Phosphorylation Signatures Of Human Heart Failure: Characterizing Dilated Cardiomyopathy-associated Signaling Pathways At The Cardiomyocyte Intercalated Disc

Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients ( n =4) vs. non-failing controls ( n =4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts ( P <0.0001). Overexpression of non-phosphorylatable hCTNNA3 in ex vivo isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an in vivo mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty; n ≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT; n =6), by echocardiography and ex vivo optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.