Dissertations / Theses on the topic 'ARRHYTHMOGENIC'

Introduzione: Recentemente, varianti minori di SNP del gene NOS1AP sono stati associati a prolungamento del QT e aumentata incidenza di morte improvvisa in pazienti LQT1. Il gene NOS1AP codifica per la proteina CAPON, che localizza NOS1 in prossimità del reticolo sarcoplasmico (SR). L' attività di NOS1 è importante per la modulazione mediate da NO di ICaL, dei canali RyR2 e di SERCA, interferendo così con la regolazione dell’handling del Ca2+ e la stabilità del SR. Perciò abbiamo ipotizzato che gli SNPs di NOS1AP possano alterare la localizzazione/funzione di NOS1 diminuendo la stabilità del SR. In questo contesto, il prolungamento del QT indotto da mutazione, potrebbe indurre un sovraccarico di Ca2+, il cui effetto pro-aritmico potrebbe essere smascherato da un’alterata localizzazione/funzione di NOS1. Scopo: Valutare l’effetto di cambiamenti nell’attività di NOS1 a livello della stabilità funzionale del SR, della ripolarizzazione e aritmogenesi in un contesto di perdita di IKs (LQT1). Metodi: In miociti ventricolari di cavia soggetti a blocco di IKs (per riprodurre il fenotipo LQT1) e a stimolazione adrenergica (Isoproterenolo, ISO), abbiamo misurato l’attività elettrica, le correnti di membrane e il Ca2+ intracellulare, in condizione basale e dopo inibizione selettiva di NOS1 (SMTC 3µM). Risultati: In condizioni basali, l’inibizione di NOS1 prolunga la durata del PA (APD) (152.6  11.7 ms vs 96.1  9.0 ms; 58.8%. p<0.01), aumenta la densità di ICaL (SMTC vs CTRL: -16.61.2 pA/pF vs -13.51.0 pA/pF; p<0.05) ma non altera ne IKs (SMTC vs CTRL: 2.50.4 pA/pF vs 2.60.2 pA/pF) nè IKr (SMTC vs CTRL: 0.840.04 pA/pF vs 0.910.05 pA/pF). L’isoproterenolo, agonista -adrenergico (ISO, 1nM), induce delayed afterdepolarizations (DADs), un indice di instabilità del SR, in una percentuale significativamente maggiore di cellule trattate con SMTC rispetto a quelle di controllo (93% per SMTC vs 22% per CTRL, p<0.01). Inoltre, il tempo medio di comparsa delle DADs è significativamente ridotto in miociti trattati con SMTC rispetto a quelli di CTRL (25.8 ± 3.8 s e 61.5 ± 15.3 s rispettivamente, p<0.01). In aggiunta, la durata del PA è importante per il verificarsi di questi eventi, poiché il passaggio, in AP clamp, da un PA lungo (140 ms) a uno corto (100 ms) durante applicazione di ISO, abolisce le “transient inward currents” (ITI). Conclusioni: Questi risultati indicano che la mancanza di NOS1 può contribuire al prolungamento dell’APD e aumentare l’influsso di Ca2+; questi effetti compromettono la stabilità del SR in presenza di stimolazione adrenergica. Gli effetti dell’inibizione di NOS1 sono tali da poter spiegare l’effetto aritmogenico dei polimorfismi di NOS1AP.
Background: Recently, minor SNP variants of the NOS1AP gene have been reported to be associated with QT prolongation and increased incidence of sudden death in LQT1 patients. The NOS1AP gene encodes for CAPON protein, that localizes NOS1 close to the sarcoplasmic reticulum (SR). NOS1 activity accounts for NO-mediated modulation of ICaL, RyR2 channels and SERCA, thus interfering with regulation of Ca2+ handling and SR stability. Therefore we hypothesize that NOS1AP SNPs might affect NOS1 localization/function to decrease SR stability. In this setting, mutation-induced QT prolongation would induce Ca2+ overload, whose proarrhythmic effect would be unveiled by abnormal NOS1 localization/function. Aim: To evaluate the effect of changes in NOS1 activity on SR functional stability, repolarization and arrhythmogenesis in the context of IKs deficiency (LQT1). Methods: In guinea-pig ventricular myocytes subjected to IKs blockade (to reproduce the LQT1 phenotype) and adrenergic stimulation (Isoproterenol, ISO), we measured electrical activity, membrane currents and intracellular Ca2+, in basal condition and under selective inhibition of NOS1 (SMTC 3µM). Results: Under basal conditions, NOS1 inhibition prolonged AP duration (APD) (152.6  11.7 ms vs 96.1  9.0 ms; 58.8%. p<0.01), enhanced ICaL density (peak current density at +10 mV, SMTC vs CTRL: -16.61.2 pA/pF vs -13.51.0 pA/pF; p<0.05) and did not affect IKs (SMTC vs CTRL: 2.50.4 pA/pF vs 2.60.2 pA/pF) and IKr (SMTC vs CTRL: 0.840.04 pA/pF vs 0.910.05 pA/pF). The -adrenergic agonist isoproterenol (ISO, 1nM) induced delayed afterdepolarizations (DADs), an index of SR instability, in a significantly greater percentage of SMTC treated cells, compared to control ones (93% for SMTC vs 22% for CTRL, p<0.01). Moreover, the average time of DADs appearance was significantly different between SMTC and CTRL myocytes, with a earlier rise after NOS1 inhibition (25.8 ± 3.8 s and 61.5 ± 15.3 s respectively, p<0.01). Furthermore, the duration of the AP is important for the occurrence of these events, as switching from a long AP (140 ms) to a short AP (100 ms) waveform under ISO application in AP clamp mode, transient inward currents (Iti) were abolished. Conclusions: These results indicate that NOS1 deficiency may contribute to APD prolongation and enhance Ca2+ influx; these effects compromise SR stability in the presence of adrenergic stimulation. The effects of NOS1 inhibition are such as to account for the arrhythmogenic effect of NOS1AP polymorphism.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease, where sudden cardiac death in young seemingly healthy persons may be the first symptom. There is a need for more sensitive and accurate diagnostic methods to detect signs of disease, at an early stage and in relatives of affected individuals. The aim of this thesis is the evaluation of new non-invasive modalities in assessment of right ventricular (RV) volume and function with focus on patients with ARVC. Clinical and non-invasive follow-up of fifteen patients with ARVC during a mean period of 8 years permitted the evaluation of disease progression. RV volume analysis by magnetic resonance imaging relies on short axis (SA) views. A new axially rotated modality acquisition was tested and its feasibility in assessment of RV volume was evaluated. This acquisition seems to be able to improve the assessment of RV volume and function by reducing the uncertainty in defining the basal slice of the RV. A third study concentrated on analysis of RV regional and general function by echocardiography, using tissue Doppler imaging as well as two dimensional (2D) longitudinal strain based on speckle tracking in patients with ARVC, their first degree relatives and in healthy subjects. 2D strain showed a good feasibility in analysis of the RV function in relatives and controls but less in ARVC patients probably due to the progressive myocardial cell death with fibro-fatty replacement of the RV wall. In order to detect and follow up echocardiographic changes an index was developed combining dimensional and functional parameters for the left and for the right ventricle. Advances in the molecular genetics of ARVC have provided new insights into the understanding of the disease. Hitherto, 9 candidate genes have been identified. A new mutation in the plakophilin 2 gene was detected in a three generation family. The clinical phenotype related to this mutation was investigated. The studies have evaluated and developed methods for studying the right ventricle with special emphasis on ARVC. With the ultimate goal of preventing sudden death in ARVC, a combination of genetic testing and improved diagnostic methods may create an improved algorithm for risk stratification and selection to prophylactic treatment.

Atrial fibrillation is the most prevalent form of cardiac arrhythmia. Studies in animal modelshave provided important insights into arrhythmia mechanisms. However, to date, we do not dispose ofanimal models of spontaneous atrial arrhythmia.Thus, we aimed to develop a model of spontaneous atrial arrhythmia in rats and to assesspathophysiological mechanisms of these arrhythmias by using a multidisciplinary approach. We alsoaimed to assess the presence and the extent of inflammation and endothelial dysfunction, incriminatedin atrial fibrillation-related complications such as stroke, in atrial fibrillation patients.The animal study describes the first animal model of spontaneous atrial arrhythmias. We alsoprovide evidence that multiple mechanisms participate in arrhythmia occurrence in this model,particularly autonomic imbalance with relative vagal hyperactivity, left atrial endocardial fibrosis, anddecreased left atrial expression of the Pitx2 gene. In our clinical study, we found high levels ofvascular endothelial growth factor and von Willebrand factor in atrial fibrillation patients compared tosinus rhythm controls. These results suggest specific thromboembolic risk patterns according to theclinical form of arrhythmia and highlight a parallel evolution of atrial fibrillation and endothelialdysfunction. These results add new insights into the understanding of atrial arrhythmias. This new animalmodel could facilitate studies of pathophysiological mechanisms involved in atrial arrhythmias andallow assessment of efficacy and toxicity of therapeutic agents in a setting that faithfully reproducesthe clinical presentation of the arrhythmia

Background Arrhythmogenic cardiomyopathy (AC) is a rare heart muscle disease characterized by fibrofatty myocardial replacement, prominent impairment of ventricular systolic function and arrhythmias. AC phenotypic spectrum was revealed wider than previously thought thanks to genotype–phenotype correlation. Combining multiple sources of clinical information, such as genetic, electrocardiographic, arrhythmic, morphofunctional, and histopathologic findings resulted the best approach to untangle the complexity of this disease. Aim The aim of our study was to assess AC genetic heterogeneity, applying appropriate genetic screening and performing a multiparametric genotype-phenotype correlation taking into account AC variants classification. Materials and Methods A total of 224 consecutive patients, with a clinical (n.192) or post mortem (n.32) diagnosis of AC, underwent genetic screening by using a 174 cardiac-related genes panel (Trusight Cardio, Illumina). The prevalence of Copy Number Variations (CNVs) and newly AC-associated genes such as FLNC and CDH2, were investigated. Detailed clinical data were obtained on 12-lead elettrocardiography, echocardiography, and cardiac magnetic resonance with the purpose of performing a multiparametric genotype-phenotype correlation. TTN variants were evaluated separately, due the magnitude of the gene, as well as rare variants in AC-unrelated genes. Finally, WES was carried out on 10 AC genotype negative patients in order to identify new candidate genes involved in the disease pathogenesis. Results We identified 95 different rare genetic variants in 97 (43%) of the 224 index cases. Of them, 79 variants were found in 5 major desmosomal genes (83%), whereas 16 in AC-related non desmosomal genes (17%). American College of Medical Genetics (ACMG)-based variant classification made evident that half of desmosomal variants (39/79, 49%) were classified as pathogenic/likely pathogenic and were predominantly radical (32/39, 78%). Comprehensive sequencing, including newly AC associated genes (FLNC and CDH2) and CNVs analysis, led to the identification of the genetic cause in 10 more patients increasing the overall yield of genetic screening from 43% to 48%. No pathogenic variants were identified in 117 (52%) patients. Overall we re-evaluated based on the current International Task Force Criteria (ITC) the clinical phenotype of 188 out of 224 AC (84%) index cases. Of these, 94 index cases received a definite AC diagnosis at the outpatient clinic, 16 were heart-transplanted (HTx) patients and 32 sudden death (SD) victims. The remaining 18 were borderline and 28 were possibly affected by AC. 78 of the 142 (55%) definite index cases carried at least a rare variant in AC related genes whereas, only 13 of the 46 (28%) borderline/possible index cases were genotype positive. Genotype analysis focusing on ventricular involvement highlighted that patients with Left Dominant variant (LDAC) were significantly less positive for desmosomal variants (11/42, 26%) compared to the “classic” AC cases (75/182, 41%)(p-value 0.0065). More in deep, 19 of the 42 (45%) LDAC patients were SD victims, of whom only 4 cases (21%) were genotype positive for desmosomal rare variants. Based on previous transcriptome studies from our laboratory we identified rare variants in LGALS3. Specifically, sequencing of 10 index cases through WES and 140 by direct sequencing, led to the identification of 5 LGALS3 rare nucleotide variants in 7 probands (4%, 5 males, mean age 39±11 years). Of note, two missense variants occurred in the protein carbohydrate recognition domain (CRD) conferring the loss of its binding site. Conclusions Comprehensive genetic analysis revealed a genetic cause in nearly half (48%) of AC patients, of which only half could be classified as P/LP. A proper phenotypic characterization increased variant finding likelihood in definite AC patients (55%). Nevertheless, half of AC patients still missed a genetic cause. Specifically, genetic testing achieved to identify a causative variant in only ~25% of LDAC cases. Finally a new candidate gene was identified in 4% of AC cases, supporting the fact that other genetic factors might be involved in disease pathogenesis. Most of identified genetic variants were variants of unknown significance (VUS), highlighting that cascade genetic screening remains mandatory to understand their significance in disease pathogenesis.

Background. Arrhythmogenic cardiomyopathy (AC) is an inherited myocardial disease characterized by fibro-fatty replacement of the myocardium and life-threatening arrhythmias. This genetically and phenotypically heterogeneous condition, caused mainly by mutations in desmosomal genes (JUP, DSP, PKP2, DSG2 and DSC2), exhibits reduced penetrance making challenging the diagnosis and the identification of a molecular mechanism underlying disease pathogenesis. Aims. (1) To identify one or more altered molecular pathways in AC carriers of desmosomal mutations; (2) to evaluate the diagnostic role of JUP immune analysis in AC; (3) to study the frequency of genetic variants in the five major AC-related genes in a healthy population of Veneto region in order to evaluate their role in the pathogenesis of the disease. Materials and methods. (1) Differential expression analysis was carried out on myocardial tissue of 7 transplanted AC patients harbouring a pathogenic mutation in desmosomal genes and 3 controls. Genetic/epigenetic interference-factors were unbiased analyzing also 3 mouse groups: 8 over-expressing NS-dsg2 mutation [TgNS], 6 over-expressing wild type dsg2 [TgWt] and 2Wt); each group was further subdivided in two age-groups (<2 weeks and >3 weeks) before and after the onset of disease. Data confirmation was obtained by quantitative-PCR. (2) Heart specimens (HS, either autopsy or transplants) and endomyocardial biopsies (EMB) formalin-fixed from 44 AC unrelated patients and 30 non-AC matched-subjects were evaluated both by conventional immunoperoxidase analysis (IPOX) and immunofluorescence (IF) using two different JUP antibody (Ab) dilutions. Reduced JUP signal level was defined as 50% reduction in distribution and/or intensity of the immunostained areas compared to the control samples. To exclude time-dependent tissue decay, control staining for N-Cadherin was also performed. (3) 200 unrelated young athletes (mean age 20 yrs, male/female ratio 3:1), eligible at the pre-participation clinical evaluation, underwent conventional genetic screening for major disease causative genes. Variants selection was based on the current ACMG guidelines and the absence or low frequency (minor allele frequency, MAF <0.0002) of the genetic variants in the general population. Results. (1) 1136 and 822 differentially expressed genes (DEGs) were respectively identified in the right and left human myocardium of AC compared to controls. 204 DEGs were identified comparing TgNS<2 weeks and TgNS>3 weeks gene expression profiling. 82 DEGs were identified comparing human and murine (TgNS>3 weeks) expression-profiling including genes most associated with canonical WNT/β-catenin and TGF-β pathways. On the contrary only 29 DEGs were identified in the comparison between TgNS<2 weeks to age-matched controls (WT and TgWt <2 weeks) mostly associated with inflammatory and pro-apoptotic process, but none with WNT and TGF-β pathways. (2) Test sensitivity (Se) of 70.6% and specificity (Sp) of 50%, with an Ab dilution 1:50.000 were found among HS, whereas with a 5-fold higher (1:250.000) Ab dilution the test Se was increased to 79.4% and the Sp decreased to 35%. Same analysis was performed on EMB samples showing different results: 40% Se; 80% Sp with 1:50.000 Ab dilution, whereas Se was 50% and Sp 70% with 1:250.000 Ab dilution. IF data were similar both with 1:1000 and 1:50.000 JUP-Ab dilution, indicating a Se of 61.8% and a Sp of 45% Sp in HS and a Se 50% and a Sp 70% in EMB samples. (3) Genetic screening identified rare genetic desmosomal variants in 20 healthy subjects (10%) reduced to 12 (6%) after appropriate filtering. Conclusions. Our findings demonstrated the interaction between WNT and TGF pathways at early disease stages, triggering cardiac remodelling. Specifically, we identified probably the ‘culprit molecules’ of disease onset. Routine IPOX and IF analysis of JUP signal is associated with low Se and limited Sp to be advocated as a diagnostic test. The absolute Se range was much higher in HS than EMB samples. The same for IF, HS specimens showed higher Se and lower Sp than EMB samples. Finally, high Ab dilutions confer higher Se but reduce test Sp. Comprehensive mutation screening and filtering in a large cohort of unrelated consecutive healthy subjects identified a lower rate of rare variants than the rates (16 and 18%) reported in literature for AC probably due to our selected cohort of healthy subjects.
Introduzione. La cardiomiopatia aritmogena (AC) è una patologia ereditaria del miocardio caratterizzata da sostituzione fibrosa e adiposa dei cardiomiociti e da aritmie potenzialmente letali. Questa condizione geneticamente e fenotipicamente eterogenea, causata principalmente da varianti genetiche a livello dei geni desmosomiali (JUP, DSP, PKP2, DSG2 e DSC2), mostra una penetranza incompleta; queste caratteristiche rendono difficile non solo la corretta diagnosi, ma anche l'identificazione di un meccanismo molecolare alla base della patogenesi della malattia. Obiettivi. (1) Identificare uno o più pathway molecolari alterati in pazienti AC portatori di mutazioni desmosomiali; (2) valutare il ruolo diagnostico dell'analisi immunologica della JUP; (3) studiare la frequenza delle varianti genetiche nei cinque principali geni correlati alla malattia in una popolazione sana della regione Veneto al fine di valutare il loro ruolo nella patogenesi dell’AC. Materiali e metodi. (1) L'analisi dell'espressione differenziale è stata condotta su tessuto miocardico di 7 pazienti AC precedentementi sottoposti a trapianto cardiaco, portatori di una variante patogena nei geni desmosomiali e 3 controlli. Al fine di eliminare i fattori di interferenza genetica/epigenetica sono stati analizzati anche 3 gruppi di un modello murino: 8 topi sovra-esprimenti la mutazione NS-dsg2 [TgNS], 6 sovra-esprimenti la dsg2 wild-type [TgWt] e 2 Wt; ciascun gruppo è stato ulteriormente suddiviso in base all’età (<2 settimane e >3 settimane) prima e dopo l'insorgenza della malattia. La conferma dei dati è stata ottenuta mediante PCR quantitativa. (2) Le analisi immunoistochimiche sono state eseguite sia su sezioni di miocardio (HS, autoptici o da trapianto) che su biopsie endomiocardiche (EMB) di 44 pazienti con diagnosi di AC e 30 soggetti non-AC. Tutti i casi sono stati valutati sia mediante colorazione con immunoperossidasi (IPOX) che mediante immunofluorescenza (IF) utilizzando due diverse diluizioni dell'anticorpo (Ab) che lega specificatamente la proteina JUP. Per escludere il decadimento tissutale tempo dipendente, è stata anche eseguita l’analisi di controllo sulla N-Caderina. (3) Lo screening genetico convenzionale per i principali geni responsabili delle patologia è stato eseguito su 200 giovani atleti (età media 20 anni, rapporto maschi / femmine 3: 1), considerati idonei all’attività sportiva. La selezione delle varianti era basata sulle attuali linee guida della ACMG e sull'assenza/bassa frequenza (frequenza allelica minore, MAF <0,0002) delle varianti genetiche nella popolazione generale. Risultati. (1) 1136 e 822 geni differenzialmente espressi (DEGs) sono stati identificati rispettivamente nel ventricolo destro e nel ventricolo sinistro dei pazienti AC. Sono stati identificati inoltre, 204 DEGs comparando il profilo di espressione genica di TgNS<2 settimane e TgNS> 3 settimane. Infine, confrontando il profilo di espressione umano con quello murino sono stati identificati 82 DEGs in comune; molti di questi geni sono associati ai pathway WNT e TGF-β. Al contrario, solo 29 DEGs sono stati identificati nel confronto tra TgNS <2 settimane e i controlli di età corrispondente (WT e TgWt <2 settimane); questi geni sono per lo più associati a processi infiammatori e pro-apoptotici, ma nessuno ai pathway WNT e TGF-β. (2) L’analisi IPOX eseguita sui campioni HS con una diluizione dell’Ab 1: 50.000 mostra una sensibilità (Se) del 70,6% e la specificità (Sp) del 50%, mentre con una diluizione Ab 5 volte più alta (1: 250.000) la Se del test aumenta al 79,4% e la Sp diminuisce al 35%. La stessa analisi eseguita su campioni EMB restituisce come risultato: 40% Se; 80% Sp con diluizione anticorpale di 1: 50.000, mentre alla diluizione 1: 250.000 la Se si attesta a 50% e la Sp al 70%. I dati risultanti dall’analisi IF sono simili sia con diluizione dell’anticorpo 1: 1000 che 1: 50.000, indicando una Se del 61.8% e una Sp del 45% per gli HS e una Se 50% e una Sp 70% nei campioni EMB. (3) Lo screening genetico dei 5 geni desmosomiali ha identificato varianti genetiche rare in 20 soggetti sani (10%) ridotti a 12 (6%) dopo un appropriato filtraggio. Conclusioni. I nostri risultati hanno dimostrato l'interazione tra WNT e TGF-β nelle fasi iniziali della malattia, determinando il rimodellamento cardiaco. Nello specifico, abbiamo identificato probabilmente le "molecole colpevoli" dell'insorgenza della malattia. L'analisi del segnale immunologico della JUP mostra in generale una bassa Se e ad una limitata Sp. Nello specifico, nell’analisi la Se risulta più elevata nei campioni HS rispetto ai campioni EMB. Alte diluizioni anticorpali conferiscono valori di Se superiori ma riducono la Sp del test. Lo screening genetico e la successiva analisi delle varianti individuate in un'ampia coorte di soggetti sani ha identificato un tasso minore di varianti rare rispetto a quanto riportato in letteratura per la AC (16 e 18%), probabilmente ciò è dovuto alla pre-selezione fatta sui soggetti inseriti nella coorte analizzata.

Background & Aims: Arrhythmogenic Cardiomyopathy (AC) is a rare inherited heart muscle disease associated with mutations in genes encoding mainly components of the cardiac desmosome. We performed a comprehensive study of genetic variants in a cohort of AC subjects and the assessment of Next Generation Sequencing (NGS) strategies for molecular diagnosis of AC. Methods: Ninety-nine unrelated index cases, of which 26 sudden cardiac death cases, underwent genetic screening for 5 desmosomal genes by denaturing high performance liquid chromatography and direct sequencing, whereas 46 probands were additionally screened for 3 extra desmosomal genes. A complementary analysis for copy number variants (CNVs) was performed by multiplex ligation-dependent probe amplification and quantitative real-time PCR in the entire cohort. A 4-step variant filtering strategy based on mutation type, frequency, evolutionary conservation and in silico analysis, was used. Whole Exome and Targeted NGS strategies were performed on Illumina platforms in order to test methods efficacy. Results: Screening of 8 AC genes and subsequent 4-step variants filtering identified 37 different point desmosomal mutations in 42 AC probands (42%). The most frequently mutated genes resulted PKP2 and DSP, with “radical” mutation type accounting for the 80% of the PKP2 variants. No pathogenic mutations were identified in the extra desmosomal genes analyzed. CNVs analysis further revealed 3 different large genomic rearrangements in 5 probands (4%), increasing to 46 (46%) the number of positively genotyped patients. PKP2 and DSP single mutation accounted respectively for 20% and 11% of the cohort, with DSP carriers showing a higher risk of sudden cardiac death. Eight multiple mutations carriers were observed (8%). NGS approaches identified 4 variants in extra desmosomal genes allowing a differential diagnosis in 4 patients. Conclusions: A fine variant filtering avoids overrepresentation of putative pathogenic mutations and shows that radical and missense mutations should be equally interpreted with great caution in the setting of clinical diagnosis. NGS and CNVs analysis increased significantly the diagnostic yield in AC genetic testing. The genetics of AC is more complex than previously appreciated, with frequent requirement for more than one ‘hit’ for penetrant disease.
Introduzione & Scopo: La Cardiomiopatia Aritmogena (AC) è una malattia rara del muscolo cardiaco associata a mutazioni a carico di geni che codificano principalmente per componenti del desmosoma cardiaco. Abbiamo realizzato l`analisi genetica in una coorte di soggetti affetti da AC e lo sviluppo di strategie in Next Generation Sequencing (NGS) per la diagnosi molecolare di AC. Metodi: Novantanove casi indice, di cui 26 soggetti di morte improvvisa, sono stati sottoposti a screening genetico per 5 geni desmosomiali mediante cromatografia denaturante in fase liquida ad alto rendimento e sequenziamento diretto, 46 probandi sono stati analizzati anche per 3 geni extra desmosomiali. Abbiamo inoltre ricercato nell`intera popolazione varianti del numero di copie (CNVs) mediante la tecnica MLPA (Multiplex Ligation-Dependent Probe Amplification) e PCR quantitativa. La strategia di filtraggio si è basata sul tipo di mutazione, frequenza, conservazione, e analisi in silico. Gli approcci NGS “Whole Exome” e “Targeted” sono stati eseguiti su piattaforme Illumina. Risultati: L`analisi di 8 geni associati alla AC e il successivo filtraggio delle varianti ha individuato 37 diverse mutazioni puntiformi in 42 soggetti (42%). I geni più frequentemente mutati sono la PKP2 e la DSP, le mutazioni “radicali” costituiscono l`80% delle varianti della PKP2. Non sono state riscontrate mutazioni patogene nei geni extra desmosomiali studiati. La ricerca di CNVs ha identificato 3 diversi riarrangiamenti cromosomici in 5 probandi (4%), portando a 46 (46%) i soggetti genotipo positivi. Si sono osservate mutazioni singole nei geni PKP2 e DSP nel 20% e nell`11% dei soggetti, i portatori di mutazioni nella DSP presentano un rischio maggiore di morte improvvisa, 8 (8%) soggetti presentano mutazioni multiple. Le analisi mediante NGS hanno indentificato 4 varianti in geni extra desmosomiali permettendo la diagnosi differenziale in 4 pazienti. Conclusioni: La diagnosi molecolare in ambito clinico rende necessaria un`attenta interpretazione del potenziale patogeno tanto delle mutazioni missenso quanto di quelle radicali soprattutto dopo il cospicuo aumento di varianti identificate con NGS e la ricerca di CNVs. La base genetica di AC è molto più complessa di quanto finora apprezzato, con una frequente presenza di più di una mutazione per una penetranza completa della malattia.

Background: Arrhythmogenic cardiomyopathy (AC) is a clinically and genetically heterogeneous myocardial disease, characterised by a progressive myocardial dystrophy with fibro-fatty replacement, and represents one of the major causes of sudden cardiac death in the young and athletes. Although half of AC patients harbour private desmosomal gene mutations, their low and age-dependent penetrance suggests the involvement of other regulatory molecules. MicroRNAs (miRNAs) are a group of endogenous short noncoding RNAs that regulate gene expression by sequence-specific recognition of their target transcripts. They have been associated with numerous pathophysiological conditions, including cardiovascular diseases; however, their role as key regulatory molecules in AC as well as their impact on the onset and progression of the disease is largely unknown. Purpose: miRNA profiling in genotype-positive AC-patients with different gene mutations in order to identify their potential as AC biomarkers. Methods: The study involved 59 subjects with a definite AC diagnosis, previously genotyped, and 14 healthy controls. 84-miRNA array was applied on 8 frozen right-ventricle (RV) myocardial tissue samples, from heart transplanted AC patients; 9 whole blood samples, from patients with definite AC diagnosis, and 6 healthy controls (HC). In the validation study, seven miRNAs were analysed on 42-AC and 8-HC blood samples. miRNA analysis was performed by qPCR, relative quantification ΔΔCt method and in silico target prediction. All data were expressed in fold-change values. Receiver operating characteristic (ROC) analysis was performed on validated miRNAs. Results: miRNA profiling on AC-tissue samples displayed a genotype-related profile, 19 miRNAs were differentially expressed in PKP2 carriers, 15 in DSP carriers and 14 in DSG2 carriers, when compared to healthy controls. A common signature between PKP2 and DSP carriers was identified with 14 miRNAs in common (PKP2/DSP profile). None of these miRNAs were shown within DSG2 profile. In silico target prediction identified Hippo Signaling Pathway as a common target for both profiles. Analysis of AC-tissue samples as a unique group confirmed 26 differentially expressed miRNAs (AC-tissue profile) with predicted targets in the AC pathway. AC-blood miRNA profiling demonstrated a 14-miRNA signature, with 10 miRNAs differentially expressed in common with AC-tissue profile. Hsa-miR-144-3p, -122-5p, -208a-3p and -494-3p as well as hsa-miR-21-5p, -155-5p and -320a were analysed on a larger cohort of 42-AC and 8-HC. Only hsa-mir-122-5p was significantly overexpressed (p-value<0,05). ROC analysis showed hsa-miR-122-5p to be a potential AC biomarker (area under the curve: 0.83). Conclusions: A genotype-related miRNA profile was observed in AC-tissue samples, as to reflect clinical variability. In addition, 10 miRNAs in common were identified between AC-tissue and AC-blood profiles, proving a specific miRNA signature for AC. These miRNA profiles targeted pathways involved in AC pathogenesis demonstrating their key roles in the onset and progression of the disease. Circulating level of hsa-miR-122-5p was significantly elevated in AC subjects, demonstrating its potential as a prognostic marker for heart failure in AC.
Introduzione. La Cardiomiopatia Aritmogena (AC) è una malattia clinicamente e geneticamente eterogenea del miocardio, caratterizzata da una progressiva distrofia miocardica con sostituzione fibro-adiposa, e rappresenta una delle principali cause di morte improvvisa nei giovani e negli atleti. Nonostante circa la metà dei pazienti affetti da AC presentino mutazioni nei geni desmosomiali, la bassa penetranza e dipendenza dall’età della patologia suggeriscono il coinvolgimento di altre molecole regolatrici. I microRNA (miRNA) sono un gruppo di molecole endogene, di RNA non codificante, che regolano l'espressione genica mediante lo specifico riconoscimento di sequenze target dei trascritti. Sono stati associati a numerose condizioni patofisiologiche, tra cui malattie cardiovascolari; tuttavia, il loro ruolo come molecole regolatrici nella AC e il loro impatto sull'insorgenza e sulla progressione della malattia è in gran parte sconosciuto. Scopo dello studio. Analizzare il profilo di espressione dei miRNA in pazienti affetti da AC genotipicamente positivi allo scopo di studiare il loro potenziale come biomarcatori prognostici. Materiali e metodi. Lo studio ha coinvolto 59 soggetti con una diagnosi clinica di AC, precedentemente genotipizzati, e 14 controlli sani (HC). Un array composto da 84-miRNA è stato testato su: 8 campioni di tessuto miocardico congelato del ventricolo destro, proveniente da pazienti trapiantati affetti da AC; 9 campioni di sangue intero congelato, da pazienti con diagnosi clinica di AC e 6 controlli sani. Nella fase di validazione sono stati analizzati sette miRNA su campioni di sangue provenienti da 42-AC e 8-HC. L'analisi è stata eseguita mediante qPCR seguita da quantificazione relativa con il metodo ΔΔCt e predizione in silico dei geni target. I risultati sono stati espressi in valori di “fold-change” e le curve ROC (Receiver Operating Characteristic) analizzate sui miRNA validati. Risultati. L’analisi dei miRNA su 8 campioni di tessuto di pazienti affetti da AC mostrava un profilo correlato al genotipo rispetto ai controlli sani, in particolare: 19 miRNA erano differenzialmente espressi nei portatori di una mutazione in PKP2, 15 nei portatori di una mutazioni in DSP e 14 nei portatori di una mutazione in DSG2. E’ stato identificato un profilo d’espressione in comune tra i portatori della mutazione in PKP2 e i portatori della mutazione in DSP, con 14 miRNA alterati (profilo PKP2/DSP). Nessuno di questi miRNA è stato trovato nel profilo DSG2. Lo studio in silico dei possibili geni target ha identificato la via di segnale “Hippo Signaling Pathway” come target comune per entrambi i profili (PKP2/DSP- DSG2). Considerando i campioni di tessuto AC come un unico gruppo indipendentemente dal gene mutato sono emersi 26 miRNA differenzialmente espressi (profilo AC-tessuto) che hanno come target geni coinvolti nel pathway AC. Lo studio dei miRNA nei 9 campioni di sangue dei pazienti affetti da AC ha dimostrato un profilo costituito 14-miRNA alterati, dei quali 10 alterati anche nel profilo AC-tessuto. Hsa-miR-144-3p, -122-5p, -208a-3p e -494-3p così come hsa-miR-21-5p, -155-5p e -320a sono stati infine validati su una coorte più ampia di 42-AC e 8-HC. Solo hsa-mir-122-5p è stato riscontrato come significativamente sovraespresso (valore p <0,05). L'analisi di curve ROC ha mostrato che hsa-miR-122-5p è un potenziale biomarcatore di AC (AUC: 0.83). Conclusione. Nei campioni AC di tessuto è stato osservato un profilo di miRNA correlato al genotipo, tale da rispecchiare la variabilità clinica della patologia. Inoltre, sono stati identificati 10 miRNA in comune tra i profili dei campioni AC di tessuto e sangue, evidenziando un profilo di espressione di miRNA specifico per AC. Entrambi i profili infatti (tessuto e sangue) hanno come target vie di segnale coinvolte nella patogenesi della AC, dimostrando un ruolo chiave nella insorgenza e la progressione della malattia. In particolare il livello di hsa-miR-122-5p in circolo era significativamente elevato nei soggetti affetti da AC, dimostrando il suo potenziale come marcatore prognostico della malattia.

Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic heart muscle disease characterized by electrical instability leading to ventricular arrhythmias and sudden cardiac death. The hallmark pathological lesion of ARVC is the transmural loss of the myocardium of the right ventricular (RV) free wall with replacement by fibro-fatty tissue. Three-dimensional electroanatomic voltage mapping (EVM) by CARTO system (Biosense-Webster, Diamond Bar, California) allows identification and characterization of low-voltage regions, i.e. "electroanatomical scars" (EAS), which in patients with ARVC correspond to areas of fibro-fatty replacement. Although the technique has been demonstrated to enhance the accuracy for diagnosing ARVC, its value for arrhythmic risk stratification remains to be established. Furthermore, the clinical utility of EVM for scar quantification and risk assessment is limited by its invasive nature, low availability and high costs. Thus, in daily clinical practice there is the need of a non-invasive test such as 12-lead electrocardiogram (ECG) for prediction of the amount of RV myocardial scar lesion and assessment of arrhythmic risk. Previous studies demonstrated an association between ECG repolarization/depolarization abnormalities and RV mechanical dilation/dysfunction. In fact, T wave inversion in right pericardial leads is the most common ECG abnormality of ARVC. However, the presence of T wave inversion in leads V1-V3, known as persistence of the juvenile pattern of repolarization, may also be observed in about 3% of healthy adults. The current perspective is that, at variance with healthy subjects, right precordial NTWs persist with exercise in ARVC patients. However, this view is not supported by systematic scientific data. Objective In this work, we aimed to further study some of the electrocardiographic features of ARVC. First, we assessed the prognostic value of EAS detected by EVM and its correlation with various non-invasive characteristics of ARVC, including abnormalities detected by surface ECG. Second, we studied the exercise-induced changes in right precordial negative T waves in patients with ARVC and in a group of healthy young individuals with persistence of the juvenile repolarization pattern Methods and results We first studied 69 consecutive ARVC patients (47 males; median age 35 years [28-45]) who underwent electrophysiological study and both bipolar and unipolar EVM. The extent of confluent bipolar (<1.5 mV) and unipolar (<6.0 mV) low-voltage electrograms was estimated using the CARTO-incorporated area calculation software. Fifty-three patients (77%) showed ≥1 RV electroanatomic scars with an estimated burden of bipolar versus unipolar low voltage areas of 24.8% (7.2-31.5) and 64.8% (39.8-95.3), respectively (P=0.009). In the remaining patients with normal bipolar EVM (n=16; 23%), the use of unipolar EVM unmasked ≥1 region of low-voltage electrogram affecting 26.2% (11.6-38.2) of RV wall. During a median follow-up of 41 (28-56) months, 19 (27.5%) patients experienced arrhythmic events. At multivariate analysis, the only independent predictor was the bipolar low-voltage electrogram burden (hazard ratio=1.6 per 5%; 95% confidence interval, 1.2-1.9; P<0.001). Patients with normal bipolar EVM had an uneventful clinical course. Then we further analyzed a subgroup including 49 patients [38 males, median age 35 years] with ARVC and an abnormal EVM by CARTO system. At univariate analysis, the presence of epsilon waves, the degree of RV dilation, the severity of RV dysfunction and the extent of negative T-waves correlated with RV-EAS% area. At multivariate analysis, the extent of negative T-waves remained the only independent predictor of RV-EAS% area (B=4.4, 95%CI 1.3-7.4, p=0.006) and correlated with the arrhythmic event-rate during follow-up (p=0.03). In a different cohort, we assessed the prevalence and relation to the clinical phenotype of exercise-induced right precordial negative T wave changes in 35 ARVC patients (19 males, mean age 22.2±6.2 years). Forty-one healthy individuals with right-precordial negative T waves served as controls. At peak of exercise, negative T waves persisted in 3 ARVC (9%) patients, completely normalized in 12 (34%) and partially reverted in 20 (57%). ARVC patients with or without negative T waves normalization showed a similar clinical phenotype. The overall prevalence of right precordial T-waves changes during exercise (normalization plus partial reversal) did not differ between ARVC patients and controls (92% versus 88%, p=1.0), while there was a statistically non significant trend towards a higher prevalence of complete normalization in controls (59% versus 34%, p=0.06). Conclusion In conclusion, our results showed that the extent of bipolar RV endocardial low-voltage area was a powerful predictor of arrhythmic outcome in ARVC independently of arrhythmic history and RV dilatation/dysfunction. A normal bipolar EVM characterized a low-risk subgroup of ARVC patients. Patients with abnormal ECG have a more severe RV EAS involvement, which is proportional to the extent of T wave inversion across ECG 12-leads and a higher arrhythmic risk. The absence of negative T waves characterizes a low-risk subgroup of ARVC patients with a more favorable clinical course because of a low rate of arrhythmic events. The results also showed that exercise-induced changes of negative T waves were unrelated to ARVC phenotypic manifestations and were of limited value for the differential diagnosis between ARVC and benign persistence of the juvenile repolarization pattern
Introduzione La cardiomiopatia aritmogena del ventricolo destro (CAVD) è una patologia genetica del muscolo cardiaco caratterizzata da instabilità elettrica che può portare a aritmie ventricolari e morte improvvisa. Dal punto di vista patologico, la CAVD si caratterizza per una progressiva perdita di tessuto miocardico della parete libera del ventricolo destro (VD) con sostituzione fibro-adiposa. Il mapaggio elettroanatomico tridimensionale (endocardial voltage mapping, EVM) col sistema CARTO (Biosense-Webster, Diamond Bar, California) consente di identificare e caratterizzare aree di basso-voltaggio, dette "cicatrici elettroanatomiche" (CEA), che in pazienti affetti da CAVD corrispondono ad aree di sostituzione fibro-adiposa. Nonostante la tecnica abbia dimostrato di migliorare l"accuratezza per la diagnosi di CAVD, il suo valore per la stratificazione del rischio aritmico rimane da dimostrate. Inoltre, l"utilità dell"EVM per la quantificazione della cicatrice e la valutazione del rischio è limitata dalla natura invasiva, bassa disponibilità ed alti costi. Quindi, nella pratica clinica quotidiana è auspicabile la disponibilità di un esame non-invasivo, come l"elettrocardiogramma (ECG), per la stima dell'estensione della CEA e la stratificazione del rischio aritmico. Studi precedenti hanno dimostrato un"associazione tra la presenza di anomalie della ripolarizzazione o della depolarizzazione all"ECG e l"entità della dilatazione e della disfunzione del VD. In particolare, l"inversione delle onde T nelle derivazioni precordiali destre V1-V3 è uno dei segni distintivi della CAVD. Tuttavia, lo stesso segno ECG può essere riscontrato come "persistenza del pattern giovanile di ripolarizzazione" fino al 3% degli adulti sani. La prospettiva attuale è che le T negative persistano con l'esercizio nei pazienti con CAVD ma non nei soggetti sani. Tuttavia, questa idea non è supportata da dati scientifici. Obbiettivo L"obbiettivo dell"attività di ricerca è stato quello di caratterizzare ulteriormente alcune delle caratteristiche ECG della CAVD. Inizialmente, abbiamo valutato il valore prognostico della CEA all"EVM e la sua correlazione con vari esami non invasivi, in particolare l"ECG. In secondo luogo, abbiamo studiato le modificazioni indotte dall"esercizio nella T negative nelle derivazioni precordiali destre in un gruppo di pazienti con CAVD ed in un gruppo di soggetti sani con "persistenza del pattern giovanile di ripolarizzazione". Metodi e risultati Sono stati studiati 69 pazienti consecutivi affetti da CAVD (47 maschi, età mediana 35 [28-45] anni) che sono stati sottoposti a studio elettrofisiologico endocavitario con mappa di voltaggio unipolare e bipolare. L"estensione delle aree contenenti elettrogrammi di basso voltaggio bipolari (<1.5 mV) e/o unipolari (<6.0 mV) è stata stimata usando un software incorporato nel sistema CARTO. Cinquantatre pazienti (77%) mostravano ≥1 CEA con un"estensione pari a 24.8% (7.2-31.5) dell"estensione del VD alla mappa bipolare e del 64.8% (39.8-95.3) alla mappa unipolare (p=0.009). Nei rimanenti 16 pazienti con mappa bipolare normale, la mappa unipolare è risultata alterata con un"estensione delle lesioni pari al 26.2% (11.6-38.2) del VD. Nel corso di un follow-up medio di 41 (28-56) mesi, 19 (27.5%) pazienti hanno avuto un evento aritmico maggiore. All'analisi multivariata, l'unico predittore indipendente di eventi aritmici è risultata l'estensione della CEA alla mappa di voltaggio bipolare (hazard ratio=1.6 per 5%; intervallo di confidenza 95%: 1.2-1.9; P<0.001). I pazienti con mappa di voltaggio bipolare negativa hanno avuto un follow-up privo di eventi. Successivamente, abbiamo analizzato un sottogruppo di 49 pazienti (38 maschi, età mediana 35 anni) con CAVD e mappa di voltaggio bipolare positiva. All'analisi univariata, la presenza di onde epsilon, il grado di dilatazione del VD, la severità della disfunzione del VD e l'estensione delle T negative all'ECG correlavano con l'estensione della CEA alla mappa bipolare. All'analisi multivariata, l'estensione delle onde T negative è rimasta l'unico predittore di estensione della CEA (B=4.4, 95%CI 1.3-7.4, p=0.006). Questo parametro si è inoltre dimostrato correlare con il rischio di eventi aritmici durante il follow-up (p=0.03). In una coorte differente, abbiamo valutato il comportamento durante test da sforzo delle T negative nelle derivazioni precordiali destre V1-V4 in 35 pazienti con CAVD (19 maschi, età media 22.2"±6.2 anni) ed in 41 controlli appaiati per età e sesso con benigna "persistenza del pattern giovanile di ripolarizzazione". Al picco dell'esercizio, le onde T negative persistevano in 3 (9%) pazienti con CAVD, normalizzavano completamente in 12 (34%) e normalizzavano parzialmente in 20 (57%). I pazienti affetti da CAVD con e senza normalizzazione delle onde T durante l'esercizio mostravano un fenotipo simile. La prevalenza di normalizzazione (parziale o completa) delle onde T era simile nei pazienti e nei controlli (92% e 88%, p=1,0), mentre si è notato un trend non significativo verso una più alta prevalenza di normalizzazione completa nei controlli sani rispetto ai pazienti con CAVD (59% e 34%, p=0,06). Conclusioni In conclusione, i nostri risultati hanno mostrato che l'estensione della CEA bipolare alla mappa di voltaggio del VD è un potente predittore di rischio aritmico nei pazienti con CAVD, indipendentemente dalla storia aritmica e dal grado di disfunzione/dilatazione del VD. Una mappa di voltaggio bipolare normale caratterizza una popolazione di pazienti con CAVD a basso rischio. Abbiamo inoltre dimostrato che l'estensione della CEA bipolare può essere stimata dall'estensione delle anomalie della ripolarizzazione (T negative) all'ECG. I pazienti che non mostrano T negative all'ECG dimostrano un basso rischio aritmico. Infine, abbiamo dimostrato che il comportamento delle T negative nelle derivazioni precordiali destre V1-V3 non è un utile strumento di diagnosi differenziale tra CAVD e benigna "persistenza del pattern giovanile di ripolarizzazione"

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable disorder characterised by progressive degeneration of the right ventricular myocardium, arrhythmias and an increased risk of sudden death at a young age. Fourteen chromosomal loci have been linked to ARVC and nine disease genes have been identified. Linkage analysis of a South African family was previously performed at ARVC loci 1 to 6. ARVC loci 1 to 5 were excluded as disease loci in this family based on lack of evidence for linkage. However, a peak lod score of 2.93 was obtained for the ARVC-6 locus which is highly suggestive of linkage. Subsequently another locus (ARVC-7) and five ARVC disease genes (ARVC loci 8 to 12) have been reported. The aim of this project was to identify the disease gene that causes ARVC in this family.

Cardiomyopathy accounts for 20-30% of acute heart failure cases in adult Africans. Several types of cardiomyopathy have been identified; this study focused primarily on the genetic causes of arrhythmogenic right ventricular cardiomyopathy (ARVC). Many genes are implicated in ARVC pathogenesis, but many remain to be identified. We investigated a South African family (ACM2) with autosomal dominant ARVC, for whom the genetic cause of disease was unknown. Extensive genetic analysis was previously performed using genome-wide linkage analysis, but no disease-causing genetic variant was identified. We subsequently performed candidate gene screening of the phospholamban (PLN) gene, genome-wide copy number variant (CNV) analysis and whole exome sequencing to identify the causal genetic variant. The ACM2 family harboured no disease-causing PLN variants. However, on screening all cardiomyopathy cases in our registry (ARVC, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy and peripartum cardiomyopathy), we identified a known pathogenic PLN variant (c.25C>T; p.R9C) in a DCM family of European descent. This variant was reported in an American DCM family of European descent. Haplotype analysis revealed independent variant origins in these families. CNV analysis revealed no disease-causing variants in the ACM2 family. Whole exome sequencing of two affected ACM2 family members revealed 38 variants shared by these individuals. Variants were verified in family members and population controls by high resolution melt analysis and Sanger sequencing, and by bioinformatics analysis to predict variant pathogenicity. A novel N-cadherin (CDH2) c.686A>C (p.Q229P) variant segregated with ARVC in the ACM2 family and was bioinformatically predicted to be deleterious. An additional pathogenic CDH2 variant (c.1219G>A (p.D407N)) was identified in another individual with ARVC after screening 85 cases. These CDH2 variants were absent in normal population controls. Furthermore, alterations in Cdh2 are known to cause cardiomyopathy in rodent models. Taken together, these findings support the causal role of N-cadherin gene variants in human cardiomyopathy.

Includes bibliographical references (leaves 84-90).
Heart failure is a major public health problem throughout the world. In South Africa 17% of mortality is attributed to cardiovascular disease (CVD). Heart failure may be either ischemic or non-ischemic in origin. A significant proportion of non-ischemic heart failur is due to cardiomyopathy. There are currently five types of cardiomyopathy recognised, of which arrhythmogenic right ventricular cardiomyopathy (ARVC) is one. ARVC is familial in 30 to 50% of cases and it is inherited in an autosomal dominant or an autosomal recessive manner. Twelve chromosomal loci have been linked to ARVC and six genes have been identified. In 2004 Asano and colleagues reported a mouse model of ARVC that established LAMRI and CBX5 as candidate genes for the human form of ARVC.

A recent proposal is that the alpha₁-adrenoceptor may mediate the arrhythmogenic effect of catecholamines during acute myocardial ischaernia. The purpose of this thesis was to explore the role of alpha₁ and alpha₂-adrenoceptor stimulation on vulnerability to ventricular fibrillation in the norrnoxic rat ventricular myocardium and further to evaluate the possible underlying cellular mechanism. The model used was the isolated perfused rat heart (Langendorff technique) in which ventricular fibrillation was electrically induced. The amount of current required to produce ventricular fibrillation was measured as the ventricular fibrillation threshold. Alpha₁-adrenoceptor stirnμlation with methoxamine to 10⁻⁶M to 10⁻⁵M increased the vulnerability to ventricular fibrillation. The arrhythmogenic effect of methoxamine could not be attributed to beta-adrenoceptor stimulation as it occurred in the setting of the beta-adrenoceptor antagonist agent, atenolol; furthermore no accumulation of cyclic AMP, the proposed arrhythmogenic second messenger of beta-adrenoceptor stimulation, occurred. Similarly no alteration in heart rate, coronary flow rate or myocardial high energy phosphate content accompanied the arrhythrnogenic effect of methoxamine. The QT interval increased with alpha₁-adrenoceptor stimulation, this being an indirect index of prolongation of the action potential duration. The arrhythmogenic action of methoxamine was associated with a positive inotropic effect. Prazosin 10⁻⁸M (an alpha₁-adrenoceptor antagonist agent) produced a tenfold displacement to the right of the log concentration response curve of the positive inotropic effect of methoxamine. Prazosin 10⁻⁸M prevented the methoxamine induced fall in ventricular fibrillation threshold. Alpha₂-adrenoceptor stimulation with B-HT 920 and B-HT 933 (azepexole), in the presence of the beta-adrenoceptor antagonist agent atenolol, did not alter the vulnerability to ventricular fibrillation. Alpha₂-adrenoceptor stimulation produced no alteration in heart rate, coronary flow rate or metabolic status. We next explored the possible mechanism underlying the arrhythmogenic effect of methoxamine. Alpha₁-adrenoceptor stimulation enhances transsarcolemmal calcium ion influx and may induce sarcoplasmic reticulum calcium release. To assess the role of transsarcolemmal calcium movement in alpha₁-adrenoceptor mediated effects experiments were undertaken with nisoldipine and low extracellular calcium. To evaluate the role of sarcoplasmic reticulum calcium release, experiments were undertaken with ryanodine (an agent reputed to inhibit sarcoplasmic reticulum calcium release without effecting the slow inward current). Nisoldipine 10⁻⁸M, reducing extracellular calcium (2.5 mM to 1.25 mM) and ryanodine 10⁻⁹M to 10⁻⁸M, prevented the arrhythmogenic and positive inotropic effect of methoxamine. Heart rate, metabolic status and cyclic AMP levels we're unchanged with these procedures. The mechanism underlying the arrhythmogenic action of alpha₁-adrenoceptor stimulation might be an increase in cytosolic calcium concentration. This increase may be secondary to (i) an enhanced transsarcolemmal calcium influx or (ii) an increase in the phasic release of calcium from the sarcoplasmic reticulum.

Background: Endocardial voltage mapping (EVM) identifies low-voltage right ventricular (RV) areas, which may represent the electroanatomic scar substrate of life-threatening tachyarrhythmias. We prospectively assessed the prognostic value of EVM in a consecutive series of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Methods: We studied 69 consecutive ARVC patients [47 males; median age 35 years(28-45)] who underwent electrophysiological study and both bipolar and unipolar EVM. The extent of confluent bipolar (<1.5mV) and unipolar (<6.0mV) low-voltage electrograms was estimated using the CARTO-incorporated area calculation software. Results: Fifty-three patients (77%) showed ≥1 RV electroanatomic scars with an estimated burden of bipolar vs unipolar low-voltage areas of 24.8% (7.2-31.5) and 64.8% (39.8-95.3), respectively (P=0.009). In the remaining patients with normal bipolar-EVM (n=16;23%), the use of unipolar EVM unmasked ≥1 region of low-voltage electrogram affecting 26.2% (11.6-38.2) of RV wall. During a median follow-up of 41 (28-56) months, 19(27.5%) patients experienced arrhythmic events, such as sudden death (n=1), appropriate ICD interventions (n=7), or sustained ventricular tachycardia (n=11). Univariate predictors of arrhythmic outcome included previous cardiac arrest or syncope (HR=3.4; 95%CI:1.4-8.8; P=0.03) and extent of bipolar low-voltage areas (HR=1.7 per 5%; 95%CI=1.5-2; P<0.001), while the only independent predictor was the bipolar low-voltage electrogram burden (HR=1.6 per 5%; 95% CI:1.2-1.9; P<0.001). Patients with normal bipolar-EVM had an uneventful clinical course. Conclusions: The extent of bipolar RV endocardial low-voltage area was a powerful predictor of arrhythmic outcome in ARVC, independently of history and RV dilatation/dysfunction. A normal bipolar-EVM characterized a low-risk subgroup of ARVC patients.
Introduzione: Il mappaggio elettroanatomico mediante sistema CARTO permette di identificare e quantificare aree di basso voltaggio del ventricolo destro che corrispondono a cicatrici elettroanatomiche, substrato di aritmie ventricolari pericolose per la vita. Lo scopo dello studio era di valutare, in modo prospettico, il valore prognostico del mappaggio elettroanatomico in una coorte di pazienti affetti da Cardiomiopatia Aritmogena del Ventricolo Destro. Materiali e Metodi: La popolazione di studio includeva 69 pazienti (47maschi; età mediana 35 anni; 28-35) affetti da Cardiomiopatia Aritmogena del Ventricolo Destro. Tutti i pazienti sono stati sottoposti ad un completo work up clinico che includeva: elettrocardiogramma, ecocardiografia, cateterismo cardiaco, studio elettrofisiologico e mappaggio elettroanatomico del ventricolo destro, utilizzando sia mappe bipolari sia unipolari. L’estensione degli elettrogrammi confluenti di basso voltaggio bipolari (<1.5 mV) e unipolari (<6.0 mV) è stata stimata usando un software incorporato nel sistema CARTO. Risultati: In cinquantatre pazienti (77%) è stata riscontrata ≥1 regione cicatriziale a carico del ventricolo destro con una percentuale stimata di aree di basso voltaggio bipolari e unipolari rispettivamente di 24.8% (7.2-31.5) e 64.8 (39.8-95.3), rispettivamente (P=0.009). In tutti pazienti con una normale mappa bipolare (n= 16; 23%) l’utilizzo del mappaggio unipolare ha identificato ≥1 regione con elettrogrammi di basso voltaggio che interessava il 26.2% (11.6-38.2) del ventricolo destro. Durante un follow-up di 41 (28-56) mesi 19 (27.5%) pazienti subirono eventi aritmici maggiori, quali morte improvvisa (n=1), intervento appropriato dell’ICD (n=7), o tachicardia ventricolare sostenuta (n=11). All’analisi univariata i predittori dell’outcome aritmico includevano: sincope (HR=3.4; 95%CI: 1.4-8.8; P=0.03), e l’estensione delle aree di basso voltaggio bipolare (HR=1.7 per 5%; 95%CI: 1.5-2; P<0.001). All’analisi multivariata, l’unico predittore indipendente risultava l’estensione delle aree di basso voltaggio al mappaggio bipolare (HR=1.6 per 5%;95% CI:1.2-1.9; P<0.001). Tutti i pazienti con un mappaggio bipolare normale presentavano un decorso clinico privo di eventi aritmici. Conclusioni: l’estensione delle aree endocardiche di basso voltaggio nel ventricolo destro risulta essere un potente predittore di eventi aritmici maligni nella Cardiomiopatia Aritmogena del Ventricolo Destro indipendentemente dalla storia clinica e dalla dilatazione/disfunzione del ventricolo destro. La presenza di un normale mappaggio elettroanatomico bipolare rapprestanta un sottogruppo di pazienti affetti da Cardiomiopatia Aritmogena del Ventricolo Destro a basso rischio aritmico.

Dissertação de Mestrado Integrado em Medicina Veterinária
Arrhythmogenic right ventricle cardiomyopathy (ARVC) is recognized in Boxers, cats and humans, is characterized clinically by ventricular tachyarrhythmias and histopathologically by fibrofatty replacement, mostly of the right ventricle. Affected dogs may have syncope, exercise intolerance or heart failure associated signs. ARVC is an inherited adult onset disorder that can lead to sudden death which can also be the first and single clinical sign. Ventricular premature complexes (VPC’s) with left bundle branch block morphology on the electrocardiogram (ECG) are typical for the disease. These VPCs can occur singly, in pairs, triplets or runs of ventricular tachycardia (VT). Routine ECG can be insensitive for the detection of ventricular arrhythmias when compared with a 24 hour Holter study. The antiarrhythmic drugs most commonly used are sotalol or mexiletine-atenolol association. A 24 hour Holter study should be repeated in order to evaluate the efficacy of treatment or detect a proarrhythmic effect. In this study, 4 out of 6 animals (66,6%) died suddenly, however owners refer an improvement in the animal’s quality of life once the treatment is initiated. The prognosis of this disease is guarded due to sudden death risk or heart failure development.
RESUMO - Cardiomiopatia Arritmogénica do Ventrículo Direito em Boxers – Estudo Retrospectivo (6 Casos) - A cardiomiopatia arritmogénica do ventrículo direito (CAVD) é uma patologia reconhecida em cães de raça Boxer, gatos e humanos, caracterizada clinicamente por taquiarritmias ventriculares e histopatologicamente por substituição fibro-adiposa, sobretudo do ventrículo direito. Os cães afectados podem apresentar síncope, intolerância ao exercício ou sinais associados a insuficiência cardíaca. CAVD é uma patologia hereditária do adulto com risco de morte súbita, que pode ser a primeira e única manifestação clínica da doença. Complexos ventriculares prematuros (CVP) com morfologia de bloqueio de ramo esquerdo são achados típicos da doença no electrocardiograma (ECG). Estes CVP podem ocorrer singularmente, em pares, em grupos de três ou em runs de taquicardia ventricular. Um ECG de rotina pode ser insensível na detecção das arritmias que normalmente são mais evidentes num estudo Holter de 24 horas. Os antiarritmicos mais frequentemente utilizados são o sotalol ou a associação mexiletinaatenolol. Para se avaliar a eficácia do tratamento e detectar um eventual efeito pro-arrítmico, um segundo Holter deve ser realizado. Neste estudo, 4 dos 6 animais (66,6%) morreram subitamente, porém, segundo os proprietários a qualidade de vida do animal melhorou com o tratamento. O prognóstico desta doença é reservado devido ao risco de morte súbita ou desenvolvimento de insuficiência cardíaca.

Ventricular arrhythmias (VA) are a frequent precursor to sudden cardiac death (SCD) in patients with structural heart disease (SHD). Patients with SHD are at risk of recurrent ventricular tachycardia (VT), which generally occurs due to re-entry within and around the presence of an arrhythmogenic scar. Therefore, scarred myocardium forms the necessary substrate for arrhythmogenesis to occur. A scar may occur due to obstructive coronary artery disease, causing ischaemic cardiomyopathy (ICM), or from cardiac injury due to several other causes, including inflammatory, infiltrative, toxin-mediated, or genetic heart disease, termed non-ischaemic cardiomyopathy (NICM). An implantable cardioverting defibrillator (ICD) can abort SCD from recurrent VAs. However, they do not stop VAs from occurring in the first place. Anti-arrhythmic drugs (AADs) may reduce the frequency and burden of VAs but have limited efficacy. Some have a narrow therapeutic window or the potential for multiorgan toxicity and can be poorly tolerated. Catheter ablation (CA) is a class I indication for treating sustained monomorphic VT refractory to AADs. CA reduces VT burden, the number of defibrillator therapies, greater freedom from recurrent ventricular arrhythmia, and improves quality of life. However, recurrences can be experienced in up to 50% of patients with SHD-related VT. Some reasons for the failure of CA include reliable identification of critical components of substrate that can harbour VAs both in sinus rhythm and during ongoing VT using electroanatomic mapping (EAM) and imaging techniques, as well as limitations in assessing intraprocedural endpoints. Further refinement of electroanatomic mapping techniques is required to improve the efficacy of CA. This thesis aims to expand on current techniques for substrate identification and methods to improve the efficacy of VA ablation procedures.

Introduction: The diagnosis of Arrhythmogenic Cardiomyopathy (AC) is challenging and often late after disease onset. It relies on a scoring system of major and minor criteria, requiring several clinical, instrumental and genetic tests. Diagnosis confirmation is often obtained by invasive procedures like endomyocardial biopsy and electroanatomical mapping. At present time, no circulating biomarkers are available for the diagnosis of AC. We hypothesized that circulating microRNAs (miRNAs), which have been already demonstrated as circulating biomarkers of many cardiac diseases (e.g. heart failure, myocardial infarction, atrial fibrillation) may be used as potential diagnostic tools in AC. Aims: 1. To screen the level of expression of miRNAs in plasma samples of AC and non-AC male subjects. 2. To assess the specificity of potential miRNAs in diagnosing AC. In particular to identify the differential expression of plasma miRNAs in AC patients vs. healthy controls (HC) and/or patients with ventricular arrhythmias of different aetiologies: idiopathic ventricular arrhythmias (IVT) and patients with ischemic ventricular arrhythmias (IC). 3. To evaluate a possible correlation between miRNAs expression and the severity of the disease in terms of ventricular function and fibro-adipose replacement of the myocardium by means of ElectoAnatomic voltage Mapping [EAM] and Late Gadolinium Enhancement (LGE) detected by Contrast Enhancement Cardiac Magnetic Resonance (CE-CMR) 4. To assess in cardiac stromal mesenchimal cells culture how different levels of expression of the identified miRNAs may regulate the onset and progression fibro-adipogenesis. Methods: All patients with a history of ventricular arrhythmias referred to Arrhythmia Center of the Cardiology Center Monzino (Milan), for transcatheter ablation were enrolled in the study as well as a control cohort of patients matched in terms of age and sex. All patients underwent: - CE-CMR evaluating the volumes and function of the right and left ventricle (RV and LV), the presence and extension of LGE; - Electroanatomic voltage mapping (CARTO) of RV and LV. - Endomyocardial biopsy, when indicated, to evaluate the presence and extension of fibro-fatty infiltration of the RV. - Blood sampling for molecular analysis of 320 miRNA expression. Blood samples (5ml) were collected in EDTA coated tubes and the total RNA was extracted from plasma. The expression of each single miRNA was evaluated using TaqMan microRNA assays (Life Technologies) following manufacturer’s instructions. Validated miRNAs expression data were analyzed using GraphPad Prism version 5.03 for Windows and reported as mean ± standard deviation of the mean (SD). Cardiac Mesenchymal Stromal Cells (C-MSCs) from AC ventricular samples have been obtained in our laboratory and have been used as in vitro model of AC and we decided to evaluate miR-320a in this model. In order to assess C-MSCs involvement during adipogenesis we planned an in vitro experiment to simulate AC development. We maintained the same plated number of C-MSCs in adipogenic medium for 3 days. Results: In the present study a total of 114 male subjects were enrolled: 35 patients were affected by AC, 35 were HC, 20 were affected by IVT and 24 were affected by IC. The level of expression of 368 miRNAs was screened in plasma of 3 symptomatic AC patients and 3 age- and sex-matched healthy donors: 150 miRNAs were found expressed in all screened plasma samples and 14 miRNAs resulted putatively regulated. Among the top 4 regulated miRNAs, considering both relative fold change and statistical significance, miR-320a was confirmed to be regulated in an initial validation step, performed by qRT-PCR in the plasma of 16 HC and 16 AC patients, as defined by current guidelines. Therefore its expression was analyzed in all HC and AC patients. MiR-320a showed a statistically significant lower expression in AC patients compared to HC (0.42±0.04, p=0.008) and with a cut-off value of ΔCt <-5.55 presented a sensitivity of 65% specificity of 80% to discriminate AC patients. We did not find any statistical significance in the level of expression of miR-320a between HC and IVT (fold 1.09±0.5 p=ns) as well as between HC and IC (fold 0.74±0.22 p=ns). In evaluating the expression of miR-320a in terms of the severity of the disease in patients with AC we did not find any statistically significant correlation with major arrhythmic events as well as no correlation with RV function. A significant correlation was found between impairment of the LV function and ΔCt expression (r2=0.20 p<0.04). Evaluating miR-320a expression in 12 patients with AC we found a trend of statistically significance with the extension of scar areas detected by unipolar electroanatomic mapping and LGE (p=0.07). The results from the in vitro study on C-MSC, showed a lower expression of miR-320a in AC C-MSCs compared to non-AC C-MSCs (0.44±0.08, p=ns). Conclusions: This is the first study that evaluates the diagnostic potential of circulating miRNAs in AC. Plasma levels of mir-320a are consistently lower in AC patients compared to HC, IVT and IC subjects and has a fairly good accuracy in discriminating AC vs. IVT patients. Low miR-320a plasma concentrations may represent a new potential biomarker for AC. Plasma concentration of miR-320a seems to demonstrate an inverse correlation with AC severity. MiR-320a regulation in a cardiac cellular model of AC during induced adipogenesis may pave the way to future mechanistic studies on the epigenetic control of AC adipogenesis.
Introduzione: La diagnosi di Cardiomiopatia Aritmogena (CA) risulta essere spesso una sfida per il cardiologo clinico e viene talvolta effettuata in ritardo rispetto all'insorgenza dei sintomi. Si basa sull’identificazione di criteri diagnostici ottenuti attraverso l’utilizzo di diverse indagini clinico-strumentali, talvolta invasive e tramite test genetici. Ad oggi nessun marcatore bioumorale circolante è stato validato e utilizzato quale criterio diagnostico. Abbiamo ipotizzato che i microRNA (miRNA) circolanti, già validati nella diagnosi di molte altre malattie cardiache (insufficienza cardiaca, infarto miocardico, fibrillazione atriale) possano essere utilizzati come potenziale strumento diagnostico nella CAVD. Obiettivi: 1. Eseguire uno screening di miRNA in campioni di plasma di soggetti maschi sani o affetti da CA. 2. Valutare la specificità e la sensibilità dei miRNA nel riconoscimento della CA. In particolare identificare i diversi livelli d’espressione dei miRNA plasmatici nei pazienti con CA rispetto ai controlli sani (CS) e/o ai pazienti con aritmie ventricolari a diversa eziologia (tachicardia ventricolare idiopatica (TVI) o conseguente a cardiopatia ischemica (CI). 3. Valutare la possibile correlazione tra l'espressione dei miRNA e la gravità della malattia espressa in termini di numerosità degli eventi aritmici maggiori, funzionalità ventricolare destra e sinistra e in termini di estensione della sostituzione fibro-adiposa ottenuta mediante mappaggio elettroanatomico e risonanza magnetica cardiaca (RMC). 4. Valutare come la differente espressione dei miRNA circolanti identificati possa regolare lo sviluppo e la progressione della sostituzione fibro-adiposa in colture di cellulle cardiache mesenchimali stromali (C-MSC). Metodi: Sono stati arruolati nello studio tutti i pazienti con aritmie ventricolari, riferiti all’Unità di Elettrofisiologia del Centro Cardiologico Monzino (Milano) per essere sottoposti ad ablazione transcatetere e controlli sani. Tutti i pazienti sono stati sottoposti a: - RMC per valutazione dei volumi e funzionalità del ventricolo destro e sinistro e per determinazione dell’estensione delle “scars”. - Mappaggio elettroanatomico intracavitario bipolare e unipolare (CARTO). - Biopsia endomiocardica, quando indicata per valutazione della presenza e l’estensione della sostituzione fibro-adiposa del ventricolo destro. - Prelievo di sangue venoso l'analisi molecolare di 320 miRNA. I campioni di sangue (5 ml) sono stati raccolti in provette EDTA e l'RNA totale è stato estratto dal plasma. L'espressione di ogni singolo miRNA è stata valutata utilizzando saggi TaqMan microRNA (Life Technologies) seguendo le istruzioni del produttore. L’espressione dei MiRNA validati è stata analizzata utilizzando GraphPad Prism versione 5.03 per Windows e riportati come media ± deviazione standard della media (SD). Le cellule stromali mesenchimali cardiache (C-MSC) sono state ottenute da un campione bioptico di pazienti con sospetta CA. Al fine di valutare il coinvolgimento C-MSC durante adipogenesi è stato eseguito un esperimento in vitro per simulare lo sviluppo della patologia. Risultati: 114 soggetti maschi sono stati arruolati nello studio: 35 soggetti affetti da CA, 35 soggetti sani, 20 affetti da TVI e 24 da CI. Il livello d’espressione di 368 miRNA circolanti è stato valutato su campioni di plasma di 6 soggetti (3 affetti da CA e soggetti sani). Di 150 miRNA espressi in tutti i campioni di plasma, 14 miRNA sono risultati regolati. Tra i 4 miRNA maggiormente regolati, il miR-320a è stato validato inizialmente mediante qRT-PCR nel plasma di 32 soggetti (16 controlli sani e 16 soggetti affetti da CA) In seguito abbiamo valutato l’espressione di miR-320a in tutti i soggetti dello studio. I livelli d’espressione del miR-320A sono risultati statisticamente inferiori nei soggetti affetti da CA rispetto ai controlli sani (0,42 ± 0,04, p = 0,008) con un valore di cut-off (ΔCt <-5.55) il miR-320a ha presentato una sensibilità del 65% e una specificità dell’ 80% nel discriminare I pazienti con CA. Non abbiamo trovato nessuna differenza statisticamente significativa nell’espressione di miR-320a tra soggetti sani e affetti da TVI (1,09 ± 0,5 p = ns) come tra soggetti sani e affetti da CI (0.74 ± 0.22 p = ns). Nei soggetti affetti da CA, non abbiamo trovato alcuna correlazione tra i livelli d’espressione di miR-320 a e il verificarsi di eventi aritmici maggiori così come con gli indici di funzionalità ventricolare. Una correlazione statisticamente significativa è stata riscontrata tra i livelli di espressione del miR-320a e la funzione ventricolare sinistra (FE)(r2=0.20 p<0.04). In 12 dei pazienti con CA abbiamo trovato un trend di significatività statistica tra i livelli di miR-320 a e le aree di “scar” al mappaggio elettroanatomico unipolari e la presenza di LGE (p=0,07). Dallo studio in vitro sulle C-MSC abbiamo evidenziato una ridotta espressione di miR-320 nelle cellule ottenute dai pazienti con CA rispetto alle cellule ottenute dagli altri soggetti (0,44 ± 0,08, p=ns) Conclusioni: Questo è il primo studio in cui sia stata valutata l’utilità diagnostica dei miRNA circolanti nella CA. I livelli plasmatici d’espressione del miR-320a sono costantemente più bassi nei soggetti affetti da CA rispetto a soggetti sani o con cardiopatia ischemica o affetti da tachicardia ventricolare idiopatica e si è dimostrato in grado di discriminare con una buona accuratezza i soggetti affetti da CA rispetto ai soggetti affetti da TVI. I nostri dati preliminari suggeriscono miR-320a quale potenziale marcatore bioumorale di CA e sembrerebbero evidenziare una correlazione inversa tra i livelli d’espressione di miR-320a e la gravità della patologia. La creazione di un modello cellulare di adipogenesi indotta in cellule mesenchimali stromali cardiache mediante regolazione di miR-320a potrebbe aprire la strada a futuri studi meccanicistici sul controllo epigenetico dell’adipogenesi nella CA.

Aims: Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, the consequences of these mutations in early disease stages are unknown. We investigated whether mutation-induced intercalated disc remodeling impacts on electrophysiological properties before the onset of cell death and replacement fibrosis. Methods and Results: Transgenic mice with cardiac overexpression of mutant Desmoglein2 (Dsg2) Dsg2-N271S (Tg-NS/L) were studied before and after the onset of cell death and replacement fibrosis. Mice with cardiac overexpression of wild-type Dsg2 and wild-type mice served as controls. Assessment by electron microscopy established that intercellular space widening at the desmosomes/adherens junctions occurred in Tg-NS/L mice before the onset of necrosis and fibrosis. At this stage, epicardial mapping in Langendorff-perfused hearts demonstrated prolonged ventricular activation time, reduced longitudinal and transversal conduction velocities, and increased arrhythmia inducibility. A reduced action potential upstroke velocity due to a lower Na+ current density was also observed at this stage of the disease. Furthermore, co-immunoprecipitation demonstrated an in vivo interaction between Dsg2 and the Na+ channel protein NaV1.5. Conclusion: Intercellular space widening at the level of the intercalated disc (desmosomes/fascia adherens junctions) and a concomitant reduction in action potential upstroke velocity, as a consequence of lower Na+ current density, leads to slowed conduction and increased arrhythmia susceptibility at disease stages preceding the onset of necrosis and replacement fibrosis. The demonstration of an in vivo interaction between Dsg2 and NaV1.5 provides a molecular pathway for the observed electrical disturbances during the early ARVC stages.
Introduzione: Mutazioni nei geni che codificano per le proteine desmosomali giocano un ruolo fondamentale nella patogenesi della cardiomiopatia aritmogena del ventricolo destro (ARVC). Tuttavia, le conseguenze di tali mutazioni negli stadi precoci della malattia sono sconosciute. Scopo del nostro studio è stato di indagare se il rimodellamento dei dischi intercalari come conseguenza di mutazioni nelle proteine desmosomali modifichi le proprietà elettrofisiologiche cardiache prima dello sviluppo di morte cellulare e fibrosi sostitutiva. Metodi e Risultati: Topi transgenici con overespressione cardiaca della proteina Desmogleina2 mutata (Dsg2) -Dsg2-N271S (Tg-NS/L)- sono stati studiati prima e dopo lo sviluppo di morte miocitaria e fibrosi sostitutiva. Come controlli, abbiamo usato topi wild-type e topi con overespressione della Dsg2 normale. Studi di microscopia elettronica hanno evidenziato la presenza di spazi intercellulari allargati in corrispondenza delle giunzioni meccaniche desmosomi/giunzioni aderenti nei topi Tg-NS/L prima dello sviluppo di necrosi e fibrosi. Contemporaneamente, il mappaggio epicardico in cuori perfusi con soluzione Langendorff ha dimostrato prolungamento del tempo di attivazione ventricolare, riduzione della velocità di conduzione longitudinale e trasversale, ed aumento della inducibilità di aritmie. Inoltre, nello stesso stadio di malattia, si osservava ridotta velocità del potenziale d’azione dovuta a minore densità della corrente del sodio. Studi di co-immunoprecipitazione, infine, dimostravano un’interazione in vivo tra la Dsg2 e la proteina NaV1.5 dei canali del sodio. Conclusioni: Un allargamento degli spazi intercellulari a livello dei dischi intercalari e una concomitante riduzione del potenziale d’azione, come conseguenza di una minore corrente del sodio, portano ad un ritardo di conduzione ed ad aumentata suscettibilità aritmica negli stadi di malattia che precedono la necrosi e la fibrosi sostitutiva. La dimostrazione di un’interazione in vivo tra Dsg2 e NaV1.5 suggerisce una spiegazione a livello molecolare dei disturbi elettrici osservati negli stadi precoci dell’ARVC.

Introduction: Little is known about the molecular genetics of cardiomyopathy in Africans. Aims: to (I) determine the prevalence of desmosomal gene mutations in arrhythmogenic right ventricular cardiomyopathy (ARVC) and dilated cardiomyopathy (DCM) in desmosomal protein genes (i.e., plakophilin 2, desmocollin 2, desmoglein 2, and plakoglobin), (2) establish the presence of a founder effect in families with a recurrent mutation in the plakophilin 2 (PKP2) gene, (3) investigate whether single nucleotide polymorphisms found in desmosomal genes affect gene expression, and (4) search for new candidate genes for ARVC in families where no causal mutation was found in desmosomal protein genes. Methods: 177 participants with cardiomyopathy were screened for desmosomal gene mutations which were confumed by Sanger sequencing. The following methods were used: in the founder effect study we used haplotyping with microsatellite markers; for total gene expression we used real time polymerase chain reaction and allelic expression imbalance and exome sequencing was used for mutation screening in two siblings with severe early onset ARVC. To all novel variants identified prediction tools were used to predict the pathogenicity of the variant in uestion. Results: 21.5% of ARVC pro bands had a disease-causing mutation in one of four desmosomal genes; no disease-causing mutation was found in the 112 OCM index cases. A recurrent PKP2 mutation occurred on a common haplotype background in four white South African probands with cardiomyopathy. Investigation of a common PKP2 polymorphism had no effect on total gene expression nor was there evidence of allelic expression imbalance. Finally, rare mutations were found in PARVA and HMGXB3 by exome sequencing of two siblings.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a genetic cardiac disease inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity. Its main feature is the progressive substitution of the myocardium with fatty or fibro-fatty tissue, involving predominantly the right ventricle. Clinically, ARVC/D is characterized by ventricular arrhythmias, often associated with syncope and sudden cardiac death, especially in the young and athletes. Up to now, several disease genes have been identified, including 5 encoding desmosomal proteins. In this study, mutation screening for desmosomal genes performed in 80 consecutive unrelated Italian probands failed to detect mutation in about half of index cases, thus suggesting further genetic heterogeneity. On the basis of alphaT-catenin (a protein of cardiac area composita) cellular localization and function, mutation screening was then performed in CTNNA3 candidate gene in 76 affected subjects, negative for mutations in desmosomal genes. Four mutations have been detected. In vitro functional studies assessed the pathogenicity for two of them. For the first time mutations in CTNNA3 have been detected in a human disease. Particularly, the recurrence of pathogenic mutations in CTNNA3 gene in ARVC/D patients expands the concept of this disease beyond desmosomes
La cardiomiopatia aritmogena del ventricolo destro (ARVC/D) è una malattia genetica cardiaca, ereditata come carattere autosomico dominante a penetranza incompleta ed espressività variabile. La sua caratteristica principale è la progressiva sostituzione del miocardio del ventricolo destro con tessuto adiposo e/o fibroadiposo, con formazione di circuiti anatomici di rientro e conseguenti aritmie e morte improvvisa, specie nei giovani. Attualmente sono noti dieci geni malattia, di cui cinque sono codificanti per le proteine desmosomali. In questo studio, lo screening di mutazioni nei geni desmosomali eseguito in 80 pazienti affetti non ha evidenziato mutazioni in circa la metà dei casi, suggerendo la presenza di ulteriore eterogeneità genetica. In virtù della funzione e della localizzazione cellulare dell’alphaT-catenina (proteina dell’area composita), è stato svolto lo screening di mutazioni nel gene candidato CTNNA3 in 76 casi indice negativi per mutazioni nei geni desmosomali. Sono state identificate quattro variazioni. Studi funzionali in vitro hanno dimostrato la patogenicità per due di esse. Per la prima volta, mutazioni nel gene CTNNA3 sono state associate a una malattia umana. In particolare, la ricorrenza di mutazioni patogene nel gene CTNNA3 in pazienti ARVC/D estende il concetto di questa malattia oltre i desmosomi

Introduction: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty replacement of myocardial tissue and high incidence of serious ventricular tachyarrhythmias. To date, ten disease-genes have been identified, five of which encoding desmosomal proteins (PKP2, DSP, DSG2, DSC2 and JUP). Aim of the study: The study described in the present thesis aimed at determining the spectrum and prevalence of desmosomal mutations in 80 Italian unrelated index cases. Moreover, the identification of novel disease loci and genes in three ARVC families was attempted by integrating different approaches, including genome-wide linkage study, Copy Number Variations (CNVs) analysis, and exome-sequencing. Methods: Mutation screening of the desmosomal ARVC genes was performed by Denaturing High-Performance Liquid Chromatography (DHPLC) and direct sequencing. In order to map novel loci and, possibly, to identify novel genes, genome-wide linkage study and CNVs analysis was performed in three independent families with recurrence of ARVC, showing no mutations in any of the desmosomal genes. In addition, in Family #2 and in Family #3 a combined strategy of linkage analysis and exome sequencing was adopted to identify novel ARVC genes. Results: Mutation screening of five desmosomal ARVC genes in 80 Italian probands identified single point mutations in 32.5% of cases and multiple mutations in 12.5%. No mutations were identified in the remaining 55% of probands. The genes most frequently involved were PKP2, DSP and DSG2. Among the index cases negative for point mutations in the desmosomal genes, three belong to independent families with recurrence of ARVC. Assuming a further genetic heterogeneity in these families, a genome-wide scan was performed in order to identify novel loci and genes. In Family #1, a CNV involving a desmosomal ARVC gene was identified in all affected family members. In Family #2, a novel ARVC locus was mapped on chromosome 19 by linkage analysis. Finally, in Family #3, a possible novel candidate gene for ARVC was detected by a combined strategy of linkage analysis and exome-sequencing. Discussion: In the present study, genetic screening of five desmosomal ARVC genes in 80 Italian probands identified causative mutations in 45% of cases, mainly involving the “big3” genes PKP2, DSP, and DSG2, according to data reported in literature. Genetic analysis of available family members confirmed the high heterogeneity in the clinical expression of ARVC mutations even among relatives. Mutation screening of desmosomal genes failed to detect causative mutations in more than 50% of index cases, suggesting that additional and still unknown genes could be involved. In this perspective, a genome-wide scan was performed in three large ARVC families, showing no mutations in any of the desmosomal genes. In Family #1, a CNV was identified in a desmosomal ARVC gene, highlighting the importance of complementing the conventional mutation screening in ARVC genes with other approaches able to detect possible structural variations. In Family #2, genome-wide linkage results provided strong evidence for a novel ARVC locus on chromosome 19, highlighting the soundness of this strategy for identifying susceptibility regions in large, highly informative families and providing the basis for the identification of a novel disease gene. Finally, in Family #3, exome-sequencing identified a novel putative candidate gene for ARVC. Identification of novel ARVC genes is of great importance for understanding the molecular pathogenesis of this disease, as well as for increasing the power of genetic screening and developing successful targeted therapies
Introduzione: La Cardiomiopatia Aritmogena del Ventricolo Destro (ARVC) è una malattia ereditaria del muscolo cardiaco caratterizzata dalla progressiva perdita e sostituzione fibro-adiposa dei cardiomiociti che costituiscono la parete libera delventricolo destro. Tale disomogeneità del tessuto cardiaco altera la normale conduzione dell'impulso elettrico, determinando l'insorgenza di aritmie che occasionalmente portano a fibrillazione ventricolare e morte improvvisa per arresto cardiaco, soprattutto nei giovani e negli atleti. Attualmente, sono noti 10 geni implicati nella determinazione genetica dell’ARVC e cinque di questi codificano per proteine costituenti il desmosoma cardiaco: Placofilina-2 (PKP2), Desmoplachina (DSP), Desmogleina-2 (DSG2), Desmocollina-2 (DSC2) e Placoblobina (JUP). Scopo dello studio: Lo studio descritto nella presente tesi mira a valutare la prevalenza e lo spettro di mutazioni nei cinque geni ARVC desmosomali in un gruppo di 80 casi indice Italiani, non imparentati tra loro. Inoltre, in tre grandi famiglie con ricorrenza di casi ARVC e in cui non sono state identificate mutazioni nei geni desmosomali, è stata effettuata un'analisi genome-wide integrando diversi approcci, quali studio di linkage, analisi di Copy Number Variations (CNVs) e sequenziamento dell’esoma. Metodi: Lo screening per la ricerca di mutazioni nei cinque geni ARVC desmosomali ha coinvolto 80 casi indice ed è stato effettuato tramite analisi DHPLC (Denaturing High-Performance Liquid Chromatography) e sequenziamento diretto del DNA.I soggetti appartenenti a ciascuna delle tre famiglie selezionate per l'analisi genome-wide sono stati genotipizzati utilizzando un pannello di marcatori ad alta densità che include più di 370.000 polimorfismi di singolo nucleotide (SNPs) (Illumina HumanCNV370-Duo BeadChip). In ciascuna famiglia è stato effettuato uno studio di linkage ed un’analisi di CNVs. Inoltre, Nella Famiglia #2 e nella #3 l'identificazione del gene malattia è stata tentata integrando i risultati dello studio di linkage con i dati ottenuti dal sequenziamento dell'esoma di due soggetti affetti. Risultati: L'analisi delle sequenze codificanti dei geni PKP2, DSP, DSG2, DSC2 e JUP in 80 casi indice Italiani ha permesso di identificare mutazioni singole nel 32.5% dei casi e mutazioni multiple nel 12.5%. Il 55% dei probandi non è risultato portatore di alcuna mutazione nei geni desmosomali. La maggior parte delle mutazioni ha coinvolto i geni PKP2, DSP, DSG2, confermando i dati riportati in letteratura. Tra i casi indice in cui non sono state identificate mutazioni nei geni desmosomali, tre appartengono a famiglie indipendenti con ricorrenza di casi ARVC. In ognuna di queste famiglie è stata effettuata un'analisi genome-wide allo scopo di identificare nuovi loci e geni malattia. Nella Famiglia #1 è stata identificata una CNV che coinvolge uno dei geni ARVC desmosomali noti e co-segrega con il fenotipo patogeno. Nella Famiglia #2, l’analisi di linkage ha permesso di identificare un nuovo locus ARVC sul cromosoma 19. Infine, nella Famiglia #3 è stato identificato un nuovo potenziale gene candidato per l’ARVC. Discussione: L’analisi genetica delle sequenze codificanti dei cinque geni ARVC desmosomali, in un gruppo di 80 probandi italiani, ha permesso di identificare mutazioni nel 45% dei casi, confermando il prevalente coinvolgimento dei tre geni PKP2, DSP e DSG2, in accordo con i dati riportati in letteratura. L'analisi genetica dei familiari dei probandi ha confermato la penetranza incompleta e l'espressività variabile della malattia, anche all'interno della stessa famiglia. L'assenza di mutazioni in più del 50% dei casi suggerisce il coinvolgimento di altri geni nella determinazione genetica dell'ARVC. In quest' ottica, un'analisi genome-wide è stata effettuata in tre famiglie con ricorrenza di casi ARVC e in cui non sono state individuate mutazioni nei geni desmosomali. Nella Famiglia #1, l’identificazione di una CNV in uno dei geni ARVC desmosomali, presente in tutti i soggetti affetti, sottolinea l'importanza di associare alle metodiche tradizionali utilizzate per lo screening di mutazioni puntiformi approcci che permettano di identificare eventuali variazioni strutturali presenti nel genoma. Nella Famiglia #2, l'analisi di linkage ha fornito una significativa evidenza dell'esistenza di un nuovo locus ARVC sul cromosoma 19, fornendo le basi per l'identificazione di nuovi geni. Infine, nella Famiglia #3, il sequenziamento dell'esoma di due soggetti affetti ha identificato un nuovo gene come un possibile candidato per l'ARVC

Recent advances in genome sequencing technologies provided unexpected opportunities to characterize individual genomic landscape and identify mutations relevant for diagnosis and therapy in clinics. Specifically, whole-exome sequencing for complex disease (such as tumor/normal matched sample) and target resequencing for Mendelian disease, using next-generation sequencing (NGS) technologies, are gaining popularity in the human genetics community due to the moderate costs and the huge quantity of information provided by each experiment. However, NGS data analysis still remains the crucial bottleneck in this approach because of the great amount of data containing millions of potential disease-causing variants and difficulties involving the integration of different sources. Here, we describe the application of a bioinformatics analysis pipeline for NGS data in two case studies about rare cardiac diseases. The case 1 is focused on the target sequencing of 158 candidate genes in 91 patients affected by Brugada Syndrome (BrS). To date the clinical phenotype is associated with mutations in the SCN5A gene but explain only the 30% of the BrS cases. Therefore we selected a panel of genes previously associated to cases of arrhythmogenic disorders in literature and we analysed them in a cohort of BrS patients, which were negative for known SCN5A mutations. We found 98 novel genetic variations and 60 clinical rs belonging to 70 genes. In particular we found 13 genes significantly mutated in our cohort compared to healthy controls in 1000 Genomes data that were not previously associated to BrS phenotype. In case study 2 we performed a whole-exome sequencing experiment of trio family where the child is affected by a severe cardiac disease with unclear diagnosis. We developed a specific bioinformatics pipeline to filter out the germline mutations. We found six genes with novel deleterious mutations that were homozygous in the affected child, and heterozygous in both parents. Among the six mutated genes the TRDN and UNC45A genes were already associated to cardiac dysfunctions in literature. Functional studies will be performed to evaluate the involvement of the mutated genes in the disease onset. In conclusion, we developed an automatic and versatile pipeline to analyse NGS data coming from whole-exome sequencing and target sequencing strategies. In addition, we integrated in the pipeline several public variation databases to evaluate and interpret the candidate mutations. The mutations found were validated by Sanger sequencing to evaluate the strength of the pipeline filters.

Mutation analysis of the recognized ARVC genes and of further candidate genes was performed on a large cohort of ARVC patients. Several novel mutations were identified and three further desmosomal genes were linked to the disease: plakophilin2, desmocollin2 and desmoglein2. Heart and skin samples from ARVC patients were subjected to microscopic examination and immunohistochemistry to study the effect of the newly-identified mutations on the structure of cell adhesion complexes.;The functional effects of a particular novel mutation were thoroughly examined in vitro. S39_K40insS is the first dominant ARVC-causing plakoglobin mutation to be reported. Yeast-two hybrid analysis was used to investigate the effect of S39_K40insS on the proteins interactions established by plakoglobin. A HEK293 cell line stably expressing the mutant protein was generated and used to study the effects of S39_K40insS on desmosomal structure, cell proliferation, cell death, subcellular localization and expression levels of proteins involved in adhesion and signalling and cellular responses to defined mechanical load. A recombinant adenovirus expressing the mutant protein was generated and used to transfect neonatal rat ventricular cardiomyocytes, whose behaviour and responses were subsequently analysed. The functional consequences of S39_K40insS were compared with those of PK215del2, a previously reported recessive plakoglobin mutation known to underlie Naxos disease, a syndromic form of ARVC.;These results point towards novel mechanisms of disease pathogenesis, that apart from weakened cell-cell adhesion involve altered protein turnover kinetics and defects in signalling pathways. Similar studies should improve our understanding of ARVC and provide a more accurate diagnostic algorithm.

The pulmonary veins are widely recognised as a source of ectopic electrical activity that can cause atrial fibrillation. While the ectopic activity likely originates in the cardiomyocytes that form an external sleeve around the veins, the underlying mechanisms are unknown. Changes in intracellular Ca2+ signalling have been proposed to play an important role in the arrhythmogenic properties of the pulmonary vein. Therefore, the aim of this thesis was to study factors that might influence Ca2+ signalling in the cardiomyocytes. This involved determining the localisation of Ca2+ handling proteins in the cardiomyocytes, examining interventions that might alter the characteristics of intracellular Ca2+ signalling, as well as looking at the arrhythmogenic effect of adrenergic stimulation. In the rat pulmonary vein, the cardiomyocytes displayed spontaneous Ca2+ transients that were usually manifest as waves, and were asynchronous in neighbouring cardiomyocytes. The frequency of spontaneous Ca2+ transients was increased following a brief period of electrical stimulation at 3 Hz or greater, and this effect was enhanced in the presence of isoprenaline, or when the external Ca2+ concentration was raised. Noradrenaline also increased the frequency of the spontaneous Ca2+ transients; however, synchronous Ca2+ transients, like those that could be evoked by electrical field stimulation, were not observed. As spontaneous Ca2+ transients are due to Ca2+ released from the sarcoplasmic reticulum through the ryanodine receptors, immunocytochemistry was used to determine their distribution. The ryanodine receptors were arranged in a striated pattern with some distribution at the periphery of the cells, which was similar to myocytes from the atria. When the sarcolemma of the pulmonary vein cardiomyocytes was labelled with Di-4 ANEPPS, they were shown to possess transverse (T)-tubules, which are involved in co-ordinating the intracellular Ca2+ transient in response to depolarisation. This differed from atrial myocytes where a T-tubule system was not observed. Furthermore, the L-type Ca2+ channels and Na+/Ca2+ exchanger (NCX), which are involved in Ca2+ influx and removal during excitation-contraction coupling, were arranged in a more striated manner in the pulmonary vein cardiomyocytes, compared to those of the atria. This could have important consequences for the contractile activity of the cardiomyocytes, as well as their ability to generate abnormal electrical activity, as the NCX is known to cause depolarisation in response to an increase in intracellular Ca2+. The contractile properties of the pulmonary vein were studied in vitro using myography techniques, where it was shown to display a negative force-frequency relationship, whereby increasing the frequency of electrical stimulation reduced the contractile amplitude. In the presence of noradrenaline, the amplitude of the electrically evoked contractions was increased and the negative force-frequency relationship was only evident at the higher stimulation frequencies (5 to 7 Hz). Noradrenaline also induced periodic bursts of contractions that occurred independently of electrical stimulation, suggesting that it had an arrhythmogenic effect. Such activity was partially inhibited by blocking the NCX with ORM-10103. This suggests a potential target for future research into selective pharmacological intervention for catecholamine based arrhythmias.

Razionale e ipotesi. La Cardiomiopatia Aritmogena (ACM) è una patologia cardiaca caratterizzata da sostituzione fibro-adiposa del miocardio ventricolare e aritmie maligne. Nonostante sia geneticamente determinata, soprattutto da mutazioni in geni desmosomiali (ad esempio mutazioni in PKP2), i fenotipi clinici ACM sono altamente variabili e i meccanismi molecolari alla base della variabilità fenotipica non sono del tutto noti. Abbiamo ipotizzato che le lipoproteine a bassa densità ossidate (oxLDL), agendo sul recettore gamma attivato dai proliferatori dei perossisomi (PPARγ), il principale regolatore del processo adipogenico nell’ACM, possano contribuire alla patogenesi della malattia. Metodi e risultati. I pazienti ACM hanno mostrato un’elevata concentrazione plasmatica di oxLDL rispetto a controlli sani (HC) matchati per sesso, età e fattori di rischio cardiovascolari, e rispetto a loro famigliari portatori della stessa mutazione causativa ma asintomatici. Inoltre, abbiamo ottenuto una maggiore perossidazione lipidica nel tessuto cardiaco di pazienti ACM rispetto a HC. Utilizzando come modelli in vitro cellule mesenchimali stromali cardiache ottenute da pazienti e cardiomiociti derivati da cellule staminali pluripotenti indotte, abbiamo dimostrato che le oxLDL e il loro componente acido 13-idrossi-octadecadienoico (13HODE) sono cofattori di adipogenesi nell’ACM. Dal punto di vista meccanicistico, l’aumento dell’accumulo lipidico dipende dall’internalizzazione di oxLDL nelle cellule attraverso il recettore CD36, provocando l’aumento di PPARγ. Inducendo l’aumento delle oxLDL plasmatiche nel modello murino ACM eterozigote knock-out per Pkp2, attraverso una dieta ad alto contenuto di grassi e colesterolo, abbiamo confermato in vivo l’effetto del metabolismo dei lipidi ossidati sull’adipogenesi cardiaca e disfunzione ventricolare. Il trattamento con atorvastatina, invece, ha prevenuto questi fenotipi. Alti livelli plasmatici di oxLDL predicono un fenotipo clinico severo, in termini di infiltrazione adiposa, disfunzione ventricolare e rischio di eventi aritmici maggiori, nei pazienti ACM. Conclusioni. Il ruolo dei lipidi ossidati nell’adipogenesi ACM, come dimostrato in vitro, in vivo e sui pazienti, rappresenta un nuovo meccanismo di patogenesi, rilevante per la stratificazione del rischio dei pazienti e per nuove strategie farmacologiche.
Rationale and hypothesis. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac condition hallmarked by ventricular tissue fibro-adipogenic alterations, contributing to progressive function deterioration and arrhythmias. Although genetically-determined, mainly by mutations in desmosomal genes (e.g. PKP2), ACM clinical phenotypes are highly variable for poorly understood reasons. More data on molecular phenotype modulators, clinical prognosticators and etiological therapies are awaited. We hypothesized that oxidized low density lipoproteins (oxLDL)-dependent activation of peroxisome proliferator-activated receptor γ (PPARγ), a recognized effector of ACM adipogenesis, may contribute to disease pathogenesis. Methods and results. ACM patients showed higher plasma concentration of oxLDL vs. matched healthy controls (HC) and vs. ACM mutation-carrier healthy relatives. Moreover, we found higher lipid peroxidation indexes in cardiac bioptic tissues of ACM vs. HC subjects. By using ACM patient-derived cardiac mesenchymal stromal cells and induced pluripotent stem cell-derived cardiomyocytes, we demonstrated that oxLDL and their component 13-hydroxy-octadecadienoic acid (13HODE) are major cofactors of cardiac adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through the scavenger receptor CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL plasma concentration in a Pkp2 heterozygous knock-out ACM mouse model, through high fat diet feeding, we confirmed in vivo the dependency of cardiac adipogenesis and right ventricle dysfunction on oxidized lipid metabolism. Conversely, atorvastatin treatment prevented these phenotypes. Importantly, high oxLDL plasma levels predict a severe clinical phenotype in terms of fat infiltration, ventricular dysfunction and risk of major arrhythmic events in ACM patients. Conclusions. The modulatory role of oxidized lipids in ACM adipogenesis, as demonstrated at cellular, mouse and patient levels, represents a novel molecular pathogenic mechanism relevant for patients’ risk stratification and for new pharmacological strategies.

The present study investigates in vitro electrophysiological effects of therapeutic concentrations of quinidine (5-10 $ mu$M) and its combination with the Class Ib agents mexiletine and tocainide in canine Purkinje fibers with the use of standard microelectrode techniques. The frequency- and voltage-dependent depression of V$ sb{ rm max}$, used as an index of peak sodium conductance, by quinidine and the combination of quinidine and tocainide (50 $ mu$M) were assessed with the kinetics of onset of, and recovery form, rate-dependent block, and the curve relating V$ sb{ rm max}$ to membrane potential. The frequency-dependence of quinidine-induced repolarization abnormalities arising from early after-depolarizations (EADs) was characterized in the presence of low (K$ sp+$) $ sb0$ (2.7 mM) and mild acidosis (pH = 7.06 $ pm$ 0.08). Acidosis was found to favor triggered activity by directly prolonging action potential duration. Quinidine induced two types of EAD-induced triggered activity, viz. arising from phase 2 and arising from phase 3, which differed in the frequency-dependence of their characteristics (activation voltage, amplitude, and coupling interval), and their sensitivities to abolition by mexiletine. Adrenaline (1 $ mu$M) decreased the minimum cycle length for triggered activity, shortened the coupling interval of triggered responses, and transformed single triggered responses into multiple. It also induced rapid activity resembling sustained triggered activity, induced triggered responses arising from phase 2, and facilitated their transmission to ventricular muscle. The combination of quinidine with a Class Ib drug proved to be beneficial by improving its antiarrhythmic effect and preventing its bradycardia-related excessive QT prolongation. Triggered activity was found to originate only in Purkinje fibers, but electrotonic influence from ventricular muscle strongly affected its manifestation. These results provide the evidence that EAD-induced triggered

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically determined heart muscle disorder that presents clinically with ventricular arrhythmias, heart failure, and sudden death. The pathological process consists of progressive loss of ventricular myocardium with fibro-fatty replacement. Right ventricle is mostly involved, but presentation of the disease with predominantly left ventricular involvement has been reported. ARVC is typically inherited as a dominant disease, although recessive variants exist and the involvement of family members often can only be detected by molecular genetic analysis (low penetrance of mutations). Genetic studies over the last few years have offered insight into the potential causes of ARVC. Early works demonstrated substantial genetic heterogeneity, and at least 12 independent loci and 7 disease-genes have now been identified. These findings also implicated desmosomal proteins or proteins involved in desmosomal function as candidate causes of the disorder. In the present study Perp was investigated as a candidate gene for ARVC because of its possible role in cell-cell adhesion, as a structural constituent of desmosomes or as a protein playing an yet unknown role in desmosome assembly. After PERP human cardiac expression was tested and confirmed, 90 ARVC index cases were screened for PERP mutations by DHPLC analysis and direct sequencing. Two variations G59R in exon1 and c.1091C>T in 3'UTR were detected in two patients, in whom a mutation in a known ARVC gene was previously identified. The missense variation G59R was detected in 1 control out of 250 screened and the variation c.1091C>T was identified in 2 controls out of 192 screened. Moreover these two novel variants involved respectively a highly conserved amino acid and a highly conserved nucleotide. Interestingly index cases, carrying two mutations (one in PERP gene and one in a known ARVC gene), showed a more severe phenotype than family members carriers for only one of these variations. It is impossible to establish whether these single PERP mutations might lead to ARVC determination, but on the other hands, in patients carrying a pathogenic mutation in a different gene involved in ARVC, PERP mutations might worsen the clinical phenotype. The idea that ARVC is due to desmosomal dysfunction was strengthened by two recent studies that reported mutations in the desmosomal desmocollin-2 (DSC2) gene as the cause of ARVC. During the present study six different DSC2 mutations were identified in seven out of sixty-four ARVC unrelated Italian index cases. Two nucleotide substitutions (c.-92G>T and c.3241A>T) in 5' and 3' UTR regions were detected in two different index cases. Neither of the nucleotide changes were found in 300 chromosomes from the same population, but to exclude that these mutations could correspond to rare polymorphisms the size of the control group should be increased to 500. Moreover in order to test whether this UTR mutations could affect the expression levels of DSC2 gene, specific in vitro functional studies are needed. Another nucleotide substitution (c.348A>G) absent among 500 control chromosomes, was detected in exon 3; although this mutation corresponds to a synonymous variation (Q116Q), it has been demonstrated that it creates a cryptic splice site, leading to a deletion of 9 nucleotides. Although skipping of 9bp in the mutant transcript doesn't alter the reading frame of DNA sequence, at protein level it leads to loss of three amino acids very conserved among species. This mutation mapped on a region important for maturation of the protein. Two heterozygous point substitutions c.304G>A and c.1034T>C were detected in other two patients. Both nucleotide changes was never found in 250 unrelated controls (500 control chromosomes). Variations c.304G>A in exon 3 and c.1034T>C in exon 8 result in predicted p.E102K and p.I345T amino acid substitutions. The mutated amino acids had completely different physico-chemical properties when compared to the wild type. Both these changes occurred in a residue highly conserved among species and are located in protein regions involved on DSC2 adhesion function. The sixth mutation c.2687_2688insGA in exon 17 was detected in two different patients and in six control subjects, suggesting the possibility of a polymorphism. This mutation would affect the C terminus of DSC2a, precisely the ICS domain, by altering 4 aa residues before a termination codon is prematurely introduced. The change occurred in the last five aa residues of the protein, which are non conserved among mammals, in contrast with the high conservation of the upstream region. The final part of this thesis work was focused on the analysis of potential pathogenic effects of the last three DSC2 mutations described above in cultured cardiomyocytes. Once human cDNAs coding for wild type, two polymorphic variants and mutant proteins were obtained, constructs containing also GFP protein were expressed by transient transfection of HL-1 cell line. In transfected HL-1 cells, wild type protein and the two polymorphic variants were detected in the cell membrane, into cell-cell contact regions since co-localised with the endogenous desmoglein which was marked with a monoclonal dsg antibody. In contrast the three mutant proteins were almost exclusively distributed throughout the cytoplasm with very scarce cell membrane localisation, affecting the normal localisation of DSC2 and suggesting the potential pathogenic effect of the mutations.

Ion channel mutations can cause defects on the electrical cellular activity, leading to arrhythmogenic diseases also known as channelopathies. Genetic analysis has been a useful tool to identify these mutations as the cause of different arrhythmogenic diseases. However, not all the members of the same family carrying a mutation present the same phenotype. This is known as incomplete penetrance. The results from this Thesis support that the incomplete penetrance of arrhythmogeic diseases is regulated by specific variants from each patient. Thus, a variant pathogenicity should be evaluated taking into account the presence of other genetic changes that could modulate its effect
Mutacions als canals iònics poden causar defectes en l’activitat elèctrica cel·lular, donant lloc a malalties aritmogèniques, també conegudes com canalopaties. Les anàlisis genètiques han sigut una eina útil per a identificar aquestes mutacions com la causa de diferents malalties aritmogèniques. Malgrat això, no tots els membres d’una família que tenen una mutació presenten la mateixa simptomatologia. Això es coneix com a penetrància incompleta. Els resultats d’aquesta Tesi recolzen que la penetrància incompleta de malalties aritmogèniques està regulada per variants específiques de cada pacient. Així doncs, la patogenicitat d’una variant hauria de ser avaluada tenint en compte la presència d’altres canvis genètics que puguin modular el seu efecte
Programa de Doctorat en Biologia Molecular, Biomedicina i Salut

Introduction. Arrhythmogenic cardiomyopathy (ACM) is a predominantly genetically determined disease characterized by fibrofatty replacement which leads to right ventricular failure, arrhythmias, and sudden cardiac death (SCD). ACM is inherited as an autosomal dominant trait with incomplete penetrance. Advances in genetic technology have revealed substantial genetic heterogeneity: at least 15 independent loci and 13 disease genes have now been identified associated with the disease, with the large involvement of the genes encoding for desmosomal and area composita proteins. Since causative mutations in ACM genes have been detected in about 60% of probands, additional and still unknown disease-genes could be involved. Aim of the study. This study aimed at detecting causative mutations underlying the disease expression in a cohort of 59 Italian unrelated index cases through a new targeted next generation sequencing (NGS) approach. Moreover, the identification of novel disease loci and genes in four families with recurrence of ACM was attempted by integrating different genetic approaches. Methods. Three different NGS approaches have been applied: targeted gene panels (TGP), whole exome sequencing (WES), and whole genome sequencing (WGS). Two custom targeted gene panels, including 56 genes associated with different cardiomyopathies or 69 known and candidate ACM genes have been used to screen both familial and sporadic cases. Genetic analysis was extended to available family members to evaluate the segregation of each mutation identified in the index case. The whole exome of 11 subjects belonging to 4 families was sequenced. In one of these families (Family#6) also WGS in 3 subjects in addition to multipoint linkage analysis was performed. Results. Targeted gene panels resulted a valuable tool for mutation screening in patients affected with ACM. At least a mutation was found in 15 out of 19 probands screened with the ‘Cardiomyopathies gene panel’. Moreover, by using the ‘ACM known and candidate genes panel’, a mutation in a candidate gene has been identified in 40.6% of probands negative for mutations in known-disease genes. Two novel missense variants in TJP1 gene (p.R265W, p.Y669C), encoding for protein ZO-1 and two in CDH2 gene (p.E493G, p.V491G), encoding for N-cadherin resulted of particular interest, other than a stop mutation in TP63 gene (p.R266*). Both TJP1 mutations affected highly conserved amino acid residues and in silico analysis of p.Y669C mutation, which segregates within the proband’s family, showed its predicted damaging effect into the protein structure. WES analysis allowed to identify a pathogenic mutation in DSP gene (p.Q1297*) not previously detected by dHPLC in Family#3, and two putative TTN mutations (p.R32573C and p.L32198M) that segregate in Family#4 and Family#5. In Family#5, a rare stop mutation in a candidate gene (CMYA5, p.K3597*) was also identified. In Family #6, a multi-step approach was applied. The availability of many affected and unaffected family members allowed to perform linkage analysis in an ‘affected only’ approach. The analysis pointed out the presence of two regions with positive pLOD score values on chromosome 19p13.3 and 11q21. The first corresponded to a 7cM region and was shared by all the affected subjects, but one. The 3cM region on chromosome 11q21 segregated in all the affected subjects exception made for two, who carried a PKP2 splice site variant (c.2578-3 T>C). WES performed in 4 patients of the family failed to identified a possible shared pathogenic mutation neither inside the critical regions, nor in the whole exome. These results were confirmed with WGS performed in 2 affected and a healthy individual. Moreover WGS pointed out the presence of a huge amount of complex variants located in repetitive intronic or intragenic regions. Discussion. The identification of disease-causing mutations facilitates timely diagnosis, allows the prevention of complications and determines the potential risk in close relatives of a proband. For hereditary cardiomyopathies, as well as for most mendelian diseases, in the last ten years NGS technology improved this process allowing to parallel sequence a large amount of genes in a time and cost-efficient manner. Since mutations in the most index cases sequenced by using a targeted gene panel have been detected, it resulted a valid approach not only for genetic testing but also to identify putative novel disease genes. In the present study, novel mutations in genes encoding for intercalated disc proteins (TJP1, CDH2) have been identified, confirming the idea that ACM has to be considered a ‘junctional disease’ rather than only a ‘desmosomal disease’. Despite TGP remains the most commonly used approach for hereditary cardiomyopathies, WES, applied in familial ACM cases, allowed to identify novel variants in candidate genes that would be not detected with TGP and could have a possible pathogenic role or a modifier effect in the phenotypic expression. Finally, even though WGS didn’t allow to make a genotype-phenotype correlation in Family #6, the huge amount of data produced represent permanent data that could be re-analysed in the future with new insights and the discovery of novel disease genes.
Introduzione. La cardiomiopatia aritmogena (ACM) è una patologia ereditaria del muscolo cardiaco, caratterizzata da una progressiva sostituzione adiposa o fibroadiposa a carico prevalentemente del miocardio del ventricolo destro. Dal punto di vista clinico, è una patologia eterogenea con ampia variabilità clinica inter- ed intra- familiare; presenta infatti sia forme completamente asintomatiche sia forme molto gravi con rischio di morte improvvisa. Questa patologia, geneticamente eterogenea, è trasmessa come carattere autosomico dominante a penetranza incompleta ed espressività variabile. Ad oggi 15 loci e 13 geni sono stati associati alla malattia, di cui gran parte codificano per proteine desmosomali e proteine della cosiddetta area composita dei dischi intercalari. Poiché sono state identificate mutazioni causative in geni noti sono nel 60% dei casi, altri geni, non ancora identificati, potrebbero essere coinvolti nella comparsa del fenotipo patologico. Scopo della ricerca. Nello studio descritto nella presente tesi, il DNA di 59 casi indice è stato analizzato attraverso due diversi pannelli di geni tramite sequenziamento di nuova generazione (NGS). Inoltre, in quattro famiglie con ricorrenza di casi di ACM, in cui non sono state identificate mutazioni nei geni desmosomali, sono state integrate diverse tecniche allo scopo di identificare nuovi loci e geni malattia. Metodi. Nell’ambito del sequenziamento di nuova generazione (NGS) tre diversi approcci sono stati utilizzati: il sequenziamento di pannelli di geni target (TGP), il sequenziamento dell’intero esoma (WES) e il sequenziamento dell’intero genoma. Due sono i pannelli di geni considerati, uno comprendente 56 geni associati a diverse cardiomiopatie, l’altro comprendente 69 geni, tra cui i 13 geni associati alla cariomiopatia aritmogena e 56 geni candidati. L’analisi genetica è stata poi estesa ai familiari dei probandi, ove disponibili, per valutare la segregazione delle mutazioni identificate. L’intero esoma è stato poi sequenziato in 11 soggetti appartenenti a 4 diverse famiglie. Infine, in una di queste famiglie (Famiglia#6) è stata eseguita un’analisi di linkage ed è stato sequenziato l’intero genoma di tre soggetti. Risultati. L’utilizzo dei pannelli di geni target si è rivelato una buona strategia per lo screening di mutazioni in pazienti affetti da ACM: almeno una mutazione è stata trovata in 15 probandi su un totale di 19 analizzati con il pannello di geni associati a diverse cardiomiopatie; inoltre, è stata identificata una mutazione in uno dei geni candidati nel 40.6% dei probandi sequenziati con il secondo pannello e risultati negativi per mutazioni nei geni noti. Tra queste mutazioni, risultano di particolare interesse 2 nuove mutazioni missenso localizzate nel gene TJP1 (p.R265W, p.Y669C), 2 mutazioni nel gene che codifica per l’N-caderina (CDH2, p.E493G, p.V491G) e una mutazione di stop nel gene TP63 (p.R266*). Entrambe le mutazioni di TJP1 riguardano aminoacidi altamente conservati, inoltre un’analisi in silico degli effetti della mutazione p.Y669C, che segrega all’interno della famiglia, evidenzia un riarrangiamento della struttura proteica della proteina che riporta la mutazione rispetto alla proteina wild-type. L’analisi dell’esoma nella Famiglia#3 ha permesso di identificare una mutazione patogena di stop nel gene DSP, che non era stato possibile individuare al precedente screening tramite dHPLC. Inoltre, tale approccio ha permesso di identificare due putative mutazioni nel gene TTN (p.R32573C and p.L32198M) che segregano nelle Famiglie #4 e #5, rispettivamente. Nella Famiglia #5 inoltre è stata identificata una rara mutazione di stop in un gene candidato (CMYA5, p.K3597*). Per lo studio della Famiglia#6, invece, sono stati utilizzati diversi approcci. Data la disponibilità di molti soggetti affetti e sani appartenenti alla famiglia, in seguito alla genotipizzazione degli stessi, è stata eseguita un’analisi di linkage. L’analisi ha evidenziato la presenza di due loci che riportano valori di lod score positivi a livello del cromosoma 19p13.3 e del cromosoma 11q21. Il primo locus corrisponde ad una regione di 7 cM ed è condiviso da tutti i soggetti affetti della famiglia, eccetto uno. La regione di 3cM nel cromosoma 11q21 invece segrega in tutti i soggetti affetti ad eccezione di due, che portano una mutazione in un sito di splicing del gene PKP2 (c.2578-3 T>C). Il sequenziamento dell’esoma in 4 soggetti affetti della famiglia non ha permesso di identificare nuove mutazioni condivise né all’interno delle due regioni critiche, né all’interno dell’intero esoma. Tali risultati sono stati confermati dal sequenziamento del genoma effettuato in due affetti e un soggetto sano della famiglia. Il sequenziamento del genoma ha inoltre messo in luce la presenza di un’enorme quantità di varianti complesse localizzate in regioni introniche o intrageniche. Discussione. L’identificazione di mutazioni causative nella cardiomiopatia aritmogena facilita la diagnosi tempestiva, permette di prevenire eventuali complicazioni e determina il rischio di sviluppare la malattia nei familiari di un soggetto affetto. Per le cardiomiopatie ereditarie, come per la maggior parte delle malattie mendeliane, negli ultimi 10 anni le tecniche NGS hanno apportato grossi miglioramenti nel processo di identificazione di mutazioni, permettendo di sequenziare una grande quantità di geni parallelamente, in modo più rapido e meno costoso. In questo studio, dal momento che sono state identificate mutazioni nella maggior parte di probandi analizzati, l’utilizzo di pannelli di geni target si è dimostrato un valido approccio non solo per un test genetico ma anche per l’identificazione di nuovi geni malattia. Sono state infatti identificate nuove mutazioni in geni che codificano per proteine dei dischi intercalari dei cardiomiociti, confermando l’idea che l’ACM deve essere considerata una ‘malattia delle giunzioni’ piuttosto che una ‘malattia dei desmosomi’. Nonostante l’utilizzo di pannelli di geni target rimanga l’approccio più comunemente usato nella ricerca di mutazioni nelle cardiomiopatie ereditarie, il sequenziamento dell’intero esoma, applicato in questo studio solo a casi familiari, ha permesso di identificare varianti in geni candidati non inclusi nei pannelli di geni e che potrebbero avere un ruolo nell'espressione del fenotipo patologico. Infine, nonostante il sequenziamento del genoma nella Famiglia#6 non abbia permesso al momento di stabilire una correlazione tra genotipo e fenotipo al momento, la mole di dati prodotti potrà essere rianalizzata in futuro alla luce di nuovi geni annotati e nuovi geni malattia identificati.

Severe remodeling processes may occur in the heart due to both genetic and non-genetic diseases. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction at different orders of magnitude and, eventually, lead to arrhythmias. In this scenario, arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease that involves ventricular dysfunction, arrhythmias, and localized replacement of contractile fibers with fibrofatty scar tissue. Unfortunately, nowadays, redicting the impact of fine structural alterations on the electrical disfunction in entire organs is challenging, due to the inefficacy of standard imaging methods in performing high-resolution three-dimensional reconstructions in massive tissues. In this work, we developed a new full-optical correlative approach to quantify and integrate the electrical dysfunctions with three-dimensional structural reconstructions of entire hearts, both in controls and in a mouse model of ACM. We combined optical mapping of the action potential propagation (APP) with advances in tissue clearing and light-sheet microscopy techniques. First, we employed an optical platform to map and analyze the APP in Langendorff-perfused hearts. Then, we optimized the SHIELD procedure for the clearing of cardiac tissue, thus converting the previously electrically characterized samples into well-preserved and fully-transparent specimens. A high-throughput light-sheet microscope has been developed allowing the reconstruction of the whole mouse heart with a micrometric resolution allowing fine quantification of myocytes alignment and fibrosis deposition across the organ. Finally, we developed a software pipeline that employs high-resolution 3D images to analyze and co-register APP maps with the 3D anatomy, contractile fibers disarray, and fibrosis deposition on each heart. We found that although fiber disarray is not involved in this remodeling process, the accumulation of fibrotic patches found in the ACM mouse model can deform the propagating wavefront potentially facilitating the formation of reentry circuits. We believe that this promising methodological framework will allow clarifying the involvement of fine structural alterations in the electrical dysfunctions, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

ARVC is a cardiac disease associated with ventricular cardiomyocyte fibro-fatty replacement and sudden death. It presents with incomplete penetrance and variable clinical expression. Desmoplakin (DP) and plakoglobin (PG) gene mutations were previously identified. This study aimed: to identify desmosomal (DS) gene mutations in an ARVC cohort by DNA sequencing study the gene transmission and disease expression in affected families and determine functional implications of three identified mutations (A733fsX740PKP-2, S140FPKP-2 and Q273fsX288DP) using wild-type and mutant cDNA plasmid cloning and cell line protein expressions. Eight PKP-2 mutations were identified of which four were novel: frame-shifts disrupting Armadillo domains (ARM) 4, 5 and 8, and a non-sense disrupting ARM 2. One missense and two frame-shift novel DP mutations occurred. Pedigree analysis showed incomplete gene penetrance and variable ARVC expression. Stable cells over-expressing A733fsX740PKP-2 showed increased cellular adhesion and apoptosis following mechanical stretch recovery, and absence of cell junction Connexin-43 (Cx43) protein without significant change in cell-input resistance. Desmosomal lengths were statistically unaltered, but intermittent pale DS coupling occurred. Truncated PKP-2 showed reduced PG interaction. Stable cells over-expressing S140FPKP-2 showed no differences in cell proliferation, adhesion, apoptosis following mechanical stretch recovery, and in cell junction Cx43 protein localization. DS lengths, however, were significantly increased, and missense PKP-2 showed reduced p-catenin interaction. Stable cells over-expressing Q273fsX288 DP showed reduced DS widths, vimentin filament retraction, and lower monolayer adhesion. PG and PKP-2 interacted normally with truncated DP. Altered DS morphologies featured prominently in all three mutant protein expressions. Reduced inter-protein interactions of truncated and missense PKP-2 proteins with PG and p-catenin respectively suggests PG and p-catenin signalling may be affected. Q273fsX288DP expression lacking DP distal domains correlated with loss of DS filament association. These data suggests that expression of mutant desmosomal proteins leads to abnormal DS formation, thus providing the substrate for arrhythmia and cardiomyopathy.

Acute coronary artery occlusion is the most common cause of sudden cardiac death. In some cases an acute myocardial infarction (MI) can immediately lead to lethal arrhythmias, but the factors that determine whether an MI precipitates arrhythmias are uncertain. In this thesis, I compare and contrast the detailed electrophysiology of hearts that develop arrhythmias post MI compared to those that do not using voltage sensitive fluorescent dyes in isolated rabbit hearts. In an attempt to improve the information from voltage mapping studies, initial work involved attempts to use ratiometric imaging of the fluorescence from the dye RH237. These identified optimal filter settings to collect voltage data at two distinct wavebands that would eliminate movement artefact and permit absolute voltage measurements. But routine implementation of this technique was prevented by additional technical issues related to uneven illumination levels and alignment of the two cameras. In initial studies the drug E-4031, a selective blocker of the delayed rectifier potassium current (IKr), was used to assess the contribution of this channel to repolarisation in rabbit ventricle, both in the steady state at a range of physiological and sub-physiological frequencies and in the transition between step frequency changes. The data suggests that IKr has a small but significant contribution to repolarisation at normal heart rates, 300ms pacing cycle length; a close to maximal concentration of E-4031 (0.03M) increased action potential duration (APD90) by 8.5 ± 1.7ms (P<0.01). This contribution is considerably larger at lower stimulation frequencies; at 1Hz E-4031 increased APD by 73.7 ± 13.7ms (P<0.05). The EC50 for E-4031 in this study was 0.01M which is similar to that reported in the literature. The recovery of the channel from inactivation appeared an important determinant of the rate of adaptation of the action potential duration. In the main experimental section, a novel snare technique was used to produce the acute coronary artery occlusion in the apical region of the left ventricle (LV) free wall. From control experiments (n=21), 47.6% of the hearts develop ventricular fibrillation (VF) within 30 minutes of coronary artery occlusion. On average, hearts with intrinsically longer epicardial action potential duration prior to ischaemia (mean APD50 168.8 ± 5.5ms) did not develop VF, and those with shorter APD (mean APD50 141.5 ± 3.5ms) during pre-occlusion period were more prone to VF (P<0.001). However, artificially prolonging the APD with the drug E-4031 (0.03 concentration) prior to coronary artery occlusion did not significantly change the incidence of arrhythmia. Brief and transient exposure to isoprenaline (0.3Mconcentration) before the occlusion shortened the average APD prior to occlusion but still did not increase the likelihood of VF. Therefore, I concluded that shorter epicardial APD values prior to ischaemia are associated with a higher incidence of arrhythmia but are not the cause. To investigate this further, a panoramic optical mapping technique was used to look at the electrophysiological properties across the entire ventricular surface of the hearts. The panoramic optical mapping study confirmed the correlation between shorter APD pre-occlusion and the incidence of VF during occlusion and indicated that the region of the LV exhibiting a shorter APD is confined to the apical half of the LV, and does not include basal LV or RV electrophysiology. Panoramic imaging also revealed a delayed activation time predominantly in the basal aspects of the LV. Both of these events – shorter APD in the apex and longer activation time in the base – were a feature of hearts that developed VF on ligation of the coronary artery. Future work will investigate the cellular/molecular basis for these differences in ventricular electrophysiology.

Includes bibliographical references.
Arrhythmogenic right ventricular cardiomyopathy / dysplasia (ARVC/D) is a genetic disease causing fibro-fatty replacement of the right ventricular myocardium, resulting in cardiac arrhythmias and sudden death. Part of the diagnostic work up for these patients includes a biopsy of the endocardium which has historically been difficult to interpret and of limited value in the early stages of disease. This study will focus on novel immunohistochemical stains of the cardiac desmosomes. These will be used to try to aid in the early diagnosis of ARVC.

Introduction - Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited heart muscle disorder that primarily affects the right ventricular myocardium early in the course of disease with later-onset left ventricular involvement. Clinically, it is characterized by ventricular arrhythmias of right ventricular origin, as noted by ventricular tachycardia with a left bundle branch block morphology, commonly associated with syncope or sudden cardiac death in particular in teenagers and in young adults. To date, mutations in 7 genes, including 5 encoding desmosomal proteins, Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin-2 (PKP2), Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2), have been identified in ARVC/D patients. The study of genetically engineered mice models of ARVC/D, generated through transgenesis and gene targeting, recapitulates the pathogenic characteristics of the disease. Methods - The study involved a cohort of 110 unrelated consecutive index cases and their available family members. Clinical diagnosis of ARVC/D was based on major and minor criteria established by an international Task Force. Mutation screening in four desmosomal protein genes (PKP2, DSP, DSG2 and DSC2) was performed by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in ARVC/D index cases. Desmin (DES) and plakophilin-4 (PKP4) candidate genes were screened in 80 ARVC/D index cases, by DHPLC and direct sequencing as well. In order to generate a knock-in mouse carrying a targeted mutation in DSG2, the mouse dsg2 gene was isolated from the l FIX II 129/SVJ library. A 7041bp genomic fragment was subcloned in the targeting vector, and three nucleotide mutations (G105R, N271S, and K299E) were introduced in mouse dsg2 exons 4, 7, and 8 by site-directed mutagenesis. Neomycin resistance cassette (Neo) and Thymidine kinase cassette (TK) were cloned in the targeting vector, thus allowing positive-negative selection of the recombination events. Results - Analysis of coding sequences of PKP2, DSP, DSG2 and DSC2 genes was performed on genomic DNA of 110 ARVC/D index cases. One PKP2 mutation was detected in 16 probands (14.6%), one DSP mutation in 11 (10.0%), one DSG2 mutation in 8 (7.3%), and one DSC2 mutation in 3 subjects (2.7%). Compound or double heterozygosity was identified in 14 probands (12.7%). Available family members of 19 index cases were screened for the detected mutations and clinical investigation showed that clinical expression of ARVC/D mutations is heterogeneous even among relatives, ranging from a complete lack of symptoms and/or clinical manifestations to severe disease phenotype. According with the hypothesis that ARVC/D is due to desmosomal defects, DES and PKP4 genes were screened in 80 ARVC/D probands. Two variations (K241E and c.736-11A>G) were detected in DES gene in two subjects. Three PKP4 variations (c.245+101A>G, A479A, and P797P) were detected in three subjects. None of the nucleotide changes was found in 300 control subjects from the same population. To establish a cause and effect relationship between DSG2 mutations and ARVC/D, a knock-in mouse model will be generated. A dsg2 genomic fragment was cloned in the targeting vector that will be used; by site-directed mutagenesis three dsg2 pathogenic mutations (G105R, N271S, and K299E) were introduced; also Neo and TK cassettes were cloned. After linearization, the vector will be transfected into the murine embryonic stem cells. Discussion - ARVC/D is a recognized cause of sudden cardiac death, which may be prevented by timely detection and intervention. Since mutations causing ARVC/D have been identified so far in genes encoding desmosomal proteins, this cardiomyopathy might be considered as "a disease of the desmosome". Mutation screening of the four desmosomal genes PKP2, DSP, DSG2 and DSC2 in 110 ARVC/D unrelated individuals allowed successful genotyping of 52 (47.3%). Emerging data suggest that an important minority of ARVC/D patients are compound heterozygous or double heterozygous (12.7%). Clinical comparison of patients carrying single and multiple mutations showed no significant differences in terms of electrocardiographic and structural abnormalities, major events and disease expression. The only significant difference was that patients carrying DSG2 mutations were found older at diagnosis and at the time of major arrhythmic symptoms than DSP and PKP2 carriers. On the ground of these data, it is impossible to clinically differentiate different forms of ARVC/D due to mutations in different genes. Since no causing mutations have been identified in more than 50% of patients, additional components of the desmosome-intermediate filament complex and associated proteins were considered the primary candidates disease-genes. The coding regions of DES and PKP4 genes were screened in 80 ARVC/D index cases. Most of the detected nucleotide changes were intronic and synonymous variations that do not change the sequence of the gene product, but might affect splicing (by activating a cryptic splice site). On the basis of present data, it is not possible to exclude the involvement of these genes in the pathogenesis of ARVC/D; thus, mutation screening in ARVC/D genes on DNA of probands should be planned only on the basis of relative prevalence of mutations in different genes. The identification of the primary genetic causes of ARVC/D has opened the possibility to generate animal models where the events underlying the pathophysiology of this disease can be studied in detail. Gene transfer technology allows the creation of specific mutant genotypes in animals thereby increasing their chance of resembling human diseases at the genetic and phenotypic levels. The generated targeting vectors will be transfected into the murine embryonic stem cells to create a DSG2 knock-in mouse model. In perspective, such models should prove useful for investigating cellular mechanisms involved in the molecular pathogenesis of ARVC/D and for assessing the effects of selected pharmacological treatments.

Cardiomyocyte contraction involves sarcolemmal depolarization causing a small influx of Ca2+ which is then amplified via a larger release from the sarcoplasmic reticulum (SR). Under certain conditions SR Ca2+ is released in the absence of depolarization - so called SR Ca2+ leak. This is thought to be a key cause of arrhythmogenesis in heart failure (HF). The aims of this thesis were to assess how SR leak changes in a rat model of HF induced by chronic myocardial infarction (MI) and the mechanism of modulation using INa blockers. Several novel methodologies were developed to do this including the use of hierarchical statistical analysis which reduced the chance of type I errors in comparison to standard techniques. Detailed assessment of the HF model showed that there was fluid retention and eccentric hypertrophic remodelling of an impaired left ventricle by 16 weeks post MI which were more marked compared with earlier timepoints. Although under basal conditions Ca2+ leak was similar in HF and control cells, leak enhancement in response to isoprenaline was more marked in HF cells and there were significant heterogeneities in leak when comparing the borderzone to remote regions. At an earlier stage (8 weeks post MI) we found more frequent Ca2+ waves even under basal conditions. Analysis of Ca2+ leak in 3-D for the first time using a novel microscopy technique showed that arrhythmogenic waves originate from regions of preserved t-tubules. Finally we explored the use of flecainide to inhibit SR leak and showed that it acts via reduction of INa, which enhances Ca2+ efflux via the Na+/Ca2+ exchanger. In conclusion this thesis has drawn on several novel methodologies to gain a deeper understanding of SR leak, both in terms of how it changes in HF and by exploring a novel mechanism by which it can be reduced.

Cardiomyopathies are a heterogeneous group of diseases of the heart muscle, associated to alterations of cardiac currents and ions handling that cause the impairment of the cardiac function leading to the insurgence of arrhythmias, heart failure and sudden death. In this project, we focus our attention on ion channel diseases. I will use different models, from expression system, to human adult cardiomyocytes isolated from septal samples, until human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) to design cardiomyopathies and used it to test the efficacy and safeness of “old” drugs in restore cardiac function. I analysed the electrophysiological feature of those models, by calcium and sodium fluorescence and by patch clamp technique to better investigate the cardiac impairment. In the cell-based model of a mixed phenotype between BrS and LQT3s obtained in HEK293 cells transfected with the wt-SCN5A gene or the 1795insD-SCN5A, together with the β-subunits, we tested the potential efficacy of Mexiletine in increase the altered peak sodium current (INa) and in reducing the late sodium current (INaL) (that were respectively reduce and increase by the mutation on the cardiac sodium channel). The double efficacy make it a possible effective treatment for the overlap syndrome. Our results confirmed this hypothesis, Mexiletine (500µM) in chronic treatment (48h incubation, washed before recordings) is able to increase INa. In cardiomyocytes isolated from human septal samples of obstructive HCM (OHCM) patients undergoing surgical myectomy, we analysed the efficacy and safeness of Disopyramide. Less is known about the complexity of its action, beside the sodium channel block, so we investigated its effects in reducing arrhythmic event by measuring action potential (AP) and cardiac ion currents by patch clamp technique and Calcium transient features, using calcium sensing fluorescent dye. Our results show that it is able to reduced AP duration, the upstroke velocity and the incidence of EAD and DAD reducing the intensity of INa-L and ICa-L and inhibiting Ik and calcium sparks from RYR. It’s action on multiple ion channel make Disopyramide a safe drug for OHCM, more than the actual treatment suggested by the guidelines. hiPSCs are a novel tool to obtain cells with preserved genetic background of healthy individuals or patients. The limitations of this model consist in the maturity of the cardiomyocytes, that are consistently different from an adult ventricular cardiomyocytes (for both morphological and electrophysiological features). To improve the maturation level of our cells we used long term culture (until 90 days post differentiation) associated to patterned surfaces. By measuring calcium handling and action potential profile at different time points (30, 60 and 90 days), we tried to understand how the electrophysiological features of these cardiomyocytes evolve in the control and DMD line. hiPSC-CM could be the future of disease modelling but the limits in the maturation protocols need to be overpass, our methods shows how is possible do that using a long term culture associated with patterned surfaces and how it could give us the chance to understand the disease progression during cardiomyocytes development and which pathway target with drugs to inhibits disease progression. Pay attention in choosing the right model for our purpose is essential, this could let our work to successfully and reliable result with high translational value. The troubles in modelling cardiomyopathy associated with elevated arrhythmic risk make the decision even harder and probably the combination of different model will be a valid option. Our results also highlight the importance of a personalized therapy for those patients, in which every subject has a different background and different phenotype manifestation; studying, not the disease, but the patient’s phenotype will help us to identify specific drug treatment suitable for that subject.