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

RATIONALE β1-adrenoceptors (β1ARs) exist at intracellular membranes and Organic Cation Transporter 3 (OCT3) 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, co-immunoprecipitation and single-molecule pulldown demonstrated a pool of β1ARs in mouse hearts that was associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local protein kinase A (PKA) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared to wild type (WT), myocytes lacking OCT3 (OCT3KO) 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 OCT3KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3KO 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 sotatol in OCT3KO 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.