Two siblings presented with cardiomyopathy, hypertension, arrhythmia, and fibrosis of the left atrium. Each had a homozygous null variant in , the gene encoding atrial natriuretic peptide (ANP)-converting enzyme. A plasma sample obtained from one of the siblings had no detectable levels of corin or N-terminal pro-ANP but had elevated levels of B-type natriuretic peptide (BNP) and one of the two protein markers of fibrosis that we tested. These and other findings support the hypothesis that BNP cannot fully compensate for a lack of activation of the ANP pathway and that corin is critical to normal ANP activity, left atrial function, and cardiovascular homeostasis.

Background:The incidence of atrial fibrillation (AF)-related stroke increases with aging. Natriuretic peptides (NPs) family, including Corin-B type natriuretic peptide (BNP)-neprilysin (NEP) protein levels increased with age and are risk markers of cardiovascular and cerebrovascular diseases, such as AF and cardioembolic stroke. Aging is also linked to epigenetics, specifically DNA methylation. However, only a few studies have investigated the effect of DNA methylation on the NP system. Thus, the present study aimed to investigate whether the Corin-BNP-NEP protein pathway is involved in the pathogenesis of AF-stroke and CpG methylation in the promoter region of the Corin protein gene has an effect on AF-related ischemic stroke. Methods:A total of 82 patients hospitalized with acute ischemic strokes were enrolled in this study. The differences in clinical information were compared between the AF-stroke ( = 37) and no AF-stroke groups ( = 45). Plasma-soluble Corin and NEP were detected using an ELISA kit. CpG methylation in the promoter region of the gene was assessed by a next-generation sequencing-based bisulfite sequencing polymerase chain reaction (BSP). Results:(1) Patients in AF-stroke were older, had higher initial NIHSS score, 90-day mRs, higher D2-dimer, INR, and APTT, and low TG, TC, and HbA1c (all < 0.05). (2) Serum levels of Corin and BNP in the AF-stroke group were significantly higher than that in the no AF-stroke group ( < 0.05). No significant difference was detected in the serum levels of NEP between the two groups. (3) The levels of CpG methylation in the promoter region of the Corin protein gene in the AF-stroke group was significantly lower than that in the no AF-stroke group ( < 0.05). The CpG sites with maximal methylation differences between the two groups were CORIN:678, CORIN:682, CORIN:694, and CORIN:700. Conclusion:The current findings raise the possibility that the Corin-BNP-NEP protein pathway may be involved in the pathogenesis of AF-related ischemic stroke. Deficient CpG methylation in the promoter region of the Corin protein gene is associated with AF-related ischemic stroke.

BACKGROUND:NPPA mutations/polymorphisms were associated with atrial fibrillation (AF), and plasma proatrial natriuretic peptide (proANP) concentrations were increased in AF patients. Corin, as a transmembrane protease that processes proANP in the heart, may play a potential role in AF. METHODS:To test whether corin concentrations are altered in AF patients, we used ELISA to measure corin and N-terminal proANP (NT-proANP) concentrations in plasma samples from control (n=127) and AF patients (n=141), including paroxysmal AF (PAF, n=83) and persistent AF (PeAF, n=58). RESULTS:In patients with AF, plasma corin concentrations were 1209±510pg/ml, which were significantly higher than in the controls (973±528pg/ml, P<0.001). The increased plasma corin concentrations were found in both male and female patients. Plasma NT-proANP concentrations in AF patients were 3.1±2.42nmol/l, which were higher than in the controls (1.77±1.04nmol/l, P<0.001). Gender (P=0.003), weight (P=0.016) and PR interval (P=0.028) were independent predictors of plasma corin concentrations in AF patients. A positive correlation was found between corin concentrations and left atrial diameter/PR interval in AF patients. CONCLUSION:High plasma corin concentrations in AF patients suggest that corin may play an important role in the pathology of AF.

Importance:Thyroid hormone levels are tightly regulated through feedback inhibition by thyrotropin, produced by the pituitary gland. Hyperthyroidism is overwhelmingly due to thyroid disorders and is well recognized to contribute to a wide spectrum of cardiovascular morbidity, particularly the increasingly common arrhythmia atrial fibrillation (AF). Objective:To determine the association between genetically determined thyrotropin levels and AF. Design, Setting, and Participants:This phenome-wide association study scanned 1318 phenotypes associated with a polygenic predictor of thyrotropin levels identified by a previously published genome-wide association study that included participants of European ancestry. North American individuals of European ancestry with longitudinal electronic health records were analyzed from May 2008 to November 2016. Analysis began March 2018. Main Outcomes and Measures:Clinical diagnoses associated with a polygenic predictor of thyrotropin levels. Exposures:Genetically determined thyrotropin levels. Results:Of 37 154 individuals, 19 330 (52%) were men. The thyrotropin polygenic predictor was positively associated with hypothyroidism (odds ratio [OR], 1.10; 95% CI, 1.07-1.14; P = 5 × 10-11) and inversely associated with diagnoses related to hyperthyroidism (OR, 0.64; 95% CI, 0.54-0.74; P = 2 × 10-8 for toxic multinodular goiter). Among nonthyroid associations, the top association was AF/flutter (OR, 0.93; 95% CI, 0.9-0.95; P = 9 × 10-7). When the analyses were repeated excluding 9801 individuals with any diagnoses of a thyroid-related disease, the AF association persisted (OR, 0.91; 95% CI, 0.88-0.95; P = 2.9 × 10-6). To replicate this association, we conducted an inverse-variance weighted average meta-analysis using AF single-nucleotide variant weights from a genome-wide association study of 17 931 AF cases and 115 142 controls. As in the discovery analyses, each SD increase in predicted thyrotropin was associated with a decreased risk of AF (OR, 0.86; 95% CI, 0.79-0.93; P = 4.7 × 10-4). In a set of AF cases (n = 745) and controls (n = 1680) older than 55 years, directly measured thyrotropin levels that fell within the normal range were inversely associated with AF risk (OR, 0.91; 95% CI, 0.83-0.99; P = .04). Conclusions and Relevance:This study suggests a role for genetically determined variation in thyroid function within a physiologically accepted normal range as a risk factor for AF. The clinical decision to treat subclinical thyroid disease should incorporate the risk for AF as antithyroid medications to treat hyperthyroidism may reduce AF risk and thyroid hormone replacement for hypothyroidism may increase AF risk.

Atrial fibrillation affects more than 33 million people worldwide and increases the risk of stroke, heart failure, and death. Fourteen genetic loci have been associated with atrial fibrillation in European and Asian ancestry groups. To further define the genetic basis of atrial fibrillation, we performed large-scale, trans-ancestry meta-analyses of common and rare variant association studies. The genome-wide association studies (GWAS) included 17,931 individuals with atrial fibrillation and 115,142 referents; the exome-wide association studies (ExWAS) and rare variant association studies (RVAS) involved 22,346 cases and 132,086 referents. We identified 12 new genetic loci that exceeded genome-wide significance, implicating genes involved in cardiac electrical and structural remodeling. Our results nearly double the number of known genetic loci for atrial fibrillation, provide insights into the molecular basis of atrial fibrillation, and may facilitate the identification of new potential targets for drug discovery.

BACKGROUND:The relationship between omega-3 fatty acids and atrial fibrillation (AF) remains controversial. OBJECTIVES:This study aimed to determine the prospective associations of blood or adipose tissue levels of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) with incident AF. METHODS:We used participant-level data from a global consortium of 17 prospective cohort studies, each with baseline data on blood or adipose tissue omega-3 fatty acid levels and AF outcomes. Each participating study conducted a de novo analyses using a prespecified analytical plan with harmonized definitions for exposures, outcome, covariates, and subgroups. Associations were pooled using inverse-variance weighted meta-analysis. RESULTS:Among 54,799 participants from 17 cohorts, 7,720 incident cases of AF were ascertained after a median 13.3 years of follow-up. In multivariable analysis, EPA levels were not associated with incident AF, HR per interquintile range (ie, the difference between the 90th and 10th percentiles) was 1.00 (95% CI: 0.95-1.05). HRs for higher levels of DPA, DHA, and EPA+DHA, were 0.89 (95% CI: 0.83-0.95), 0.90 (95% CI: 0.85-0.96), and 0.93 (95% CI: 0.87-0.99), respectively. CONCLUSIONS:In vivo levels of omega-3 fatty acids including EPA, DPA, DHA, and EPA+DHA were not associated with increased risk of incident AF. Our data suggest the safety of habitual dietary intakes of omega-3 fatty acids with respect to AF risk. Coupled with the known benefits of these fatty acids in the prevention of adverse coronary events, our study suggests that current dietary guidelines recommending fish/omega-3 fatty acid consumption can be maintained.

BACKGROUND:Some, but not all, large-scale randomized controlled trials (RCTs) investigating the effects of marine ɷ-3 fatty acids supplementation on cardiovascular outcomes have reported increased risks of atrial fibrillation (AF). The potential reasons for disparate findings may be dose-related. METHODS:The MEDLINE and Embase databases were searched for articles and abstracts published between January 1, 2012, and December 31, 2020, in addition to a meta-analysis of large cardiovascular RCTs published in 2019. RCTs of cardiovascular outcomes of marine ɷ-3 fatty acids that reported results for AF, either as a prespecified outcome, an adverse event, or a cause for hospitalization, with a minimum sample size of 500 patients and a median follow-up of at least 1 year were included. RCTs specifically examining shorter-term effects of ɷ-3 fatty acids on recurrent AF in patients with established AF or postoperative AF were not included. The hazard ratio (HR) for the reported AF outcomes within each trial was meta-analyzed using random effects model with Knapp-Hartung adjustment and evaluated a dose-response relationship with a meta-regression model. RESULTS:Of 4049 screened records, 7 studies were included in the meta-analysis. Of those, 5 were already detected in a previous meta-analysis of cardiovascular RCTs. Among the 81 210 patients from 7 trials, 58 939 (72.6%) were enrolled in trials testing ≤1 g/d and 22 271 (27.4%) in trials testing >1 g/d of ɷ-3 fatty acids. The mean age was 65 years, and 31 842 (39%) were female. The weighted average follow-up was 4.9 years. In meta-analysis, the use of marine ɷ-3 fatty acid supplements was associated with an increased risk of AF (n=2905; HR, 1.25 [95% CI, 1.07-1.46]; =0.013). In analyses stratified by dose, the HR was greater in the trials testing >1 g/d (HR, 1.49 [95% CI, 1.04-2.15]; =0.042) compared with those testing ≤1 g/d (HR, 1.12 [95% CI, 1.03-1.22]; =0.024; for interaction <0.001). In meta-regression, the HR for AF increased per 1 g higher dosage of ɷ-3 fatty acids dosage (HR, 1.11 [95% CI, 1.06-1.15]; =0.001). CONCLUSIONS:In RCTs examining cardiovascular outcomes, marine ɷ-3 supplementation was associated with an increased risk of AF. The risk appeared to be greater in trials testing >1 g/d.

Atrial fibrillation (AF) is a common cardiac arrhythmia resulting in increased risk of stroke. Despite highly heritable etiology, our understanding of the genetic architecture of AF remains incomplete. Here we performed a genome-wide association study in the Japanese population comprising 9,826 cases among 150,272 individuals and identified East Asian-specific rare variants associated with AF. A cross-ancestry meta-analysis of >1 million individuals, including 77,690 cases, identified 35 new susceptibility loci. Transcriptome-wide association analysis identified IL6R as a putative causal gene, suggesting the involvement of immune responses. Integrative analysis with ChIP-seq data and functional assessment using human induced pluripotent stem cell-derived cardiomyocytes demonstrated ERRg as having a key role in the transcriptional regulation of AF-associated genes. A polygenic risk score derived from the cross-ancestry meta-analysis predicted increased risks of cardiovascular and stroke mortalities and segregated individuals with cardioembolic stroke in undiagnosed AF patients. Our results provide new biological and clinical insights into AF genetics and suggest their potential for clinical applications.

BACKGROUND:Cytokines and growth factors have been implicated in the initiation and propagation of vascular disease. Observational studies have shown associations of their circulating levels with stroke. Our objective was to explore whether genetically determined circulating levels of cytokines and growth factors are associated with stroke and its etiologic subtypes by conducting a 2-sample Mendelian randomization (MR) study. METHODS:Genetic instruments for 41 cytokines and growth factors were obtained from a genome-wide association study of 8293 healthy adults. Their associations with stroke and stroke subtypes were evaluated in the MEGASTROKE genome-wide association study data set (67 162 cases; 454 450 controls) applying inverse variance-weighted meta-analysis, weighted-median analysis, Mendelian randomization-Egger regression, and multivariable Mendelian randomization. The UK Biobank cohort was used as an independent validation sample (4985 cases; 364 434 controls). Genetic instruments for monocyte chemoattractant protein-1 (MCP-1/CCL2) were further tested for association with etiologically related vascular traits by using publicly available genome-wide association study data. RESULTS:Genetic predisposition to higher MCP-1 levels was associated with higher risk of any stroke (odds ratio [OR] per 1 SD increase, 1.06; 95% CI, 1.02-1.09; P=0.0009), any ischemic stroke (OR, 1.06; 95% CI, 1.02-1.10; P=0.002), large-artery stroke (OR, 1.19; 95% CI, 1.09-1.30; P=0.0002), and cardioembolic stroke (OR, 1.14; 95% CI, 1.06-1.23; P=0.0004), but not with small-vessel stroke or intracerebral hemorrhage. The results were stable in sensitivity analyses and remained significant after adjustment for cardiovascular risk factors. Analyses in the UK Biobank showed similar associations for available phenotypes (any stroke: OR, 1.08; 95% CI, 0.99-1.17; P=0.09; any ischemic stroke: OR, 1.07; 95% CI, 0.97-1.18; P=0.17). Genetically determined higher MCP-1 levels were further associated with coronary artery disease (OR, 1.04; 95% CI, 1.00-1.08; P=0.04) and myocardial infarction (OR, 1.05; 95% CI, 1.01-1.09; P=0.02), but not with atrial fibrillation. A meta-analysis of observational studies showed higher circulating MCP-1 levels in patients with stroke in comparison with controls. CONCLUSIONS:Genetic predisposition to elevated circulating levels of MCP-1 is associated with higher risk of stroke, in particular with large-artery stroke and cardioembolic stroke. Whether targeting MCP-1 or its receptors can lower stroke incidence requires further study.

Atrial fibrillation (AF) affects more than 33 million individuals worldwide and has a complex heritability. We conducted the largest meta-analysis of genome-wide association studies (GWAS) for AF to date, consisting of more than half a million individuals, including 65,446 with AF. In total, we identified 97 loci significantly associated with AF, including 67 that were novel in a combined-ancestry analysis, and 3 that were novel in a European-specific analysis. We sought to identify AF-associated genes at the GWAS loci by performing RNA-sequencing and expression quantitative trait locus analyses in 101 left atrial samples, the most relevant tissue for AF. We also performed transcriptome-wide analyses that identified 57 AF-associated genes, 42 of which overlap with GWAS loci. The identified loci implicate genes enriched within cardiac developmental, electrophysiological, contractile and structural pathways. These results extend our understanding of the biological pathways underlying AF and may facilitate the development of therapeutics for AF.

Cardiovascular diseases exist across all developed countries. Biomarkers that can predict or diagnose diseases early in their pathogeneses can reduce their morbidity and mortality in afflicted individuals. microRNAs are small regulatory RNAs that modulate translation and have been identified as potential fluid-based biomarkers across numerous maladies. We describe the current state of cardiovascular disease biomarkers across a range of diseases, including myocardial infarction, acute coronary syndrome, myocarditis, hypertension, heart failure, heart transplantation, aortic stenosis, diabetic cardiomyopathy, atrial fibrillation, and sepsis. We present the current understanding of microRNAs as possible biomarkers in these categories and where their best opportunities exist to enter clinical practice.

Atrial fibrillation (AF) is an all-too-common and often challenging reality of clinical care. AF leads to significant morbidity and mortality; however, currently available treatments for AF have modest efficacy and high recurrence rates. In recent years, genetic therapy approaches have been explored in preclinical models of AF, and offer potential as a treatment modality with targeted delivery, tissue specificity, and therapy tailored to address mechanisms underlying the arrhythmia. However, many challenges remain before gene therapy can advance to a clinically relevant AF treatment. In this review, we summarize the available published data on gene therapy and discuss the challenges, opportunities, and limitations of this approach.

Autonomic neuromodulation therapies (ANMTs) (ie, ganglionated plexus ablation, epicardial injections for temporary neurotoxicity, low-level vagus nerve stimulation [LL-VNS], stellate ganglion block, baroreceptor stimulation, spinal cord stimulation, and renal nerve denervation) constitute an emerging therapeutic approach for arrhythmias. Very little is known about ANMTs' preventive potential for postoperative atrial fibrillation (POAF) after cardiac surgery. The purpose of this review is to summarize and critically appraise the currently available evidence. Herein, the authors conducted a systematic review of 922 articles that yielded 7 randomized controlled trials. In the meta-analysis, ANMTs reduced POAF incidence (OR: 0.37; 95% CI: 0.25 to 0.55) and burden (mean difference [MD]: -3.51 hours; 95% CI: -6.64 to -0.38 hours), length of stay (MD: -0.82 days; 95% CI: -1.59 to -0.04 days), and interleukin-6 (MD: -79.92 pg/mL; 95% CI: -151.12 to -8.33 pg/mL), mainly attributed to LL-VNS and epicardial injections. Moving forward, these findings establish a base for future larger and comparative trials with ANMTs, to optimize and expand their use.

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

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of , a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

Because of suboptimal therapeutic strategies, restoration of sinus rhythm in symptomatic atrial fibrillation (AF) often requires in-hospital delivery of high-voltage shocks, thereby precluding ambulatory AF termination. Continuous, rapid restoration of sinus rhythm is desired given the recurring and progressive nature of AF. Here, we present an automated hybrid bioelectronic system for shock-free termination of AF that enables the heart to act as an electric current generator for autogenous restoration of sinus rhythm. We show that local, right atrial delivery of adenoassociated virus vectors encoding a light-gated depolarizing ion channel results in efficient and spatially confined transgene expression. Activation of an implanted intrathoracic light-emitting diode device allows for termination of AF by illuminating part of the atria. Combining this newly obtained antiarrhythmic effector function of the heart with the arrhythmia detector function of a machine-based cardiac rhythm monitor in the closed chest of adult rats allowed automated and rapid arrhythmia detection and termination in a safe, effective, repetitive, yet shock-free manner. These findings hold translational potential for the development of shock-free antiarrhythmic device therapy for ambulatory treatment of AF.

Atrial fibrillation (AF), the most common sustained heart rhythm disorder worldwide, is linked to dysfunction of the intrinsic cardiac autonomic nervous system (ICNS). The role of ICNS damage occurring during catheter-based treatment of AF, which is the therapy of choice for many patients, remains controversial. We show here that the neuronal injury marker S100B is expressed in cardiac glia throughout the ICNS and is released specifically upon catheter ablation of AF. Patients with higher S100B release were more likely to be AF free during follow-up. Subsequent in vitro studies revealed that murine intracardiac neurons react to S100B with diminished action potential firing and increased neurite growth. This suggests that release of S100B from cardiac glia upon catheter-based treatment of AF is a hallmark of acute neural damage that contributes to nerve sprouting and can be used to assess ICNS damage.

BACKGROUND:Excessive binge alcohol drinking has acute cardiac arrhythmogenic effects, including promotion of atrial fibrillation (AF), which underlies "Holiday Heart Syndrome." The mechanism that couples binge alcohol abuse with AF susceptibility remains unclear. We previously reported stress-activated c-Jun N-terminal kinase (JNK) signaling contributes to AF development. This is interesting because JNK is implicated in alcohol-caused organ malfunction beyond the heart. OBJECTIVES:The purpose of this study was to detail how JNK promotes binge alcohol-evoked susceptibility to AF. METHODS:The authors found binge alcohol-exposure leads to activated JNK, specifically JNK2. Furthermore, binge alcohol induces AF (24- vs. 1.8-Hz burst pacing-induced episodes per attempt per animal), higher incidence of diastolic intracellular Ca activity (Ca waves, sarcoplasmic reticulum [SR] Ca leakage), and membrane voltage (V) and systolic Ca release spatiotemporal heterogeneity (Δt). These changes were completely eliminated by JNK inhibition both in vivo and in vitro. calmodulin kinase II (CaMKII) is a proarrhythmic molecule known to drive SR Ca mishandling. RESULTS:The authors report for the first time that binge alcohol activates JNK2, which subsequently phosphorylates the CaMKII protein, enhancing CaMKII-driven SR Ca mishandling. CaMKII inhibition eliminates binge alcohol-evoked arrhythmic activities. CONCLUSIONS:Our studies demonstrate that binge alcohol exposure activates JNK2 in atria, which then drives CaMKII activation, prompting aberrant Ca waves and, thus, enhanced susceptibility to atrial arrhythmia. Our results reveal a previously unrecognized form of alcohol-driven kinase-on-kinase proarrhythmic crosstalk. Atrial JNK2 function represents a potential novel therapeutic target to treat and/or prevent AF.

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.

BACKGROUND:Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K current (), called ; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. METHODS:To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. RESULTS:First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating by a mechanism involving frequency-dependent activation of PKC (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of . Attenuated membrane translocation of PKC appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. CONCLUSIONS:NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific -mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in -deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca-dependent pathway for AF risk.

BACKGROUND:Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS:We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS:We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( I channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of I channel function by increasing the basal current, even in the absence of m muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective I channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS:The I channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant I channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective I channel blocker. Thus, the I channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the I channel.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

BACKGROUND:Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS:Using an knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS:There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS:These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.

BACKGROUND:Conditions affecting the right heart, including diseases of the lungs and pulmonary circulation, promote atrial fibrillation (AF), but the mechanisms are poorly understood. OBJECTIVES:This study sought to determine whether right heart disease promotes atrial arrhythmogenesis in a rat model of pulmonary hypertension (PH) and, if so, to define the underlying mechanisms. METHODS:PH was induced in male Wistar rats with a single intraperitoneal injection of 60 mg/kg of monocrotaline, and rats were studied 21 days later when right heart disease was well developed. AF vulnerability was assessed in vivo and in situ, and mechanisms were defined by optical mapping, histochemistry, and biochemistry. RESULTS:Monocrotaline-treated rats developed increased right ventricular pressure and mass, along with right atrial (RA) enlargement. AF/flutter was inducible in 32 of 32 PH rats (100%) in vivo and 11 of 12 (92%) in situ, versus 2 of 32 (6%) and 2 of 12 (17%), respectively, in control rats (p < 0.001 vs. PH for each). PH rats had significant RA (16.1 ± 0.5% of cross-sectional area, vs. 3.0 ± 0.6% in control) and left atrial (LA: 11.8 ± 0.5% vs. 5.4 ± 0.8% control) fibrosis. Multiple extracellular matrix proteins, including collagen 1 and 3, fibronectin, and matrix metalloproteinases 2 and 9, were up-regulated in PH rat RA. Optical mapping revealed significant rate-dependent RA conduction slowing and rotor activity, including stable rotors in 4 of 11 PH rats, whereas no significant conduction slowing or rotor activity occurred in the LA of monocrotaline-treated rats. Transcriptomic analysis revealed differentially enriched genes related to hypertrophy, inflammation, and fibrosis in RA of monocrotaline-treated rats versus control. Biochemical results in PH rats were compared with those of AF-prone rats with atrial remodeling in the context of left ventricular dysfunction due to myocardial infarction: myocardial infarction rat LA shared molecular motifs with PH rat RA. CONCLUSIONS:Right heart disease produces a substrate for AF maintenance due to RA re-entrant activity, with an underlying substrate prominently involving RA fibrosis and conduction abnormalities.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the locus, one intronic (RE(int)) and one downstream (RE(down)) of . In both lines, we observed a modest (30%) increase of in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor was induced in cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased expression were normalized when reducing cardiac expression in mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.

BACKGROUND:The aldosterone inhibitor eplerenone (EPL) has been shown to reduce the incidence of atrial fibrillation (AF) in patients with systolic heart failure, but the mechanism is unknown. OBJECTIVES:This study hypothesized that by reducing atrial dilation and fibrosis in the absence of heart failure, EPL also reduces AF burden and prevents AF perpetuation. METHODS:The authors conducted a randomized controlled study in 34 sheep that were atrially tachypaced (13 ± 1 week). They compared daily oral EPL (n = 19) versus sugar pill (SP) treatment (n = 15) from the start of tachypacing. The endpoint was a continuous 7-day stretch of persistent AF (n = 29) or completion of 23 weeks tachypacing (n = 5). RESULTS:EPL significantly reduced the rate of left atrial dilation increase during AF progression. Atria from EPL-treated sheep had less smooth muscle actin protein, collagen-III expression, interstitial atrial fibrosis, and cell hypertrophy than SP-treated sheep atria did. However, EPL did not modify the AF-induced increase in the rate of dominant frequency and ion channel densities seen under SP treatment, but rather prolonged the time to persistent AF in 26% of animals. It also reduced the degree of fibrillatory conduction, AF inducibility, and AF burden. CONCLUSIONS:In the sheep model, EPL mitigates fibrosis and atrial dilation, modifies AF inducibility and AF complexity, and prolongs the transition to persistent AF in 26% of animals, but it does not prevent AF-induced electrical remodeling or AF persistence. The results highlight structural remodeling as a central upstream target to reduce AF burden, and the need to prevent electrical remodeling to avert AF perpetuation.

Changes of intracellular Ca concentration regulate many aspects of cardiac myocyte function. About 99% of the cytoplasmic calcium in cardiac myocytes is bound to buffers, and their properties will therefore have a major influence on Ca signaling. This article considers the fundamental properties and identities of the buffers and how to measure them. It reviews the effects of buffering on the systolic Ca transient and how this may change physiologically, and in heart failure and both atrial and ventricular arrhythmias, as well. It is concluded that the consequences of this strong buffering may be more significant than currently appreciated, and a fuller understanding is needed for proper understanding of cardiac calcium cycling and contractility.

Atrial fibrillation is a highly prevalent cardiac arrhythmia and the most important cause of embolic stroke. Although genetic studies have identified an increasing assembly of AF-related genes, the impact of these genetic discoveries is yet to be realized. In addition, despite more than a century of research and speculation, the molecular and cellular mechanisms underlying AF have not been established, and therapy for AF, particularly persistent AF, remains suboptimal. Current antiarrhythmic drugs are associated with a significant rate of adverse events, particularly proarrhythmia, which may explain why many highly symptomatic AF patients are not receiving any rhythm control therapy. This review focuses on recent advances in AF research, including its epidemiology, genetics, and pathophysiological mechanisms. We then discuss the status of antiarrhythmic drug therapy for AF today, reviewing molecular mechanisms, and the possible clinical use of some of the new atrial-selective antifibrillatory agents, as well as drugs that target atrial remodeling, inflammation and fibrosis, which are being tested as upstream therapies to prevent AF perpetuation. Altogether, the objective is to highlight the magnitude and endemic dimension of AF, which requires a significant effort to develop new and effective antiarrhythmic drugs, but also improve AF prevention and treatment of risk factors that are associated with AF complications.

BACKGROUND:Elevated intracardiac pressure attributable to heart failure induces electrical and structural remodeling in the left atrium (LA) that begets atrial myopathy and arrhythmias. The underlying molecular pathways that drive atrial remodeling during cardiac pressure overload are poorly defined. The purpose of this study is to characterize the response of the ETV1 (ETS translocation variant 1) signaling axis in the LA during cardiac pressure overload in humans and mouse models and explore the role of ETV1 in atrial electrical and structural remodeling. METHODS:We performed gene expression profiling in 265 left atrial samples from patients who underwent cardiac surgery. Comparative gene expression profiling was performed between 2 murine models of cardiac pressure overload, transverse aortic constriction banding and angiotensin II infusion, and a genetic model of cardiomyocyte-selective knockout (). RESULTS:Using the Cleveland Clinic biobank of human LA specimens, we found that expression is decreased in patients with reduced ejection fraction. Consistent with its role as an important mediator of the NRG1 (Neuregulin 1) signaling pathway and activator of rapid conduction gene programming, we identified a direct correlation between expression level and , , , and levels in human LA samples. In a similar fashion to patients with heart failure, we showed that left atrial ETV1 expression is downregulated at the RNA and protein levels in murine pressure overload models. Comparative analysis of LA RNA sequencing datasets from transverse aortic constriction and angiotensin II-treated mice showed a high Pearson correlation, reflecting a highly ordered process by which the LA undergoes electrical and structural remodeling. Cardiac pressure overload produced a consistent downregulation of , , , and and upregulation of profibrotic gene programming, which includes , and numerous collagen genes. mice displayed atrial conduction disease and arrhythmias. Correspondingly, the LA from mice showed downregulation of rapid conduction genes and upregulation of profibrotic gene programming, whereas analysis of a gain-of-function ETV1 RNA sequencing dataset from neonatal rat ventricular myocytes transduced with showed reciprocal changes. CONCLUSIONS:ETV1 is downregulated in the LA during cardiac pressure overload, contributing to both electrical and structural remodeling.

Atrial fibrillation (AF) contributes to morbidity and mortality of millions of individuals. Its molecular, cellular, neurohumoral, and hemodynamic pathophysiological mechanisms are complex, and there is increasing awareness that a wide range of comorbidities can contribute to AF-promoting atrial remodeling. Moreover, recent research has highlighted that AF risk is not constant and that the temporal variation in concomitant conditions contributes to the complexity of AF dynamics. In this review, we provide an overview of fundamental AF mechanisms related to established and emerging comorbidities or risk factors and their role in the AF-promoting effects. We focus on the accumulating evidence for the relevance of temporally dynamic changes in these risk factors and the consequence for AF initiation and maintenance. Finally, we highlight the important implications for future research and clinical practice resulting from the dynamic interaction between AF risk factors and mechanisms.

As an excitatory transmitter system, the glutamatergic transmitter system controls excitability and conductivity of neurons. Since both cardiomyocytes and neurons are excitable cells, we hypothesized that cardiomyocytes may also be regulated by a similar system. Here, we have demonstrated that atrial cardiomyocytes have an intrinsic glutamatergic transmitter system, which regulates the generation and propagation of action potentials. First, there are abundant vesicles containing glutamate beneath the plasma membrane of rat atrial cardiomyocytes. Second, rat atrial cardiomyocytes express key elements of the glutamatergic transmitter system, such as the glutamate metabolic enzyme, ionotropic glutamate receptors (iGluRs), and glutamate transporters. Third, iGluR agonists evoke iGluR-gated currents and decrease the threshold of electrical excitability in rat atrial cardiomyocytes. Fourth, iGluR antagonists strikingly attenuate the conduction velocity of electrical impulses in rat atrial myocardium both in vitro and in vivo. Knockdown of GRIA3 or GRIN1, two highly expressed iGluR subtypes in atria, drastically decreased the excitatory firing rate and slowed down the electrical conduction velocity in cultured human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocyte monolayers. Finally, iGluR antagonists effectively prevent and terminate atrial fibrillation in a rat isolated heart model. In addition, the key elements of the glutamatergic transmitter system are also present and show electrophysiological functions in human atrial cardiomyocytes. In conclusion, our data reveal an intrinsic glutamatergic transmitter system directly modulating excitability and conductivity of atrial cardiomyocytes through controlling iGluR-gated currents. Manipulation of this system may open potential new avenues for therapeutic intervention of cardiac arrhythmias.

BACKGROUND:The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS:We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome () and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS:We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS:Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

BACKGROUND:Patients with metabolic syndrome (MetS) have an increased risk of atrial fibrillation (AF). Impaired Ca homeostasis and mitochondrial dysfunction have emerged as an arrhythmogenic substrate in both patients and animal models of MetS. Whether impaired mitochondrial Ca handling underlies AF associated with MetS remains poorly explored. OBJECTIVES:The aim of this study was to determine the initial mechanisms related to AF susceptibility and mitochondrial dysfunction encountered in metabolic cardiomyopathy. METHODS:A total of 161 mice and 34 patients were studied. Mitochondrial Ca and mitochondrial Ca uniporter complex (MCUC) were investigated in right atrial tissue of patients with (n = 18) or without (n = 16) MetS and of C57Bl/6J mice fed with a high-fat sucrose diet (HFS) for 2 (n = 42) or 12 (n = 39) weeks. Susceptibility to AF was evaluated in isolated sinoatrial tissue and in vivo in mice. RESULTS:Increased expression of the MICUs subunits of the MCUC (1.00 ± 0.33 AU vs 1.29 ± 0.23 AU; P = 0.034) was associated with impaired mitochondrial Ca uptake in patients (168.7 ± 31.3 nmol/min/mg vs 127.3 ± 18.4 nmol/min/mg; P = 0.026) and HFS mice (0.10 ± 0.04 ΔF/F0 × ms vs 0.06 ± 0.03 ΔF/F0 × ms; P = 0.0086, and 0.15 ± 0.07 ΔF/F0 × ms vs 0.046 ± 0.03 ΔF/F0 × ms; P = 0.0076 in 2- and 12-week HFS mice, respectively). HFS mice elicited a 70% increased susceptibility to AF. The MCUC agonist kaempferol restored MCUC activity in vitro and abolished the occurrence of AF in HFS mice. CONCLUSIONS:Impaired MCUC activity and mitochondrial Ca homeostasis from the early stage of metabolic cardiomyopathy in mice lead to AF. Given that similar defects in cardiac mitochondrial Ca handling are present in MetS patients, the modulation of the MCUC activity represents an attractive antiarrhythmic strategy.

BACKGROUND:Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. METHODS:Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. RESULTS:HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. CONCLUSIONS:These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β-treated human AEECs, cardiac-specific TGF-β-transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β-treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β-transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

BACKGROUND:Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS:We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS:We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; <0.0001). CONCLUSIONS:These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

BACKGROUND:Atrial fibrillation (AF) is a highly prevalent condition that can cause or exacerbate heart failure, is an important risk factor for stroke, and is associated with pronounced morbidity and death. Genes uniquely expressed in the atria are known to be essential for maintaining atrial structure and function. Atrial tissue remodeling contributes to arrhythmia recurrence and maintenance. However, the mechanism underlying atrial remodeling remains poorly understood. This study was designed to investigate whether other uncharacterized atrial specific genes play important roles in atrial physiology and arrhythmogenesis. METHODS:RNA-sequencing analysis was used to identify atrial myocyte specific and angiotensin II-responsive genes. Genetically modified, cardiomyocyte-specific mouse models (knockout and overexpression) were generated. In vivo and in vitro electrophysiological, histology, and biochemical analyses were performed to determine the consequences of CIB2 (calcium and integrin binding family member 2 protein) gain and loss of function in the atrium. RESULTS:Using RNA-sequencing analysis, we identified CIB2 as an atrial-enriched protein that is significantly downregulated in the left atria of patients with AF and mouse models of AF from angiotensin II infusion or pressure overload. Using cardiomyocyte-specific C knockout () and atrial myocyte-specific -overexpressing mouse models, we found that loss of C enhances AF occurrence, prolongs AF duration, and correlates with a significant increase in atrial fibrosis under stress. Conversely, overexpression mitigates AF occurrence and atrial fibrosis triggered by angiotensin II stress. Mechanistically, we revealed that CIB2 competes with and inhibits CIB1-mediated calcineurin activation, thereby negating stress-induced structural remodeling and AF. CONCLUSIONS:Our data suggest that CIB2 represents a novel endogenous and atrial-enriched regulator that protects against atrial remodeling and AF under stress conditions. Therefore, CIB2 may represent a new potential target for treating AF.

To identify genetic variation underlying atrial fibrillation, the most common cardiac arrhythmia, we performed a genome-wide association study of >1,000,000 people, including 60,620 atrial fibrillation cases and 970,216 controls. We identified 142 independent risk variants at 111 loci and prioritized 151 functional candidate genes likely to be involved in atrial fibrillation. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans (GATA4, MYH6, NKX2-5, PITX2, TBX5), or near genes important for striated muscle function and integrity (for example, CFL2, MYH7, PKP2, RBM20, SGCG, SSPN). Pathway and functional enrichment analyses also suggested that many of the putative atrial fibrillation genes act via cardiac structural remodeling, potentially in the form of an 'atrial cardiomyopathy', either during fetal heart development or as a response to stress in the adult heart.

As the most prevalent form of arrhythmia, atrial fibrillation (AF) increases the risk of heart failure, thromboembolism, and stroke, contributing to the raising mortality and morbidity in patients with cardiovascular diseases. Despite the multifaceted nature of AF pathogenesis and complexity of AF pathophysiology, a growing body of evidence indicates that the NLRP3 inflammasome activation contributes to onset and progression of AF. Herein, the authors aim at reviewing the current literature on the role of inflammasome signaling in AF pathogenesis, and novel therapeutic options in the management of AF.