In mouse cardiomyocytes, intracellular β1-adrenergic receptors at the sarcoplasmic reticulum, requiring OCT3 for catecholamine transport, mediated phospholamban phosphorylation and contractility.
Intracellular β1-adrenergic receptors at the sarcoplasmic reticulum, accessed via OCT3-mediated transport, are critical for catecholamine-induced cardiac contractility.
Rationale: β 1 ARs (β 1 -adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) 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, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β 1 ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca 2+ -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 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.
Wang et al. (Fri,) reported a other. OCT3 knockout and beta-adrenergic agonists/antagonists vs. Wild-type myocytes was evaluated on PKA activation, phospholamban phosphorylation, and cardiac contractility. In mouse cardiomyocytes, intracellular β1-adrenergic receptors at the sarcoplasmic reticulum, requiring OCT3 for catecholamine transport, mediated phospholamban phosphorylation and contractility.
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