Monoamine oxidase A and organic cation transporter 3 coordinate intracellular β1AR signaling to calibrate cardiac contractile function

Fig. 1. Corticosterone suppresses the MAOi-mediated enhancement of inotropic response in mouse hearts. M-mode echocardiography was recorded for 2 min at baseline and for 8 min after each drug administration. The maximal cardiac response was reported. A Representative echocardiography images of WT mouse before and after injection of EPI or DOB (100 µg/kg, i.p.) with and without MAOi pretreatment (0.4 mg/kg, i.p., 5 min). B, C Quantification of mouse cardiac EF and HR as treated in A. D Representative echocardiography images of WT mouse in response to EPI after pretreatment with MAOi and CORTI (200 μg/kg, i.p., 5 min). E, F Quantification of maximal EF and HR in D. Data are shown as mean ± SD of individual mice. n = 9/group, p values were obtained by row-matched (RM) one-way ANOVA followed by Tukey’s test

Fig. 2. Corticosterone prevents MAOi from enhancing catecholamine stimulation of excitation–contraction coupling in WT AVMs. AVMs were loaded with Ca2+ indicator (5 µmol/L Fluo-4 AM) and paced at 1 Hz. AVMs was pretreated with MAOi or CORTI for 5 min and followed by 6-min incubation of β-agonists (NE, EPI, or DOB). SS and Ca2+ were recorded for 2 min at baseline and for 6 min after agonist stimulation. The maximal SS and Ca2+ were reported. A, B Representative and quantification of SS% kinetics at the baselines and after stimulation with NE, EPI, or DOB in the absence or presence of MAOi. Dot plots represent the mean ± SD of the indicated number of AVMs from 6 WT mice. C Ca2+ transient (CaT) amplitude and decay (Tau) in response to NE, EPI, or DOB in the absence or presence MAOi. D, E Representative and quantification of SS% in response to EPI, MAOi, and CORTI cotreatment. Dot plots represent the mean ± SD of the indicated number of AVMs from 6 WT mice. F, G Representative CaT dynamics and quantification of CaT amplitude and Tau after EPI, MAOi, and CORTI cotreatment. Dot plots represent the mean ± SD of the indicated number of AVMs from 6 mice. NE = 0.1 µmol/L, EPI = 1 µmol/L, DOB = 1…

Fig. 3. Corticosterone antagonizes the positive effects of MAOi on promoting SR-localized β1AR signaling and PLB phosphorylation. Mouse AVMs expressing AKAR3 biosensors were treated with drugs as indicated. YFP/CFP FRET ratio was recorded before and after agonist stimulation for total 400 s. The maximal FRET response after drug stimulation was plotted. AVMs was pretreated with MAOi or CORTI for 5 min and followed by 5-min incubation of β-agonists (EPI or DOB) for western blot. A Schematic of intracellular catecholamine hemostasis and local β1AR activation is modulated by OCT3 and MAO-A. B, C WT AVMs were stimulated with EPI after pretreatment with vehicle control (Ctrl) or MAOi. Time courses of FRET dynamics and quantification of local PKA activities at the PM and SR after EPI and MAOi administration were plotted. Dot plots represent the mean ± SD of the indicated number of AVMs from 4 WT mice. D, E Immunoblots show detection of phosphorylated Ca2+ handling proteins (p-Ser 16 of PLB, p-Ser 1928 of LTCC, and p-Ser 2808 of RyR2) in response to EPI in the presence of MAOi. n ≥ 3 WT mice. Data were shown as mean ± SD. F Schematic depicting the effects of CORTI on subcellular β1AR activation…

Fig. 4. Cardiac-specific deletion of MAO-A increases myocardium catecholamine contents and enhances contractile function. A Quantitative measurements of endogenous NE in mouse heart, plasma, and brain (n ≥ 6 mice per group). B Quantification of cyclic AMP levels in MAO-A-FF and CKO heart tissues (n = 8 FF, 7 CKO). C Quantification of ROS in mice hearts (n = 10 mice per group). D Heart weight/body weight ratio in MAO-A-FF and CKO hearts. n = 8. E, F Western blots show the phosphorylation of PLB (p-Ser16), TnI (p-Ser23/24), RyR2 (p-Ser2808), and LTCC (p-Ser1928) in MAO-A-FF and CKO heart tissues (n = 6/group). G Schematic shows that deletion of MAO-A enhances local β1AR signaling at the SR but not PM microdomain. H M-mode echocardiography was recorded for 2 min at baselines and for 8 min after each drug administration. The maximal cardiac response was reported. Representative echocardiography images of MAO-A-FF and CKO mice before and after EPI and DOB stimulation (100 µg/kg, i.p.). I Quantification of EF and HR before and after EPI or DOB stimulation n = 9. Data are shown as mean ± SD. p values were obtained by unpaired Student’s t test (A–D, and F) or one-way ANOVA with Tukey’s multiple…

Fig. 5. Cardiac-specific deletion of MAO-A selectively enhances PKA signals at the SR microdomain. MAO-A-FF and CKO AVMs expressing AKAR3 biosensors were treated with drugs as indicated. YFP/CFP FRET ratio was recorded before and after agonist stimulation for total 400 s. A–D Time courses of SR-PKA or PM-PKA FRET ratio changes after 1 µmol/L EPI (top) or DOB (bottom) stimulation in MAO-A-FF and CKO AVMs. The maximal increases in SR-PKA and PM-PKA in AVMs were plotted as mean ± SD of the indicated number of AVMs from 4 MAO-A-FF and 5 CKO mice. E, F Western blots show detection and quantification of p-Ser 16 of PLB and p-Ser 1928 of LTCC in isolated AVMs after EPI and DOB stimulation (1 µmol/L, 5 min). Dot plots represent mean ± SD of at least three independent experiments in AVMs from MAO-A-FF and CKO mice. Data were shown as mean ± SD. p values were obtained by unpaired Student’s t test (A–D) or non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test (E, F)

Fig. 6. Deletion of MAO-A sensitizes myocyte E–C coupling response to catecholamine. MAO-A-FF and CKO AVMs were loaded with Ca2+ indicator, 5 µmol/L Fluo-4 AM, and paced at 1 Hz. AVMs SS and Ca2+ were recorded at the baseline and for 6 min after agonist stimulation. The maximal response was reported. A, C Representative and quantification of SS% at the baseline and after stimulation with 1 µmol/L EPI or DOB. Dot plots represent the mean ± SD of the indicated number of AVMs from 4 MAO-A-FF and 6 CKO mice. B, D Representative traces and quantification of CaT and Tau at the baseline and after stimulation with 1 µmol/L EPI or DOB. Dot plots represent the mean ± SD of the indicated number of AVMs from 4 MAO-A-FF and 6 CKO mice. E, F Agonist dose–response curves of SS% to EPI (EC50, CKO 82.9 ± 12.9 nmol/L, FF 62.9 ± 7.8 nmol/L) or DOB (EC50, CKO 42.5 ± 7.9 nmol/L, FF 20.8 ± 7.3 nmol/L) stimulation were analyzed in MAO-A-FF and CKO AVMs. Data represent the mean ± SD of the AVMs from 6 MAO-A-FF and 8 CKO mice. p values were obtained by one-way ANOVA (C, D) or two-way ANOVA (E, F) test followed by Tukey’s multiple comparison correction

Fig. 7. Inhibiting OCT3 by corticosterone abrogates the amplification of SR-localized β1AR signaling in MAO-A-CKO. MAO-A-FF and CKO AVMs expressing AKAR3 biosensors were treated with drugs as indicated. YFP/CFP FRET ratio was recorded before and after agonist stimulation for total 400 s. A–D Time courses and quantification of changes in SR-PKA FRET ratio after EPI or DOB (1 µmol/L) stimulation in the presence of CORTI pretreatment (2 µmol/L, 5 min). E–H Time courses and quantification of changes in PM-PKA FRET ratio after EPI or DOB (1 µmol/L) stimulation in the presence of CORTI pretreatment (2 µmol/L, 5 min). Dot plots represent the mean ± SD of the indicated number of MAO-A-FF and CKO AVMs from 4 MAO-A-FF and 5 CKO mice. Data were shown as mean ± SD. p values were obtained by one-way ANOVA test followed by Tukey’s test

Fig. 8. Corticosterone abolishes the efficacy of MAO-A deletion in promoting contractility and Ca2+ cycling. AVMs were incubated with 5 µmol/L Fluo-4 AM and paced at 1 Hz. SS and Ca2+ were recorded at baseline and for 6 min after agonist stimulation. The maximal SS and Ca2+ responses were reported. A, B Representative traces and maximal increases in SS% after EPI (1 µmol/L) stimulation with or without CORTI pretreatment (5 min). C, D Ca2+ dynamics and quantification of CaT and Tau before and after EPI (1 µmol/L) stimulation with or without CORTI pretreatment (2 µmol/L, 5 min). Dot plots represent the mean ± SD of the indicated number of AVMs 4 MAO-A-FF and 7 CKO mice. E, F Representative SS% curve and maximal SS% before and after DOB (1 µmol/L) stimulation with or without CORTI pretreatment (5 min). G, H Ca2+ dynamics and quantification of CaT and Tau before/after DOB (1 µmol/L) stimulation with or without CORT pretreatment (2 µmol/L, 5 min). Dot plots represent the mean ± SD of the indicated number of AVMs 4 MAO-A-FF and 7 CKO mice. p values were obtained by one-way ANOVA followed by Tukey’s test

Fig. 9. MAO-A and OCT3 coordinate cardiac fight-or-flight response. M-mode echocardiography was recorded for 2 min at baselines and for 8 min after drug administration. The maximal cardiac response was reported. A Representative echocardiography images of MAO-A-CKO mouse heart before and after EPI stimulation (100 µg/kg, i.p.) with and without CORTI pretreatment (200 µg/kg, i.p., 5 min). B The schematic depicts that inhibiting OCT3 by CORTI suppresses the SR-localized β1ARs activation in MAO-A-CKO hearts. C The EF and HR of MAOA-A-FF and CKO mice before and after treated with EPI and CORTI described in A. n = 8 mice in each condition. D Representative echocardiography images of OCT3-KO mouse heart before and after EPI stimulation (100 µg/kg, i.p.) with and without MAOi (0.4 mg/kg, i.p.). E The schematic depicts that deletion of OCT3 prevents the effects of MAOi on enhancing the SR-localized β1AR signaling. F The EF and HR in WT and OCT3-KO mice before and after EPI and MAOi treatment described in D. n = 8 KO mice. Data were shown as mean ± SD. p values were obtained by one-way ANOVA followed by Tukey’s test