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, 103 (1), 178-87

Impaired Local Regulation of Ryanodine Receptor Type 2 by Protein Phosphatase 1 Promotes Atrial Fibrillation

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Impaired Local Regulation of Ryanodine Receptor Type 2 by Protein Phosphatase 1 Promotes Atrial Fibrillation

David Y Chiang et al. Cardiovasc Res.

Abstract

Aims: Altered Ca(2+) handling in atrial fibrillation (AF) has been associated with dysregulated protein phosphatase 1 (PP1) and subcellular heterogeneities in protein phosphorylation, but the underlying mechanisms remain unclear. This is due to a lack of investigation into the local, rather than global, regulation of PP1 on different subcellular targets such as ryanodine receptor type 2 (RyR2), especially in AF.

Methods and results: We tested the hypothesis that impaired local regulation of PP1 causes RyR2 hyperphosphorylation thereby promoting AF susceptibility. To specifically disrupt PP1's local regulation of RyR2, we used the spinophilin knockout (Sp(-/-)) mice (Mus musculus) since PP1 is targeted to RyR2 via spinophilin. Without spinophilin, the interaction between PP1 and RyR2 was reduced by 64%, while RyR2 phosphorylation was increased by 43% at serine (S)2814 but unchanged at S2808. Lipid bilayer experiments revealed that single RyR2 channels isolated from Sp(-/-) hearts had an increased open probability. Likewise, Ca(2+) spark frequency normalized to sarcoplasmic reticulum Ca(2+) content was also enhanced in Sp(-/-) atrial myocytes, but normalized by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-93 and AIP and also by genetic inhibition of RyR2 S2814 phosphorylation. Finally, Sp(-/-) mice exhibited increased atrial ectopy and susceptibility to pacing-induced AF, both of which were also prevented by the RyR2 S2814A mutation.

Conclusion: PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels and may represent a potential drug target of AF.

Keywords: Atrial fibrillation; Ca2+/calmodulin-dependent kinase II; Protein phosphatase 1; Ryanodine receptor type 2; Spinophilin.

Figures

Figure 1
Figure 1
Decreased association of PP1 with RyR2 in the absence of spinophilin. Representative western blots and quantification showing that with spinophilin ablation. (A) The protein levels of RyR2, PP1, and PP2A are unaltered. (B) The protein levels of other key Ca2+-handling proteins are also unchanged and (C) the association between RyR2 and PP1, but not between RyR2 and PP2A, is significantly decreased. ***P < 0.001 vs. WT.
Figure 2
Figure 2
Impaired local regulation by PP1 leads to increased phosphorylation of RyR2 at S2814. Representative western blots and quantification showing (A) increased phosphorylation of RyR2 at S2814 (pS2814) but not at S2808 (pS2808) in atria from Sp−/− mouse, (B) unchanged global protein levels of PKA and CaMKII, and (C) unaltered level of CaMKII in the RyR2 complex as determined by co-immunoprecipitation. *P < 0.05 vs. WT.
Figure 3
Figure 3
Increased RyR2 open probability with impaired local regulation by PP1. (A) Representative single-channel recordings of RyR2 from WT and Sp−/− mouse hearts. (B) Increased open probability, (C) increased mean open time, and (D) decreased mean close time of single RyR2 channels from Sp−/− hearts. Numbers within the bars indicate the number of channels studied from three animals per group. *P < 0.05, **P < 0.01 vs. WT.
Figure 4
Figure 4
Impaired local regulation by PP1 increased CaSF in atrial myocytes. (A) Representative confocal line-scan images of atrial myocytes loaded with Fluo4-AM. (B) Bar graph summarizing the ratio of CaSF normalized to SR load for atrial myocytes isolated from WT and Sp−/− mice, in the presence or absence of CaMKII inhibitors KN-93 or AIP, or control drug KN-92. (C) Bar graph comparing the ratio of CaSF to SR load in atrial myocytes paced at 1 Hz (same data as from panel B) or at 3 Hz. Numbers in the bars indicate the number of cells studied from 2 to 12 mice. **P < 0.01 vs. WT. #P < 0.05, ##P < 0.01 vs. Sp−/−.
Figure 5
Figure 5
Impaired regulation of RyR2 by PP1 increased atrial ectopy in mice. (A) Representative telemetry ECG recordings of conscious, freely roaming mice. Arrow points to an atrial ectopic beat in a Sp−/− mouse. (B) Bar graph summarizing the number of atrial ectopic beats per hour in WT, Sp−/−, and Sp−/−:S2814A mice. Numbers under the bars indicate the number of mice studied. *P < 0.05 vs. WT. #P < 0.05 vs. Sp−/−.
Figure 6
Figure 6
Impaired regulation of RyR2 by PP1 increased AF susceptibility in mice. (A) Representative simultaneous recordings of surface ECG and intracardiac electrograms (atrial and ventricular) after burst pacing. (B) Bar graph summarizing the incidence of pacing-induced AF in WT, Sp−/−, and Sp−/−:S2814A mice. Numbers in the bars indicate the number of mice studied. *P < 0.05 vs. WT. #P < 0.05 vs. Sp−/−.
Figure 7
Figure 7
Overexpression of spinophilin decreases RyR2 phosphorylation at S2814. (A) Representative western blots and summary bar graphs showing overexpression of spinophilin in HEK293 cells co-transfected with spinophilin (Sp), RyR2, and CaMKII. Empty, empty pcDNA3.1 vector. (B) Representative western blots and summary bar graphs showing a significant decrease in RyR2 phosphorylation at S2814 but not at S2808 in HEK293 cells co-transfected with spinophilin, RyR2, and CaMKII. Numbers in the bars indicate the number of independent co-transfection experiments. *P < 0.05 vs. WT.

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