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Genetic characterization of biopsy-proven myocarditis: a pilot study Background Myocarditis is characterized by the presence of an inflammatory infiltrate in the myocardium with degenerative/necrotic changes of cardiomyocytes, not-related to ischemic damage. Its clinical presentation is extremely heterogenous. Endomyocardial Biopsy (EMB) is the diagnostic gold standard and provides etiopathogenetic diagnosis. Biopsy-proven myocarditis may be infectious, mainly viral, toxic or non-infectious immune-mediated/autoimmune. A complex interplay between genetic factors, environmental triggers (i.e. viral infection) and the immune response of the host is postulated at the basis of different disease evolution. Purpose To determine the prevalence of pathogenic/likely pathogenic (P/LP) variants in cardiomyopathy-related genes in a well-characterized cohort of biopsy-proven myocarditis. Methods Seventy-three biopsy-proven myocarditis-affected patients (mean age 51±9, 41 males) underwent screening of 200 genes related to inherited cardiomyopathies. Definite/moderate gene association with Dilated Cardiomyopathy (DCM) and Arrhythmogenic Cardiomyopathy was based on the ClinGen framework. Variant prioritization was carried out using American College of Medical Genetics and Genomics rules. Correlation with presence of virus on endomyocardial biopsy by polymerase chain reaction, family history and serum anti-heart autoantibodies (AHA) and/or aintiintercalated-disk autoantibodies (AIDA), among other clinical features, was appraised. Results Nineteen of the 73 biopsy-proven myocarditis patients (26%) carried a P/LP variant in cardiac-related genes. Titin (TTN) was the most overrepresented gene accounting for 11% of cases (8/73), followed by Myosin Binding Protein C3 (MYBPC3) and Desmoplakin (DSP), each accounting for 3% of cases (2/73), respectively. No differences were observed in the distribution of disease onset, heart failure classification (NYHA class), ejection fraction, presence of virus and presence of anti-heart autoantibodies between genotype-positive and genotype-negative cohorts. However, 12 of the 19 genotype-positive index cases (63%) referred family history for cardiomyopathy and/or sudden cardiac death; whereas only 4 of the 54 genotype-negative declared positive family history, demonstrating a statistically significant difference. On the other hand, 37% of genotype positive patients declared negative family history, suggesting a non-familial or sporadic form of myocarditis. Conclusion The prevalence of clinically actionable P/LP variants in cardiomyopathy-related genes is nearly one third of biopsy-proven viral or autoimmune myocarditis patients, most of them associated with DCM, but clinical parameters do not differ between genotype-positive and genotype-negative patients. On the other hand, positive family history in genotype-positive patients indicate that family history assessment is mandatory to identify the genetic substrate of inherited inflammatory cardiomyopathies.

Background: In the last few years, a phenotypic variant of arrhythmogenic cardiomyopathy (ACM) labeled arrhythmogenic left ventricular cardiomyopathy (ALVC) has been defined and researched. This type of cardiomyopathy is characterized by a predominant left ventricular (LV) involvement with no or minor right ventricular (RV) abnormalities. Data on the specific risk and management of pregnancy in women affected by ALVC are, thus far, not available. We have sought to characterize pregnancy course and outcomes in women affected by ALVC through the evaluation of a series of childbearing patients. Methods: A series of consecutive female ALVC patients were analyzed in a cross-sectional, retrospective study. Study protocol included 12-lead ECG assessments, 24-h Holter ECG evaluations, 2D-echocardiogram tests, cardiac magnetic resonance assessments, and genetic analysis. Furthermore, the long-term disease course of childbearing patients was compared with a group of nulliparous ALVC women. Results: A total of 35 patients (mean age 45 ± 9 years, 51% probands) were analyzed. Sixteen women (46%) reported a pregnancy, for a total of 27 singleton viable pregnancies (mean age at first childbirth 30 ± 9 years). Before pregnancy, all patients were in the NYHA class I and none of the patients reported a previous heart failure (HF) episode. No significant differences were found between childbearing and nulliparous women regarding ECG features, LV dimensions, function, and extent of late enhancement. Overall, 7 patients (20%, 4 belonging to the childbearing group) experienced a sustained ventricular tachycardia and 2 (6%)—one for each group—showed heart failure (HF) episodes. The analysis of arrhythmia-free survival patients did not show significant differences between childbearing and nulliparous women. Conclusions: In a cohort of ALVC patients without previous episodes of HF, pregnancy was well tolerated, with no significant influence on disease progression and degree of electrical instability. Further studies on a larger cohort of women with different degrees of disease extent and genetic background are needed in order to achieve a more comprehensive knowledge regarding the outcome of pregnancy in ALVC patients.

Aims To study the impact of genotype on the performance of the 2019 risk model for arrhythmogenic right ventricular cardiomyopathy (ARVC). Methods and results The study cohort comprised 554 patients with a definite diagnosis of ARVC and no history of sustained ventricular arrhythmia (VA). During a median follow-up of 6.0 (3.1,12.5) years, 100 patients (18%) experienced the primary VA outcome (sustained ventricular tachycardia, appropriate implantable cardioverter defibrillator intervention, aborted sudden cardiac arrest, or sudden cardiac death) corresponding to an annual event rate of 2.6% [95% confidence interval (CI) 1.9–3.3]. Risk estimates for VA using the 2019 ARVC risk model showed reasonable discriminative ability but with overestimation of risk. The ARVC risk model was compared in four gene groups: PKP2 (n = 118, 21%); desmoplakin (DSP) (n = 79, 14%); other desmosomal (n = 59, 11%); and gene elusive (n = 160, 29%). Discrimination and calibration were highest for PKP2 and lowest for the gene-elusive group. Univariable analyses revealed the variable performance of individual clinical risk markers in the different gene groups, e.g. right ventricular dimensions and systolic function are significant risk markers in PKP2 but not in DSP patients and the opposite is true for left ventricular systolic function. Conclusion The 2019 ARVC risk model performs reasonably well in gene-positive ARVC (particularly for PKP2) but is more limited in gene-elusive patients. Genotype should be included in future risk models for ARVC.

In recent years a phenotypic variant of Arrhythmogenic cardiomyopathy has been described, characterized by predominant left ventricular (LV) involvement with no or minor right ventricular abnormalities, referred to as Arrhythmogenic left ventricular cardiomyopathy (ALVC). Different disease-genes have been identified in this form, such as Desmoplakin (DSP), Filamin C (FLNC), Phospholamban (PLN) and Desmin (DES). The main purpose of this critical systematic review was to assess the level of knowledge on genetic background and clinical features of ALVC. A search (updated to April 2022) was run in the PubMed, Scopus, and Web of Science electronic databases. The search terms used were “arrhythmogenic left ventricular cardiomyopathy” OR “arrhythmogenic cardiomyopathy” and “gene” OR “arrhythmogenic dysplasia” and “gene”. The most represented disease-gene turned out to be DSP, accounting for half of published cases, followed by FLNC. Overall, ECG abnormalities were reported in 58% of patients. Major ventricular arrhythmias were recorded in 26% of cases; an ICD was implanted in 29% of patients. A total of 6% of patients showed heart failure symptoms, and 15% had myocarditis-like episodes. DSP is confirmed to be the most represented disease-gene in ALVC patients. An analysis of reported clinical features of ALVC patients show an important degree of electrical instability, which frequently required an ICD implant. Moreover, myocarditis-like episodes are common.

Desmoplakin (DSP) genetic variants have been reported in arrhythmogenic cardiomyopathy with particular regard to predominant left ventricular (LV) involvement.The purpose of this study was to improve our understanding of clinical phenotype and outcome of DSP variant carriers.The clinical picture and outcome of 73 patients (36% probands) harboring a pathogenic/likely pathogenic DSP variant were evaluated.The phenotype during follow-up (mean 11 years; range 1-39 years) changed in 25 patients (35%), arrhythmogenic LV cardiomyopathy (ALVC) forms being the most frequent (n = 26 [36%]), followed by biventricular (BIV; n = 20 [27%]) and arrhythmogenic right ventricular cardiomyopathy (ARVC; n = 16 [22%]) forms. Major ventricular arrhythmias were detected in 21 patients (29%), and they were more common in ARVC (n = 6, 56%) and BIV forms (n = 8, 40%) than in ALVC forms (n = 4, 15%). In patients with ALVC, major ventricular arrhythmias occurred in the setting of a normal/mildly reduced systolic function. Heart failure (HF) occurred in 6 patients (8%); none affected with ALVC. Females showed more commonly LV involvement, while ARVC forms were more frequently detected in males (21 [61%] vs 15 [38%]; P = .147). Males showed a higher incidence of major ventricular arrhythmias (18 [52%] vs 9 [24%]; P = .036), HF (11 [31%] vs 1 [3%]; P = .004), and cardiac death (11 [31%] vs 0 [0%]; P.001).The clinical phenotype in pathogenic/likely pathogenic DSP variant carriers is wide. Although most patients show LV involvement, 16 (22%) has right ventricular abnormalities in keeping with a "classical" arrhythmogenic cardiomyopathy form. In ALVC, HF and major ventricular arrhythmias seem less common than in right ventricular and BIV variants. Females show more frequently LV involvement and a better outcome.

Background Mutations in filamin-C (FLNC) are involved in the pathogenesis of arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy (DCM), and have been associated with a left ventricular (LV) phenotype, characterized by nonischemic LV fibrosis, ventricular arrhythmias, and sudden cardiac death (SCD). Objective The purpose of this study was to investigate the prevalence of FLNC variants in a gene-negative ACM population and to evaluate the clinical phenotype and SCD risk factors in FLNC-associated cardiomyopathies. Methods ACM probands who tested negative for mutations in ACM-related genes underwent FLNC genetic screening. Clinical and genetic data were collected and pooled together with those of previously published FLNC-ACM and FLNC-DCM patients. Results In a cohort of 270 gene-elusive ACM probands, 12 (4.4%) had FLNC variants, and 13 additional family members carried the same mutation. Eighteen FLNC variant carriers (72%) had a diagnosis of ACM (72% male; mean age 45 years). On pooled analysis, 145 patients with FLNC-associated cardiomyopathies were included. Electrocardiographic (ECG) low QRS voltages were detected in 37%, and T-wave inversion (TWI) in inferolateral/lateral leads in 24%. Among 67 patients who had cardiac magnetic resonance (CMR), LV nonischemic late gadolinium enhancement (LGE) was found in 75%. SCD occurred in 28 patients (19%), 15 of whom showed LV nonischemic LGE/fibrosis. Compared with patients with no SCD, those who experienced SCD more frequently had inferolateral/lateral TWI (P = .013) and LV LGE/fibrosis (P = .033). Conclusion Clinical phenotype of FLNC cardiomyopathies is characterized by late-onset presentation and typical ECG and CMR features. SCD is associated with the presence of LV LGE/fibrosis but not with severe LV systolic dysfunction.

Background Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disorder, mainly caused by mutations in genes encoding desmosomal proteins, and accounts for most stress-related arrhythmic sudden cardiac deaths (SCD) in the young and athletes. The AC myocardium is hallmarked by cardiomyocyte (CM) death and fibro-fatty replacement, which generate a pro-arrhythmogenic substrate. Several pathogenetic factors in AC remain obscure and better understanding of the disease mechanisms is required to develop novel efficacious therapies to prevent SCD, which are sorely missing. The lexical analogy between desmosomes and desmosomal proteins has originally biased AC research towards CMs, the paradigmatic desmosome-bearing cells in heart. However, the myocardium is composed by different cell types, many of which express desmosomal proteins, albeit in the absence of desmosomes, including CMs, sympathetic neurons, vascular cells and fibroblasts. Notably, AC mutations are transmitted at germline, and thus may manifest in all cell types expressing desmosomal proteins. This might explain why the majority of preclinical AC models, using CM specific over-expression or deletion of the disease-causing mutation, failed to fully recapitulate the human disease phenotype. Hypothesis On these bases, we aimed to generate a knock-in (KI) AC mouse model for comprehensively studying AC pathogenesis. Methods As Desmoplakin (DSP) mutations occur in a large part of the Italian AC population, we used CRISP/Cas9 to generate a KI mouse strain harboring the Serine-to-Alanine substitution of S311, the murine homolog of human S299 [Bauce et al, 2005]. We successfully obtained DSPS311A/WT KI founders, which were viable and fertile and after backcrossing for >10 generations, used to expand the new mouse strain. Mouse cardiac phenotype was characterized, at different stages (1,2,4,6,9 mo.) by functional (i.e. ECHO, telemetry-ECG, chronic exercise) and structural (i.e. EM, standard histology, confocal IF, TUNEL assay) analyses. Molecular/biochemical analyses probed the state of the main pathways involved in AC. Results Our analyses showed that, starting from 4 mo., DSP homozygous KI mice display contractile dysfunction, worsening during aging, and fibrotic myocardial remodelling with focal fatty lesions, accompanied by frequent arrhythmic beats, which become sustained ventricular arrhythmias upon Noradrenaline administration. Hearts showed desmosome alterations, particularly at advanced disease stages, and lateralization of cx43, which corresponded to the phenotype of human AC hearts. Heterozygous mice showed similar alterations, which only took longer to appear. Exercise accelerated disease progression and increased the incidence of SCD (DSPS311A: SCD=63%, n=11; ctrls: SCD=8%, n=12). Conclusion Our KI mice replicate the clinical and pathological phenotype of DSP-linked biventricular AC and are thus suited for the mechanistic study of the multicellular origin of the disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PRIN Miur 2015