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

Abstract

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.

Conclusion:

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.

Graphical Abstract

INTRODUCTION

Plasma membrane receptors are major mechanisms by which cells respond to extracellular stimuli, allowing cells to adapt to their surrounding environments and to modulate tissue/organ function in reaction to changes of neurohormones. Non-steroid hormones signal mainly through receptors at the plasma membrane (PM) to regulate cellular function. This includes sympathetic regulation of cardiac function via adrenergic receptors (ARs) on the PM of cardiomyocytes. Recently, a complementary thread of studies has found that neurotransmitters, peptide hormones, and growth factors also act on intracellular membrane receptors.^1–4 Accumulating data indicate that intracellular G-protein coupled receptors (GPCRs) located at different organelles, e.g., mitochondria,⁵ endoplasmic reticulum membranes⁶ and Golgi,⁷ can be activated insides the cells and induce location-specific effects. The development of these observations helps expanding the current understanding of hormonal regulation of cardiac function.

β₁ARs, a prototype GPCR, acts as a linchpin of sympathetic regulation in the heart and has been demonstrated to be localized and activated intracellularly in cardiomyocytes.⁸ However, the role of intracellular β₁ARs in the regulation of cardiac excitation-contraction is still unknown. During the fight or flight response, activation of the sympathetic nerve and adrenal glands releases catecholamines (epinephrine, EPI and norepinephrine, NE) to activate βARs on myocytes, and thus enhance heart rate, cardiac contraction and relaxation.^{9, 10} These effects involve βARs stimulation-dependent cAMP and subsequent activation of protein kinase A (PKA) to enhance substrate phosphorylation. The PKA phosphorylation of different substrates occurs in different spatiotemporal nanodomains including L-type Ca²⁺ channel (LTCC) at the PM, phospholamban (PLB) and ryanodine receptor type 2 (RyR2) on the SR, and troponin I (TnI) and myosin binding protein C (MYBP-C) on the myofilaments.^{11, 12}

Both NE and EPI are vital neurohormones in physiological stress responses. However, EPI and NE have extremely hydrophilic octanol/water partition coefficients (LogP =−1.37 and −1.24, respectively, https://pubchem.ncbi.nlm.nih.gov). After release from nerve termini, a small portion of catecholamine can enter myocytes, primarily mediated by corticosterone-sensitive organic cation transporter 3 (OCT3, also named extra-neuronal monoamine transporter, EMT).^{2, 13} OCT3-mediated NE uptake is necessary for activation of intracellular ARs in cardiomyocytes, including both nuclear α₁ARs and Golgi-localized β₁ARs.^{2, 8} Intriguingly, decreased NE uptake into myocardium and cardiac NE contents in heart failure patients are associated with significantly blunted inotropy response.¹⁴ In dilated cardiomyopathy patients, OCT2 expression is reduced and predicts the impairment of cardiac function.¹⁵ However, the functional role of NE uptake facilitated by OCT3 in adrenergic stimulation of cardiac contractile is unclear.

Activation of β₁ARs induces spatial, temporal segregated cAMP-PKA signals in heart.^{12, 16} The application of Förster resonance energy transfer (FRET)-based biosensors have been instrumental in the investigation and dissection of compartmentalized cAMP/PKA signaling on the nanodomain scale.^{12, 17, 18} By using genetically encoded biosensors, previous studies from our group and others found that compartmentalized cAMP/PKA signal generated in response to βAR stimulation differs at the PM and SR local domains in both amplitude and kinetics.^{12, 17} The mechanism underlying this heterogeneity is still unclear. We hypothesize that a population of β₁ARs is located near or at the SR and plays an important role in modulating calcium (Ca²⁺) cycling and cardiac contractility. In this study, we tested the expression, activation and functional effects of SR-localized β₁AR in cardiac contractility. Our results provide novel insights into the localization and regulation of intracellular β₁AR, therefore, offering potential and precise target for β₁AR-associated therapeutic strategies.

METHODS

Data Availability.

The data, analytic methods, and study materials are made available to other researchers for purposes of reproducing the results or replicating the procedure. Please see the Major Resources Table and Expanded Materials & Methods in the Online Data Supplement.

All experiments including animal feeding, treatment, and tissue collection were approved by the Institutional Animal Care and Use Committees (protocol: 20234 and 20957) of the University of California at Davis and follow NIH guidelines. Male and female 2–4-month-old C57/BL6 and OCT3KO, newborn β₁AR-KO, β₂AR-KO and β₁β₂AR-KO mice were described previously.^{17, 19} Male Sprague Dawley outbred rats and New Zealand white rabbits were used in this study.

RESULTS

Existence and distribution of a distinct pool of β₁AR at the SR in cardiomyocytes.

To examine the existence of different subcellular pools of β₁ARs in adult mouse hearts, we fractionated heart tissues to separate the PM from intracellular membrane compartments. Sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) and ryanodine receptor 2 (RyR2), which are SR resident proteins, were used as markers for the intracellular membrane fraction (non-PM), and insulin receptor (InsR) and lack of SERCA2/RyR2 were used to identify PM fraction. β₁ARs were clearly detected in both PM and intracellular membrane fractions (non-PM) in WT mouse hearts, but not in β₁AR-KO mouse hearts (Figure 1A and Online Figure IA), indicating that endogenous β₁AR exists in at least two different pools in mouse hearts. In addition, quantitative radioligand-binding assay measured the functional β₁ARs that bind to [I¹²⁵] radio-labeled cyanopindolol in both PM and intracellular membrane fractions (Figure 1B), supporting the existence of an intracellular pool of functional β₁ARs in hearts. We also applied a fluorescence dye BODIPY-tagged βAR antagonist propranolol (FL-Prop) to stain endogenous β₁ARs in mouse adult ventricle myocytes (AVMs). Confocal images of FL-Prop fluorescent staining in WT mouse AVMs showed a distribution of βARs on both surface membrane and intracellular space (Online Figure IB), which was abrogated by addition of 1 μmol/L and 10 μmol/L propranolol (Online Figure IB). Moreover, compared with WT and β₂AR-KO cardiomyocytes, the fluorescent dots and intensity of FL-Prop were greatly reduced in the β₁AR-KO and β₁β₂AR-KO cardiomyocytes (Online Figure IC–D), indicating that Fl-Prop mainly stains endogenous β₁AR.

Figure 1. Intracellular β₁AR associates with SERCA2 but not RyR2 in hearts and isolated mouse adult ventricular cardiomyocytes (AVMs).

(A) WT and β₁AR-KO mouse heart lysates were fractionated to assess cellular distribution of β₁AR. Representative blots and the percentages of β₁AR in the plasma membrane (PM) and intracellular membrane fractions (non-PM) over the total β₁AR in WT and β₁AR-KO mouse hearts. Data are shown in mean ± S.E.M. N = 9 mice. p values were obtained by student t-test. (B) β₁AR densities in the PM and non-PM fractions of WT mouse hearts were determined by quantitative radioligand binding assay. Data are shown in mean ± S.E.M. N = 6 mice. p values were obtained by student t-test. (C-D) Representative confocal images showing distribution of β₁AR, SERCA2, and RyR2 in isolated mouse AVMs. β₁AR was overexpressed in AVMs by infection with recombinant adenovirus. Scale bar = 5 μm. (E) The overlap between staining from confocal images was evaluated by Pearson’s correlation coefficient using ImageJ. Data are shown in mean ± S.E.M; AVM (in the parenthesis) and mouse numbers are indicated in the figure. p value was obtained by t-test. (F) Schematic of in situ proximity ligation assay (PLA), exemplary fluorescence images (the whole cell images are in Online Figure II) and quantification of PLA signals after labeling with antibodies against β₁AR and IgG, β₁AR and SERCA2, and β₁AR and RyR2 in AVMs, respectively. Positive PLA signal (red), DAPI (blue). Scale bar = 5 μm. Data are shown in mean ± S.E.M; AVM (in the parenthesis) and mouse numbers are indicated. p values were obtained by one-way ANOVA followed by Tukey multiple comparison test. (G) Representative images and quantitative assessment of PLB, SERCA2 and RyR2 coimmunoprecipitated with β₁AR in WT mouse hearts. A.U. = arbitrary unit is defined as the ratio of intensity of proteins over inputs. Data are shown in mean ± S.E.M. (N = 5); p values were obtained by student t-test; (H) Schematic of SiMPull assay and representative images of SiMPull assay after endogenous β₁AR, SERCA2 complex were pulled down with anti-β₁AR or control IgG antibodies. The images were quantified using MATLAB. Scale bar = 5 μm. Data are shown in mean ± S.E.M. N = 5 mice. p values were obtained by t-test.

SR localized Ca²⁺ cycling proteins such as SERCA2 and RyR2 are crucial for the modulation of cardiac contractility in response to βAR activation.¹¹ We hypothesize that a population of intracellular β₁ARs are present at the SR. We examined a potential colocalization of overexpressed β₁AR with SERCA2 or RyR2. β₁AR displayed specific colocalization with SERCA2, but not RyR2, in mouse AVMs (Figure 1C–E and Online Figure IIA). As controls, overexpressed β₂AR did not display overlap with either SERCA2 or RyR2 (Online Figure IIIA–B). Next, we applied proximity ligation assay (PLA) to further explore the proximal relationship between β₁AR and SERCA2. We detected robust PLA signals in mouse AVMs co-stained for β₁AR and SERCA2, suggesting that these β₁ARs and SERCA2 are at 40 nm or less apart. Conversely, PLA signals were not detected after co-staining with antibodies against β₁AR and IgG or against β₁AR and RyR2 and only a small amount of PLA signals was detected after co-staining for β₁AR and junctophilin 2 (JP2) (Figure 1F and Online Figure IIB–D). Moreover, endogenous β₁AR was pulled down with β₁AR antibody (Figure 1G). We found that both SERCA2 and its regulatory protein, phospholamban (PLB), were co-immunoprecipitated with the endogenous β₁ARs. In contrast, RyR2 was not observed in the immunoprecipitation. Furthermore, we applied a super-sensitive single molecule pull down (SiMPull) assay²⁰ to visualize individual β₁AR-SERCA2 complex in heart lysate. Figure 1H shows that β₁AR antibody was able to pull down a significant amount of SERCA2 molecules compared to control IgG antibody. Together, these data demonstrate that a population of β₁ARs is localized at the SR and forms a complex with SERCA2 in AVMs.

Organic Cation Transporter 3 (OCT3) is required to activate the pool of β₁ARs by catecholamine at the SR.

β-adrenoceptor stimulation results in activation of PKA, a key mediator for phosphorylating multiple Ca²⁺ handling proteins (e.g. PLB).²¹ To directly assess PKA activity induced by the SR-localized β₁AR, we employed a genetically encoded Förster resonance energy transfer (FRET)-based A kinase activity reporter (AKAR) that is anchored at the intracellular SERCA2 complex on the SR (SR-AKAR3).^{17, 18} In comparison, the PKA activity induced by the PM-localized β₁AR was assessed by an AKAR3 anchored on the PM (PM-AKAR3).

Previous studies showed that OCT3-mediated transportations of EPI or NE into cells are essential for activating intracellular adrenergic receptors in cardiomyocytes.^{2, 8} Here, we used mice with genetic deletion of OCT3 to examine the impacts of OCT3 on subcellular PKA activity induced by PM- or SR-localized β₁AR (PM-β₁AR or SR-β₁AR). Unlike endogenous catecholamines, isoproterenol (ISO, LogP = 1.4) and dobutamine (LogP = 3.6), two membrane permeant β agonists, activate internal βAR via passive diffusion into myocytes (Figure 2A–B).⁷ Therefore, deleting OCT3 should selectively block the access of NE and EPI but not ISO and Dob to SR-localized β₁AR (Figure 2A–B). NE, EPI, ISO and Dob all induced robust PKA activity at both the PM and SR in WT myocytes (Online Figure IVA–D). The kinetics of ligand induced PKA activity at the PM were comparable but were faster than those at the SR (Online Figure IVA–E). Among the SR-PKA activity, the Dob induced increases were faster than those by ISO, EPI and NE, consistent with its faster membrane permeability (Online Figure IV A–E). As predicted, the ISO-induced PKA activity at both the PM and SR was not affected by deletion of OCT3 (Figure 2C–D). However, deletion of OCT3 selectively attenuated NE-induced PKA activity at the SR without affecting PKA activity at the PM (Figure 2E–F). Moreover, inhibition of β₁AR with CGP20712a completely abolished PKA activity induced by NE and ISO at both PM and the SR in AVMs whereas inhibition of β₂AR with ICI118551 did not affect these PKA activities (Online Figure IV F–I). These observations were not due to change in β₁AR expression in OCT3-KO hearts (Online Figure V). Additionally, we have examined PKA activity at the myofilaments (MF) with an AKAR3 anchored on the troponin complex (MF-AKAR3¹⁸), which is adjacent to the SR. Our data show deletion of OCT3 selectively attenuated NE but not ISO induced PKA activity on the myofilaments (Online Figure VA). To explore specific PKA substrate affected by SR-localized β₁AR activation, we then examined PKA phosphorylation of phospholemman (PLM, Ser⁶³) and LTCC (Ser¹⁹²⁸) at the PM, PLB (Ser¹⁶) and RyR2 (Ser²⁸⁰⁸) at the SR, TnI (Ser^23/24) and MyBPC (Ser²⁸², Ser²⁷³, Ser³⁰²) at myofilament. Deletion of OCT3 selectively attenuated NE-induced PKA phosphorylation of PLB and TnI without affecting PKA phosphorylation of PLM, LTCC, RyR2, and MyBPC (Figure 2G–H and Online Figure V B–H). In comparison, ISO-induced PKA phosphorylation of both PLM and PLB were not affected by OCT3 deficiency (Figure 2G–H). These data suggest that deletion of OCT3 blocks catecholamine-dependent activation of the β₁ARs at the SR and the myofilament and downstream PKA-dependent PLB and TnI phosphorylation.

Figure 2. Differential local regulation of NE-induced β₁AR/PKA activities in WT and OCT3KO AVMs.

(A-B) Schematics of local activation of β₁AR-induced PKA activity at subcellular locations and detection using FRET based biosensors (plasma membrane, PM-AKAR3 and sarcoplasmic reticulum, SR-AKAR3) in WT and OCT3KO AVMs. Isoproterenol (ISO), norepinephrine (NE). FRET assay was analyzed with F/F0 of YFP/CFP ratio. (C-D) Concentration-response curves of changes in YFP/CFP ratio after ISO stimulation in WT and OCT3KO AVMs expressing PM-AKAR3 or SR-AKAR3. Data were from 6 WT and 6 OCT3KO mice. (E-F) Concentration-response curves of changes in YFP/CFP ratio after NE stimulation in WT and OCT3KO AVMs expressing PM-AKAR3 or SR-AKAR3. Data were from 8 WT and 7 OCT3KO mice. Data are shown in mean ± S.E.M. p values were obtained by two-way ANOVA with Tukey’s multiple comparison test when comparing WT to OCT3-KO. (G-H) Detection of phosphorylation of phospholamban (PLB) at serine 16 and phosphorylation of phospholemman (PLM) at serine 63 in mouse AVMs after stimulation with 100 nmol/L ISO or 100 nmol/L NE. Data are shown in mean ± S.E.M. (N = 5). A.U. (arbitrary unit) is defined as the ratio of intensity of phosphorylated proteins over intensity of total proteins. p values were obtained by two-way ANOVA with Tukey’s multiple comparison test.

Functional consequences of the internal pool of β₁ARs on adult ventricular myocyte.

We then assessed whether the deletion of OCT3 affects catecholamine induced Ca²⁺ handling and sarcomere shortening (SS) in isolated AVMs. Compared with basal conditions, stimulation with different βAR agonists (ISO, Dob, NE, and EPI) led to increased SS and Ca²⁺ transient amplitude, and decreased Ca²⁺ decay tau in both WT and OCT3KO AVMs (Figure 3A–H and Online Figure VIA–K). Dob induced a faster inotropic response than ISO and NE in WT AVMs. The increases in E-C coupling were blocked by β₁AR antagonist CGP20712a, but not affected by β₂AR inhibition with ICI118551 (Online Figure VII). Deletion of OCT3 did not affect ISO- or Dob-induced inotropic responses (Figure 3A–D and Online Figure VID–K). Conversely, the NE- and EPI-promoted responses in contraction, Ca²⁺ transients and [Ca²⁺]_i decay tau were significantly attenuated in OCT3KO vs WT AVMs (Figure 3E–H and Online Figure VIJ–K). Deletion of OCT3 also shifted the NE dose-response curve of SS to the right without affecting ISO-induced dose-response curve (Figure 3I–J).

Figure 3. Deletion of Organic Catecholamine Transporter3 (OCT3) attenuates stimulation of myocyte contractility with norepinephrine and epinephrine.

(A-B) Representative traces show sarcomere shortening (SS) before (close line) and after (dash line) stimulation with ISO (100 nmol/L) in the WT and OCT3KO mouse AVMs. The peak SS were quantified. (C-D) Representative traces show FS before (close line) and after (dash line) the application of dobutamine (Dob, 1μmol/L) in WT and OCT3KO AVMs. The peak SS were quantified. (E-F) Representative traces show SS before (close line) and after (dash line) the application of norepinephrine (NE, 100 nmol/L) in WT and OCT3KO AVMs. The peak SS were quantified. (G-H) Representative traces show SS before (close line) and after (dash line) the application of epinephrine (EPI, 100 nmol/L) in WT and OCT3KO AVMs. The peak SS were quantified. For panel A-H, data are shown in mean ± S.E.M. AVM (in the parenthesis) and animal numbers are indicated. p values were obtained by two-way ANOVA analysis followed with Tukey’s multiple comparison test. (I-J) Dose response curves of SS in AVMs after stimulation with ISO (7 WT and 7 OCT3-KO mice) or NE (6 WT and 8 OCT3-KO mice). p values were obtained by two-way ANOVA with Tukey’s multiple comparison test when comparing OCT3-KO to WT.

To independently test the role of OCT3 in the SR-located β₁AR/PKA signaling, we applied an OCT3/EMT inhibitor corticosterone (CORTI) to prevent NE transport into myocytes (Figure 4A–B). While CORTI had minimal effect on NE-induced PKA activity at the PM domain, it significantly attenuated NE-induced PKA activity at the SR domain (Figure 4C–D). CORTI did not change ISO-induced PKA activity at either the PM or SR domain in rat AVM (Figure 4C–D). In agreement, CORTI significantly reduced NE- but not ISO-induced PKA phosphorylation of PLB at Ser¹⁶ (Figure 4E–F). CORTI also significantly attenuated NE-induced increases in contractility, Ca²⁺ transient amplitude, and rate of Ca²⁺ transient decay (Figure 4G–I). However, these CORTI effects were absent in the responses to ISO stimulation (Figure 4J–L). Together, these data suggest that OCT3-dependent transport is required for stimulation of SR-localized β₁AR/PKA/PLB signal cascade for optimal increases in Ca²⁺ handling and contractility.

Figure 4. Inhibition of organic catecholamine transporters reduces norepinephrine-promoted SR-localized β₁AR signal and myocardial contractility.

(A-B) Schematics show detection of β₁AR-induced PKA activity at different subcellular locations with and without OCT3 inhibitor corticosterone (CORTI). (C-D) Rat AVMs expressing PM-AKAR3 or SR-AKAR3 FRET biosensor were pretreated with CORTI (1 μmol/L) before stimulation with NE (100 nmol/L) or ISO (100 nmol/L). FRET was analyzed as F/F0 of YFP/CFP ratio. Data show the maximal increases in YFP/CFP ratios in mean ± S.E.M. AVM (in the parenthesis) and rat numbers are indicated. p values were obtained by One-way ANOVA with Tukey’s multiple comparison test. (E-F) Representative immunoblot detection and quantification of phosphorylated serine 16 (pPLB) and total PLB in rat AVMs. Cells were treated with 5-minute incubation with ISO (100 nmol/L) or NE (100 nmol/L) in the absence and presence of CORTI pretreatment. A.U. (arbitrary units) is defined as the ratio of intensity of phosphorylated proteins over intensity of total proteins. Data are shown in mean ± S.E.M. (N = 3). p values were obtained by One-way ANOVA with Tukey’s multiple comparison test. (G-L) Rat AVMs were loaded with Ca²⁺ indicator, 5 μmol/L Fluo-4 AM and pretreated with CORTI (1 μmol/L) before stimulation with NE (100 nmol/L) or ISO (100 nmol/L). Sarcomere shortening (SS) and calcium transient amplitude and tau were recorded with 1Hz pacing. Data are shown in mean ± S.E.M. AVM (in the parenthesis) and animal numbers are indicated in figures. p values were obtained by One-way ANOVA with Tukey’s multiple comparison test.

Activation of the internal β₁ARs at the SR was not blocked by membrane impermeant β-blockers.

Sotalol, a membrane impermeant β-blocker (LogP = 0.24), has limited access to intracellular β₁AR.²² To further dissect the physiological function of the SR-β₁AR, we applied sotalol to selectively block the PM-β₁AR but permit access and activation of SR-β₁AR by agonists. In contrast, propranolol, a membrane permeant β-blocker (LogP = 3.48), is expected to block βARs both on the PM and at intracellular membranes (Online Figure VIIIA). We tested the accessibilities of sotalol and propranolol to the β₁ARs by examining their abilities to compete with fluorescent BODIPY-tagged propranolol (Fl-Prop) from binding to β₁ARs. Confocal images of Fl-Prop fluorescent staining in WT mouse AVMs showed a distribution of βAR on both surface and intracellular membranes (Online Figure VIIIB). Sotalol pretreatment did not block the intracellular staining (Online Figure VIIIB); whereas addition of propranolol effectively competed against Fl-Prop from binding to β₁AR in AVMs (Online Figure VIIIB). Addition of ISO and NE also effectively competed against Fl-Prop from binding to β₁AR in AVMs. Thus, intracellular β₁AR can be blocked by membrane permeant β-blockers but are less accessible to membrane impermeant β-blockers.

Sotalol was then applied to isolate PKA activity induced by local compartmentalized β₁AR at the SR (Figure 5A–C). In WT, β₁AR-KO and β₂AR-KO mouse neonatal myocytes and WT mouse, rat, and rabbit AVMs, sotalol readily inhibited ISO induced and β₁AR mediated PKA activity at the PM, but not those at the SR (Figure 5D–G, Online Figure IXA–D and Online Figure X). In rat AVMs, the IC50 of sotalol for inhibition of ISO-induced PKA activity at the SR was about 10-fold higher than that of PKA activity at the PM (Figure 5G, PM-AKAR3 IC₅₀ = 2.22 ± 0.06 μmol/L; SR-AKAR3 IC₅₀ = 21.5 ± 0.06 μmol/L). In contrast, the membrane permeant β-blocker propranolol inhibited ISO-induced PKA activity at both the PM and SR in a concentration-dependent manner (Figure 5F, PM-AKAR3 IC₅₀ = 74.0 ± 0.05 nmol/L, SR-AKAR3 IC₅₀ = 99.2 ± 0.06 nmol/L). Another poorly permeant β-blocker, atenolol (LogP = 0.6), acted similarly to sotalol by selectively inhibiting ISO-induced PKA activity only at the PM in rat AVMs (Online Figure IXE–F). In comparison, membrane permeant carvedilol (LogP = 4.19) inhibited ISO-induced PKA activity at both the PM and the SR (Online Figure IXE–F). Together, these data support that local activation of SR-β₁AR-PKA signaling was minimally suppressed by membrane impermeant β-blockers, a conserved mechanism throughout different cardiac myocytes from different species. This also indicates that the β₁AR at the SR are not activated at the PM and then translocated to the SR.

Figure 5. Activation of β₁AR in the SR is essential for maximal stimulated contractility in AVMs.

(A-C) Schematics show detection of β₁AR-induced PKA activity at different subcellular locations (PM and SR) with FRET based biosensors in the presence of membrane impermeant β-blocker sotalol (SOTA) or the membrane permeant β-blocker propranolol (PROP). (D, E) Rat AVMs expressing PM-AKAR3 or SR-AKAR3 were stimulated with 100 nmol/L ISO with or without 5-mintue pretreatment of 25 μmol/L SOTA (blue) or 1 μmol/L PROP (red). Representative time courses show FRET response of PM-AKAR3 or SR-AKAR3 in AVMs. FRET was analyzed as F/F0 of YFP/CFP ratio. Data are shown in mean ± S.E.M. AVM (in the parenthesis) and rat numbers are indicated. p values were obtained by one-way ANOVA analysis with Tukey’s multiple comparison test. (F, G) Dose-dependent inhibition curves of PROP or SOTA on ISO-induced increases in FRET responses in AVMs expressing PM-AKAR3 or SR-AKAR3. Data show YFP/CFP ratios normalized against the maximal increases induced by ISO in the absence of β-blocker (N = 5 rats). p values were obtained by two-way ANOVA analysis followed by Tukey’s multiple comparison test when compared to OCT3-KO. (PM-AKAR3: PROP, IC50 = 7.399×10⁻⁸ mol/L, SR-AKAR3: PROP, IC50 = 9.92×10⁻⁸ mol/L; PM-AKAR3: SOTA, IC50 = 2.216×10⁻⁶ mol/L; SR-AKAR3, STOA, IC50 = 2.149×10⁻⁵ mol/L). (H-J) Rat AVMs were incubated with Ca²⁺ indicator (5 μM Fluo-4 AM) before 1Hz pacing. After pretreatment with SOTA (25 μmol/L) or PROP (1 μmol/L), sarcomere shortening (SS) and calcium transient were recorded before and after stimulation with 100 nmol/L ISO. The peak SS, amplitude of Ca²⁺ transient, and rate of Ca²⁺ decay (Tau) are shown as mean ± S.E.M. Animal numbers and cell numbers are indicated in figures. p values were obtained by one-way ANOVA analysis with Tukey’s multiple comparison test.

Activation of the β₁AR at the SR promotes optimal cardiac contractility and Ca²⁺ handling in AVMs.

Consistent with the PKA activity detected at the SR, sotalol blocked ISO-induced PKA phosphorylation of LTCC at Ser¹⁹²⁸ but did not affect ISO-induced PKA phosphorylation of PLB at Ser¹⁶ in mouse, rat or rabbit AVMs (Online Figure XI). On the other hand, propranolol abolished ISO-induced phosphorylation of PLB and LTCC (Online Figure XI). These data support that activation of the SR-β₁AR signaling promotes SR-localized PKA phosphorylation of PLB in AVMs. Accordingly, ISO-induced increases in sarcomere shortening (SS), Ca²⁺ transient amplitude and deceases in tau, were partially reduced by sotalol but completely abolished by propranolol in rat AVMs (Figure 5H–J). These data suggest that phosphorylation of PLB by PKA is mediated by localized β₁AR signaling at the SR and promotes myocyte contractility.

Similarly, sotalol selectively inhibited NE-induced PKA activity at the PM yet permitted increases in PKA activity in the SR local domain (Figure 6A–D and Online Figure XIIA–F). We then assessed whether OCT3 controls the NE-induced local PKA activity at the SR in the presence of sotalol. Deletion of OCT3 abolished PKA activity at the SR domain induced by NE in the presence of sotalol (Figure 6A–B and Online Figure XIIA–F). In contrast, ISO-induced PKA activity at the SR was not affected by OCT3KO and sotalol pretreatment (Figure 6C–D). In the presence of sotalol, deleting OCT3 completely blocked NE-induced increases in sarcomere shortening, Ca²⁺transient, and decreases in Ca²⁺ decay tau (Figure 6E–G) but did not affect the responses induced by ISO (Figure 6H–J). These data show that in the presence membrane impermeant β-blocker sotalol, NE can selectively stimulate SR-β₁AR signaling to promote local PKA activity and enhance myocyte contractility. These activities can be blocked by deletion of OCT3 that is responsible for uptake of NE into AVMs.

Figure 6. Activation of β₁AR at the SR promotes myocyte calcium cycling and contractility.

(A, B) WT and OCT3KO AVMs were used to express PM-AKAR3 or SR-AKAR3 biosensors. FRET was analyzed as F/F0 of YFP/CFP ratio. Representative curves and maximal changes show subcellular PKA FRET responses to NE (100 nmol/L) stimulation in AVMs pretreated with sotalol (SOTA, 25 μmol/L). Animal numbers and cell numbers (in the parenthesis) are indicated. p values were obtained by two-way ANOVA analysis followed with Tukey’s multiple comparison test when compared to OCT3-KO. (C, D) WT and OCT3KO mouse AVMs were used to express PM-AKAR3 or SR-AKAR3 biosensors. Representative curves and maximal changes show subcellular PKA FRET responses to ISO (100 nmol/L) stimulation in AVMs pretreated with sotalol (SOTA, 25 μmol/L). Animal numbers and cell numbers (in the parenthesis) are indicated. p values were obtained by two-way ANOVA analysis followed with Tukey’s multiple comparison test when compared to OCT3-KO. (E-G) AVMs from WT and OCT3 hearts were stimulated with NE (100 nmol/L) in the presence of SOTA (25 μmol/L) pretreatment. Data show maximal changes in sarcomere shortening (SS), Ca²⁺ transient amplitude, and rate of Ca²⁺ decay (Tau) as mean ± S.E.M. Animal numbers and cell numbers (in the parenthesis) are indicated in figures. p values were obtained by two-way ANOVA analysis with Tukey’s multiple comparison test. (H-J) AVMs from WT and OCT3 hearts were stimulated with ISO (100 nmol/L) in the presence of SOTA (25 μmol/L) pretreatment. Data show maximal changes in SS, Ca²⁺ transient amplitude, and Tau as mean ± S.E.M. Animal numbers and cell numbers (in the parenthesis) are indicated. p values were obtained by two-way ANOVA analysis with Tukey’s multiple comparison test.

OCT3 deficiency impairs catecholamine transport in heart and attenuates epinephrine induced inotropy and heart rate in vivo.

Finally, we sought to understand the impacts of intracellular β₁AR activation on adrenergic regulation of cardiac function in vivo. We observed no differences in heart weight/body weight ratios of OCT3KO and WT mice (Figure 7A), indicating a grossly normal cardiac structure in OCT3KO hearts. However, deletion of OCT3 reduced endogenous NE concentration in hearts compared to WT controls, whereas OCT3KO and WT mice had comparable NE concentrations in the plasma (Figure 7B–C). Consequently, OCT3KO mice show lower cAMP concentrations in hearts relative to WT controls (Figure 7D). These data indicate disrupted catecholamine transport and potential intracellular adrenergic receptor activation by OCT3 deletion. Compared to WT, OCT3KO mice showed no difference in cardiac function, including ejection fraction (EF) and heart rate (HR) at baseline conditions (Figure 7E–I). These observations obtained by echocardiography are probably relevant to adaptive gene expressions in myocardium due to OCT3 deletion. The expression of PLB and PLB/SERCA2 ratio were decreased whereas expression of RyR2 was increased in OCT3KO hearts (Online Figure XIIIA–B). Furthermore, we detected cardiac β-adrenergic response with 10 μg/kg EPI or ISO injection in vivo. Compared to WT, OCT3KO mice showed no difference in cardiac function after 10 μg/kg ISO administration (Figure 7G–I). However, OCT3KO displayed significantly blunted EF and HR responses to EPI injection relative to WT (Figure 7G–I). These data show that OCT3 is essential for enhancing myocardial contractility induced by epinephrine in vivo.

Figure 7. Deletion of OCT3 reduces catecholamine uptake, cAMP signal and inotropic response in response to adrenergic stimulation of mouse hearts.

(A) Heart weight/body weight ratio (HW/BW %) in WT and OCT3-KO mice. Data are shown as mean ± S.E.M. (N = 8). p values were obtained by student t-test. (B, C) Quantitative determination of endogenous NE in cardiac tissues and the plasma. Data are shown as mean ± S.E.M. (N = 8). p values were obtained by student t-test. (D) Quantitative determination of cAMP levels in cardiac tissues. Data are shown as mean ± S.E.M. (N = 4). p values were obtained by student t-test. (E) Representative echocardiographic images of WT (N = 7) and OCT3KO (N = 6) mice at baseline and after intraperitoneal injection of 10 μg/kg isoproterenol (ISO) or epinephrine (EPI). (F-I) Cardiac ejection fraction (EF) of individual mice before and after intraperitoneal injection of ISO or EPI in WT (N = 7) and OCT3KO (N = 6). Quantification of EF and heart rate (HR) in WT and OCT3KO mice before and after injection with ISO or EPI. Data are shown as mean ± S.E.M. p values were obtained by two-way ANOVA analysis with Tukey’s multiple comparison test.

DISCUSSION

Adrenergic signaling is one of the most important mechanisms for regulating cardiac function and is typically blunted/disturbed in cardiac diseases including HF.²³ In this study, we provide evidence to support the presence of a pool of β₁AR at the SR that is essential for enhancing contractility in cardiomyocytes. We also show that monoamine transporter OCT3/EMT mediated catecholamine uptake is essential for activation of the SR-β₁AR and for promoting phosphorylation of PLB by PKA and cardiac contractility. Furthermore, our data suggest that different clinically used β-blockers differ significantly in their ability to suppress internal β₁AR-PKA signaling due to different membrane permeability. While membrane-permeant β blockers such as propranolol and carvedilol are able to block activation of intracellular β₁ARs, membrane impermeant β-blockers such as sotalol and atenolol cannot readily access intracellular β₁AR. Therefore, this study opens a novel avenue to optimize therapeutic strategies for targeting cardiac β₁ adrenergic signaling in different clinical settings.

Intracellular adrenergic receptor signaling at the SR.

Accumulating evidence suggests the presence of GPCRs and G proteins inside mammalian cells.²⁴ Increasing number of intracellular GPCRs have been identified in animal hearts, including angiotensin³ and adrenergic receptors.^{2, 8} In cardiomyocytes, previous studies have characterized both β₁ and α₁ARs, but not β₂AR on the nuclear envelope.^{1, 2} β₁ARs were also found to reside in the Golgi apparatus and initiate an internal Gs-cAMP signal from the Golgi apparatus, and this internal signal is independent of receptors at the PM.^{7, 8} However, whether ARs are localized at other subcellular locations and their functional implications in cardiac contractility remain to be elucidated. Here, we used a combination of sophisticated molecular probes, genetically modified mice and pharmacological inhibitors to uncover important details of the intracellular β₁AR-cAMP-PKA pathway at the SR. We provide solid evidence of the presence of intracellular β₁ARs at the SR in cardiomyocytes, which is associated with SERCA2 and PLB, consistent with a recent report that exogenously expressed β₁ARs bind to SERCA2 in fibroblasts.²⁵ Meanwhile, our data do not support the presence of a cardiac β₂AR-SERCA2 complex at the SR membrane. This identification of SR-localized β₁ARs provides a novel possibility to explain precisely compartmentalized β₁AR signaling in cardiomyocytes. Upon βAR activation the locally anchored PKA is activated and promotes phosphorylation of targets within a nanodomain, including LTCC at the PM, PLB on the SR, and troponin I and myosin binding protein C on the myofilaments.^{12, 18, 26} The present work shows that activation of the intracellular β₁ARs at the SR is necessary for promoting local PKA activity to phosphorylate PLB and TnI in cardiomyocytes, and probably plays a more critical role in rate-limiting cardiac outputs during the physiological fight-or-flight responses. The intracellular β₁AR and PKA activity was essential to optimize enhancement of Ca²⁺ transients and cardiac contractility. Yet, much remains to be learnt about the composition, regulation, and function of this emerging β₁AR signaling machinery at the SR in hearts.

Requirement of catecholamine-uptake for intracellular β₁ARs activation.

Based on lipophilicity, different βARs ligands access intracellular β₁AR either by passive diffusion or transporter-mediated uptake. For instance, the current and prior studies show that hydrophilic catecholamine NE and epinephrine require OCT3/EMT for cellular uptake,² whereas hydrophobic ligands (e.g. ISO) can passively cross the PM.^{7, 8} During sympathetic stimulation, catecholamine is released from nerve termini to stimulate adrenergic receptors on cardiomyocytes. Extracellular catecholamines are quickly reduced mainly by re-uptake at nerve termini.²⁷ However, cardiomyocytes can also take up a portion of catecholamines via OCT3/EMT.²⁸ A key question is raised by these observations: what is the function of the catecholamine uptake into cardiomyocytes? Traditionally, it is considered as a mechanism to clear excess catecholamines to prevent overstimulation of cardiac adrenergic signaling.¹⁴ However, recent research revealed that intracellular NE activates Golgi-localized β₁ARs to promote PI4P hydrolysis.⁸ In this work, for the first time, we show that inhibition of NE uptake by OCT3/EMT reduces intracellular PKA activity at the SR as well as contractility in cardiomyocytes. Accordingly, OCT3/EMT dependent-catecholamine uptake is essential for optimal βAR-induced enhancement of contractility in cardiac muscles. Deletion of OCT3 attenuated EPI-induced increases in cardiac EF. Meanwhile, deletion of OCT3 also blunted EPI-induced increases in cardiac HR, supporting a notion that SR Ca²⁺ content contributes to sinoatrial nodal pacemaker cells for physiological heart rate increases.²⁹ Further studies may be merited to address impacts of intracellular β₁ARs activation on SR Ca²⁺ content and Ca²⁺ handling in myocytes. It also remains to be examined whether other transporters such as OCT2 may affect activation of intracellular β₁ARs in hearts. These observations highlight the transporter-dependent uptake of catecholamines as a potential drug target for clinical applications in a variety of cardiovascular conditions.

Similarly, clinically used β-blockers differ significantly in their membrane permeability. Carvedilol is a commonly used β-blocker in HF therapy, and it can permeate the PM and inhibit β₁AR at both the PM and intracellular membrane compartments. Thus, it may potentially inhibit detrimental effects of chronic activation of the intracellular β₁AR such as nuclear PKA activity for maladaptive gene expression. In comparison, membrane impermeant β-blockers such as sotalol and atenolol, which should mainly block β₁AR only at the PM,^{7, 8} are not widely used for HF therapy. Nonetheless, these β-blockers are effective as anti-arrhythmic drugs,³⁰ probably in part due to their ability to inhibit β₁AR on the PM. Our study thus provides a conceptual platform to evaluate and assess individual β-blockers for more efficacious therapy in different clinical applications.

Integrity of β₁ARs at the cell surface and subcellular membrane.

Recent and previous studies indicate that the activation of intracellular βAR/PKA signal generates localization-specific and substrate specific effects.^{7, 8} Membrane localization of βARs and their associated complexes precisely fine-tune βAR activation, which is essential to maintain the cellular function and appropriate responses to extracellular stimuli, such as catecholamines.^{16, 26} More importantly, we found that the integral regulation of β₁AR at both cell surface and the SR is required to achieve maximal enhancement of cardiac contraction via optimally coordinating PKA-dependent phosphorylation of substrates in multiple loci. Impaired integrity of β₁AR/cAMP/PKA signaling and blunted cardiac contractile response to β-adrenergic stimulation have been implicated in pathological stress conditions.²⁶ It has been reported that in HF βAR activation induced PKA activity is significantly inhibited or abolished at the SR and at the myofilaments, but well-preserved at the PM,^{12, 18} suggesting the dysregulation of intracellular βAR/PKA signal is involved in the pathogenesis of HF. Besides, a large body of studies revealed that in human HF, NE reuptake was impaired.³¹ Although cardiac NE release by sympathetic nerves was increased in HF patients, cardiac NE uptake and cardiac NE contents were significantly reduced. Accordingly, OCT transporters expression is reduced in dilated cardiomyopathy patients.¹⁵ Those observations are consistent with our findings in OCT3KO mice that disrupted catecholamine uptake leads to decreased NE concentrations and cAMP in the myocardium. Meanwhile, OCT3KO mice show attenuated inotropic response to catecholamines. Taken together, these findings suggest a decreased activation of intracellular β₁AR at the SR due to dysregulated OCT3 may contribute to the decreased PKA activity and phosphorylation of PLB associated with cardiac dysfunction. The integrity β₁ARs at intracellular membranes and their role in development of HF remains to be further explored.

In summary, this study uncovers a novel signaling paradigm in which a SR-localized intracellular β₁AR is functionally crucial in adrenergic stimulation of cardiac contractility. Moreover, the data not only help to broaden our current understanding of the general role of β₁AR signaling in cardiac regulation, but also open up a novel platform to evaluate and optimize β-blockers in clinical applications based on their “biased” properties in selective inhibition of cardiac β₁AR at distinct subcellular locations.

NOVELTY AND SIGNIFICANCE.

What Is Known?

• β₁-adrenoceptors (β₁ARs) are the major subtype driving cardiac contractility in response to catecholamine stimulation.

• β₁-adrenoceptors (β₁ARs) exist in intracellular membranes.

• Organic Cation Transporter 3 (OCT3) mediates norepinephrine entry into cardiomyocytes.

What New Information Does This Article Contribute?

• A pool of β1AR are associated with the sarco-endoplasmic reticulum calcium ATPase (SERCA2) complex at the sarcoplasmic reticulum (SR).

• Activation of the SR localized β1AR is essential to promote phosphorylation of phospholamban.

• Organic Cation transporter 3 is required for catecholamines (norepinephrine and epinephrine) to enter cells and stimulate the β1AR at the SR.

• Activation of intracellular SR β1AR is essential to promote cardiac contractility.

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.

For decades, catecholamines have been shown to signal via βARs on the plasma membrane to regulate heart contractile functions. Little is known about the presence and function of intracellular βARs in cardiomyocytes. We show the presence of of intracellular β₁AR associated with SERCA2 on the SR. Activation of this SR-localized β₁AR is essential for promoting PKA-dependent phosphorylation of PLB and optimizing the cardiac contraction response. Intracellular β₁AR activation is regulated by catecholamine uptake via the OCT3 transporter or membrane permeant β-blockers in cardiomyocytes. These findings provide new mechanistic insight into the composition, localization and regulation of β₁AR signaling. Targeting intracellular β₁AR and its regulators could have potential therapeutic utility in diseases related to cardiac contractile function.

Nonstandard Abbreviations and Acronyms:

AR

Adrenergic receptors

AVMs

Adult ventricular cardiomyocytes

AKARs

A kinase activity reporters

FL-Prop

BODIPY-tagged propranolol

Ca²⁺

Calcium

CORTI

Corticosterone

Dob

Dobutamine

EF

Ejection fraction

EPI

Epinephrine

FRET

Förster resonance energy transfer

FS

Fractional shortening

ISO

Isoproterenol

NE

Norepinephrine

OCT3/EMT

Organic Cation Transporter 3/extraneuronal monoamine transporter

PLB

Phospholamban

PLM

Phospholemman

PM

Plasma membrane

PKA

Protein kinase A

PLA

Proximity ligation assay

RyR2

Ryanodine receptor 2

JP2

Junctophilin 2

SS

Sarcomere Shortening

SERCA2

Sarcoplasmic endoplasmic reticulum Ca^2+-ATPase 2

SR

Sarcoplasmic reticulum

WT

Wild type

cAMP

Cyclic AMP

OCT3KO

OCT3 knockout

LTCC

L-type calcium channel