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

An appropriate intensity and duration of increased AC-cAMP-PKA-Ca2+ signaling is the quintessential component of acute adaptation to cardiac stress. Our previous 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 in order to determine the role of altered protein phosphorylation in the chronic adaptive heart performance and protection profile of left ventricle (LV) of adult 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, including protein kinase A (PKA) signaling, PI3K signaling, and AMPK signaling, predicted upstream regulators, e.g., PDPK1, PAK1 and PTK2B, and downstream functions, e.g., cell viability, protein quality control, and metabolism. 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. In order 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 (by dividing the protein phosphorylation abundance by protein abundance). In functional enrichment analysis of 229 normalized phosphoproteins that were differentially regulated by genotype, enriched IPA canonical pathways in the phosphoproteome of TGAC8 LV before and after normalization showed high consistency. The combination of aforementioned analyses indicated that many stress response signaling pathways, i.e., PI3K/AKT signaling and ubiquitin labeling, were consistently enriched at transcriptional, translational and PTM levels, and that PI3K/AKT signaling was also activated in the TGAC8 LV. Thus, PTM of protein by phosphorylation, per se, contributes immensely to the heart performance and protection profile of TGAC8 LV, and is coordinately regulated with altered transcription and translation in the TGAC8 to activate critical stress response pathways.