAP100) controls cyclic AMP / PKA signals downstream by binding to PKA, PDE4D3, and PP2A. mAKAP is a master scaffold that governs cardiac hypertrophic pathways. SNPs in different genes encoding essential cardiac proteins have been proven to increase the risk of CVDs in humans. Polymorphic analysis of disease-causing human mAKAP has been unexplored so far. The goal of this work is to study human mAKAP SNPs found in CVDs databases (S1653R and E2124G). We performed immunoprecipitation analysis to see whether SNPs affect binding of mAKAP to PKA, PDE4D3, and PP2A. In immunoprecipitation studies, S1653R mutant exhibited increased binding with PDE4D3 at baseline but significantly reduced binding after stimulation as compared to WT. E2124G mutant displayed significantly lower PKA binding at baseline and higher binding after stimulation. Both the mutants show no change in binding with PP2A as compared to WT. Intracellular cAMP was significantly lower at baseline but higher after stimulation in S1653R mutant cells as compared to the WT. PKA activity assay revealed identical results for the S1653R mutant. E2124G expressed cells showed no change in cAMP levels. PKA activity was significantly lower at baseline but the sharp increase was observed after stimulation. PDE activity assay showed significantly higher activity in S1653R mutant before stimulation which was considerably lowered after stimulation as compared to WT. E2124G mutant showed similar changes as WT with respect to PDE activity. Immunoblotting revealed altered expression of PKA-mediated hypertrophic markers in mAKAP mutants. Notably, E2124G mutant also showed increased intracellular calcium after stimulation as compared with WT in red fluorescence quantification assay. In summary, human mAKAP SNPs might predispose individuals to the risk of developing CVDs by altering AKAP-PKA signaling and affecting cAMP and/or calcium dynamics. mAKAP can be further targeted as a candidate gene for identifying disease-causing SNPs in CVDs.



A-kinase anchoring proteins, Cardiovascular diseases, Single nucleotide polymorphisms, Protein kinase A