Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

Ying Wang, Qian Shi, Minghui Li, Meimi Zhao, Raghavender Reddy Gopireddy, Jian Peng Teoh, Bing Xu, Chaoqun Zhu, Kyle E. Ireton, Sanghavi Srinivasan, Shaoliang Chen, Paul J. Gasser, Julie B C Bossuyt, Johannes W. Hell, Donald M. Bers, Yang K. Xiang

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

RATIONALE: β1ARs (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. OBJECTIVE: Test localization and function of intracellular β1AR on cardiac contractility. METHODS AND RESULTS: Membrane 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. CONCLUSIONS: Functional 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.

Original languageEnglish (US)
Pages (from-to)246-261
Number of pages16
JournalCirculation research
Volume128
Issue number2
DOIs
StatePublished - Jan 22 2021

Keywords

  • catecholamine
  • intracellular membrane
  • norepinephrine
  • phospholamban
  • phosphorylation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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