Dual ablation of the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases propensity for pro‐arrhythmic spontaneous Ca2+ releases

• Published in: The Journal of physiology Vol. 602; no. 20; pp. 5179 - 5201
• Main Authors: Janicek, Radoslav, Camors, Emmanuel M., Potenza, Duilio M., Fernandez‐Tenorio, Miguel, Zhao, Yanting, Dooge, Holly C., Loaiza, Randall, Alvarado, Francisco J., Egger, Marcel, Valdivia, Hector H., Niggli, Ernst
• Format: Journal Article
• Language: English
• Published: London Wiley Subscription Services, Inc 01-10-2024
• Subjects: Ablation; Adrenergic receptors; Arrhythmia; Ca2+/calmodulin-dependent protein kinase; Ca2+‐induced Ca2+ release; Calcium (reticular); Calcium channels; Calcium imaging; Calcium release channels; Calcium signalling; Calmodulin; Cardiac muscle; Cardiomyocytes; dephosphorylation; Electrophysiology; excitation–contraction coupling; Heart; Kinases; Muscle contraction; Myocytes; Phenotypes; Phosphorylation; Protein kinase A; protein phosphatase; Receptor mechanisms; ryanodine receptor 2; Ryanodine receptors; Sarcoplasmic reticulum
• ISSN: 0022-3751; 1469-7793; 1469-7793
• DOI: 10.1113/JP286453

During exercise or stress, the sympathetic system stimulates cardiac contractility via β‐adrenergic receptor (β‐AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation–contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type‐2‐specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock‐in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2‐S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β‐ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β‐AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2‐S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2‐Ser2808 and RyR2‐S2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β‐AR‐mediated sensitization of RyRs.
Key points
Phosphorylation of cardiac sarcoplasmic reticulum Ca2+‐release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function.
Ablation of both the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs.
The intra‐sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2‐double‐knock‐in cells.
The RyR2 from double‐knock‐in cells exhibits increased Ca2+ sensitivity.
Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel.
Phosphorylation of Ser2030 might be sufficient for a β‐adrenergic response.
figure legend Immunocytochemistry reveals the ablation of ryanodine receptor (RyR2)‐S2808 and S‐2814 phosphorylation sites in cardiac myocytes from transgenic mice. This leads to increased susceptibility to Ca2+ waves and arrhythmias in double‐knock‐in (RyR‐DKI) compared to wild‐type (WT) mice. Spark recovery kinetics are accelerated in control conditions and in the presence of isoprenaline (Iso), suggesting elevated Ca2+ sensitivity of the RyRs harbouring the mutations. A simple model implementing an increase of the open probability (Po) for both RyR2 hyper‐ and hypophosphorylation can explain our findings.