Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation

Abstract

Background: Abnormal calcium (Ca²⁺) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca²⁺ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN.

Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca²⁺ imaging.

Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca²⁺ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF.

Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca²⁺ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

Keywords: atrial fibrillation; calcium release activated calcium channels; protein phosphatase 1; ryanodine receptor 2; ryanodine receptor calcium release channel.

Figure 1.. Validation of binding between RyR2, PPP1R3A and PP1c in mouse heart.

Representative Western blots confirming the interaction between RyR2, PPP1R3A, and PP1c in WT mouse heart lysates immunoprecipitated with (A) RyR2 antibody or (B) PPP1R3A antibody. Confocal and STED imaging of co-immunostained ventricular (C) and atrial (D) mouse cardiomyocytes. STED, but not confocal imaging, resolves the PPP1R3A (red) and RyR2 (green) signals, which enables image segmentation for regional nanodomain visualization (right). Of note, RyR2 and PPP1R3A clusters show considerable differences in their subcellular distribution between ventricular and atrial myocytes. Scale bars 10 μm (left, cell overview) or 1 μm (image panels). (E) Left: Magnified view (as indicated in D). Center: zoom-in showing the local association of PPP1R3A with RyR2 clusters at nanometric scale. The white triangles indicate the nanodomain orientation used for signal intensity profiling. Right: The PPP1R3A (red) and RyR2 (green) signal distribution confirms sub-cluster areas exhibiting co-localized signals (yellow). Scale bars 500 nm (left) or 200 nm (center). Dashed boxes indicate magnified views in (D) and (E).

Figure 2.. Genetic ablation of PPP1R3A impairs binding of PP1c to both RyR2 and PLN.

Representative Western blots and corresponding dot plots showing that with PPP1R3A ablation, (A and B) the association between RyR2 and PP1c is significantly reduced with no change in the association between RyR2 and PP2A, and (C and D) the association between PLN and PP1c is significantly reduced. Data represent mean±SEM and were analyzed using unpaired 2-tailed Student’s t-test. (*P<0.05 vs. WT).

Figure 3.. Loss of local PP1c regulation in the absence of PPP1R3A increases RyR2 and PLN phosphorylation.

Representative Western blots and corresponding dot plots showing (A and B) increased phosphorylation of RyR2 at S2808 but not at S2814 in Ppp1r3a-KO (KO) mouse atria compared with wild-type (WT) atria. (C and D) increased phosphorylation of PLN at both S16 and T17 in Ppp1r3a-KO mouse atria. Data represent mean±SEM and were analyzed using unpaired 2-tailed Student’s t-test (*P<0.05, **P<0.01 vs. WT).

Figure 4.. Ppp1r3a-KO mice exhibit increased susceptibility to pacing-induced AF.

A, Representative recordings of surface ECG lead II showing no change in baseline ECG parameters comparing 4-month old Ppp1r3a-KO mice and WT littermates. B, Simultaneous recordings from surface and intracardiac (atrial and ventricular) leads demonstrating AF in Ppp1r3a-KO mice (right) and normal sinus rhythm in WT littermate (left) following atrial burst pacing. C, Bar graph summarizing the incidence of inducible AF in Ppp1r3a-KO mice. Data were analyzed using Fisher’s exact test (*P<0.05 vs. WT).

Figure 5.. Ca²⁺ handling is altered in atrial cardiomyocytes from Ppp1r3a-KO mice due to increased activities of RyR2 and SERCA2a.

A, Representative confocal line-scan images of atrial myocytes from Ppp1r3a-KO mice and WT littermates. B, Dot plots summarizing spontaneous Ca²⁺ spark frequency (CaSF). C, Summary of SR-Ca²⁺ load and (D) CaSF normalized to caffeine-induced SR-Ca²⁺ load. E, Representative tracings of Ca²⁺ transient recordings during 1Hz pacing and after exposure to 10mM caffeine. F, Dot plot summarizing Ca²⁺ transient amplitude. G, SERCA2a activity calculated as the difference between the decay of the pacing-induced transient and the caffeine-induced transient. H, NCX activity calculated from the decay of the caffeine-induced transient. Abbreviations: CaSF, Ca²⁺ spark frequency; CaT, Ca²⁺ transient; Tyr, Tyrode’s buffer; Caff, caffeine. Data represent mean±SEM and were analyzed using the Generalized Estimating Equation function in SPSS (*P<0.05; **P<0.01 vs. WT).

Figure 6.. Complexome profiling reveals PPP1R3A as a protein within a novel high molecular weight RyR2/PLN/SERCA2a complex.

A, Heat map and B-C, migration profiles of PLN, SERCA2a, RyR2 and PPP1R3A in cardiomyocytes from PLN wildtype (WT) mice. Arrows indicate high-molecular weight complexes. D, Heat map and E-F, migration profiles of SERCA2a, RyR2, and PPP1R3A in cardiomyocytes from PLN-KO mice.

Figure 7.. PPP1R3A protein expression is reduced in atria of cAF patients.

Representative Western blots and corresponding dot plots from human atrial biopsy samples showing (A and B) slight but non-significant decrease in PPP1R3A expression levels (bottom two bands) in early stage (paroxysmal) AF patients and (C and D) significant decrease in PPP1R3A expression levels in late stage (chronic) AF patients. Abbreviations: pAF, paroxysmal AF; cAF, chronic AF; SR, sinus rhythm. Data represent mean±SEM and were analyzed using unpaired 2-tailed Student’s t-test (**P<0.01 vs. SR control).

Figure 8.. Higher molecular complexes in human atrial tissue are disrupted in atrial fibrillation.

A, Heat map representation of the migration profile of PLN, SERCA2a, RyR2, and JPH2 in human atrial tissue (total membrane fraction). Atrial samples were obtained from patients with sinus rhythm (SR), paroxysmal AF (pAF), or chronic AF (cAF). B, In SR (pseudo-control; see Supplemental Table 2), profiling shows comigration of PLN/SERCA2a and RyR2 in higher molecular complexes (>1 MDa) and additionally JPH2 (≤1 MDa). In contrast, in paroxysmal AF the RyR2 channel was nearly abolished in higher complexes and only monomeric JPH2 exists. In contrast, in cAF the abundance of higher molecular complexes was decreased and JPH2 was not detected any longer.