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.

Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1β, a main effector of NLR family pyrin domain–containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1β levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1β signaling in the pathogenesis of CKD-induced AF, Nlrp3 –/– and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1β levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3 –/– mice or neutralizing anti–IL-1β antibodies effectively reduced IL-1β levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1β antibodies may be beneficial in preventing CKD-induced AF.

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This article reviews progress in the field of cardiac genome editing, in particular, its potential utility in treating cardiac arrhythmias. First, we discuss genome editing methods by which DNA can be disrupted, inserted, deleted, or corrected in cardiomyocytes. Second, we provide an overview of in vivo genome editing in preclinical models of heritable and acquired arrhythmias. Third, we discuss recent advancements in cardiac gene transfer, including delivery methods, gene expression optimization, and potential adverse effects associated with therapeutic somatic genome editing. While genome editing for cardiac arrhythmias is still in its infancy, this approach holds great promise, especially for inherited arrhythmia syndromes with a defined genetic defect.