BACKGROUND:The strongest genetic risk factors for atrial fibrillation (AF) are DNA variants on chromosome 4q25 near the transcription factor gene PITX2 (Pitx2:Paired-like homeodomain transcription factor 2). Mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, although the molecular mechanism(s) remains controversial. Pitx2 encodes a transcription factor that activates an antioxidant response to promote cardiac repair. Increased reactive oxygen species causing oxidation of polyunsaturated fatty acids generates reactive lipid dicarbonyl moieties that adduct to proteins and other macromolecules to promote cellular injury. We tested the hypothesis that oxidative stress, and specifically isolevuglandins, the most reactive lipid dicarbonyls identified, are increased in the setting of Pitx2 deficiency to promote proarrhythmic remodeling and AF.METHODS:Pitx2+/- and Pitx2+/+ wild-type littermate control mice were treated orally with vehicle, the lipid dicarbonyl scavenger 2-hydroxybenzylamine, or an inactive control compound at weaning, until study at age 16 to 18 weeks.RESULTS:Pitx2+/- mice demonstrated increased P wave duration indicative of slowed atrial conduction, as well as increased inducible AF burden and sustained AF, compared with wild type, and these abnormalities were prevented by 2-hydroxybenzylamine. Both reactive oxygen species and isolevuglandin protein adducts were elevated in Pitx2+/- atria with reduced expression of reactive oxygen species-protective genes. High-resolution respirometry demonstrated impaired mitochondrial function in Pitx2+/- atria, with disruption of mitochondrial integrity and cell-cell junctions with connexin lateralization, as well as decreased mitochondrial biogenesis gene expression. Proarrhythmic ionic current remodeling in Pitx2+/- atrial myocytes included elevated resting membrane potential, abbreviated action potential duration, and reduced maximum phase 0 upstroke velocity compared with wild type. Most of these abnormalities were ameliorated or prevented by 2-hydroxybenzylamine.CONCLUSIONS:These results demonstrate a critical role for lipid dicarbonyl mediators of oxidative stress in the proarrhythmic remodeling and AF susceptibility that occurs with Pitx2 deficiency, implying the possibility of genotype-specific therapy to prevent AF.

Naxos disease is a rare autosomal recessive condition combining arrhythmogenic right ventricular cardiomyopathy, woolly hair, and palmoplantar keratoderma. The first identified causative variant was in the gene encoding the desmosomal protein plakoglobin. Naxos disease exhibits fibro-fatty myocardial replacement with immunohistological abnormalities in cardiac protein and signaling pathways, highlighting the role of inflammation and potential anti-inflammatory treatments. Childhood cutaneous signs precede cardiac features, which are diagnosed by familial and genetic evaluation, electrocardiography and cardiac imaging. Disease progression necessitates holistic care with risk management and lifestyle adjustments, often needing treatment for arrhythmia and heart failure. Similar phenotypes have been linked to desmoplakin and rarely desmocollin2 gene variants, highlighting the importance of familial and genetic evaluation. This document summarizes current knowledge on Naxos disease and related cardiocutaneous syndromes and initiates an international endeavor to collect and study all global cases, aiming to improve understanding, treatment, and patient care through shared data and research.

Aims:Plasminogen activator inhibitor-1 (PAI-1) regulates plasmin-mediated proteolysis, thereby influencing vascular stability and tissue remodeling. Angiotensin II (AngII) induces an increase of PAI-1 during the development of ascending thoracic aortic aneurysm (ATAA). The purpose of this study was to determine whether deletion of PAI-1 influenced the development of ATAA. Methods and results:AngII was infused for 4 weeks in whole-body PAI-1 deficient (PAI-1-/-) mice and their wild-type littermates (PAI-1+/+) to examine the role of PAI-1 in ATAA. PAI-1 deficiency did not alter AngII-induced aortopathy but revealed a striking cardiac phenotype characterized by replacement fibrosis predominantly within the epicardium and posterior septum. Ferric iron, indicative of prior hemorrhage, was coincident with fibrosis. Similar phenotypes were observed in PAI-1-/- mice infused with norepinephrine for 4 weeks. To define the pathological events preceding cardiac fibrosis, either AngII or norepinephrine was infused for 1 week in PAI-1+/+ or -/- mice. Both infusions induced extensive epicardial hemorrhage and posterior septal fibrosis in PAI-1-/- mice. To explore the initiation of hemorrhage and fibrosis, mice were infused with AngII for approximately 1 day, resulting in diffuse hemorrhage and cardiomyocyte loss localized to the posterior septum of PAI-1-/- mice. Conclusions:These findings support that, under hemodynamic stress, PAI-1 deficiency promotes early cardiac hemorrhage and cardiomyocyte loss, implicating plasmin-mediated proteolysis as an initiator of cardiac injury and fibrosis.

We analyzed the role of pro- and anti-inflammatory eicosanoids in the pathogenesis of arrhythmogenic cardiomyopathy (ACM). Lipidomics revealed reduced levels of anti-inflammatory oxylipins in plasma and increased levels of pro-inflammatory eicosanoids in hearts of Dsg2mut/mut mice, a preclinical model of ACM. Disease features were reversed in vitro in rat ventricular myocytes expressing mutant JUP by the anti-inflammatory epoxyeicosatrienoic acid 14-15-EET, whereas 14,15-EEZE, which antagonizes the 14,15-EET receptor, intensified nuclear accumulation of the desmosomal protein plakoglobin. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that converts anti-inflammatory EETs into polar, less active diols, prevented progression of myocardial injury in Dsg2mut/mut mice and promoted recovery of contractile function. This was associated with reduced myocardial expression of genes involved in innate immune signaling and fewer injurious macrophages expressing CCR2. These results suggest that pro-inflammatory eicosanoids contribute to the pathogenesis of ACM. Inhibition of sEH may be an effective, mechanism-based therapy for ACM patients.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

Arrhythmogenic cardiomyopathy (ACM) is a genetic form of heart failure that affects 1 in 5,000 people globally and is caused by mutations in cardiac desmosomal genes including PKP2, DSP, and DSG2. Individuals with ACM suffer from ventricular arrhythmias, sudden cardiac death, and heart failure. There are few effective treatments and heart transplantation remains the best option for many affected individuals. Here we performed single nucleus RNA sequencing and spatial transcriptomics on myocardial samples from patients with ACM and control donors. We identified disease-associated spatial niches characterized by coexistence of fibrotic and inflammatory cell types and failing cardiac myocytes. The inflammatory-fibrotic niche colocalized to areas of cardiac myocyte loss and comprised FAP (fibroblast activation protein) and POSTN (periostin) expressing fibroblasts, macrophages that expressed NLRP3, and nuclear factor κB activated genes. Using homozygous Dsg2 mutant (Dsg2mut/mut) mice, we identified analogous populations of Postn-expressing fibroblasts and inflammatory macrophage populations that co-localized within diseased areas. Detailed single nucleus RNA-sequencing analysis of inflammatory macrophage subsets that were increased in ACM samples revealed high levels of Il1b expression. To delineate the possible benefit of targeting IL1B in ACM, we treated Dsg2mut/mut mice with an anti-IL1B neutralizing antibody and observed attenuated fibrosis, reduced levels of inflammatory cytokines and chemokines, preserved cardiac function, and diminished conduction slowing and automaticity, key mechanisms of arrhythmogenesis. These results suggest that currently approved therapeutics that target IL1B or IL1 signaling may improve outcomes for patients with ACM.

BACKGROUND:Patients with arrhythmogenic cardiomyopathy due to pathogenic variants in PKP2, the gene for the desmosomal protein plakophilin-2, are being enrolled in gene therapy trials designed to replace the defective allele via adeno-associated viral transduction of cardiac myocytes. Evidence from experimental systems and patients indicates that ventricular myocytes in PKP2 arrhythmogenic cardiomyopathy have greatly reduced electrical coupling at gap junctions and reduced Na+ current density. In previous adeno-associated viral gene therapy trials, <50% of ventricular myocytes have generally been transduced. METHODS:We used established computational models of ventricular cell electrophysiology to define the effects of varying levels of successful gene therapy on conduction in patients with PKP2 arrhythmogenic cardiomyopathy. Conduction velocity and development of conduction block were analyzed in tissue constructs composed of cells with levels of electrical coupling and Na+ current density observed in experimental studies. RESULTS:We observed a nonlinear relationship between conduction velocity and the proportion of transduced cells. Conduction velocity increased only modestly when up to 40% of myocytes were transduced. Conduction block did not occur in tissue constructs with moderate levels of uncoupling (0.10 or 0.15 of normal) as this degree of coupling was sufficient to allow electrotonic current to pass through diseased cells. Thus, low levels of transduction, likely to occur in phase 1 clinical trials, do not seem to pose a major safety concern. However, our models did not incorporate the potential effects of fibrosis and inflammation, both of which are presumably present in PKP2 arrhythmogenic cardiomyopathy patients undergoing gene therapy and could impact arrhythmogenesis. CONCLUSIONS:The extent of successful ventricular myocyte transduction anticipated to be achieved in PKP2 adeno-associated viral gene therapy trials will likely not restore conduction velocity to levels sufficient to decrease the risk of reentrant arrhythmias. Transduction efficiency of 60% to 80% would be required to restore conduction velocity to 50% of normal.

Inhibition of nuclear factor kappa-B (NFκB) signaling prevents disease in Dsg2 mut/mut mice, a model of arrhythmogenic cardiomyopathy (ACM). Moreover, NFκB is activated in ACM patient-derived iPSC-cardiac myocytes under basal conditions in vitro . Here, we used genetic approaches and sequencing studies to define the relative pathogenic roles of immune signaling in cardiac myocytes vs. inflammatory cells in Dsg2 mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2 mut/mut mice. It does this by mobilizing cells expressing C-C motif chemokine receptor-2 (CCR2+ cells) to the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2 mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA sequencing and cellular indexing of transcriptomes and epitomes (CITE-seq) studies revealed marked pro-inflammatory changes in gene expression and the cellular landscape in hearts of Dsg2 mut/mut mice involving cardiac myocytes, fibroblasts and CCR2+ cells. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2 mut/mut mice were modulated by actions of CCR2+ cells. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.BRIEF SUMMARY:We have uncovered a therapeutically targetable innate immune mechanism regulating myocardial injury and cardiac function in a clinically relevant mouse model of Arrhythmogenic Cardiomyopathy (ACM).

Objectives:We sought to determine if persistent innate immune signaling via NFκB occurs in cardiac myocytes in patients with arrhythmogenic cardiomyopathy and if this is associated with myocardial infiltration of pro-inflammatory cells expressing CCR2. We also determined if buccal mucosa cells from young subjects with inherited disease alleles exhibit NFκB signaling. Background:NFκB signaling in cardiac myocytes causes disease in a mouse model of arrhythmogenic cardiomyopathy by mobilizing CCR2-expressing macrophages which promote myocardial injury and arrhythmias. Buccal mucosa cells exhibit pathologic features similar to those seen in cardiac myocytes in patients with arrhythmogenic cardiomyopathy. Methods:We analyzed myocardium from arrhythmogenic cardiomyopathy patients who died suddenly or required cardiac transplantation. We also analyzed buccal mucosa cells from young subjects with inherited disease alleles. The presence of immunoreactive signal for RelA/p65 in nuclei of cardiac myocytes and buccal cells was used as a reliable indicator of active NFκB signaling. We also counted myocardial CCR2-expressing cells. Results:NFκB signaling was seen in cardiac myocytes in 34 of 36 cases of arrhythmogenic cardiomyopathy but in none of 19 age-matched controls. Cells expressing CCR2 were increased in patient hearts in numbers directly correlated with the number of cardiac myocytes showing NFκB signaling. NFκB signaling also occurred in buccal cells in young subjects with active disease. Conclusions:Patients with clinically active arrhythmogenic cardiomyopathy exhibit persistent innate immune responses in cardiac myocytes and buccal mucosa cells reflecting an inflammatory process that fails to resolve. Such individuals may benefit from anti-inflammatory therapy. CONDENSED ABSTRACT:NFκB signaling in cardiac myocytes causes arrhythmias and myocardial injury in a mouse model of arrhythmogenic cardiomyopathy by mobilizing pro-inflammatory CCR2-expressing macrophages to the heart. Based on these new mechanistic insights, we analyzed hearts of arrhythmogenic cardiomyopathy patients who died suddenly or required cardiac transplantation. We observed active NFκB signaling in cardiac myocytes associated with marked infiltration of CCR2-expressing cells. We also observed NFκB signaling in buccal mucosa cells obtained from young subjects with active disease. Thus, anti-inflammatory therapy may be effective in arrhythmogenic cardiomyopathy. Screening buccal cells may be a reliable way to identify patients most likely to benefit. HIGHLIGHTS:Inflammation likely contributes to the pathogenesis of arrhythmogenic cardiomyopathy but the responsible mechanisms and the roles of specific classes of immune cells remain undefined.NFκB signaling in cardiac myocytes is sufficient to cause disease in a mouse model of arrhythmogenic cardiomyopathy by mobilizing injurious myeloid cells expressing CCR2 to the heart.Here, we provide evidence of persistent NFκB signaling in cardiac myocytes and increased CCR2-expressing cells in hearts of patients with arrhythmogenic cardiomyopathy. We observed a close correlation between the number of cardiac myocytes with active NFκB signaling and the number of CCR2-expressing cells in patient hearts.We also provide evidence of active NFκB signaling in buccal mucosa cells associated with initial onset of disease and/or disease progression in young subjects with arrhythmogenic cardiomyopathy alleles.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

BACKGROUND: Imaging evaluation of arrhythmogenic right ventricular cardiomyopathy (ARVC) remains challenging. Myocardial strain assessment by echocardiography is an increasingly utilized technique for detecting subclinical left ventricular (LV) and right ventricular (RV) dysfunction. We aimed to evaluate the diagnostic and prognostic utility of LV and RV strain in ARVC. METHODS: Patients with suspected ARVC (n = 109) from a multicenter registry were clinically phenotyped using the 2010 ARVC Revised Task Force Criteria and underwent baseline strain echocardiography. Diagnostic performance of LV and RV strain was evaluated using the area under the receiver operating characteristic curve analysis against the 2010 ARVC Revised Task Force Criteria, and the prognostic value was assessed using the Kaplan-Meier analysis. RESULTS: Mean age was 45.3±14.7 years, and 48% of patients were female. Estimation of RV strain was feasible in 99/109 (91%), and LV strain was feasible in 85/109 (78%) patients. ARVC prevalence by 2010 ARVC Revised Task Force Criteria is 91/109 (83%) and 83/99 (84%) in those with RV strain measurements. RV global longitudinal strain and RV free wall strain had diagnostic area under the receiver operating characteristic curve of 0.76 and 0.77, respectively (both P <0.001; difference NS). Abnormal RV global longitudinal strain phenotype (RV global longitudinal strain > −17.9%) and RV free wall strain phenotype (RV free wall strain > −21.2%) were identified in 41/69 (59%) and 56/69 (81%) of subjects, respectively, who were not identified by conventional echocardiographic criteria but still met the overall 2010 ARVC Revised Task Force Criteria for ARVC. LV global longitudinal strain did not add diagnostic value but was prognostic for composite end points of death, heart transplantation, or ventricular arrhythmia (log-rank P =0.04). CONCLUSIONS: In a prospective, multicenter registry of ARVC, RV strain assessment added diagnostic value to current echocardiographic criteria by identifying patients who are missed by current echocardiographic criteria yet still fulfill the diagnosis of ARVC. LV strain, by contrast, did not add incremental diagnostic value but was prognostic for identification of high-risk patients.