Treatment with beta-blockers normalizes RyR2 phosphorylation and calcium spark activity in atrial myocytes from patients with atrial fibrillation

Background Atrial fibrillation has been associated with an increase in ryanodine receptor (RyR2) phosphorylation and local calcium release (calcium sparks). Carvedilol, a nonselective beta-adrenergic receptor blocker also inhibits the cardiac ryanodine receptor (RyR2), but it has been suggested that the enantiomer R-carvedilol only inhibits RyR2 activity and hence has the potential to inhibit calcium sparks without affecting RyR2 phosphorylation. Purpose This study aimed to determine the ability of the enantiomers R- and S-carvedilol to reverse RyR2 phosphorylation at s2808 and calcium sparks induced by the β2-adrenergic agonist fenoterol, in order to determine the relationship between RyR2 phosphorylation at s2808 and calcium spark frequency, and to assess the efficacy of R- and S-carvedilol. Methods Human right atrial myocytes were isolated and subjected to immunofluorescent labelling of total and s2808 phosphorylated RyR2, or loaded with fluo-4 and subjected to confocal calcium imaging. Beta-adrenergic receptors were first activated with 3μM fenoterol and then inhibited by different concentrations of carvedilol R- or S-enantiomers. Results Incubation of myocytes with fenoterol increased the s2808/RyR2 ratio from 0.32±0.03 to 0.66±0.05 (n=18, p<0.001). Incubation with 0.1, 0.3, 1 or 3μM R-carvedilol in the presence of fenoterol changed the s2808/RyR2 ratio to 0.64±0.05, 0.44±0.04, 0.34±0.07 and 0.28±0.05 (p<0.01) respectively. For comparison 3μM S-carvedilol reduced the s2808/RyR2 ratio to 0.23±0.06 in myocytes from 5 patients (p<0.01). Confocal calcium imaging revealed that fenoterol increased the spark density from 0.28±0.04 to 1.24±0.25 events/s/1000μm2 (n=9, p<0.01) and addition of 0.1, 0.3, or 1μM R-carvedilol changed the frequency to 1.32±0.52, 0.38±0.05, and 0.15±0.05 events/s/1000μm2 (p<0.01) respectively. Analysis of atrial myocytes from patients without atrial fibrillation revealed that the s2808/RyR2 ratio was similar in 25 patients treated with beta-blockers (0.39±0.04) and 57 that did not receive beta-blockers (0.44±0.03, p=0.33) while the s2808/RyR2 ratio was significantly smaller in 16 patients with atrial fibrillation receiving beta-blockers (0.43±0.08) than in 5 patients that did not (0.80±0.19, p<0.05). Conclusions R-carvedilol reverses the effects of beta-adrenergic stimulation on s2808 phosphorylation and calcium sparks in human atrial myocytes, and treatment with beta-blockers reduces excessive RyR2 phosphorylation at s2808 in patients with atrial fibrillation to levels observed in those without the arrhythmia, pointing to beta-adrenergic receptors as a target for controlling RyR2 phophorylation and activity 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 & Spanish Ministry of Health and Consume

Pathological phosphorylation of the ryanodine receptor at s2808 increases the number of individual clusters activated per calcium spark and the calcium released per cluster

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<0.05), were wider (1.43±0.03 vs. 1.13±0.04 μm, p<0.05), and lasted longer at half maximum (59.8±5.2 vs. 44.4±2.4 ms, p<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<0.01). Interestingly, sparks lasted longer (79±5 vs. 61±4 ms, p<0.001) were wider (3.0±0.2 vs. 2.2±0.1 μm, p>0.001) and had bigger mass (31.5±3.3 vs. 21.9±3.3 a.u, p<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<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<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<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<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