Background Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+-leak can promote atrial fibrillation (AF). The mechanisms leading to DADs in AF-patients have not been defined. Methods and Results Protein levels (Western-blot), membrane-currents and action-potentials (patch-clamp), and [Ca2+]i (Fluo-3) were measured in right-atrial samples from 77 sinus-rhythm (Ctl) and 69 chronic-AF (cAF) patients. Diastolic [Ca2+]i and SR-Ca2+-content (integrated INCX during caffeine-induced-Ca2+-transient [cCaT]) were unchanged, whereas diastolic SR Ca2+-leak, estimated by blocking RyR2 with tetracaine, was ~50% higher in cAF vs. Ctl. Single-channel recordings from atrial RyR2 reconstituted into lipid-bilayers revealed enhanced open-probability in cAF-samples, providing a molecular basis for increased SR Ca2+-leak. Calmodulin-expression (+60%), CaMKII-autophosphorylation at Thr287 (+40%) and RyR2-phosphorylation at Ser2808 (PKA/CaMKII-site, +236%) and Ser2814 (CaMKII-site, +77%) were increased in cAF. The selective CaMKII-blocker KN-93 decreased SR Ca2+-leak, the frequency of spontaneous Ca2+-release events and RyR2 open-probability in cAF, whereas PKA-inhibition with H-89 was ineffective. Knock-in mice with constitutively-phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+-sparks and increased susceptibility to pacing-induced AF vs. controls. The relationship between [Ca2+]i and INCX-density revealed INCX-upregulation in cAF. Spontaneous Ca2+-release events accompanied by inward INCX-currents and DADs/triggered-activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF vs. Ctl. Conclusions Enhanced SR Ca2+-leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+-release and increased diastolic [Ca2+]i–voltage coupling gain, cause AF-promoting atrial DADs/triggered-activity in cAF patients.

Background Electrical, structural and Ca2+-handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic-reticulum Ca2+-release events (SCaEs) in longstanding persistent AF, but the occurrence and mechanisms of SCaEs in paroxysmal AF (pAF) are unknown. Method and Results Right-atrial appendages from control sinus-rhythm patients (Ctl) or patients with pAF (last episode median 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (Fluo-3) and membrane-currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, as well as Western blot. Action potential duration, L-type Ca2+-current and Na+/Ca2+-exchange current were unaltered in pAF, indicating absence of AF-induced electrical remodeling. In contrast, there was an increased incidence of delayed afterdepolarizations (DADs) in pAF. Ca2+-transient (CaT)-amplitude and sarcoplasmic-reticulum Ca2+-load (caffeine-induced CaT-amplitude, integrated membrane current) were larger in pAF. CaT-decay was faster in pAF but decay of caffeine-induced CaT was unaltered, suggesting increased Serca2a function. In agreement, phosphorylation (inactivation) of the Serca2a-inhibitor protein phospholamban was increased in pAF. Ryanodine-receptor (RyR2) fractional phosphorylation was unaltered in pAF, whereas RyR2-expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both RyR2 dysregulation and enhanced Serca2a activity promote increased sarcoplasmic-reticulum Ca2+-leak and SCaEs, causing DADs/triggered activity in pAF. Conclusions Increased diastolic sarcoplasmic-reticulum Ca2+-leak and related DADs/triggered activity promote cellular arrhythmogenesis in pAF-patients. Biochemical, functional and modeling studies point to a combination of increased sarcoplasmic-reticulum Ca2+-load related to phospholamban-hyperphosphorylation and RyR2 dysregulation as underlying mechanisms.

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Atrial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca 2+ handling. Diastolic Ca 2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2 R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice.

The proinflammatory cytokine interleukin (IL)-1 signals exclusively through the type I IL-1 receptor (IL-1RI). IL-1 expression is markedly induced in the infarcted heart; however, its role in cardiac injury and repair remains controversial. We examined the effects of disrupted IL-1 signaling on infarct healing and cardiac remodeling using IL-1RI؊/؊ mice. After reperfused infarction IL-1RI-null mice exhibited decreased infiltration of the infarcted myocardium with neutrophils and macrophages and reduced chemokine and cytokine expression. In the absence of IL-1 signaling, suppressed inflammation was followed by an attenuated fibrotic response. Infarcted IL-1RI ؊/؊ mice had decreased myofibroblast infiltration and reduced collagen deposition in the infarcted and remodeling myocardium. IL-1RI deficiency protected against the development of adverse remodeling; however, infarct size was comparable between groups suggesting that the beneficial effects of IL-1RI gene disruption were not attributable to decreased cardiomyocyte injury. Reduced chamber dilation in IL-1RI-null animals was associated with decreased collagen deposition and attenuated matrix metalloproteinase (MMP)-2 and MMP-3 expression in the peri-infarct area, suggesting decreased fibrotic remodeling of the noninfarcted heart. IL-1␤ stimulated MMP mRNA synthesis in wild-type, but not in IL-1RI-null cardiac fibroblasts. In conclusion, IL-1 signaling is essential for activation of inflammatory and fibrogenic pathways in the healing infarct, playing an important role in the pathogenesis of remodeling after infarction. Thus, interventional therapeutics targeting the IL-1 system may have great benefits in myocardial infarction. Infarct healing is dependent on induction of an inflammatory cascade that ultimately results in formation of a collagen-based scar.1 The healing response is closely intertwined with ventricular remodeling, a complex process that involves both the infarcted and noninfarcted myocardium resulting in dilation, hypertrophy, and enhanced sphericity of the ventricle. 2 The extent of remodeling after infarction is an important predictor of mortality and adverse outcome after infarction, 3,4 and depends on the size of the infarct and on the mechanical and structural characteristics of the healing wound. Inflammatory mediators may be critically involved in the pathogenesis of cardiac remodeling by modulating cell behavior in the infarcted heart and by regulating extracellular matrix metabolism. 5-7Interleukin (IL)-1 plays a central role in regulating inflammatory and fibrotic responses by inducing synthesis of proinflammatory mediators, by promoting leukocyte infiltration and activation, and by modulating fibroblast function. IL-1 binds to two distinct receptors on the cell membrane: the type I IL-1 receptor (IL-1RI) is sufficient to mediate all IL-1 actions, 8 whereas the type II receptor (IL-1RII) serves as a decoy target, 9 trapping and scavenging IL-1 molecules, 10 thus reducing IL-1 concentration available for interaction with the IL-1RI signa...

-Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The "NACHT, LRR and PYD domain containing protein 3" (NLRP3)-inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells, but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3-inflammasome in AF. -NLRP3-inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal (pAF) or long-standing persistent (chronic) AF (cAF). To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knock-in mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electrical activation pattern, Ca spark frequency (CaSF), atrial effective refractory period (AERP), and morphology of atria were evaluated in CM-KI mice and WT littermates. -NLRP3-inflammasome activity was increased in atrial CMs of pAF and cAF patients. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3-inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic-reticulum Ca-release, AERP shortening and atrial hypertrophy. Adeno-associated virus subtype-9 mediated CM-specific knockdown of suppressed AF development in CM-KI mice. Finally, genetic inhibition of prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. -Our study establishes a novel pathophysiological role for CM NLRP3-inflammasome signaling with a mechanistic link to the pathogenesis of AF, and establishes inhibition of NLRP3 as a potential novel AF-therapy approach.

Elevated resting heart rate is associated with greater risk of cardiovascular disease and mortality. In a 2-stage meta-analysis of genome-wide association studies in up to 181,171 individuals, we identified 14 new loci associated with heart rate and confirmed associations with all 7 previously established loci. Experimental downregulation of gene expression in Drosophila melanogaster and Danio rerio identified 20 genes at 11 loci that are relevant for heart rate regulation and highlight a role for genes involved in signal transmission, embryonic cardiac development and the pathophysiology of dilated cardiomyopathy, congenital heart failure and/or sudden cardiac death. In addition, genetic susceptibility to increased heart rate is associated with altered cardiac conduction and reduced risk of sick sinus syndrome, and both heart rate–increasing and heart rate–decreasing variants associate with risk of atrial fibrillation. Our findings provide fresh insights into the mechanisms regulating heart rate and identify new therapeutic targets.

Background Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.