Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response

Figures

• 

  Figure 1.. ECC gain is decreased in S2030A+/+ myocytes after β-adrenergic stimulation and matched SR Ca2+ loading. (A) Depolarization of voltage-clamped cells from −40 to −25 mV (left) or to 0 mV (right) in control conditions and during β-adrenergic stimulation with 100 nmol/liter Iso (N = 5, n = 11 for both WT and S2030A+/+ animals). The figure shows representative line profiles of the Ca2+ transients (ΔF/F0; black traces for control and red for Iso), confocal line-scan images, and ICa traces for both WT and S2030A+/+ cells. (B and C) ECC gain was obtained from the ratio of the maximal Ca2+ transient amplitude and the peak ICa density (ΔF/F0)/(pA/pF), normalized to the SR Ca2+ content. (D) The ECC gain was obtained in conditions of matched SR Ca2+ load and ICa between all cell groups. The gain was minimally influenced by Iso in WT cells. However, the mutation affected the ECC gain ratio in Iso, mostly because of a decreased Ca2+ release during β-adrenergic stimulation (see Table S2). Please note that in this experiment, the SR Ca2+ content was experimentally matched between control and Iso. *, P < 0.05, Iso versus control; †, P < 0.05, S2030A versus WT. The whiskers cover the range from…

• 

  Figure 2.. RyR2-S2030 site phosphorylation level is increased by Iso stimulation. (A) Representative blot showing total RyR2 expression levels in WT (N = 8) and S2030A+/+ (N = 10) animals. (B) Representative blot showing the level of dephosphorylation at RyR2-S2030 site in WT animals (N = 4), before (C, control) and after 100 nmol/liter Iso application. The numbers 1 and 2 identify different animals. The level of the phosphorylation was expressed as the ratio of the dephospho-RyR2-S2030 and the total RyR ODs, both normalized for the GAPDH signal. Please note that a low level of dephosphorylation corresponds to a high level of phosphorylation. (C) Representative blot showing the maximal level of dephosphorylation at the RyR2-S2030 site in WT animals (N = 4) obtained with a high dose of Iso (1 µmol/liter) combined with the phosphatase inhibitor OA (10 µmol/liter). The dephosphorylation levels were normalized to the control (100%). Iso at the concentration used in all experiments (100 nmol/liter) resulted in a decrease in the signal corresponding to an increased level of phosphorylation (69 ± 6% compared with the control). The combination of high Iso concentration and OA further decreased the…

• 

  Figure 3.. S2030 site ablation limits the Iso-dependent increase in CaSpF. (A) Confocal line-scan images showing Ca2+ sparks in intact cells in control and after 3 min of 100 nmol/liter Iso treatment for WT (N = 4, n = 12), S2030A+/+ (N = 4, n = 13), and S2808A+/+ (N = 3, n = 10) myocytes. (B) The basal CaSpF (number of sparks per 100 µm/s) was similar among the animal groups; after application of Iso, the CaSpF in S2030A+/+ myocytes was significantly lower compared with WT and S2808A+/+ cells. (C) SR Ca2+ content was not significantly different between the mouse models. Please note that unlike in the patch-clamp experiments, the SR Ca2+ load here was not matched between control and Iso. *, P < 0.05 versus control; †, P < 0.05 versus WT. The whiskers cover the range from 10% to 90%.

• 

  Figure 4.. Maximal SERCA stimulation unmasks a limited cAMP-dependent increase in CaSpF. (A) Representative confocal line-scan images showing Ca2+ sparks in β-escin permeabilized cells before and after 5 min in 5 µmol/liter cAMP. (B) Basal CaSpF was similar for WT (N = 5, n = 12) and S2030A+/+ (N = 4, n = 10; 2.98 ± 0.34 vs. 3.39 ± 0.47 sparks per 100 µm/s, respectively; i). cAMP enhanced SpF in both cell types. SR Ca2+ content was increased after treatment with cAMP (ii). (C) Confocal line-scan images showing Ca2+ sparks in myocytes pretreated with 100 µg/ml 2D12-Fab (15 min) before and after cAMP application. (D) 2D12-Fab increased basal CaSpF (6.01 ± 0.41 vs. 5.51 ± 0.56 sparks per 100 µm/s, respectively) for WT (N = 6, n = 10) and S2030A+/+cells (N = 4, n = 10); cAMP further increased CaSpF only in WT cells. *, P < 0.05 versus control; **, P < 0.05 versus 2D12-Fab; †, P < 0.05 versus WT. The whiskers cover the range from 10% to 90%.

• 

  Figure 5.. Ca2+ spark-to-spark delay shortening is blunted during β-adrenergic stimulation in S2030A+/+ cells. (A) Representative line-scan image in control conditions (i). A very low concentration of ryanodine (50 nmol/liter) entrains repetitive sparks after binding to only a single monomeric RyR channel subunit in an entire couplon (ii). The spark-to-spark delay and the relative spark amplitudes were measured to investigate Ca2+ spark restitution (iii). (B) Histograms of spark-to-spark delays for control WT (328.8 ms, 95% CI, 313.8–348.3 ms; N = 10, n = 36, 937 spark pairs; i), Iso WT (261.8 ms, 95% CI, 254.5–269.4 ms; N = 11, n = 44, 1,783 spark pairs; ii), control S2030A+/+ (308.3 ms, CI 95%: 291.6–329.2 ms; N = 6, n = 20, 488 spark pairs; iii), and Iso S2030A+/+ cells (299.6 ms, CI 95%: 286.0–310.9 ms; N = 7, n = 27, 929 spark pairs; iv). Red lines identify the median values.

• 

  Figure 6.. Iso does not modify the latency of the first spontaneous Ca2+ release in S2030A+/+ myocytes. (A) Line-scan images were recorded in the end of a train of electrical stimulations at 1 Hz for 30 s. Each cell was recorded in control and after 3 min of 100 nmol/liter Iso treatment. The figure also shows line profiles of the triggered Ca2+ transients and the spontaneous Ca2+ releases (ΔF/F0; black traces for the control and red for Iso). The latency was calculated considering the last electrical stimulus and the first spontaneous Ca2+ release (arrows). (B) SCaW latency (17.72 ± 3.42 s vs. 8.08 ± 1.16 s), respectively, for control and after Iso stimulation for WT (18.04 ± 1.95 s for control vs. 14.27 ± 2.51 s for Iso in S2030A+/+). Latency values were not paired, because not all cells displayed SCaW. (C) Occurrence of spontaneous release (percentage of cells that show at least one Ca2+ wave; N = 4, n = 16 for WT; N = 4, n = 15 for S2030A+/+). (D) Comparison of wave speed (WT: N = 4, n = 11; 78.42 ± 5.05 µm/s [control] vs. 99.31 ± 4.03 µm/s [Iso]); S2030A+/+: N = 4, n = 15; 83.49 ± 5.56 µm/s [control] vs. 85.40 ± 4.64 µm/s [Iso]). In all experiments shown, the SR Ca2+ load was not…

• 

  Figure 7.. S2030 ablation generates adaptive changes at RyR2-S2808 and S2814 phosphorylation sites in mutant cells. (A) Representative blot showing the phosphorylation of RyR2 at the S2808 and S2814 sites in both WT and S2030A+/+ cells in control (C) and during Iso stimulation. (B and C) The level of the phosphorylation was expressed as the ratio of the respective phospho- and total RyR ODs, both normalized to the GAPDH signal (N = 8 mice for both WT and S2030A+/+ cells were used to compare baseline phosphorylation; between these, N = 4 animals were also assessed after Iso application). These data were tested for paired statistical difference. *, P < 0.05 versus control; †, P < 0.05 versus WT. The whiskers cover the range from 10% to 90%.