Identification Of Novel Phosphoprotein Signaling Pathways In Human Dilated Cardiomyopathy By Integrative Proteomic And Phosphoproteomic Analysis

Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure, yet the majority of the underlying signaling mechanisms remain poorly characterized. Protein phosphorylation is a key regulatory element with profound effects on the activity and function of signaling networks; however, there is a lack of comprehensive phosphoproteomic studies in human DCM patients. We assessed the hypothesis that an integrative phosphoproteomics analysis of human DCM would reveal novel phosphoprotein candidates involved in disease pathophysiology. Combined proteomic and phosphoproteomic analysis of explanted left ventricular tissue samples from DCM patients ( n =4) and non-failing controls ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. From these analyses, we identified αT-catenin as a unique candidate protein with a cluster of 4 significantly hyperphosphorylated sites in DCM hearts ( P <0.0001), with no change in total αT-catenin expression at the protein level. Bioinformatics analyses of human datasets and confocal imaging of human and mouse cardiac tissue show highly cardiac-enriched expression of αT-catenin, localized to the cardiomyocyte intercalated disc. High resolution 3-dimensional reconstruction shows elongated intercalated disc morphology in DCM hearts (10.07±0.76 μm in controls vs. 17.20±1.87 μm in DCM, P <0.05, n =3/group), with significantly increased colocalization of αT-catenin with the intercalated disc membrane protein N-cadherin (Pearson’s coefficient 0.55±0.04 in controls vs. 0.71±0.02 in DCM, P <0.05, n =3/group). To investigate the functional role of cardiac αT-catenin phosphorylation, we overexpressed WT protein vs. non-phosphorylatable forms based on the loci identified in DCM hearts, in adult mouse cardiomyocytes using lentiviral transduction. Confocal imaging revealed significant internalization of the phospho-null form, as compared to the prominent intercalated disc staining of the WT protein (17.78±0.79% of WT vs. 9.25±0.49% of 4A mutant, P <0.0001, n =50 cells/group). Together, these findings suggest a critical role for αT-catenin phosphorylation in maintaining cardiac intercalated disc organization in human DCM.