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. Author manuscript; available in PMC: 2022 Jan 22.
Published in final edited form as: Circ Res. 2020 Nov 13;128(2):246–261. doi: 10.1161/CIRCRESAHA.120.317452

Figure 5. Activation of β1AR in the SR is essential for maximal stimulated contractility in AVMs.

Figure 5.

(A-C) Schematics show detection of β1AR-induced PKA activity at different subcellular locations (PM and SR) with FRET based biosensors in the presence of membrane impermeant β-blocker sotalol (SOTA) or the membrane permeant β-blocker propranolol (PROP). (D, E) Rat AVMs expressing PM-AKAR3 or SR-AKAR3 were stimulated with 100 nmol/L ISO with or without 5-mintue pretreatment of 25 μmol/L SOTA (blue) or 1 μmol/L PROP (red). Representative time courses show FRET response of PM-AKAR3 or SR-AKAR3 in AVMs. FRET was analyzed as F/F0 of YFP/CFP ratio. Data are shown in mean ± S.E.M. AVM (in the parenthesis) and rat numbers are indicated. p values were obtained by one-way ANOVA analysis with Tukey’s multiple comparison test. (F, G) Dose-dependent inhibition curves of PROP or SOTA on ISO-induced increases in FRET responses in AVMs expressing PM-AKAR3 or SR-AKAR3. Data show YFP/CFP ratios normalized against the maximal increases induced by ISO in the absence of β-blocker (N = 5 rats). p values were obtained by two-way ANOVA analysis followed by Tukey’s multiple comparison test when compared to OCT3-KO. (PM-AKAR3: PROP, IC50 = 7.399×10−8 mol/L, SR-AKAR3: PROP, IC50 = 9.92×10−8 mol/L; PM-AKAR3: SOTA, IC50 = 2.216×10−6 mol/L; SR-AKAR3, STOA, IC50 = 2.149×10−5 mol/L). (H-J) Rat AVMs were incubated with Ca2+ indicator (5 μM Fluo-4 AM) before 1Hz pacing. After pretreatment with SOTA (25 μmol/L) or PROP (1 μmol/L), sarcomere shortening (SS) and calcium transient were recorded before and after stimulation with 100 nmol/L ISO. The peak SS, amplitude of Ca2+ transient, and rate of Ca2+ decay (Tau) are shown as mean ± S.E.M. Animal numbers and cell numbers are indicated in figures. p values were obtained by one-way ANOVA analysis with Tukey’s multiple comparison test.