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
No
J. Physiol.-London
Article
Científica
4.4
01/10/2024
001321217100001
During exercise or stress, the sympathetic system stimulates cardiac contractility via beta-adrenergic receptor (beta-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Ca2+ signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A).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 beta-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 beta-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 beta-AR-mediated sensitization of RyRs.
Ca2+-induced Ca2+ release; cardiac muscle; dephosphorylation; excitation-contraction coupling; protein phosphatase; ryanodine receptor 2