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Effects of atrial fibrillation on the distribution and frequency of spontaneous calcium release events in human atrial myocytes (AF in Human Myocytes)
Start date: Apr 2, 2013, End date: Apr 1, 2015 PROJECT  FINISHED 

Atrial fibrillation (AF) is the most common cardiac arrhythmia and currently there are no efficient treatments. The host laboratory has linked AF to abnormal Ca2+ handling by increased spontaneous sarcoplasmic reticulum (SR) Ca2+ release in human atrial myocytes (HAMs), and that an underlying mechanism is an increased expression and activation of A2A receptors. We hypothesize that the spatial distribution and frequency of spontaneous Ca2+ releases are altered in myocytes from patients with AF because of changes in the activity and distribution of the SR Ca2+ release channel (RyR2) or in proteins that modulate its activity. Then, the project has four aims: Spatial remodelling of spontaneous Ca2+ release in AF; Distribution of proteins that modulate RyR2 activity in AF; Role of adenosine receptor heteromerization in AF; and Adenosine receptor-mediated modulation of the electrical signal. To do that we shall use: Fast confocal to determine if AF is associated with changes in the spatial distribution of spontaneous Ca2+ release sites; Immunofluorescent labeling, confocal and fluorescence lifetime imaging (FLIM) to detect spatial changes in the RyR2distribution; Westernblot, IP and bimolecular fluorescence complementation to determine expression and heteromerization of A1-A2A in HAMs from patients with and without AF; and the effects of adenosine receptor heteromerization on [Ca2+]i handling shall be visualized in cultured atrial HL-1 myocytes using bimolecular fluorescent complementation and fast confocal simultaneously. Patch-clamp shall be used to quantify corresponding changes in ICa,L, Ca2+ content of the SR and frequency of spontaneous INCX. This study will allows us the identification of changes in the distribution of RyR2 or proteins that modulate its activity as a novel mechanism that potentiates the arrhythmogenic effect of spontaneous Ca2+ release and to elucidate the role of A1-A2A receptor heterodimers to abnormal Ca2+ handling in patients with AF.
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