BioID‐Based Network Analysis to Probe Alterations and Functional Consequences of Adenylyl Cyclase‐Containing Macromolecular Complexes in Cardiomyocytes

Abstract

Numerous cardiac functions rely on the proper formation of macromolecular complexes that regulate cyclic AMP (cAMP) signaling. Localized cAMP production by adenylyl cyclase (AC) is coordinated by scaffolding proteins such as A-kinase anchoring proteins (AKAPs) that anchor AC, the cAMP effector protein kinase A (PKA), along with downstream targets of PKA and other regulators. Alteration of AKAP complexes can promote hypertrophy and changes in contractility, leading to cardiac remodeling and cardiomyopathy. However, the composition and dynamics of AC-containing complexes in heart disease remain mostly unknown. We have therefore carried out a comprehensive study of AC complexes in heart using BioID (proximity-dependent identification of near-neighbor interaction) proteomics technique to probe alterations in complex formation resulting from hypertrophic cardiomyopathy. We have fused the mini-Turbo biotin ligase with three different AC isoforms, two AKAPs, and control GFP and have expressed these fusion proteins in neonatal cardiomyocytes using adenoviruses. Neonatal cardiomyocytes were subsequently treated with β adrenergic receptor agonist norepinephrine (NE) for 48 hours, incubated with biotin for 3 hours, and harvested for streptavidin pull-down and mass spectrometry. Norepinephrine treatment induced hypertrophy, leading to a 229% increase in cell size and a 295% increase in atrial natriuretic factor expression. Clear differences in biotinylation patterns were observed with and without NE by western blot analysis for each of the mini-Turbo fusions. Biotin-labeled proteins resulting from AC or AKAP expression were sorted by Significance Analysis of INTeractome (SAINT) and bioinformatics analysis identified unique protein patterns with different AC and AKAP isoforms under physiological and hypertrophic conditions. Our comprehensive network analysis of localized complex formation is expected to provide important insights for understanding the functional changes of signalosomes and signaling pathways in disease states.