A new prediction model for ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy.

AIMS: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is characterized by ventricular arrhythmias (VAs) and sudden cardiac death (SCD). We aimed to develop a model for individualized prediction of incident VA/SCD in ARVC patients. METHODS AND RESULTS: Five hundred and twenty-eight patients with a definite diagnosis and no history of sustained VAs/SCD at baseline, aged 38.2 ± 15.5 years, 44.7% male, were enrolled from five registries in North America and Europe. Over 4.83 (interquartile range 2.44-9.33) years of follow-up, 146 (27.7%) experienced sustained VA, defined as SCD, aborted SCD, sustained ventricular tachycardia, or appropriate implantable cardioverter-defibrillator (ICD) therapy. A prediction model estimating annual VA risk was developed using Cox regression with internal validation. Eight potential predictors were pre-specified: age, sex, cardiac syncope in the prior 6 months, non-sustained ventricular tachycardia, number of premature ventricular complexes in 24 h, number of leads with T-wave inversion, and right and left ventricular ejection fractions (LVEFs). All except LVEF were retained in the final model. The model accurately distinguished patients with and without events, with an optimism-corrected C-index of 0.77 [95% confidence interval (CI) 0.73-0.81] and minimal over-optimism [calibration slope of 0.93 (95% CI 0.92-0.95)]. By decision curve analysis, the clinical benefit of the model was superior to a current consensus-based ICD placement algorithm with a 20.3% reduction of ICD placements with the same proportion of protected patients (P < 0.001). CONCLUSION: Using the largest cohort of patients with ARVC and no prior VA, a prediction model using readily available clinical parameters was devised to estimate VA risk and guide decisions regarding primary prevention ICDs (www.arvcrisk.com).

Arrhythmogenic Cardiomyopathy: Exercise Pitfalls, Role of Connexin-43, and Moving beyond Antiarrhythmics.

Arrhythmogenic Cardiomyopathy (ACM), a Mendelian disorder that can affect both left and right ventricles, is most often associated with pathogenic desmosomal variants that can lead to fibrofatty replacement of the myocardium, a pathological hallmark of this disease. Current therapies are aimed to prevent the worsening of disease phenotypes and sudden cardiac death (SCD). Despite the use of implantable cardioverter defibrillators (ICDs) there is no present therapy that would mitigate the loss in electrical signal and propagation by these fibrofatty barriers. Recent studies have shown the influence of forced vs. voluntary exercise in a variety of healthy and diseased mice; more specifically, that exercised mice show increased Connexin-43 (Cx43) expression levels. Fascinatingly, increased Cx43 expression ameliorated the abnormal electrical signal conduction in the myocardium of diseased mice. These findings point to a major translational pitfall in current therapeutics for ACM patients, who are advised to completely cease exercising and already demonstrate reduced Cx43 levels at the myocyte intercalated disc. Considering cardiac dysfunction in ACM arises from the loss of cardiomyocytes and electrical signal conduction abnormalities, an increase in Cx43 expression-promoted by low to moderate intensity exercise and/or gene therapy-could very well improve cardiac function in ACM patients.

Efficacy and Safety of Angiotensin Receptor Blockers in a Pre-Clinical Model of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is a familial heart disease, characterized by contractile dysfunction, ventricular arrhythmias (VAs), and the risk of sudden cardiac death. Currently, implantable cardioverter defibrillators and antiarrhythmics are the mainstays in ACM therapeutics. Angiotensin receptor blockers (ARBs) have been highlighted in the treatment of heart diseases, including ACM. Yet, recent research has additionally implicated ARBs in the genesis of VAs and myocardial lipolysis via the peroxisome proliferator-activated receptor gamma (PPARγ) pathway. The latter is of particular interest, as fibrofatty infiltration is a pathological hallmark in ACM. Here, we tested two ARBs, Valsartan and Telmisartan, and the PPAR agonist, Rosiglitazone, in an animal model of ACM, homozygous Desmoglein-2 mutant mice (Dsg2mut/mut). Cardiac function, premature ventricular contractions (PVCs), fibrofatty scars, PPARα/γ protein levels, and PPAR-mediated mRNA transcripts were assessed. Of note, not a single mouse treated with Rosiglitazone made it to the study endpoint (i.e., 100% mortality: n = 5/5). Telmisartan-treated Dsg2mut/mut mice displayed the preservation of contractile function (percent ejection fraction [%EF]; 74.8 ± 6.8%EF) compared to Vehicle- (42.5 ± 5.6%EF) and Valsartan-treated (63.1 ± 4.4%EF) mice. However, Telmisartan-treated Dsg2mut/mut mice showed increased cardiac wall motion abnormalities, augmented %PVCs, electrocardiographic repolarization/depolarization abnormalities, larger fibrotic lesions, and increased expression of PPARy-regulated gene transcripts compared to their Dsg2mut/mut counterparts. Alternatively, Valsartan-treated Dsg2mut/mut mice harbored fewer myocardial scars, reduced %PVC, and increased Wnt-mediated transcripts. Considering our findings, caution should be taken by physicians when prescribing medications that may increase PPARy signaling in patients with ACM.

Therapeutic Modulation of the Immune Response in Arrhythmogenic Cardiomyopathy.

BACKGROUND: Inflammation is a prominent feature of arrhythmogenic cardiomyopathy (ACM), but whether it contributes to the disease phenotype is not known. METHODS: To define the role of inflammation in the pathogenesis of ACM, we characterized nuclear factor-κB signaling in ACM models in vitro and in vivo and in cardiac myocytes from patient induced pluripotent stem cells. RESULTS: Activation of nuclear factor-κB signaling, indicated by increased expression and nuclear accumulation of phospho-RelA/p65, occurred in both an in vitro model of ACM (expression of JUP2157del2 in neonatal rat ventricular myocytes) and a robust murine model of ACM (homozygous knock-in of mutant desmoglein-2 [Dsg2mut/mut]) that recapitulates the cardiac manifestations seen in patients with ACM. Bay 11-7082, a small-molecule inhibitor of nuclear factor-κB signaling, prevented the development of ACM disease features in vitro (abnormal redistribution of intercalated disk proteins, myocyte apoptosis, release of inflammatory cytokines) and in vivo (myocardial necrosis and fibrosis, left ventricular contractile dysfunction, electrocardiographic abnormalities). Hearts of Dsg2mut/mut mice expressed markedly increased levels of inflammatory cytokines and chemotactic molecules that were attenuated by Bay 11-7082. Salutary effects of Bay 11-7082 correlated with the extent to which production of selected cytokines had been blocked. Nuclear factor-κB signaling was also activated in cardiac myocytes derived from a patient with ACM. These cells produced and secreted abundant inflammatory cytokines under basal conditions, and this was also greatly reduced by Bay 11-7082. CONCLUSIONS: Inflammatory signaling is activated in ACM and drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM.

Exercise restriction is protective for genotype-positive family members of arrhythmogenic right ventricular cardiomyopathy patients.

AIMS: In arrhythmogenic right ventricular cardiomyopathy (ARVC) patients, exercise worsens disease course, so exercise restriction is recommended. However, recommendations for genotype-positive ARVC family members is incompletely resolved. We aimed to provide evidence for exercise recommendations for genotype-positive ARVC family members. METHODS AND RESULTS: Arrhythmogenic right ventricular cardiomyopathy family members inheriting a pathogenic desmosomal variant were interviewed about exercise history from age 10. Exercise was characterized by duration, intensity, and dose (duration*intensity). Associations between exercise and (i) diagnosis by 2010 Task Force Criteria and (ii) development of sustained ventricular arrhythmias were examined. The study included 101 family members (age: 40.5 ± 19.3 years, male: 41%, Plakophilin-2 variant: 81%). Forty-four individuals (44%) met diagnostic criteria and 16 (16%) experienced sustained ventricular arrhythmia. Individuals who met diagnostic criteria had significantly higher average exercise duration and dose, but not peak intensity than those who did not. Only one individual who exercised below the American Heart Association recommended minimum (650 metabolic equivalent of task-hours/year) met diagnostic criteria or experienced sustained ventricular arrhythmia as opposed to 50% of individuals who exceeded it (adjusted odds ratio = 0.03, 95% confidence interval 0.003-0.26). The difference in exercise exposure between affected and unaffected individuals was more striking in females than in males. Females who had done high-dose exercise in adolescence had the worst survival free from diagnosis (P < 0.01). CONCLUSIONS: In phenotype-negative ARVC family members with a pathogenic desmosomal variant, athletic activities should be limited, particularly exercise dose. Exercise may play a greater role in promoting disease in female family members.

A new prediction model for ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy.

AIMS: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is characterized by ventricular arrhythmias (VAs) and sudden cardiac death (SCD). We aimed to develop a model for individualized prediction of incident VA/SCD in ARVC patients. METHODS AND RESULTS: Five hundred and twenty-eight patients with a definite diagnosis and no history of sustained VAs/SCD at baseline, aged 38.2 ± 15.5 years, 44.7% male, were enrolled from five registries in North America and Europe. Over 4.83 (interquartile range 2.44-9.33) years of follow-up, 146 (27.7%) experienced sustained VA, defined as SCD, aborted SCD, sustained ventricular tachycardia, or appropriate implantable cardioverter-defibrillator (ICD) therapy. A prediction model estimating annual VA risk was developed using Cox regression with internal validation. Eight potential predictors were pre-specified: age, sex, cardiac syncope in the prior 6 months, non-sustained ventricular tachycardia, number of premature ventricular complexes in 24 h, number of leads with T-wave inversion, and right and left ventricular ejection fractions (LVEFs). All except LVEF were retained in the final model. The model accurately distinguished patients with and without events, with an optimism-corrected C-index of 0.77 [95% confidence interval (CI) 0.73-0.81] and minimal over-optimism [calibration slope of 0.93 (95% CI 0.92-0.95)]. By decision curve analysis, the clinical benefit of the model was superior to a current consensus-based ICD placement algorithm with a 20.6% reduction of ICD placements with the same proportion of protected patients (P < 0.001). CONCLUSION: Using the largest cohort of patients with ARVC and no prior VA, a prediction model using readily available clinical parameters was devised to estimate VA risk and guide decisions regarding primary prevention ICDs (www.arvcrisk.com).

Basic and translational mechanisms in inflammatory arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a familial, nonischemic heart disease typically inherited via an autosomal dominant pattern (Nava et al., [1]; Wlodarska et al., [2]). Often affecting the young and athletes, early diagnosis of ACM can be complicated as incomplete penetrance with variable expressivity are common characteristics (Wlodarska et al., [2]; Corrado et al., [3]). That said, of the five desmosomal genes implicated in ACM, pathogenic variants in desmocollin-2 (DSC2) and desmoglein-2 (DSG2) have been discovered in both an autosomal-recessive and autosomal-dominant pattern (Wong et al., [4]; Qadri et al., [5]; Chen et al., [6]). Originally known as arrhythmogenic right ventricular dysplasia (ARVD), due to its RV prevalence and manifesting in the young, the disease was first described in 1736 by Giovanni Maria Lancisi in his book "De Motu Cordis et Aneurysmatibus" (Lancisi [7]). However, the first comprehensive clinical description and recognition of this dreadful disease was by Guy Fontaine and Frank Marcus in 1982 (Marcus et al., [8]). These two esteemed pathologists evaluated twenty-two (n = 22/24) young adult patients with recurrent ventricular tachycardia (VT) and RV dysplasia (Marcus et al., [8]). Initially, ARVD was thought to be the result of partial or complete congenital absence of ventricular myocardium during embryonic development (Nava et al., [9]). However, further research into the clinical and pathological manifestations revealed acquired progressive fibrofatty replacement of the myocardium (McKenna et al., [10]); and, in 1995, ARVD was classified as a primary cardiomyopathy by the World Health Organization (Richardson et al., [11]). Thus, now classifying ACM as a cardiomyopathy (i.e., ARVC) rather than a dysplasia (i.e., ARVD). Even more recently, ARVC has shifted from its recognition as a primarily RV disease (i.e., ARVC) to include left-dominant (i.e., ALVC) and biventricular subtypes (i.e., ACM) as well (Saguner et al., [12]), prompting the use of the more general term arrhythmogenic cardiomyopathy (ACM). This review aims to discuss pathogenesis, clinical and pathological phenotypes, basic and translational research on the role of inflammation, and clinical trials aimed to prevent disease onset and progression.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

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.

Vasopressin-induced constriction of the isolated rat occipital artery is segment dependent.

BACKGROUND: Circulating factors delivered to the nodose ganglion (NG) by the occipital artery (OA) have been shown to affect vagal afferent activity, and thus the contractile state of the OA may influence blood flow to the NG. METHODS: OA were isolated and bisected into proximal and distal segments relative to the external carotid artery. RESULTS: Bisection highlighted stark differences between maximal contractile responses and OA sensitivity. Specifically, maximum responses to vasopressin and the V1 receptor agonist were significantly higher in distal than proximal segments. Distal segments were significantly more sensitive to 5-hydroxytryptamine (5-HT) and the 5-HT2 receptor agonist than proximal segments. Angiotensin II (AT)2, V2 and 5-HT(1B/1D) receptor agonists did not elicit vascular responses. Additionally, AT1 receptor agonists elicited mild, yet not significantly different maximal responses between segments. CONCLUSION: The results of this study are consistent with contractile properties of rat OA being mediated via AT1, V1 and 5-HT2 receptors and dependent upon the OA segment. Furthermore, vasopressin-induced constriction of the OA, regardless of a bolus dose or a first and second concentration-response curve, retained this unique segmental difference. We hypothesize that these segmental differences may be important in the regulation of blood flow through the OA in health and disease.

A Novel Disability Advocacy and Awareness Program for Training Future Healthcare Professionals on Care for Patients with Disabilities.

In a poll of 714 US physicians, it was revealed that only 40.7% felt very confident in their ability to provide the same quality of care, overall, to patients with disabilities (PWDs) compared with patients without disabilities. It was also found that only 56.5% strongly agreed that they welcomed PWDs into their practice as healthcare providers. This suggests a systemic issue of inequity in medicine, which affects both physicians and patients. If this problem is not corrected, our healthcare system will continue to lack in providing adequate care to PWDs. A key component of this issue is that the lack of confident care for PWDs appears to be a result of insufficient exposure to PWDs during the formative years in medical schools. Although medical students are taught extensive clinical skills and bedside manners, there appears to be little mention of how to make adaptations to basic patient encounters to accommodate PWDs. Further, the lack of representation of PWDs in the medical community results in minimal experience among trainees and the perpetuation of unjust biases in the healthcare system. Changes to the medical field must start with shaping future physicians and filling the void in medical education. As a solution, we at Florida State University (FSU) College of Medicine (COM) propose a program called the Disability Advocacy and Awareness Program (DAAP). Two interactive sessions were designed, and students were offered an immersive experience in which they were not only provided with information through well-crafted presentations but also encouraged to engage in direct interactions with PWDs and a myriad of assistive devices. We believe a great deal of the program's success stemmed from the two-phase interactive model that allowed students to undergo a truly immersive experience that a textbook cannot endow. Although we cannot expect every provider to be an expert on all disabilities, all physicians should have an understanding of how a disability may impact a patient's life and medical care. Improved knowledge and awareness surrounding disability and the barriers faced by the PWD population will provide insights that will allow for the most equitable, patient-centered care for the disabled community.

Correction: A Novel Disability Advocacy and Awareness Program for Training Future Healthcare Professionals on Care for Patients with Disabilities.

[This corrects the article DOI: 10.7759/cureus.33881.].

Mechanisms of Innate Immune Injury in Arrhythmogenic Cardiomyopathy.

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).

Platelet activation and endothelial dysfunction biomarkers in acute coronary syndrome: the impact of PCSK9 inhibition.

AIMS: Platelet activation and endothelial dysfunction contribute to adverse outcomes in patients with acute coronary syndromes (ACS). The goals of this study were to assess the impact of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition on markers of platelet activation and endothelial dysfunction in ACS patients and the interaction among PCSK9, platelets, and endothelial cells (ECs) on left internal mammary artery (LIMA) vascular endothelium using specimens obtained during coronary artery bypass surgery (CABG). METHODS AND RESULTS: Acute coronary syndromes patients enrolled in the Evolocumab in ACS trials were randomized to placebo or a single dose of 420 mg evolocumab within 24 h of hospitalization. Serum samples for analysis of platelet factor 4 (PF4) and P-selectin, markers of platelet activation, and von Willebrand factor (vWF), a marker of endothelial dysfunction, were obtained at baseline and 30 days. Additionally, LIMA segments obtained during CABG from patients who were and were not receiving evolocumab were immunostained with PCSK9; CD61, a platelet-specific marker; and CD31, an endothelial cell-specific marker. Forty-six participants were randomized to placebo or to evolocumab. Controlling for baseline levels, PF4 and vWF were significantly lower in the evolocumab, than in the placebo, group at 30 days. Immunostaining of LIMA specimens from twelve participants undergoing CABG revealed colocalization of PCSK9, CD61, and CD31 at the vascular endothelium. Administration of evolocumab was associated with decreased overlap of PCSK9, CD61, and CD31. CONCLUSIONS: Proprotein Convertase Subtilisin/Kexin 9 inhibition decreases markers of platelet activation and endothelial dysfunction in ACS patients. PCSK9 is associated with platelets and vascular ECs in LIMA segments and PCSK9 inhibition decreases that interaction.

Innate Immune Signaling in Hearts and Buccal Mucosa Cells of Patients with Arrhythmogenic Cardiomyopathy.

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.

Innate immune signaling in hearts and buccal mucosa cells of patients with arrhythmogenic cardiomyopathy.

Background: Nuclear factor κB (NF-κB) signaling in cardiac myocytes causes disease in a mouse model of arrhythmogenic cardiomyopathy (ACM) by mobilizing CCR2-expressing macrophages that promote myocardial injury and arrhythmias. Buccal mucosa cells exhibit pathologic features similar to those seen in cardiac myocytes in patients with ACM. Objectives: We sought to determine if persistent innate immune signaling via NF-κB occurs in cardiac myocytes in patients with ACM and if this is associated with myocardial infiltration of proinflammatory cells expressing CCR2. We also determined if buccal mucosa cells from young subjects with inherited disease alleles exhibit NF-κB signaling. Methods: We analyzed myocardium from ACM 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 NF-κB signaling. We also counted myocardial CCR2-expressing cells. Results: RelA/p65 signal was seen in numerous cardiac myocyte nuclei in 34 of 36 cases of ACM but not in 19 age-matched control individuals. Cells expressing CCR2 were increased in patient hearts in numbers directly correlated with the number of cardiac myocytes showing NF-κB signaling. NF-κB signaling was observed in buccal cells in young subjects with active disease. Conclusions: Patients with clinically active ACM exhibit persistent innate immune responses in cardiac myocytes and buccal mucosa cells, reflecting a local and systemic inflammatory process. Such individuals may benefit from anti-inflammatory therapy.

Arrhythmogenic Right Ventricular Cardiomyopathy Presenting as Clinical Myocarditis in Women.

Patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) classically initially present with ventricular arrhythmias or, less commonly, heart failure. Myocardial inflammation has been implicated in pathogenesis, but clinical myocarditis in ARVC is less described. We therefore studied clinical myocarditis as an initial ARVC presentation, and hypothesized that these patients have distinct clinical and genetic characteristics. Using the Johns Hopkins ARVC Registry, we identified 12 patients (all female, median age 20) referred between 2014 and 2019 diagnosed with myocarditis at presentation who were subsequently diagnosed with ARVC by Task Force Criteria. Majority presented with chest pain (n = 7, 58%) or ventricular arrhythmia (n = 3, 25%). All patients had troponin elevations and left ventricular (LV) function was reduced in 5 (42%). Magnetic resonance imaging demonstrated LV delayed gadolinium enhancement and/or pericardial enhancement in 10 (83%); only 3 (25%) patients had right ventricular abnormalities. Pathogenic genetic variants were identified in 11 (92%) patients: 10 desmoplakin (DSP) and 1 desmoglein-2 (DSG2). Thus, nearly 1/3 (10/32, 31%) of overall DSP ARVC patients were originally diagnosed with myocarditis. Patients were diagnosed with ARVC 1.8 years (IQR 2.7 years) after presentation and 8 (75%) patients did not meet Task Force Criteria without genetic testing. ARVC diagnosis led to an additional 5 (42%) patients referred for implantable cardiac defibrillator and 17 family member diagnoses. In conclusion, ARVC may initially present as myocarditis and these patients have distinct characteristics including female gender, LV involvement and DSP gene variants. Genetic testing is key to ARVC diagnosis and should be considered in select myocarditis patients.

Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart condition which causes fibro-fatty myocardial scarring, ventricular arrhythmias, and sudden cardiac death. Most cases of ARVC can be linked to pathogenic mutations in the cardiac desmosome, but the pathophysiology is not well understood, particularly in early phases when arrhythmias can develop prior to structural changes. Here, we created a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of ARVC from a patient with a c.2358delA variant in desmoglein-2 (DSG2). These DSG2-mutant (DSG2Mut) hiPSC-CMs were compared against two wildtype hiPSC-CM lines via immunostaining, RT-qPCR, Western blot, RNA-Seq, cytokine expression and optical mapping. Mutant cells expressed reduced DSG2 mRNA and had altered localization of desmoglein-2 protein alongside thinner, more disorganized myofibrils. No major changes in other desmosomal proteins were noted. There was increased pro-inflammatory cytokine expression that may be linked to canonical and non-canonical NFκB signaling. Action potentials in DSG2Mut CMs were shorter with increased upstroke heterogeneity, while time-to-peak calcium and calcium decay rate were reduced. These were accompanied by changes in ion channel and calcium handling gene expression. Lastly, suppressing DSG2 in control lines via siRNA allowed partial recapitulation of electrical anomalies noted in DSG2Mut cells. In conclusion, the aberrant cytoskeletal organization, cytokine expression, and electrophysiology found DSG2Mut hiPSC-CMs could underlie early mechanisms of disease manifestation in ARVC patients.

Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disorder at high risk of arrhythmic sudden death in the young and athletes. AC is hallmarked by myocardial replacement with fibro-fatty tissue, favoring life-threatening cardiac arrhythmias and contractile dysfunction. The AC pathogenesis is unclear, and the disease urgently needs mechanism-driven therapies. Current AC research is mainly focused on 'desmosome-carrying' cardiomyocytes, but desmosomal proteins are also expressed by non-myocyte cells, which also harbor AC variants, including mesenchymal stromal cells (MSCs). Consistently, cardiac-MSCs contribute to adipose tissue in human AC hearts. We thus approached AC as a multicellular disorder, hypothesizing that it also affects extra-cardiac bone marrow (BM)-MSCs. Our results show changes in the desmosomal protein profile of both cardiac- and BM- MSCs, from desmoglein-2 (Dsg2)-mutant mice, accompanied with profound alterations in cytoskeletal organization, which are directly caused by AC-linked DSG2 downregulation. In addition, AC BM-MSCs display increased proliferation rate, both in vitro and in vivo, and, by using the principle of the competition homing assay, we demonstrated that mutant circulating BM-MSCs have increased propensity to migrate to the AC heart. Taken altogether, our results indicate that cardiac- and BM- MSCs are additional cell types affected in Dsg2-linked AC, warranting the novel classification of AC as a multicellular and multiorgan disease.

Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with ß-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.