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

Abstract

Rationale: β 1 -adrenoceptors (β 1 ARs) exist at intracellular membranes and Organic Cation Transporter 3 (OCT3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β 1 AR in cardiac contractility remains to be elucidated. Objective: Test localization and function of intracellular β 1 AR on cardiac contractility. Methods and Results: Membrane fractionation, super-resolution imaging, proximity ligation, co-immunoprecipitation and single-molecule pulldown demonstrated a pool of β 1 ARs in mouse hearts that was associated with sarco/endoplasmic reticulum Ca 2+ -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 β 1 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 1 ARs requires catecholamine transport via OCT3.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury

“…More recently, however, reduced oral bioavailability and attenuated pharmacological response to certain substrate drugs such as metformin have been detected in OCT3 knockout mice, and a 3′-untranslated region variant of OCT3 was found to be associated with reduced metformin response in humans, suggesting that OCT3 may significantly contribute to the distribution of certain substrate drugs to OCT3expressing tissues (32). Studies involving OCT3 knockout mice have also highlighted that OCT3 is an important organic cationic transporter in the heart (26,28,33,34), where it may regulate the cardiac uptake of xenobiotic substrates. This prior knowledge is consistent with our current observation that OCT3 is the sole cationic transporter driving the cardiac uptake of doxorubicin and its subsequent injury and provides a mechanistic explanation for the observation that the expression of the OCT3 gene is particularly highly up-regulated in hiPSC-CMs from pediatric cancer patients who experienced a clinically annotated cardiotoxic event after treatment with doxorubicin.…”

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2–Deficient Hearts

“…Ventricular myocytes were preincubated for 5 minutes with antagonists before ISO incubation and Ca 2+ transients examined accordingly, following published protocols. 18 The RyR2 blocker ent -verticilide ( ent -vert) 19 was used in isolated cells (25 µmol/L) and in anesthetized mice (30 mg/kg). Preparation and methods of administration are described in the Supplemental Material.…”

QR code model: a new possibility for GPCR phosphorylation recognition

“…(3) Intracellular activation of GPCR. β 1 -AR is distributed in both cell and organelle membranes and both can signal through the cAMP-PKA pathway [ 122 , 123 ]. However, how the activated GPCR on the organelle membrane is desensitized and endocytosed remains to be further investigated.…”