Differential Downregulation of ß1-Adrenergic Receptor Signaling in the Heart.

BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of ß1 adrenergic receptor (ß1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ß1AR signaling in the heart. METHODS AND RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine ß1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular ß1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced ß1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the ß1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the ß1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced ß1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular ß1AR signaling in the heart under chronic adrenergic stimulation.

Structure and dynamics determine G protein coupling specificity at a class A GPCR.

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The ß2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a new Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

Phosphodiesterase in heart and vessels: from physiology to diseases.

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides are critical secondary messengers in the neurohormonal regulation in the cardiovascular system. PDEs precisely control spatiotemporal subcellular distribution of cyclic nucleotides in a cell- and tissue-specific manner, playing critical roles in physiological responses to hormone stimulation in the heart and vessels. Dysregulation of PDEs has been linked to the development of several cardiovascular diseases, such as hypertension, aneurysm, atherosclerosis, arrhythmia, and heart failure. Targeting these enzymes has been proven effective in treating cardiovascular diseases and is an attractive and promising strategy for the development of new drugs. In this review, we discuss the current understanding of the complex regulation of PDE isoforms in cardiovascular function, highlighting the divergent and even opposing roles of PDE isoforms in different pathogenesis.

Loss of Myeloid Cell-Specific ß2-Adrenergic Receptor Expression Ameliorates Cardiac Function and Remodeling after Acute Ischemia.

Myeloid cells, including neutrophils, monocytes and macrophages, accumulate quickly after ischemic injury in the heart where they play integral roles in the regulation of inflammation and repair. We previously reported that deletion of ß2-adrenergic receptor (ß2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact on myeloid cells specifically. To investigate this, we crossed floxed ß2AR (F/F) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific ß2AR knockout mice (LB2) and subjected them to myocardial infarction (MI). Via echocardiography and immunohistochemical analyses, LB2 mice displayed better cardiac function and less fibrotic remodeling after MI than the control lines. Despite similar accumulation of myeloid cell subsets in the heart at 1-day post-MI, LB2 mice displayed reduced numbers of Nu by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for Nu efferocytosis. Indeed, bone marrow-derived macrophage (BMDM)-mediated efferocytosis of Nu was enhanced in LB2-versus F/F-derived cells in vitro. Mechanistically, several pro-efferocytosis-related genes were increased in LB2 myeloid cells, with annexin A1 ( Anxa1 ) in particular elevated in several myeloid cell types following MI. Accordingly, shRNA-mediated knockdown of Anxa1 in LB2 bone marrow prior to transplantation into irradiated LB2 mice reduced Mac-induced Nu efferocytosis in vitro and prevented the ameliorative effects of myeloid cell-specific ß2AR deletion on cardiac function and fibrosis following MI in vivo. Altogether, our data reveal a previously unrecognized role for ß2AR in the regulation of myeloid cell-dependent efferocytosis in the heart following injury.

Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart.

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac ß2 adrenoreceptor (ß2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac ß2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of ß2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

Silencing of circCacna1c Inhibits ISO-Induced Cardiac Hypertrophy through miR-29b-2-5p/NFATc1 Axis.

Pathological cardiac hypertrophy is one of the notable causes of heart failure. Circular RNAs (circRNAs) have been studied in association with cardiac hypertrophy; however, the mechanisms by which circRNAs regulate cardiac hypertrophy remain unclear. In this study, we identified a new circRNA, named circCacna1c, in cardiac hypertrophy. Adult male C57BL/6 mice and H9c2 cells were treated with isoprenaline hydrochloride (ISO) to establish a hypertrophy model. We found that circCacna1c was upregulated in ISO-induced hypertrophic heart tissue and H9c2 cells. Western blot and quantitative real-time polymerase chain reaction showed that silencing circCacna1c inhibited hypertrophic gene expression in ISO-induced H9c2 cells. Mechanistically, circCacna1c competitively bound to miR-29b-2-5p in a dual-luciferase reporter assay, which was downregulated in ISO-induced hypertrophic heart tissue and H9c2 cells. MiR-29b-2-5p inhibited the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) to control hypertrophic gene expression. After silencing circCacna1c, the expression of miR-29b-2-5p increased, which reduced hypertrophic gene expression by inhibiting NFATc1 expression. Together, these experiments indicate that circCacna1c promotes ISO-induced pathological hypertrophy through the miR-29b-2-5p/NFATc1 axis.

Transfer of nuclear and ribosomal material from Sox10-lineage cells to neurons in the mouse brain.

Material transfer is an essential form of intercellular communication to exchange information and resources between cells. Material transfer between neurons and from glia to neurons has been demonstrated to support neuronal survival and activity. Understanding the extent of material transfer in the healthy nervous system is limited. Here we report that in the mouse central nervous system (CNS), neurons receive nuclear and ribosomal material of Sox10-lineage cell (SOL) origin. We show that transfer of SOL-derived material to neurons is region dependent, establishes during postnatal brain maturation, and dynamically responds to LPS-induced neuroinflammation in the adult mouse brain. We identified satellite oligodendrocyte-neuron pairs with loss of plasma membrane integrity between nuclei, suggesting direct material transfer. Together, our findings provide evidence of regionally coordinated transfer of SOL-derived nuclear and ribosomal material to neurons in the mouse CNS, with potential implications for the understanding and modulation of neuronal function and treatment of neurological disorders.

Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory.

G protein-coupled receptors (GPCRs) promote the expression of immediate early genes required for learning and memory. Here, we showed that ß2-adrenergic receptor (ß2AR) stimulation induced the nuclear export of phosphodiesterase 4D5 (PDE4D5), an enzyme that degrades the second messenger cAMP, to enable memory consolidation. We demonstrated that the endocytosis of ß2AR phosphorylated by GPCR kinases (GRKs) mediated arrestin3-dependent nuclear export of PDE4D5, which was critical for promoting nuclear cAMP signaling and gene expression in hippocampal neurons for memory consolidation. Inhibition of the arrestin3-PDE4D5 association prevented ß2AR-induced nuclear cAMP signaling without affecting receptor endocytosis. Direct PDE4 inhibition rescued ß2AR-induced nuclear cAMP signaling and ameliorated memory deficits in mice expressing a form of the ß2AR that could not be phosphorylated by GRKs. These data reveal how ß2AR phosphorylated by endosomal GRK promotes the nuclear export of PDE4D5, leading to nuclear cAMP signaling, changes in gene expression, and memory consolidation. This study also highlights the translocation of PDEs as a mechanism to promote cAMP signaling in specific subcellular locations downstream of GPCR activation.

Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein.

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (ß2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to ß2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from ß2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-ß2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance between the Gsα and ß2AR, which may indicate a partial dissociation of Gsα The distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of ß2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological ß-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

Probing spatiotemporal PKA activity at the ryanodine receptor and SERCA2a nanodomains in cardomyocytes.

Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of ß adrenoceptor (ßAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac ß1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the ß2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent ß2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows ß2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes. Video abstract.

Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 (Mfn2), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Monoamine oxidase A and organic cation transporter 3 coordinate intracellular ß1AR signaling to calibrate cardiac contractile function.

We have recently identified a pool of intracellular ß1 adrenergic receptors (ß1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular ß1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized ß1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local ß1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-ß1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-ß1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Subcellular Propagation of Cardiomyocyte ß-Adrenergic Activation of Calcium Uptake Involves Internal ß-Receptors and AKAP7.

ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, ß-AR agonists or high extracellular [Ca] were applied locally at one end, to measure ß-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca]o, increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted. Local isoproterenol (ISO) or norepinephrine (NE) increased CaT amplitude and SR Ca uptake, that spread along the myocyte to the unexposed end. Thus, local [Ca]i decline kinetics reflect spatio-temporal progression of ß-AR end-effects in myocytes. To test whether intracellular ß-ARs contribute to this response, we used ß-AR-blockers that are membrane permeant (propranolol) or not (sotalol). Propranolol completely blocked NE-dependent CaT effects. However, blocking surface ß-ARs only (sotalol) suppressed only ∼50% of the NE-induced increase in CaT peak and rate of [Ca]i decline, but these changes spread more gradually than NE alone. We also tested whether A-kinase anchoring protein 7γ (AKAP7γ; that interacts with phospholamban) is mobile, such that it might contribute to intracellular spatial propagation of ß-AR signaling. We found AKAP7γ to be highly mobile using fluorescence recovery after photobleach of GFP tagged AKAP7γ, and that PKA activation accelerated AKAP7γ-GFP wash-out upon myocyte saponin-permeabilization, suggesting increased AKAP7γ mobility. We conclude that local ß-AR activation can activate SR Ca uptake at remote myocyte sites, and that intracellular ß-AR and AKAP7γ mobility may play a role in this spread of activation.

Gα12 and Gα13: Versatility in Physiology and Pathology.

G protein-coupled receptors (GPCRs), as the largest family of receptors in the human body, are involved in the pathological mechanisms of many diseases. Heterotrimeric G proteins represent the main molecular switch and receive cell surface signals from activated GPCRs. Growing evidence suggests that Gα12 subfamily (Gα12/13)-mediated signaling plays a crucial role in cellular function and various pathological processes. The current research on the physiological and pathological function of Gα12/13 is constantly expanding, Changes in the expression levels of Gα12/13 have been found in a wide range of human diseases. However, the mechanistic research on Gα12/13 is scattered. This review briefly describes the structural sequences of the Gα12/13 isoforms and introduces the coupling of GPCRs and non-GPCRs to Gα12/13. The effects of Gα12/13 on RhoA and other signaling pathways and their roles in cell proliferation, migration, and immune cell function, are discussed. Finally, we focus on the pathological impacts of Gα12/13 in cancer, inflammation, metabolic diseases, fibrotic diseases, and circulatory disorders are brought to focus.

Deciphering cellular signals in adult mouse sinoatrial node cells.

Sinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct ß-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.

Carvedilol induces biased ß1 adrenergic receptor-nitric oxide synthase 3-cyclic guanylyl monophosphate signalling to promote cardiac contractility.

AIMS: ß-blockers are widely used in therapy for heart failure and hypertension. ß-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by ß-blockers in animal hearts. METHODS AND RESULTS: Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure ß-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes ß1 adrenergic receptor (ß1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote ß1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust ß1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced ß1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts. CONCLUSION: These observations present a novel ß1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

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.