Pathological phosphorylation of the ryanodine receptor at s2808 increases the number of individual clusters activated per calcium spark and the calcium released per cluster
Bibliographic record
Abstract
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<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
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".