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Background: Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β 1 AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β 1 AR signaling in the heart., Methods and Results: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β 1 AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β 1 AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β 1 AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P <0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P <0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P <0.05). However, the β 1 AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P <0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β 1 AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β 1 AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening., Conclusions: This study reveals distinct mechanisms for the downregulation of subcellular β 1 AR signaling in the heart under chronic adrenergic stimulation.

We have recently identified a pool of intracellular β 1 adrenergic receptors (β 1 ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular β 1 AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized β 1 AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local β 1 AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca 2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-β 1 AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-β 1 AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

BackgroundChronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.Methods and resultsWe applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P

We have recently identified a pool of intracellular β1 adrenergic receptors (β1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular β1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized β1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local β1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-β1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-β1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of β adrenoceptor (βAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac β1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the β2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent β2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows β2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes. Video abstract.

Rationaleβ1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated.ObjectiveTest localization and function of intracellular β1AR on cardiac contractility.Methods and resultsMembrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.ConclusionsFunctional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3.