Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation

Overview

Journal Circulation

Specialty Cardiology & Vascular Diseases

Date 2020 Jul 21

PMID 32683896

Citations 41

Authors

Hannah M Campbell

Ann P Quick

Issam Abu-Taha

David Y Chiang

Carlos F Kramm

Tarah A Word

Soren Brandenburg

Mohit Hulsurkar

Katherina M Alsina

Hui-Bin Liu

Brian Martin

Dennis Uhlenkamp

Oliver M Moore

Satadru K Lahiri

Eleonora Corradini

Markus Kamler

Albert J R Heck

Stephan E Lehnart

Dobromir Dobrev

Xander H T Wehrens

Affiliations

Soon will be listed here.

Abstract

Background: Enhanced diastolic calcium (Ca) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca/calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis.

Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca leak in atrial cardiomyocytes was assessed using confocal Ca imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9.

Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca spark frequency in atrial cardiomyocytes with Ca imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca leak, as well. 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 sarcoplasmic reticulum Ca release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin 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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