Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs 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 ßAR 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 ßAR-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 ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation.

Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of Gi protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP3. By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP3-dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP3 that leads to pronounced alterations of cytosolic cAMP levels.

Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes.

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes.