Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ 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 Ca 2+ 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-Ca 2+ 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-Ca 2+ 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.

Background: Enhanced diastolic calcium (Ca 2+ ) release via ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum (SR) Ca 2+ leak is caused by increased RyR2 phosphorylation by protein kinase A (PKA) or Ca 2+ /calmodulin-dependent kinase-II (CaMKII) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus sought to determine the role of 'striated muscle preferentially expressed protein kinase' (SPEG), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from paroxysmal (p)AF patients. SPEG atrial knock-out (aKO) mice were generated using adeno-associated virus 9 (AAV9). In mice, AF inducibility was determined using intracardiac programmed electrical stimulation (PES), and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phospho-proteomics studies and western blotting were used to measure RyR2 phosphorylation. In order to test the effects of RyR2-S2367 phosphorylation, knock-in mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from pAF patients in comparison to patients in sinus rhythm. SPEG aKO mice exhibited increased susceptibility to pacing-induced AF by PES and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phospho-proteomics in hearts from SPEG cardiomyocyte knock-out mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Additionally, western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in pAF patients. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF as well as aberrant atrial SR Ca 2+ leak. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated SR Ca 2+ -release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with pAF. Studies in S2367 knock-in mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

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This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.

Rationale: Autosomal-dominant mutations in ryanodine receptor type-2 (RYR2) are responsible for ~60% of all catecholaminergic polymorphic ventricular tachycardia (CPVT). Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue CPVT. Objective: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct CPVT arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+). Methods and Results: Guide RNAs (gRNA) were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the Staphylococcus aureus Cas9 (SaCas9) genome editing system. AAV vectors based on serotype 9 (AAV9) were used to deliver Cas9 and gRNA to neonatal mice by a single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wildtype littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wildtype Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients. Conclusions: AAV9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders such as CPVT.