Pathological phosphorylation of the ryanodine receptor at s2808 increases the number of individual clusters activated per calcium spark and the calcium released per cluster
Authors: Nolla-Colomer C , Tarifa C , Llach A , Jimenez-Sabado V , Vallmitjana A , Casabella S , Colino H , Izquierdo P , Casellas S , Rodriguez-Font E , Cinca J , Chen S.R.W , Benitez R , Hove-Madsen L 
Published: November 2020

Abstract

Background
Atrial fibrillation (AF) has been associated with an increase in ryanodine receptor (RyR2) phosphorylation and local calcium release (sparks), but it is not known how calcium dynamics of individual RyR2 clusters affect spark dimensions and properties.

Purpose
This study aimed to test the hypothesis that pathological alterations in the phosphorylation of individual RyR2 clusters at s2808 facilitate the fusion of spontaneous calcium release events from neighboring RyR2 clusters.

Methods
Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to confocal calcium imaging or immunofluorescent labelling of total and s2808 phosphorylated RyR2. Calcium signals were measured at a frame rate of 240 Hz in a 0.5 x 0.5 μm region of interest (ROI) for each GFP-tagged RyR2 cluster and spontaneous calcium release events were detected using a custom-made algorithm.

Results
Calcium sparks recorded in 41 myocytes with GFP-tagged RyR2s was due to the spontaneous opening of a single RyR2 cluster in 91.2±2.2% of the cells and two neighbouring clusters in (6.2±1.6%) of the cells. Events with two clusters had bigger amplitude (0.14±0.01 vs. 0.10±0.01, p&lt;0.05), were wider (1.43±0.03 vs. 1.13±0.04 μm, p&lt;0.05), and lasted longer at half maximum (59.8±5.2 vs. 44.4±2.4 ms, p&lt;0.01). Consequently, the calcium spark mass, measured as the time integral of the spark in each ROI increased from 9.2±1.6 for 1 cluster to 17.8±3.5 a.u. for 2 clusters (p&lt;0.01). Interestingly, sparks lasted longer (79±5 vs. 61±4 ms, p&lt;0.001) were wider (3.0±0.2 vs. 2.2±0.1 μm, p&gt;0.001) and had bigger mass (31.5±3.3 vs. 21.9±3.3 a.u, p&lt;0.01) in atrial myocytes from 21 patients with AF than in 27 without. Because phosphorylation of RyR2 clusters at s2808 (s2808/total RyR2) was higher in patients with than without AF (0.80±0.19 vs. 0.44±0.03, p&lt;0.05), we tested how stimulation of RyR2 phosphorylation at s2808 with the β2-adrenergic agonist fenoterol (3μM) affected calcium release in individual RyR2 clusters. Fenoterol increased s2808 phosphoryaltion from 0.39±0.05 to 0.79±0.16 (p&lt;0.05, n=9). It also increased the mass of sparks with 1 RyR2 cluster (from 9.2±1.1 to 16.0±2.3 a.u., p&lt;0.01) and sparks with 2 clusters from 17.8±3.5 to 23.6±2.7 a.u. Moreover, it increased the fraction of sparks with 2 clusters from 6.2±1.6% to 19.3±3.3% (p&lt;0.01) and sparks with 3 clusters reached 6.3±1.9% in the presence of fenoterol.

Conclusions
The calcium spark mass recorded in patients without AF is comparable to that recorded during activation of calcium release from one or two GFP-tagged RyR2 clusters. The larger mass and slower kinetics of sparks recorded in patients with AF is compatible with an increase in the calcium released from each RyR2 cluster and a 3-fold increase in sparks with 2 or 3 RyR2 clusters observed in GFP-tagged RyR2s when phosphorylation at s2808 is increased to levels observed in atrial fibrillation.

Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation; Generatlitat de Catalunya