Zebrafish models for ARVC8 analysis and drug discovery

INTRODUCTION: Desmoplakin is one the most abundant desmosomal proteins in cardiac and epithelial tissues. In humans, dominat mutations in the desmoplakin gene (DSP) cause Arrhythmogenic Right Ventricular Cardio​myopathy 8 (ARVC8), a dominant cardiomyopathy, frequently involved in juvenile sudden death. Current ARVC models are based on cell lines and transgenic mice. In this context, it has been shown that suppression of DSP expression leads to a reduction in canonical Wnt signaling, suggesting that this pathway could be a molecular target for ARVC therapeutic intervention. In order to address this issue, the present study aims to evaluate the pathogenic mechanisms of DSP mutations in vivo, using zebrafish (Danio rerio) as an innovative model for this disease. In zebrafish, the desmoplakin gene is present with two isoforms, dspa and dspb, both orthologous to the single DSP in humans. PURPOSE: The purpose of this study is the generation and the phenotypic characterization of transient ARVC8 zebrafish models using a morpholino-mediated knock-down strategy. In addition, by taking advantage of zebrafish pathway reporter lines, we aim to verify if Wnt signaling and/or other molecular cascades might be involved in ARVC8 pathogenesis. The final goal is the assessment of our ARVC8 model as a suitable tool for molecularly-targeted drug discovery. METHODS: To evaluate the expression of dspa and dspb during zebrafish embryonic development and adulthood, we used whole-mount in situ hybridization (WISH) and semi quantitative RT_PCR. Knockdown of zebrafish dspa and dspb genes was obtained by a morpholino (MO)-based antisense strategy. Specifically, we injected anti-dspa and anti-dspb MO oligos in both wild types and pathway-specific lines reporting the activity of Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. RESULTS: We found that both dspa and dspb are expressed during zebrafish embryonic development, while the molecular analysis of cDNAs from different adult tissues demonstrates that both dspa and dspb are highly expressed in heart and skin, with dspa more strongly detectable compared to dspb. MO-mediated knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and, particularly in dspb knock-down embryos, decreased heart rate. TEM analysis of cardiac and skin tissues under dspa+dspb simultaneous knock-down shows reduced and disorganized desmososmes. As far as concerns the analysis of previously mentioned signaling pathways, we observed a specific reduction of Wnt signaling responsiveness in the cardiac region of both dspa and dspb knock- down embryos (Fig. 1). CONCLUSION: Our results show that transient knock-down of zebrafish desmoplakin genes is able to phenocopy some ARVC8 features, such as cardiac and cutaneous desmosomal defects, heart rate alteration and Wnt signaling reduction, pointing to zebrafish as a suitable ARVC8 model for in vivo screening of molecularly-targeted drugs

Arrhythmogenic Right Ventricular Cardiomyopathy: Characterization of Left Ventricular Phenotype and Differential Diagnosis With Dilated Cardiomyopathy

Background This study assessed the prevalence of left ventricular (LV) involvement and characterized the clinical, electrocardiographic, and imaging features of LV phenotype in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Differential diagnosis between ARVC-LV phenotype and dilated cardiomyopathy (DCM) was evaluated. Methods and Results The study population included 87 ARVC patients (median age 34\ua0years) and 153 DCM patients (median age 51\ua0years). All underwent cardiac magnetic resonance with quantitative tissue characterization. Fifty-eight ARVC patients (67%) had LV involvement, with both LV systolic dysfunction and LV late gadolinium enhancement (LGE) in 41/58 (71%) and LV-LGE in isolation in 17 (29%). Compared with DCM, the ARVC-LV phenotype was statistically significantly more often characterized by low QRS voltages in limb leads, T-wave inversion in the inferolateral leads and major ventricular arrhythmias. LV-LGE was found in all ARVC patients with LV systolic dysfunction and in 69/153 (45%) of DCM patients. Patients with ARVC and LV systolic dysfunction had a greater amount of LV-LGE (25% versus 13% of LV mass; P<0.01), mostly localized in the subepicardial LV wall layers. An LV-LGE 6520% had a 100% specificity for diagnosis of ARVC-LV phenotype. An inverse correlation between LV ejection fraction and LV-LGE extent was found in the ARVC-LV phenotype (r=-0.63; P<0.01), but not in DCM (r=-0.01; P=0.94). Conclusions LV involvement in ARVC is common and characterized by clinical and cardiac magnetic resonance features which differ from those seen in DCM. The most distinctive feature of ARVC-LV phenotype is the large amount of LV-LGE/fibrosis, which impacts directly and negatively on the LV systolic function

Morphofunctional abnormalities of mitral annulus and arrhythmic mitral valve prolapse

Background\u2014Arrhythmic mitral valve prolapse (MVP) is characterized by myxomatous leaflets and left ventricular (LV) fibrosis of papillary muscles and inferobasal wall. We searched for morphofunctional abnormalities of the mitral valve that could explain a regional mechanical myocardial stretch. Methods and Results\u2014Thirty-six (27 female patients; median age: 44 years) arrhythmic MVP patients with LV late gadolinium enhancement on cardiac magnetic resonance and no or trivial mitral regurgitation, and 16 (6 female patients; median age: 40 years) MVP patients without LV late gadolinium enhancement were investigated by morphofunctional cardiac magnetic resonance. Mitral annulus disjunction (median: 4.8 versus 1.8 mm; P1.5 (22 [61%] versus 4 [25%]; P=0.016) were higher in MVP patients with late gadolinium enhancement than in those without. A linear correlation was found between mitral annulus disjunction and curling (R=0.85). A higher prevalence of auscultatory midsystolic click (26 [72%] versus 6 [38%]; P=0.018) was also noted. Histology of the mitral annulus showed a longer mitral annulus disjunction in 50 sudden death patients with MVP and LV fibrosis than in 20 patients without MVP (median: 3 versus 1.5 mm; P<0.001). Conclusions\u2014Mitral annulus disjunction is a constant feature of arrhythmic MVP with LV fibrosis. The excessive mobility of the leaflets caused by posterior systolic curling accounts for a mechanical stretch of the inferobasal wall and papillary muscles, eventually leading to myocardial hypertrophy and scarring. These mitral annulus abnormalities, together with auscultatory midsystolic click, may identify MVP patients who would need arrhythmic risk stratification

Nonischemic left ventricular scar as a substrate of life-threatening ventricular arrhythmias and sudden cardiac death in competitive athletes

Background\u2014The clinical profile and arrhythmic outcome of competitive athletes with isolated nonischemic left ventricular (LV) scar as evidenced by contrast-enhanced cardiac magnetic resonance remain to be elucidated. Methods and Results\u2014We compared 35 athletes (80% men, age: 14\u201348 years) with ventricular arrhythmias and isolated LV subepicardial/midmyocardial late gadolinium enhancement (LGE) on contrast-enhanced cardiac magnetic resonance (group A) with 38 athletes with ventricular arrhythmias and no LGE (group B) and 40 healthy control athletes (group C). A stria LGE pattern with subepicardial/midmyocardial distribution, mostly involving the lateral LV wall, was found in 27 (77%) of group A versus 0 controls (group C; P<0.001), whereas a spotty pattern of LGE localized at the junction of the right ventricle to the septum was respectively observed in 11 (31%) versus 10 (25%; P=0.52). All athletes with stria pattern showed ventricular arrhythmias with a predominant right bundle branch block morphology, 13 of 27 (48%) showed ECG repolarization abnormalities, and 5 of 27 (19%) showed echocardiographic hypokinesis of the lateral LV wall. The majority of athletes with no or spotty LGE pattern had ventricular arrhythmias with a predominant left bundle branch block morphology and no ECG or echocardiographic abnormalities. During a follow-up of 38\ub125 months, 6 of 27 (22%) athletes with stria pattern experienced malignant arrhythmic events such as appropriate implantable cardiac defibrillator shock (n=4), sustained ventricular tachycardia (n=1), or sudden death (n=1), compared with none of athletes with no or LGE spotty pattern and controls. Conclusions\u2014Isolated nonischemic LV LGE with a stria pattern may be associated with life-threatening arrhythmias and sudden death in the athlete. Because of its subepicardial/midmyocardial location, LV scar is often not detected by echocardiography

A Systematic Analysis of the Clinical Outcome Associated with Multiple Reclassified Desmosomal Gene Variants in Arrhythmogenic Right Ventricular Cardiomyopathy Patients

The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy. Graphical Abstract

Correction to:The genetic architecture of Plakophilin 2 cardiomyopathy (Genetics in Medicine, (2021), 23, 10, (1961-1968), 10.1038/s41436-021-01233-7)

Due to a processing error Cynthia James, Brittney Murray, and Crystal Tichnell were assigned to the wrong affiliation. Cynthia James, Brittney Murray, and Crystal Tichnell have as their affiliation 5 Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA. In addition Hana Zouk, Megan Hawley, and Birgit Funke were assigned only to affiliation 3; they also have affiliation 4 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. The original article has been corrected

Importance of genotype for risk stratification in arrhythmogenic right ventricular cardiomyopathy using the 2019 ARVC risk calculator

none41siTo study the impact of genotype on the performance of the 2019 risk model for arrhythmogenic right ventricular cardiomyopathy (ARVC).Protonotarios, Alexandros; Bariani, Riccardo; Cappelletto, Chiara; Pavlou, Menelaos; García-García, Alba; Cipriani, Alberto; Protonotarios, Ioannis; Rivas, Adrian; Wittenberg, Regitze; Graziosi, Maddalena; Xylouri, Zafeirenia; Larrañaga-Moreira, José M; de Luca, Antonio; Celeghin, Rudy; Pilichou, Kalliopi; Bakalakos, Athanasios; Lopes, Luis Rocha; Savvatis, Konstantinos; Stolfo, Davide; Dal Ferro, Matteo; Merlo, Marco; Basso, Cristina; Freire, Javier Limeres; Rodriguez-Palomares, Jose F; Kubo, Toru; Ripoll-Vera, Tomas; Barriales-Villa, Roberto; Antoniades, Loizos; Mogensen, Jens; Garcia-Pavia, Pablo; Wahbi, Karim; Biagini, Elena; Anastasakis, Aris; Tsatsopoulou, Adalena; Zorio, Esther; Gimeno, Juan R; Garcia-Pinilla, Jose Manuel; Syrris, Petros; Sinagra, Gianfranco; Bauce, Barbara; Elliott, Perry MProtonotarios, Alexandros; Bariani, Riccardo; Cappelletto, Chiara; Pavlou, Menelaos; García-García, Alba; Cipriani, Alberto; Protonotarios, Ioannis; Rivas, Adrian; Wittenberg, Regitze; Graziosi, Maddalena; Xylouri, Zafeirenia; Larrañaga-Moreira, José M; de Luca, Antonio; Celeghin, Rudy; Pilichou, Kalliopi; Bakalakos, Athanasios; Lopes, Luis Rocha; Savvatis, Konstantinos; Stolfo, Davide; Dal Ferro, Matteo; Merlo, Marco; Basso, Cristina; Freire, Javier Limeres; Rodriguez-Palomares, Jose F; Kubo, Toru; Ripoll-Vera, Tomas; Barriales-Villa, Roberto; Antoniades, Loizos; Mogensen, Jens; Garcia-Pavia, Pablo; Wahbi, Karim; Biagini, Elena; Anastasakis, Aris; Tsatsopoulou, Adalena; Zorio, Esther; Gimeno, Juan R; Garcia-Pinilla, Jose Manuel; Syrris, Petros; Sinagra, Gianfranco; Bauce, Barbara; Elliott, Perry

A Systematic Analysis of the Clinical Outcome Associated with Multiple Reclassified Desmosomal Gene Variants in Arrhythmogenic Right Ventricular Cardiomyopathy Patients

The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy. [Figure not available: see fulltext.]