The reprogrammed mouse heart phosphoproteome in response to the chronic stress of markedly high cardiac-specific adenylyl cyclase type 8 overexpression.

Abstract

An appropriate intensity and duration of increased AC-cAMP-PKA-Ca2+ signaling is the quintessential component of acute adaptation to cardiac stress. Our another work demonstrated the existence of an exquisite heart performance and protection profile in response to chronically increased cardiac AC-cAMP-PKA-Ca2+ signaling in young adult mice with cardiac-specific overexpression of adenylyl cyclase type 8 (AC8) (TGAC8). Here, we conducted an unbiased phosphoproteomics analysis to determine the role of altered protein phosphorylation in the chronic adaptive heart performance and protection profile of left ventricle (LV) of TGAC8 at 3-4 months of age. We identified 12,963 phospho-peptides and 3646 phosphoproteins, including 4990 upregulated phosphopeptides and 781 differentially regulated (741 Up and 40 Down) phosphoproteins in TGAC8 vs. WT. Based on differentially regulated phosphoproteins by genotype, we discovered the enrichment of numerous stress response canonical pathways within reprogrammed TGAC8 LV, and predicted upstream regulators and downstream functions. In addition to PKA, numerous kinases (83) and phosphatases (30) were hyper-phosphorylated in TGAC8 vs. WT. By combining the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome, we determined which cardiac performance and adaptive protection profiles were coordinately regulated at post-translational modification (PTM) (phosphorylation), translational and transcriptional levels. To assess the impact of phosphorylation on mRNA transcription, we determined the phosphorylation states of transcriptional factors (TFs) that differed by genotype. Hyper-phosphorylated TFs in TGAC8 were associated with increased mRNA transcription, and immune responses and metabolism pathways. To determine the coordination of proteome and phosphoproteome in reprogramming the TGAC8 LV, we normalized its phosphoproteome to its proteome. These analyses indicated that many stress response signaling pathways were consistently enriched at transcriptional, translational and PTM levels, and that PI3K/AKT signaling was also activated in TGAC8 LV.