Alterations in the Interactome of Serine/Threonine Protein Phosphatase Type-1 in Atrial Fibrillation Patients

Abstract

BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown. OBJECTIVES The purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits. METHODS Cardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation. RESULTS A total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone. CONCLUSIONS Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

Regulating the regulator: Insights into the cardiac protein phosphatase 1 interactome

“…[5] For example, the phosphorylation levels of myosin binding protein-C and of the L-type Ca 2+ channel were decreased whereas the phosphorylation levels of phospholamban (PLN) and ryanodine receptor 2 (RyR2) were increased. [4, 5, 8] On the other hand, in patients with paroxysmal AF, there is no significant change in the expression levels of PP1c,[9] yet the phosphorylation of at least one target protein, PLN, is increased. [9, 10] Finally, in experimental AF models, both unchanged PP1c levels with or without increased PP1c activity have been reported, and again with inconsistent changes in the phosphorylation of its known targets.…”

“…[4, 5, 8] On the other hand, in patients with paroxysmal AF, there is no significant change in the expression levels of PP1c,[9] yet the phosphorylation of at least one target protein, PLN, is increased. [9, 10] Finally, in experimental AF models, both unchanged PP1c levels with or without increased PP1c activity have been reported, and again with inconsistent changes in the phosphorylation of its known targets. [6, 8, 11–14]…”

“…Motivated by this study, we undertook a large-scale unbiased study to map changes in PP1’s interactome in the setting of paroxysmal AF, discussed in a later section. [9]…”

“…[46] Using PP1 co-IP followed by MS, we also found an interaction between PP1 and phostensin in both mouse ventricles and human atria but did not detect Hepp1. [9] In any case, the precise functional roles of these proteins in the heart will need to be explored and defined in future studies.…”

Cardiovascular proteomics in the era of big data: experimental and computational advances

“…Chiang et al recently elucidated the interactome of the protein phosphtase 1 catalytic subunit (PP1c), identifying 78 interacting partners in human heart. The proteomics results found increased binding to PDE5A in paroxysmal atrial fibrillation patients to impair proteins involved in electrical and calcium remodeling, a result that has implications in the understanding and treatment of atrial fibrillation [80]. Waldron et al identified the TBX5 interactome in the developing heart to discover its interactions with the repressor complex NuRD, elucidating the mechanisms by which TBX5 mutations can influence cardiac development and confer congenital heart diseases.…”