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Vettor , Giulia (2016) Mir-320a as a potential novel circulating biomarker of Arrhythmogenic Cardiomyopathy. [Tesi di dottorato]

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Abstract (inglese)

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.

Abstract (italiano)

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.

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Tipo di EPrint:Tesi di dottorato
Relatore:Basso, Cristina
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > SCIENZE MEDICHE, CLINICHE E SPERIMENTALI > SCIENZE CARDIOVASCOLARI
Data di deposito della tesi:31 Gennaio 2016
Anno di Pubblicazione:31 Gennaio 2016
Informazioni aggiuntive:in collaboration with Arrhythmia Center Centro Cardiologico Monzino, Milan Italy
Parole chiave (italiano / inglese):Arrhythmogenic Cardiomyopathy, miRNA, biomarker, electroanatomical voltage mapping, endomiocardial biopsy, adipogenesis
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/11 Malattie dell'apparato cardiovascolare
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari
Codice ID:9484
Depositato il:06 Ott 2016 15:02
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