One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction, which is regulated by a family of scaffolding proteins, i.e., A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase 4D3 (PDE4D3), among other proteins, and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Single-nucleotide polymorphisms (SNPs) in various proteins have especially been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter the cAMP/PKA pathway, influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease-related patients with the highest predicted deleterious effects, Ser 1653 Arg (S1653R) and Glu 2124 Gly (E2124G). Coimmunoprecipitation data in human embryonic kidney-293T cells showed that the S1653R SNP, present in the PDE4D3-binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and that the E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity, and cytosolic PDE activity compared with the wild type before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be upregulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling, supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions. NEW & NOTEWORTHY We found that single-nucleotide polymorphisms in muscle-specific A-kinase anchoring protein found in human patients with cardiovascular diseases significantly affect the cAMP/PKA signaling pathway. Our results showed, for the first time, that human muscle-specific A-kinase anchoring protein polymorphisms might alter the susceptibility of individuals to develop cardiovascular diseases with known underlying molecular mechanisms.

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.