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 Ca(2+) remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

Keywords: PP1 regulatory subunits; atrial fibrillation; label-free quantification; mass spectrometry; protein phosphatase 1; proteomics.

FIGURE 1. A Novel Unbiased Method for Profiling PP1c Interactors

(A) Schematic showing the major methodological steps. (B) Western blot validation of the coimmunoprecipitation of PP1c and 1 known R-subunit (PPP1R7) from mouse ventricular lysates. (C) Western blot demonstrating equal efficiency of anti-PP1c antibody in pulling down mouse (Mo) and human (Hu) PP1c. LC = liquid chromatography; LFQ = label-free quantification; MS/MS = tandem mass spectrometry; XIC = extracted-ion chromatogram.

FIGURE 2. Identification and LFQ of PP1c Interactors

Cardiac lysates made from mouse ventricles (A) or human atria (B) were immunoprecipitated with anti-PP1c versus isotype-control IgGs and analyzed by mass spectrometry. The y-axis plots the label-free quantification (LFQ) signals of each protein from the anti-PP1c pull-down. The x-axis plots the ratios of the LFQ signals from the anti-PP1c pull-down over that from the control pull-down. Proteins with a ratio >3 are considered PP1c interactors. Proteins with an infinite ratio (∞) have no LFQ signals from the control pull-down. Known PP1c interactors are labelled with their gene names, whereas the baits Ppp1ca and PPP1CA are marked by a black circle.

FIGURE 3. Bioinformatic Analyses of PP1c Interactors

Relative binding of known PP1 regulatory subunits to PP1c on the basis of LFQ signal intensities normalized to sequence length of each protein for mouse ventricles (A) and human atria (B). (C) Sequence LOGOs of the 3 most common PP1c docking motifs (KVxF, MyPhoNE, and SILK) (9) generated from validated PP1c interactors using MEME. The E-values of the 3 motifs are 5.9E–4, 2.3E–6, and 3.8E3, respectively. PP1c interactors that have at least 1 of these motifs were plotted in Venn diagrams for mouse ventricles (D) and human atria (E). LFQ = label-free quantification.

FIGURE 4. In Vitro Validation of MS Findings

Coimmunoprecipitation followed by Western blots were performed using lysates from HEK293 cells that were cotransfected with 1 of 3 putative PP1c interactors, SEC31A (A), VCP (B), and CSDA (C), and HA-tagged PP1c. SEC31A and CSDA were FLAG tagged, whereas VCP was EGFP tagged. CSDA = cold-shock domain protein A; LFQ = label-free quantification; MS = mass spectrometry; SEC31A = protein transport protein Sec31A; VCP = valosin-containing protein.

FIGURE 5. Comparison of Binding Between PP1c and Known or Novel Interactors in SR versus PAF Patients

(A) Known interactors that are unchanged in their binding to PP1c. (B) Interactors that are changed in their binding to PP1c in paroxysmal atrial fibrillation (PAF) patients (n = 5), with CSDA and PDE5A being novel PP1c interactors. Quantification is on the basis of LFQ signals, and ACTC1 is used as an internal control. For SR patients, n = 7 to 8. ACTC1 = actin-1; PDE5A = phosphodiesterase type-5A; SR = sinus rhythm; TNS1 = tensin-1; other abbreviations as in Figure 4.

FIGURE 6. Global Levels of Select PP1c Interactors in SR Versus PAF Patients

(A and B) PP1c immunoprecipitation followed by Western blots confirming the increase in binding between PP1c and PPP1R7 in paroxysmal atrial fibrillation (PAF) patients. (C and D) Representative Western blots and quantification showing the global protein levels of the 3 interactors (PPP1R7, CSDA, and PDE5A) relative to that of PP1c. *p < 0.05 versus sinus rhythm (SR). Numbers in the bars indicate the numbers of patients per group. Abbreviations as in Figures 4 and 5.

FIGURE 7. Colocalization of Ppp1ca (PP1c) and Its Interactors

Atrial myocytes isolated from adult mice were costained with different antibodies to show the colocalization between PPP1CA (PP1c) and 3 of its interactors: PPP1R7 (A), CSDA (B), and PDE5A (C). Representative images were chosen from 10 to 12 cells from 2 to 3 mice. Scale bar = 20 mm. Abbreviations as in Figures 4 and 5.

FIGURE 8. Subcellular Targeting of PP1c by CSDA

HeLa cells were transfected with either FLAG-CSDA or empty vector and costained with anti-FLAG and anti-PP1c antibodies. White arrows in the middle panels point to nuclear or perinuclear aggregations of PP1c, which are absent without CSDA and which colocalize perfectly with CSDA as shown in the bottom panels. Scale bar = 20 mm. CSDA = cold-shock domain protein A.

CENTRAL ILLUSTRATION. PP1c Interactome in Cardiomyocytes

Localization of novel PP1 regulatory (R)-subunits is on the basis of immunocytochemistry data (Figure 7, Online Figure 1) and previous studies that used noncardiac tissues. Online Table 5 contains the definitions of the abbreviations used in the Central Illustration.