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%.