Probing spatiotemporal PKA activity at the ryanodine receptor and SERCA2a nanodomains in cardomyocytes

Fig. 1. Development of biosensors to detect local PKA activity in AVMs. A Schematics of genetically encoded FRET-based FKBP-AKAR3 and SR-AKAR3 biosensors and the subcellular distribution in AVMs. B Confocal images show FKBP-AKAR3 and SR-AKAR3 colocalize with RyR2 and SERCA2a, respectively, in rabbit AVMs. C Western blot shows PKA phosphorylation of FKBP-AKAR3 and endogenous RyR2 expressed in rabbit AVMs after stimulation with 100 nmol/L of ISO. The phosphorylation of RyR2 at serine 2807 and 2814 and FKBP-AKAR3 at the PKA substrate site were detected with specific antibodies and quantified in dot plots (N = 5). D and E AVMs with and without expressing FKBP-AKAR3 were stimulated with 100 nmol/L of ISO, sarcomere shortening (SS) and calcium transient of rabbit AVMs were measured before and after ISO stimulation. The maximal increases in SS and calcium transient are summarized in dot plots. Data represent mean ± SEM of indicated number of AVMs from rabbits. F Images show the YFP/CFP ratio in rabbit AVMs before and after stimulation with different concentrations of ISO, the representative curve shows the time course of YFP/CFP ratio after stimulation. G A dose response curve of the maximal…

Fig. 2. Biosensors detect distinct local adrenergic signaling at the RyR2 and SERCA2 nanodomains in mouse, rat, and rabbit AVMs. A and B FKBP-AKAR3 and SR-AKAR3 biosensors were expressed in mouse AVMs. Time courses show changes in YFP/CFP ratio of FKBP-AKAR3 and SR-AKAR3 in AVMs after stimulation with ISO (100 nmol/L) in the presence of β1AR antagonist CGP20712a (CGP, 300 nmol/L) and β2AR antagonist ICI118551 (ICI, 100 nmol/L). The maximal changes in FRET ratio after drug treatment were plotted. Data represent mean ± SEM of indicated number of AVMs from rats. C The maximal increases in YFP/CFP ratio of FKBP-AKAR3 and SR-AKAR3 in rat AVMs after stimulation with ISO (100 nmol/L) or in the presence of β1AR antagonist CGP (300 nmol/L) or β2AR antagonist ICI (100 nmol/L). Data represent mean ± SEM of indicated number of AVMs from rats. D The maximal increases in YFP/CFP ratio of FKBP-AKAR3 and SR-AKAR3 in rabbit AVMs after stimulation with ISO (100 nmol/L) or in the presence of CGP or ICI. Data represent mean ± SEM of indicated number of AVMs from rabbits. p values were obtained after one-way ANOVA analysis followed by Tukey’s test

Fig. 3. Phosphodiesterases and phosphatases control the baseline PKA activity at the RyR2 and SERCA2 nanodomains in AVMs. Rat AVMs expressing FKBP-AKAR3 and SR-AKAR3 were treated with drugs as indicated. A and B Time courses show the changes in YFP/CFP ratio before and after addition of inhibitor of PDE2 erythro-9-(2-Hydroxy-3-nonyl) adenine hydrochloride (EHNA 10 µmol/L), PDE3 (cilostamide, Clio, 1 µmol/L), PDE4 (rolipram, Roli, 10 µmol/L), or both Cilo and Roli. The maximal increases in YFP/CFP ratio before and after drug treatment were plotted. C and D Time courses show the changes in YFP/CFP ratio before and after addition of phosphatase inhibitor (1 nmol/L and 100 nmol/L, okadaic acid, OA) as indicated. The maximal increases in YFP/CFP ratio of FKBP-AKAR3 and SR-AKAR3 in AVMs after drug treatment were plotted. E and F co-treated with ISO (10 nmol/L) together with phosphatase inhibitor OA (100 nmol/L) as indicated. Time courses show the changes in YFP/CFP ratio before and after addition of drugs. The maximal increases in YFP/CFP ratio of FKBP-AKAR3 and SR-AKAR3 in AVMs before and after stimulation were plotted. Dot plots represent mean ± SEM of indicated number of AVMs from mice. p…

Fig. 4. Phosphodiesterases restrict β2AR signaling at the RyR2 and SERCA2 nanodomains in AVMs. Rat AVMs expressing Cyto-AKAR3, FKBP-AKAR3, and SR-AKAR3 were treated with drugs as indicated. A and B Time courses show changes in YFP/CFP ratio of Cyto-AKAR3 in rat AVMs induced by ISO (100 nmol/L) after 5 min pretreatment of β1AR antagonist CGP (300 nmol/L) and β2AR antagonist ICI (100 nmol/L). The maximal changes in FRET ratio after drug treatment were plotted. C–E Time courses show changes in YFP/CFP ratio of FKBP-AKAR3 induced by ISO (100 nmol/L) after 5 min pretreatment with β1AR antagonist CGP (300 nmol/L), which was then followed with addition of inhibitor of PDE4 (Roli, 100 nmol/L and 1 µmol/L) and PDE3 (Cilo, 1 and 10 µmol/L) as indicated. The maximal changes in FRET ratio after drug treatment were plotted. F–H Time courses show changes in YFP/CFP ratio of SR-AKAR3 induced by ISO (100 nmol/L) after 5 min pretreatment with β1AR antagonist CGP (300 nmol/L), which was then followed with addition of inhibitor of PDE4 (Roli, 100 nmol and 1 µmol/L) and PDE3 (Cilo, 1 and 10 µmol/L) as indicated. The maximal changes in FRET ratio after drug treatment were plotted. Dot plots represent mean ±…

Fig. 5. Inhibitory G protein selectively restricts β2AR signaling at the RyR2 nanodomains in AVMs. Rat AVMs expressing FKBP-AKAR3 and SR-AKAR3 were treated with drugs as indicated. A and B Time courses show changes in YFP/CFP ratio of FKBP-AKAR3 in AVMs after drug treatments. AVMs were pretreated with Gi inhibitor pertussis toxin (PTX, 300 ng/ml, 3 h) and β1AR antagonist CGP (300 nmol/L, 10 min) before stimulation with ISO (100 nmol/L), and followed with inhibitor of PDE4 (Roli, 100 nmol and 1 µmol/L) as indicated. The maximal changes in FRET ratio after drug treatment were plotted. C and D Time courses show changes in YFP/CFP ratio of SR-AKAR3 in AVMs after drug treatments. AVMs were pretreated with Gi inhibitor PTX (300 ng/ml, 3 h) and β1AR antagonist CGP (300 nmol/L, 10 min) before stimulation with ISO (100 nmol/L), and followed with inhibitor of PDE4 (rolipram, 100 nmol and 1 µmol/L) as indicated. The maximal changes in FRET ratio after drug treatment were plotted. Dot plots represent mean ± SEM of indicated number of AVMs from rats. p values were obtained by one-way ANOVA analysis followed with Tukey’s multiple comparison test