Figure 3. Protein kinase A (PKA)-dependent phosphorylation also promotes ryanodine receptor (RyR) cluster fragmentation during β-adrenergic receptor (β-AR) activation.
(a) Representative Ca2+ release units (CRUs) indicate that PKA inhibition (H89, 10 µM) reverses isoproterenol-induced cluster dispersion. (b) Direct activation of PKA using 6MB-cAMP (100 µM) can also induce cluster dispersion and is similarly reversed when followed by H89 application. (c–e) Data comparing cluster size, CRU size, and cluster density between groups (control: ncells = 26, nhearts = 3; Iso: ncells = 37, nhearts = 5; Iso + H89: ncells = 30, nhearts = 4; 6MB: ncells = 26, nhearts = 3; 6MB + H89: ncells = 44, nhearts = 4). (f) Representative CRUs indicate that 6MB-cAMP treatment effectively disperses RyR clusters in S2814A cardiomyocytes. (g) PKA inhibition also reverses isoproterenol-induced RyR dispersion in S2814A cells. Quantified experimental data are shown in (h–j). (WT: ncells = 49, nhearts = 5; S2814A: ncells = 51, nhearts = 5; S2814A + 6MB: ncells = 36, nhearts = 3; S2814A + Iso: ncells = 17, nhearts = 2; S2814A + Iso + H89: ncells = 38, nhearts = 3). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar. Scale bars: 100 nm.
Figure 3—figure supplement 1. Ryanodine receptor (RyR) cluster organization and Ca2+ spark characteristics following AIP, H89, or dual kinase inhibition in normal rat cardiomyocytes.
