Intracellular β 1 -Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

• Published in: Circulation research 2021-01, Vol.128 (2), p.246-261
• Main Authors: Wang, Ying, Shi, Qian, Li, Minghui, Zhao, Meimi, Reddy Gopireddy, Raghavender, Teoh, Jian-Peng, Xu, Bing, Zhu, Chaoqun, Ireton, Kyle E, Srinivasan, Sanghavi, Chen, Shaoliang, Gasser, Paul J, Bossuyt, Julie, Hell, Johannes W, Bers, Donald M, Xiang, Yang K
• Format: Article
• Language: English
• Subjects: Adrenergic beta-Agonists - pharmacology; Adrenergic beta-Antagonists - pharmacology; Animals; Calcium-Binding Proteins - metabolism; Cell Membrane - metabolism; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases - metabolism; Female; Heart Rate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Contraction - drug effects; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Organic Cation Transport Proteins - genetics; Organic Cation Transport Proteins - metabolism; Phosphorylation; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta-1 - genetics; Receptors, Adrenergic, beta-1 - metabolism; Receptors, Adrenergic, beta-2 - genetics; Receptors, Adrenergic, beta-2 - metabolism; Sarcoplasmic Reticulum - metabolism; Signal Transduction
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.120.317452

β ARs (β -adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β AR in cardiac contractility remains to be elucidated. Test localization and function of intracellular β AR on cardiac contractility. Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca -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. Functional β ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β ARs requires catecholamine transport via OCT3.