Subcellular compartmentalization of proximal Gα_q-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes

Abstract

G protein–coupled receptors that signal through Gα_q (G_q receptors), such as α₁-adrenergic receptors (α₁-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cβ1 (PLCβ1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂) to produce inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. Despite these common proximal signaling mechanisms, G_q receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, G_q receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation–transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP₃-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of G_q-receptor signaling as a mechanistic basis for unique G_q receptor–induced hypertrophic phenotypes in cardiac myocytes. We show that α₁-ARs co-localize with PLCβ1 and PIP₂ at the nuclear membrane. Further, nuclear α₁-ARs induced intranuclear PLCβ1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCβ1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α₁-AR–induced transcription. In summary, these results link G_q-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation–transcription coupling in adult cardiac myocytes that accounts for differential G_q-receptor localization and better explains distinct physiological functions of G_q receptors.

Introduction

G protein–coupled receptors that signal through Gα_q (G_q receptors)² share a common proximal signaling pathway through the activation of phospholipase Cβ1 (PLCβ1), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) to produce inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (1). However, in any cell, simultaneous activation of multiple proximal G_q-receptor signaling events would preclude the cell's ability to process different signals and produce a unique outcome. Despite this commonality, each G_q receptor mediates distinct physiologic processes, but how this specificity is achieved is unclear in many cases. Compartmentalization of signaling provides one mechanism through which a cell could handle a multitude of potentially overlapping signals. Cardiac myocytes offer several examples of compartmentalized receptor signaling (e.g. the ability to discriminate β-adrenergic (β-AR)-G_s-induced calcium signals that augment contractility from G_q receptor–mediated calcium signals that induce hypertrophy) (2). In this case, inositol-sensitive calcium release occurs in the nucleus based on the localization of the inositol trisphosphate receptor to the inner nuclear membrane. However, this ultimately raises questions about how G_q receptors, traditionally thought to localize to the cell surface, might activate a nuclear calcium signal to regulate hypertrophy.

In cardiac myocytes, G_q receptors, including α₁-adrenergic, endothelin, and angiotensin receptors (α₁-AR, ET-R, and AT-R, respectively), regulate vital signaling pathways controlling hypertrophy, cell survival, and inotropy that impact heart failure (HF) (3). Early studies in cell and animal models established the long-held convention that G_q signaling is maladaptive and exacerbates HF (4–6). However, several studies have challenged the convention that G_q-receptor signaling universally worsens HF. Clinical studies indicate that antagonists targeting G_q receptors in human HF do not uniformly improve HF outcomes. Although AT-R antagonists are standard of care (7), α₁-AR antagonists worsen HF (3). In mice, 8-fold cardiac myocyte-specific overexpression of G_q induces cardiac myocyte cell death and HF (6). However, G_q levels are increased only 2-fold in human HF (8, 9), which in mice produces no obvious phenotype (6). Our laboratory previously demonstrated that α₁-ARs are cardioprotective, as defined by their ability to initiate adaptive hypertrophy, survival signaling, and positive inotropy (reviewed in Ref. 3). The finding that α₁-ARs are cardioprotective agrees with the clinical data indicating that α₁-antagonists worsen HF. Collectively, these data suggest that G_q receptors are functionally unique, some protective, like α₁-ARs, and others maladaptive, like AT-Rs.

How can these apparently divergent data on cardiac G_q-receptor function be reconciled? A potentially important clue is our finding that cardiac α₁-ARs primarily localize to and signal at the nucleus unlike other G_q receptors (reviewed in Refs. 3 and 10). Using fluorescent ligands and binding assays in fractionated adult cardiac myocytes, we previously demonstrated that endogenous α₁-ARs localize primarily to the nucleus. We also found that α₁-ARs contain nuclear localization sequences (NLSs), and that whereas reconstitution of WT α₁-ARs in α₁-knockout cardiac myocytes restores α₁-signaling, reconstitution of α₁-NLS mutant receptors does not, demonstrating a requirement for α₁-nuclear localization (11). Further, we showed that α₁-ARs activate intranuclear signaling in adult cardiac myocytes based on our observations that the α₁-agonist phenylephrine activates protein kinase C in isolated nuclei and that blockade of nuclear export inhibits α₁-mediated signaling for contractile function (11). Finally, we found that organic cation transporter 3 (OCT3) mediates rapid and specific catecholamine uptake in cardiac myocytes to facilitate α₁-signaling, and others have demonstrated that OCT3 knockout mice phenocopy the small heart phenotype seen in α₁-AR knockout animals (12, 13). In total, we identified an entirely novel model for nuclear α₁-cardioprotective signaling in cardiac myocytes distinct from the classical model of maladaptive G_q-receptor signaling.

Based on these findings, we hypothesized that unique G_q-receptor function is dictated by receptor localization. To test this hypothesis, we examined the relationship between subcellular compartmentalization of proximal G_q-receptor signaling and the activation of hypertrophic signaling pathways in adult cardiac myocytes. The current model of G_q-receptor hypertrophic signaling is largely based on ET-R signaling and suggests that sarcolemmal G_q receptors produce IP₃-dependent intranuclear calcium release, activation of calmodulin kinase, phosphorylation and nuclear export of histone deacetylases (HDACs), and derepression of transcription (2, 14). This model is notable for explaining how cytosolic calcium transients required for contraction are segregated from IP₃-dependent nuclear calcium signals required for hypertrophic signaling. However, it is not entirely clear how this model might reconcile the data suggesting that G_q receptors induce unique physiology or our model of nuclear α₁-cardioprotective signaling. Here, we report for the first time that in adult cardiac myocytes, α₁-ARs and AT-Rs localize to and activate PLCβ1 in unique subcellular compartments. Further, we demonstrate for the first time that these compartmentalized proximal signals induce differential activation of nuclear hypertrophic signaling pathways to produce unique hypertrophic transcriptomes. Finally, these data suggest an entirely new model of excitation–transcription coupling that accounts for differential localization of G_q receptors and better explains the distinct physiologic function of G_q receptors in adult cardiac myocytes.

Results

α₁-ARs localize to the nuclei and AT-Rs localize to the sarcolemma in adult cardiac myocytes

Here, we sought to define the subcellular localization of α₁-ARs and AT-Rs in adult cardiac myocytes (Figs. 1 and 2). Previously, we demonstrated that endogenous α₁-ARs localize to the nucleus in adult mouse ventricular myocytes (AMVM) (11, 12, 15). However, reagents typically employed to localize receptors are generally unreliable, especially α₁-AR subtype–specific antibodies, which lack specificity (16), or fluorescent ligands, which are no longer commercially available, but nonetheless had suboptimal binding kinetics (12). To overcome these shortcomings, we developed a novel α₁-AR ligand composed of 2-piperazinyl-4-amino-6,7-dimethoxyquinazoline, the common pharmacophore of α₁-AR antagonists such as terazosin and doxazosin, attached to a PEG linker with a terminal biotin moiety fused to a streptavidin-coated fluorescent quantum dot (QDot) with an emission wavelength of 565 nm (Fig. 1A) (17). We validated the α₁-QDot-565 by infecting WT AMVM with a GFP-labeled α₁A-AR (α₁A-GFP), incubating infected myocytes with the α₁-QDot, and co-localizing the GFP and QDot fluorescent signals as an indication of receptor binding (Fig. 1B, nuclei indicated with white arrows). In AMVM expressing α₁A-GFP, the α₁-QDot-565 fluorescent signal co-localized with the GFP fluorescent signal at the nucleus, and pretreatment with the α₁-AR antagonist prazosin diminished QDot fluorescence to nearly undetectable levels, demonstrating specificity (Fig. 1C). We employed the same method as above in uninfected WT AMVM, and the α₁-QDot-565 bound to endogenous α₁-ARs at the nuclei in WT AMVM in the absence of prazosin but was blocked by pretreatment with prazosin, replicating our previous findings (11, 12, 15) (Fig. 1, D and E; quantified in F). Further, the α₁-QDot-565 bound endogenous α₁-ARs in nuclei isolated from WT AMVM (Fig. 1G), and this signal could be blocked by prazosin (Fig. 1H), indicating the presence of α₁-ARs at the nuclear membrane, again replicating our previous findings (11, 12, 15). Therefore, the α₁-QDot-565 fluorescent ligand identified endogenous α₁-ARs at the nucleus in AMVM and outperformed prior fluorescent α₁-ligands by improving kinetics (30 min) at lower concentrations (25 nm) (12).

Figure 1.

α₁-ARs localize to the nuclei in adult cardiac myocytes. A, synthesis of α₁-AR-QDot-565. The structural backbone of the piperazole class of α₁-AR antagonists, 2-piperazinyl-4-amino-6,7-dimethoxyquinazoline, was attached to biotinylated PEG (N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane). The resulting α₁-antagonist-PEG–biotinylated molecule was then coupled to streptavidin-conjugated QDot-565 to make α₁-QDot-565. B, validation of α₁-QDot-565. To define the specificity of the α₁-QDot-565, WT AMVM were infected with an adenovirus expressing α₁A-GFP and then treated with 25 nm α₁-AR-QDot-565. Cells were fixed and imaged by confocal microscopy. A representative myocyte is shown, and the channels are as follows: α₁A-GFP signal (green), QDot-565 signal (yellow), and co-localization were determined in post-processing (white). Scale bar, 10 μm. C, specificity of α₁-QDot-565. Myocytes expressing α₁A-GFP were pretreated with an excess of unlabeled prazosin (5 μm) and then treated with 25 nm α₁-AR-QDot-565. Shown are α₁A-GFP signal (green), QDot-565 signal (yellow), and colocalization determined in post-processing (white). Scale bar, 10 μm. D, α₁-ARs localize to the nuclei in AMVM. α₁-QDot-565 was added to cultured WT AMVM (25 nm, 30 min, 37 °C). Myocytes were fixed and imaged by confocal microscopy (×60 oil immersion). A representative myocyte is shown; arrows indicate nuclei. The inset is intentionally overexposed for visualization of the whole cell. Scale bar, 10 μm. E, specificity of α₁-QDot-565 in WT AMVM. To demonstrate the specificity of the α₁-QDot-565, WT AMVM were pretreated with an excess of prazosin (5 μm, 30 min, 37 °C) and then incubated with α₁-AR-QDot-565 (25 nm, 30 min, 37 °C). One representative myocyte is shown. Scale bar, 10 μm. F, quantification of α₁-QDot-565 localization. The QDot fluorescent intensity from all cardiac myocytes was quantified using FIJI (the number of cardiac myocytes (n) is indicated). QDot-565–alone images not shown. Praz, prazosin. Data are presented as mean ± S.E. (error bars). Data were analyzed by one-way ANOVA, and p < 0.05 was considered significant: α₁-QDot 565 (n = 12 myocytes from four hearts); α₁-QDot 565 + prazosin (n = 3 myocytes from two hearts); QDot 565 alone (n = 4 myocytes from one heart). Scale bar, 10 μm. G, α₁-QDot-565 labels nuclei isolated from AMVM. Nuclei were isolated from WT AMVM and incubated with α₁-AR-QDot-565 (25 nm, 15 min, 37 °C). Nuclei were stained with the nuclear marker DRAQ5, fixed, and imaged as in B. One representative nucleus is shown. Scale bar, 10 μm. H, specificity of α₁-QDot-565 in nuclei isolated from AMVM. To demonstrate the specificity of the α₁-QDot-565, nuclei from WT AMVM were pretreated with an excess of prazosin (5 μm, 15 min, 37 °C) and then incubated with α₁-AR-QDot-565 (25 nm, 15 min, 37 °C). Nuclei were stained with DRAQ5, fixed, and imaged as in B. One representative nucleus is shown. Scale bar, 10 μm.

Figure 2.

AT-Rs localize to the sarcolemma in adult cardiac myocytes. A and B, AT-Rs localize to the sarcolemma in AMVM. AngII-TAMRA (1 μm, 5 min, 37 °C) was added to WT AMVM. Raw optical sections of the middle (top left) and top (bottom left) of the myocytes were captured by confocal microscopy (×60 oil immersion). Deconvolution of optical sections from the middle (top center) and surface (bottom center) was achieved using AutoQuant X3. Arrows indicate AT-R localization at the sarcolemma. 3D surface images of deconvolved images of the middle (top right) and surface (bottom right) were created using FIJI to demonstrate receptor density. Scale bar, 10 μm. C, AngII-TAMRA is specific for AT-Rs in AMVM. WT AMVM were pretreated with an excess of unlabeled AngII (50 μm, 2 min, 37 °C) and then incubated with AngII-TAMRA (1 μm, 5 min, 37 °C). Signal was undetectable, indicating specificity. Scale bar, 10 μm. D, volume plot of AngII-TAMRA–treated AMVM showing surface localization. 3D volume plot was created using FIJI.

Similar to α₁-ARs, the lack of validated antibodies for AT-Rs led us to employ another fluorescent ligand to define the localization of endogenous AT-Rs in AMVM. In this case, we used angiotensin II (AngII), the endogenous ligand of AT-Rs, labeled with the red fluorophore tetramethylrhodamine (TAMRA). We incubated AMVM with AngII-TAMRA in the absence or presence of unlabeled AngII to demonstrate the specificity of AngII-TAMRA (Fig. 2, A–C). In AMVM, AngII-TAMRA produced a distinct localization in confocal sections from the myocyte surface (Fig. 2B, left), whereas AngII-TAMRA showed much less specific binding in confocal sections from the middle of the myocyte (Fig. 2A, left). Pretreatment with unlabeled AngII abolished the AngII-TAMRA signal, indicating specificity (Fig. 2C). To clarify receptor localization, slices from the top and middle of the cell were deconvolved, and 3D surface plots were created that identified AngII-TAMRA signal predominantly at the myocyte surface (Fig. 2, A versus B, center and right; arrows indicate localization). Finally, a volume rendering was created from a confocal stack of a myocyte labeled with AngII-TAMRA, demonstrating that the majority of the AngII-TAMRA signal localized to the myocyte surface (Fig. 2D).

Previously, a small population of AT-Rs was identified at the nucleus in AMVM based on immunochemical detection and functional assays, although the overall physiologic significance of this population of AT-Rs is still unclear (18). Further, this population of nuclear AT-Rs, which is activated by intracrine AngII, represents only a fraction of total AT-Rs (18). Our results with AngII-TAMRA indicate that the majority of ligand-accessible AT-Rs exist at the sarcolemma, and failure to detect nuclear AT-Rs here is probably because neither AngII nor AT-R antagonists readily cross the sarcolemma (19). In summary, AngII-TAMRA identified endogenous AT-Rs on the sarcolemma, and combined with results from α₁-QDot-565 identification of endogenous α₁-ARs at the nucleus, the data indicate that endogenous α₁-ARs and AT-Rs localize to different subcellular compartments in adult cardiac myocytes.

PLCβ1 and its substrate PIP₂ localize to the nuclei in adult cardiac myocytes

Proximal G_q-receptor signaling involves activation of PLCβ1 and cleavage of PIP₂ into IP₃ and DAG. Whereas it is generally accepted that G_q receptors activate PLCβ1 to hydrolyze PIP₂ at the sarcolemma (20), it is not clear whether this occurs at the nucleus in AMVM. Although G_q receptors localize to the nucleus in AMVM, including α₁-ARs (Fig. 1) (11, 12, 15) as well as small populations of AT-Rs (18) and ET-Rs (21), it is not certain that either PLCβ1 or PIP₂ localizes to the nucleus (22). In other cells, PIP₂ hydrolysis is observed in the nuclear matrix but is reported to be independent of G_q-receptor signaling (23). However, nuclear α₁-ARs activate intranuclear PKCδ in nuclei isolated from AMVM (11), suggesting production of DAG from a yet to be defined nuclear phosphatidylinositol species. Further, both AT-Rs and ET-Rs induce intranuclear signaling dependent on the production of inositol phosphates in AMVM (14, 18).

Based on prior demonstrations of G_q-receptor localization and signaling at the nucleus, we sought to clarify whether PLCβ1 is localized to the nucleus in adult cardiac myocytes. First, we sought to validate potential PLCβ1 antibodies. Preferably, we would employ AMVM from PLCβ1 knockout mice, but these mice exhibit spontaneous seizures and high mortality around 3 weeks of age (24). Alternatively, we attempted to use siRNA technology to knock down PLCβ1 in AMVM but were unable to achieve significant PLCβ1 mRNA knockdown by 40 h, probably due to low turnover of PLCβ1 in cultured AMVM. Subsequently, we attempted to validate potential PLCβ1 antibodies using siRNA technology in the N38 embryonic mouse hypothalamic cell line due to PLCβ1 enrichment in the brain (25). To assess PLCβ1 knockdown, N38 cells were transfected with either PLCβ1 siRNA or scramble siRNA for 72 h. We observed knockdown of PLCβ1 mRNA (Fig. 3C; quantified in D), and knockdown of PLCβ protein was visualized by decrease in staining of the PLCβ1 antibody (Fig. 3A; quantified in B). Finally, we stained WT AMVM with the PLCβ1 antibody, and our results indicate that PLCβ1 localizes to the sarcolemma, T-tubules, and nuclear envelope (Fig. 3E, nuclei indicated with white arrows).

Figure 3.

PLCβ1 localizes to the nuclear membrane in adult cardiac myocytes. A, visualization of siRNA-mediated PLCβ1 protein knockdown. N38 cells (embryonic mouse hypothalamic cells) were transfected with 80 pmol of either PLCβ1 siRNA or scrambled siRNA using Lipofectamine RNAiMAX for 72 h at 37 °C. Cells were fixed and stained with a primary antibody against PLCβ1. Secondary antibody was conjugated to Alexa Fluor 594. Cells were imaged using confocal microscopy (×20). Scale bar, 10 μm. B, quantification of siRNA-mediated PLCβ1 protein knockdown. Fluorescence intensity was measured using FIJI (n = 2 cultures; four optical areas were measured for scramble, and eight optical areas were measured for PLCβ1 siRNA). Data are represented as mean ± S.E. (error bars). Data were analyzed by paired student's t test, and p < 0.05 was considered significant. C, siRNA-mediated PLCβ1 mRNA knockdown. N38 cells were transfected as in A. After 72 h, RNA was harvested using an RNAEasy kit, and PLCβ1 mRNA levels were measured by RT-PCR. Results of densitometry analysis of PLCβ1 mRNA levels are shown in D. D, quantification of siRNA-mediated PLCβ1 mRNA knockdown. The ratio of glyceraldehyde-3-phosphate dehydrogenase to PLCβ1 was quantified using densitometry (n = 3 cultures). Data are represented as mean ± S.E. E, endogenous PLCβ1 localizes to the sarcolemma and nuclear membrane in AMVM. WT AMVM were isolated, cultured for 24 h, fixed, permeabilized, and stained with the primary antibody against PLCβ1. Secondary antibody was conjugated to Alexa Fluor 488. Myocytes were imaged by confocal microscopy (×60 oil immersion). Arrows, nuclear membrane. Images were cropped (white lines indicate cropped area) and horizontally aligned. Two representative images are shown. Scale bar, 10 μm.

PIP₂, the substrate for PLCβ1, localizes to the sarcolemma in AMVM (22, 26, 27), yet identifying a population of nuclear PIP₂ has been elusive because either 1) PIP₂ is not present at the nucleus or 2) the methods used to detect PIP₂ have been insufficient. To address this conundrum, we isolated AMVM nuclei using differential centrifugation and analyzed samples by MS to determine whether PIP₂ is present in nuclear membranes in AMVM. To detect the presence of PIP₂ species, we ran two blanks and two AMVM nuclear samples, adding one of each with commercially available 36:2 PIP₂, PIP₂(4′,5′)(18:1(9Z)/18:1(9Z)). Using electrospray ionization-MS scan with precursor ion m/z 281 (carboxylated anion of oleic acid; C18:1), we were able identify PIP₂ 36:2 species in all of the samples except for the blank (Fig. 4A, left). Spectra of the peak showing a parent ion m/z 1021 (Fig. 4A, right) confirmed the identity of the commercially available 36:2 phosphatidylinositol 4,5-bisphosphate in the PIP₂-added blank sample, with ion fragments at m/z 259, 281, 339, and 419 corresponding to the inositol phosphate (IP), the oleic acid backbone, inositol diphosphate (IP₂), and inositol triphosphate (IP₃) components of the fragmented 36:2 PIP₂.

Figure 4.

PIP₂ localizes to the nuclear membrane in adult cardiac myocytes. A, detection and identification of PIP₂(4′,5′)(18:1(9Z)/18:1(9Z)) in AMVM nuclear fractions. Blank and nuclear extracts were analyzed with and without PIP₂(4′,5′)(18:1(9Z)/18:1(9Z)). The yellow peak (not detected) represents blank sample with no PIP₂ addition, the green peak represents the blank sample with PIP₂ addition, the blue peak represents the nuclear fraction with no PIP₂ addition, and the red peak represents the nuclear fraction with PIP₂ addition (left). Electrospray ionization-MS scans of the PIP₂-added blank sample with precursor ion m/z 281 show m/z 1021, identifying the compound as 36:2 PIP2 (right). B, identification of PIP₂ species in AMVM nuclear fractions. Product ions of m/z 1045 (38:4 PIP₂) of the unmixed nuclear fraction sample.

Similar to previous PIP₂ analyses (28), we used negative ion MS/MS to analyze the product ions of m/z 1045 (38:4 PIP₂), the most abundant PIP₂ species in the nuclear fraction sample without the PIP₂ addition (Fig. 4B). Ions at m/z 259, 283, 303, 339, and 419 correspond to IP, carboxylate anion fatty acyl chains of stearic acid, the arachidonic acid backbone, IP₂, and IP₃, all portions of the fragmented 38:4 PIP₂ spectrum. Our results indicate that PIP₂ localizes to membranes within the nuclei of AMVM, which to our knowledge is the first such demonstration. In total, our findings reveal that PLCβ1 and its substrate, PIP₂, are found in the same subcellular compartments as both the α₁-AR (nuclear) and AT-R (sarcolemma), suggesting the potential for compartmentalized G_q-receptor signaling in adult cardiac myocytes.

AT-Rs, but not α₁-ARs, activate PLCβ1 at the sarcolemma in adult cardiac myocytes

Based on the observed distinct subcellular compartmentalization of α₁-ARs and AT-Rs (Figs. 1 and 2), we reasoned that AT-Rs would activate proximal signaling at the sarcolemma and α₁-ARs at the nuclei. To test this, we measured the compartmentalization of G_q-receptor activation of PLCβ1 with the PLCβ1 activity sensor GFP-C1-PLCδ-PH (GFP-PHD; Fig. 5A). GFP-PHD is composed of GFP fused to the N terminus of the pleckstrin homology domain of PLCδ1, which preferentially binds PIP₂ over other membrane phosphatidylinositols in vitro (29). In general, GFP-PHD associates with PIP₂ in membranes in the basal state, and upon G_q-receptor stimulation, PLCβ1 hydrolyzes PIP₂, and as PIP₂ is depleted, GFP-PHD dissociates from the membrane, as illustrated in Fig. 5B. In AMVM expressing GFP-PHD, the probe localized to the sarcolemma and T-tubules in the basal state, in agreement with previous reports (22). More importantly, the α₁-agonist phenylephrine (PE), in the absence or presence of prazosin, produced no change in the localization of GFP-PHD compared with vehicle (Fig. 5C; quantified in D). Conversely, AngII induced a marked dissociation of GFP-PHD from the membrane compared with vehicle, which was blocked by the nonselective AT-R antagonist losartan, indicating a receptor-specific effect (Fig. 5E; quantified in F). These results demonstrate that AT-Rs, but not α₁-ARs, activate PLCβ1 at the sarcolemma in adult cardiac myocytes, consistent with the subcellular compartmentalization of each receptor.

Figure 5.

AT-Rs, but not α₁-ARS, activate PLCβ1 at the sarcolemma in adult cardiac myocytes. A, schematic representation of GFP-PHD. GFP-PHD consists of the pleckstrin homology domain of phospholipase Cδ attached to the C terminus of GFP. The PH domain of PLCδ has high affinity for PIP₂, and in the basal state, GFP-PHD binds to membrane PIP₂, and cleavage of PIP₂ by PLCβ1 releases the probe from the membrane, decreasing membrane GFP fluorescence (33). B, GFP-PHD function. In the basal state (Basal), GFP-PHD localizes to the sarcolemma bound to PIP₂, and upon activation of PLCβ1 (Activated), PIP₂ is cleaved, and the probe moves into the cytoplasm. C and E, α₁-ARs (C) do not activate PLCβ1 at the sarcolemma, but AT-Rs do (E). WT AMVM expressing GFP-PHD were treated with vehicle (left) or PE (20 μm, 5 min, 37 °C) in the absence and presence of prazosin (1 mm, 30-min pretreatment, 37 °C; middle and right panels, respectively) or with AngII (100 nm, 5 min, 37 °C) in the absence and presence of losartan (5 μm, 30-min pretreatment, 37 °C, middle and right panels, respectively). Images were cropped (white lines indicate cropped area) and horizontally aligned. 3D surface images were created with FIJI to demonstrate GFP-PHD fluorescence intensity. Scale bar, 10 μm. D and F, quantification of sarcolemmal PLCβ1 activity downstream of α₁-ARs (D) and AT-Rs (F). Myocytes were classified by another investigator blinded to treatment group as responders defined by GFP-PHD movement off the sarcolemma or nonresponders defined by no movement of GFP-PHD either at baseline or following agonist treatment. Data were analyzed for cells treated with vehicle or drug (PE/AngII) by χ², and p < 0.05 was considered significant. Data are represented as mean ± S.E. (error bars): vehicle (n = 30 myocytes from seven hearts); PE (n = 24 myocytes from four hearts); AngII (n = 29 myocytes from four hearts). ns, not significant.

α₁-ARs, but not AT-Rs, activate PLCβ1 at the nuclear envelope in adult cardiac myocytes

Interestingly, GFP-PHD was not detected at the nuclear membrane in the basal state (Fig. 5). We suggest there are two explanations for this observation: 1) PIP₂ is not present in the nuclear membrane, despite our identification of PIP₂ in nuclear membranes (Fig. 4), or 2) GFP-PHD, which lacks an NLS, is unable to target the nucleus to bind nuclear PIP₂. To clarify this and additionally determine whether G_q receptor–mediated activation of PLCβ1 possibly occurs at the nucleus, we inserted an NLS sequence at the N terminus of GFP-PHD to create NLS-GFP-PHD (Fig. 6A). Insertion of an NLS promoted nuclear localization of GFP-PHD (Fig. 6, C and E), suggesting that PIP₂ is found in the nucleus, in agreement with detection of nuclear PIP₂ by MS (Fig. 4). NLS-GFP-PHD associates with PIP₂ in the nuclear membrane in the basal state, and upon nuclear G_q-receptor stimulation, PLCβ1 hydrolyzes PIP₂, and as PIP₂ is depleted, NLS-GFP-PHD dissociates from the nuclear membrane and moves into the nucleoplasm, as illustrated in Fig. 6B. Using the same experimental conditions as our experiments with GPF-PHD, the α₁-agonist PE induced a marked dissociation of NLS-GFP-PHD from the nuclear membrane, which was blocked by prazosin (Fig. 6C; quantified in D). Conversely, AngII, in the absence or presence of losartan, produced little change in the localization of NLS-GFP-PHD (Fig. 6E; quantified in F). These results demonstrate that α₁-ARs, but not AT-Rs, primarily activate PLCβ1 at the nuclear membrane in AMVM. The combined results from experiments using GFP-PHD and NLS-GFP-PHD indicate that α₁-AR– and AT-R–mediated proximal signaling is confined to distinct subcellular compartments, consistent with receptor localization.

Figure 6.

α₁-ARs and, to a lesser extent, AT-Rs activate PLCβ1 at the nuclear membrane in adult cardiac myocytes. A, schematic representation of NLS-GFP-PHD. NLS-GFP-PHD consists of GFP-PHD tagged with an N-terminal NLS from the SV40 large T-antigen to promote localization of the probe to the nucleus. NLS-GFP-PHD is thought to operate in the same manner as GFP-PHD (Fig. 3), but at the nuclear membrane (33). B, GFP-PHD function. In the basal state (Basal), GFP-PHD-NLS localizes primarily to the nuclear membrane bound to PIP₂, and upon activation of PLCβ1 (Activated), PIP₂ is cleaved, and the probe moves into the nucleoplasm. C and E, α₁-ARs (C) activate PLCβ1 at the nuclear membrane, but AT-Rs do not (E). WT AMVM expressing NLS-GFP-PHD were treated with either vehicle (left panels) or PE (20 μm, 5 min, 37 °C) in the absence or presence of prazosin (1 mm, 30-min pretreatment, 37 °C; middle and right panels, respectively) or AngII (100 nm, 5 min, 37 °C) in the absence and presence of losartan (5 μm, 30-min pretreatment, 37 °C; middle and right panels, respectively). Arrows indicate nuclear membrane activation of PLCβ1 by α₁-ARs. Images were cropped (white lines indicate cropped area) and horizontally aligned. 3D surface images were created with FIJI to demonstrate NLS-GFP-PHD fluorescence intensity. Scale bar, 10 μm. D and F, quantification of nuclear PLCβ1 activity downstream of α₁-ARs (D) and AT-Rs (F). Myocytes were classified by another investigator blinded to treatment group as responders defined by NLS-GFP-PHD movement off the nuclear membrane or nonresponders defined by no movement of NLS-GFP-PHD either at baseline or following agonist treatment. Data were analyzed for cells treated with vehicle or drug (PE/AngII) by χ², and p < 0.05 was considered significant. Data are represented as mean ± S.E. (error bars): vehicle (n = 30 myocytes from seven hearts); PE (n = 14 myocytes from four hearts); AngII (n = 16 myocytes from four hearts). ns, not significant.

α₁-ARs, but not AT-Rs, induce IP₃-dependent nuclear export of HDAC5 in adult cardiac myocytes

G_q receptors are thought to induce hypertrophy through a mechanism known as excitation–transcription coupling (2). Conventionally, sarcolemmal G_q receptor–mediated production of IP₃ elicits intranuclear calcium release from the nuclear envelope, activation of calmodulin kinase type II, and phosphorylation and nuclear export of HDAC5 (2). Nuclear compartmentalization of IP₃-dependent calcium release allows myocytes to distinguish calcium required for contraction from calcium required for transcriptional signaling. Here, we examined whether differentially localized G_q receptors would have the same effect on HDAC5 export. In AMVM expressing HDAC5-GFP, PE but not AngII induced a moderate, but significant, export of HDAC5 at 30 min (Fig. 7A; quantified in B). By 1 h, PE significantly induced HDAC5 nuclear export, whereas AngII did not (Fig. 7C; quantified in D), consistent with previous reports for PE (30). To determine whether PE-induced HDAC5 nuclear export was IP₃-dependent, AMVM expressing HDAC5-GFP were pretreated with the IP₃R inhibitor 2-aminoethoxydiphenyl borate (2-APB). Pretreatment with 2-APB abolished the PE-mediated HDAC5 nuclear export (Fig. 7E; quantified in F). Taken together, these results indicate that α₁-ARs, but not AT-Rs, activate IP₃-dependent HDAC5 nuclear export in AMVM.

Figure 7.

α₁-ARS induce HDAC5 export, whereas AT-Rs do not. A and C, α₁-ARs activate HDAC5 nuclear export, whereas AT-Rs do not. WT AMVM expressing HDAC5-GFP were treated with either vehicle (left panels), 20 μm PE (middle panels; insets of zoomed-in nuclei), or 100 nm AngII (right panels) for either 30 min (A) or 1 h (C) at 37 °C. Scale bar, 10 μm. B and D, quantification of HDAC5 export induced by α₁-ARs and AT-Rs. Cytoplasmic and nuclear GFP fluorescence was quantified using FIJI, and the ratio of cytoplasmic to nuclear GFP was plotted as an indication of HDAC5 nuclear export at 30 min (B) and 1 h (D). Data are represented as mean ± S.E. (error bars) Data were analyzed by one-way ANOVA, and p < 0.05 was considered significant: vehicle (n = 12 myocytes from six hearts); PE (n = 12 myocytes from six hearts); AngII (n = 12 myocytes from six hearts). E, HDAC5 export is inhibited in the presence of IP₃R inhibitor, 2-APB. WT AMVM expressing HDAC5-GFP were treated with 2-APB (2 μm, 30 min at 37 °C) before treatment with PE (1 h at 37 °C; middle). Scale bar, 10 μm. F, quantification of HDAC5 export in the presence of IP₃R inhibitor, 2-APB. The ratio of cytoplasmic to nuclear GFP was calculated and plotted. Data are represented as mean ± S.E. (error bars). Data were analyzed by one-way ANOVA, and p < 0.05 was considered significant: vehicle (n = 12 myocytes from six hearts); 2-APB (n = 12 myocytes from six hearts); PE (n = 12 myocytes from six hearts); PE + 2-ABP (n = 12 myocytes from six hearts).

α₁-ARs and AT-Rs induce unique transcriptomes in adult cardiac myocytes

Physiologically, α₁-ARs and AT-Rs have diametrically opposed effects on the heart. α₁-ARs induce physiologic hypertrophy, survival signaling, and positive inotropy and are not associated with fibrosis (3). AT-Rs induce pathologic hypertrophy, myocyte cell death, and negative inotropy and are pro-fibrotic (31, 32). We hypothesized that these differences in the physiologic function of cardiac α₁-ARs and AT-Rs would be revealed in their transcriptomes and reflective of their distinct subcellular localization. To evaluate α₁-AR and AT-R transcriptomes, we treated mice with vehicle, PE (30 mg/kg/day), or AngII (0.5 mg/kg/day) continually for 3 days using osmotic minipumps, at which point we isolated cardiac myocytes and performed RNA-Seq. The doses of PE and AngII that we used are known to induce hypertrophy without a concomitant increase in blood pressure (33–36). Consistent with our HDAC5 results and with the fact that HDAC5 activation relieves transcriptional repression, α₁-ARs induced a larger transcriptional response, with a total of 806 genes changed (increased or decreased) 1.7-fold versus vehicle, whereas AT-Rs induced a much smaller response, with only 173 genes changed 1.7-fold versus vehicle. 1.7-fold expression over vehicle was used as the threshold because adult cardiac myocytes are postmitotic and generally do not induce large transcriptional responses. Interestingly, between α₁-ARs and AT-Rs, 155 genes were changed by both agonists, indicating that α₁-ARs induced 651 unique genes, whereas AT-Rs induced only 18 unique genes (Fig. 8A). These results suggest that the AT-R transcriptome is largely a subset of the α₁-AR transcriptome in cardiac myocytes.

Figure 8.

α₁-ARs robustly activate transcriptional responses in adult cardiac myocytes whereas AT-ARs do not. A, Venn diagram of α₁-AR- and AT-R-induced transcriptomes. WT adult male mice were treated for 3 days with minipumps perfusing vehicle, PE, or AngII. Myocytes were then isolated, RNA was isolated, and RNA-Seq was performed. The gene list generated by RNA-Seq was filtered based on a minimum 1.7× absolute -fold change and false discovery rate–corrected p < 0.05. PE treatment altered 801 transcripts (655 unique) by at least 1.7-fold, whereas AngII only altered 173 (18 unique), with 155 common between the two treatments. B, gene ontology analysis of α₁-AR– and AT-R–induced transcriptomes. G_q receptors regulate hypertrophy, survival signaling, inotropy, and fibrosis. Therefore, using nonoverlapping search terms unique to these different biologic functions, genes were sorted into subclasses of hypertrophy, survival signaling, inotropy, and fibrosis. The total number of genes altered (increased and decreased) in common or by PE or AngII alone are plotted in the graph. Search terms used to sort genes associated with hypertrophy were as follows: NF-κB, GATA4, MEF2, NFAT, ANF, TGF, hypertrophy, myosin heavy chain, MHC, c-myc, c-fos, cell growth, and natriuretic. Search terms for survival signaling were ERK, MAPK, apoptotic, apoptosis, survival, and cell death. Search terms for inotropy were calcium, contraction, sarcoplasmic reticulum, troponin, myosin, actin, ryanodine, protein kinase C, and sarcomere. Search terms for fibrosis were collagen, matrix metalloprotease, fibrosis, fibroblast, extracellular matrix, and matrix. C, principle component analysis of α₁-AR– and AT-R–induced transcriptomes. Principle component analysis of fragments per kilobase of exon per million reads mapped (FPKM). PC1 determined 66% of all variance between samples. PC2 determined 22% of all variance between samples. D, top 25 genes determining PC1 and PC2. Top 25 genes for PC1 and PC2 were determined using FPKM loadings.

To parse the RNA-Seq results further, we initially attempted to utilize the Ingenuity Pathway Analysis (IPA) software, but the majority of the database is derived from oncogenic studies and lacks cardiac myocyte specific pathways. Thus, we derived our own analysis and sorted genes that were regulated by α₁-ARs alone, AT-Rs alone, or were in common between the two into gene ontologies corresponding to G_q-receptor biology: hypertrophy, survival signaling, inotropy, and fibrosis (Fig. 8B and Tables S1–S4). α₁-ARs most robustly altered genes in all categories as compared with common genes and AT-R–only genes. Whereas it is surprising that α₁-ARs altered more fibrotic genes than AT-Rs, these genes are not classically associated with alterations in the extracellular matrix leading to fibrosis (Table S4). Finally, principle component analysis was performed to determine the degree of difference between vehicle-, AngII-, and PE-treated samples (Fig. 8C). Principle component 1 (PC1) accounted for 66% of the variance and aligned with the α₁-AR transcriptomes, whereas PC2 accounted for 22% of the variance and aligned with the AT-R transcriptome. The AngII-treated samples also closely grouped with the vehicle-treated samples, indicating that AT-Rs do not induce a highly distinct transcriptome from control, consistent with our gene ontology results (Fig. 8, A and B) The top 25 genes determining PC1 and PC2 are presented in Fig. 8D. Taken together, α₁-ARs robustly activate transcription in adult cardiac myocytes, whereas AT-Rs minimally activate transcription. These results identify distinct differences in the transcriptomes induced by α₁-ARs and AT-Rs that align with their distinct subcellular localization and activation of proximal signaling to produce differential activation of nuclear hypertrophic signaling pathways.

Discussion

Here, we identified an entirely novel mechanistic explanation for unique G_q-receptor function in adult cardiac myocytes predicated upon subcellular compartmentalization of proximal G_q-receptor signaling and propose a novel model of excitation–transcription coupling. We found that α₁-ARs localize to the nucleus and induce intranuclear activation of PLCβ1, stimulate IP₃-dependent nuclear export of HDAC5, and activate a robust and unique transcriptome associated with hypertrophic, survival, inotropic, and (anti-)fibrotic gene programs. Conversely, we observed that AT-Rs primarily localize to and activate PLCβ1 at the sarcolemma but have little effect on nuclear export of HDAC5 and induce a small transcriptome that is a subset of the α₁-transcriptome. More importantly, these findings are consistent with our hypothesis that G_q-receptor localization dictates function by showing that compartmentalization of proximal G_q-signaling is correlated with phenotypic outcome in adult cardiac myocytes.

The excitation–transcription model of G_q receptor–mediated hypertrophic signaling in adult cardiac myocytes proposes that sarcolemmal G_q receptors induce IP₃ production and IP₃-sensitive intranuclear calcium release from perinuclear calcium stores to activate calmodulin kinase, phosphorylate and induce nuclear export of HDAC5, and thereby activate transcription (Fig. 9A). Whereas the current model of excitation–transcription coupling suggests that G_q receptors induce IP₃ production at the sarcolemma leading to activation of IP₃-dependent calcium release at the nucleus to induce HDAC5 export and promote gene transcription, it fails to explain how G_q receptors might produce unique physiological function in cardiac myocytes.

Figure 9.

Novel model of excitation–transcription coupling in adult cardiac myocytes. A, conventional model of excitation–transcription coupling in adult cardiac myocytes. All Gα_q-receptors localize to the sarcolemma in adult cardiac myocytes. Upon ligand binding, sarcolemmal PLCβ1 is activated and cleaves PIP₂ into DAG and IP₃. DAG goes on to activate protein kinase C isoforms (PKC) and induce contraction. IP₃ traverses the myocyte and binds to the IP₃R on the inner nuclear membrane, inducing intranuclear calcium release. Calcium activates calmodulin (CaM) and calcium-calmodulin dependent protein kinase II (CaMKII), which phosphorylates HDAC5. HDAC5 phosphorylation triggers HDAC5 nuclear export and derepression of transcription. B, updated model of excitation–transcription coupling in adult cardiac myocytes. α₁-ARs (nuclear) and AT-Rs (sarcolemmal) are differentially localized in adult cardiac myocytes (Figs. 1 and 2). Upon ligand binding to either α₁-ARs or AT-Rs, PLCβ1 is activated either at the nucleus (α₁-AR; Fig. 6) or at the sarcolemma (AT-R, Fig. 5). AT-R–induced PLCβ1 activation at the sarcolemma fails to induce HDAC5 nuclear export, whereas α₁-AR–induced PLCβ1 activation at the nucleus induces HDAC5 nuclear export. Furthermore, α₁-AR–induced HDAC5 nuclear export is IP₃-dependent (blocked by 2-APB) (Fig. 7). Consistent with α₁-induced, IP₃-dependent HDAC5 nuclear export, α₁-ARs induce a robust transcriptional response, whereas AT-Rs, which fail to induce HDAC5 export, produce a transcriptional response that is largely a subset of α₁-AR–induced transcriptional responses (potentially suggesting a different non-HDAC dependent mechanism of transcription) (Fig. 8).

Both ET-Rs and insulin-like growth factor receptors conform to the traditional excitation–transcription model (2, 14, 37). Our data indicate that α₁-ARs might support this model as well, but interestingly, AT-Rs do not. Although we observed AT-R–mediated activation of PLCβ1, we failed to detect AT-R–mediated nuclear export of HDAC5 and found a much smaller transcriptional response. One interpretation of this result is that close proximity to the nucleus is required for G_q receptor–mediated activation of IP₃-dependent hypertrophic signaling. In support of this interpretation, α₁-ARs localize to the inner nuclear membrane (11), and ET-Rs and insulin-like growth factor receptors localize to the bottom of T-tubules in close apposition to the nucleus in adult cardiac myocytes (37, 38). Further, the failure of AT-Rs to induce nuclear export of HDAC5 suggests that AT-R–mediated activation of PLCβ1 at the sarcolemma either fails to generate enough IP₃ to reach the nucleus or that IP₃ is degraded before it reaches the nucleus. The potential degradation of IP₃ before reaching the nucleus might be analogous to the compartmentalization of cAMP signaling in cardiac myocytes (39).

Here, we propose a new model of excitation–transcription coupling that is based on compartmentalization of G_q receptors that explains distinct physiologic function of G_q receptors in adult cardiac myocytes. Our model suggests that G_q receptor–induced IP₃ production is compartmentalized and that IP₃ produced inside the nucleus (or possibly in close proximity to the nucleus) induces HDAC5 export to promote gene transcription, whereas IP₃ produced at a distance from the nucleus (at the sarcolemma) has a different and smaller effect on transcriptional regulation (Fig. 9B). In summary, we suggest that G_q-receptor compartmentalization has a large influence on the transcriptomes induced by differentially localized G_q receptors, illustrating the fundamental physiologic importance of G_q-receptor compartmentalization.

With regard to our model of nuclear α₁-induced HDAC5 export, previous work indicated that HDAC5 export downstream of α₁-ARs occurs mainly through activation of protein kinase D, and our results do not exclude activation of this signaling cascade (40). Nevertheless, in agreement with our results, Luo et al. (41) demonstrated that α₁-ARs activate IP₃-dependent nuclear calcium transients in cardiac myocytes, consistent with our finding that α₁-ARs induce IP₃-sensitive HDAC nuclear export. At this time, the discrepancy between these findings is not entirely clear.

The consensus view of cardiac G_q-receptor function has been that G_q receptors mediate pathologic remodeling, promoting maladaptive hypertrophy, myocyte cell death, and negative inotropic responses (4). However, in clinical trials of hypertension (Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT)) and HF (Vasodilator Heart Failure Trial (V-HeFT)), α₁-AR antagonists worsened outcomes (42, 43). Further, our studies indicate that α₁-ARs are cardioprotective, promoting adaptive or physiologic hypertrophy, prevention of cell death, and positive inotropic effects identifying a mechanistic basis for the negative results of α₁-AR antagonists in clinical trials (3). In short, the cardioprotective nature of α₁-ARs stands in stark contrast to the consensus view of maladaptive G_q-receptor function. Here, our data suggest that compartmentalization of G_q receptors could explain differences in G_q-receptor function. We demonstrated that although both α₁-ARs and AT-Rs activate PLCβ1, they do so in different subcellular compartments, which has a profound effect on activation of intranuclear hypertrophic signaling and transcriptional activation. Therefore, we suggest that nuclear G_q-receptor signaling, typified by α₁-ARs, is cardioprotective. Aside from α₁-ARs, a small population of functional ET-Rs and AT-Rs localize to the nucleus as well (18, 21), although their physiologic significance is unclear. Conversely, we observed AT-Rs primarily at the sarcolemma, but not in close proximity to the nucleus, which might suggest that G_q-receptor signaling at the sarcolemma is pathologic, which is supported by studies with AT-Rs (31, 32). In support of this concept, adenoviral mediated expression of the PLCβ1b at the sarcolemma induces contractile dysfunction (44). In summary, our hypothetical model of compartmentalized G_q-receptor signaling, where nuclear G_q-receptor signaling is cardioprotective, suggests a more nuanced view of G_q-receptor function in cardiac myocytes.

The concept of GPCR compartmentalization in cardiac myocytes is not without precedence. In co-cultures of sympathetic ganglionic neurons and neonatal rat cardiac myocytes, β₁-ARs localize to regions of axonal contact rich in SAP97, AKAP97, catenins, and cadherins, whereas β₂-ARs are excluded from these domains (45). In adult cardiac myocytes, β₁-ARs are distributed over the entire sarcolemma, whereas β₂-ARs are restricted deep within T-tubules (46). Finally, platelet-derived growth factor receptors, which also signal through G_s, localize to caveolae in cardiac myocytes and do not induce an inotropic response (47). These examples demonstrate that cardiac myocytes compartmentalize G_s-mediated GPCR signaling, analogous to our findings with G_q receptors. In conclusion, our findings support a model of compartmentalized G_q-receptor signaling in adult cardiac myocytes and suggest a revision of the classic model of excitation–transcription coupling. Our new model, largely based on our identification of cardioprotective nuclear α₁-AR signaling, provides a plausible mechanistic basis to explain the unique function of cardiac G_q receptors. Additionally, this model suggests a reexamination of the classic paradigm of maladaptive G_q signaling in cardiac myocytes in favor of a more nuanced view of compartmentalized G_q-receptor signaling.

Experimental procedures

Experimental models: Mice

In this study, male and female C57BL/6J mice (10–15 weeks of age) were used for primary adult cardiac myocytes. Male FVB/NJ mice (10–11 weeks of age) were used for infusion of agonist to measure G_q receptor–induced hypertrophic transcriptional responses. All animals were sourced from Jackson Laboratories. The use of all animals in this study conformed to the United States Public Health Service Guide for Care and Use of Laboratory Animals and was approved by the University of Minnesota institutional animal care and use committee.

Method details

Isolation and culture of AMVM

This was carried out as described previously (48).

Isolation and labeling of nuclei from AMVM

This was carried out as described previously (49). Labeling is described in the supporting information.

Adenoviral production

The α₁A-GFP adenovirus was described previously (50). Production of the GFP-C1-PLCδ-PH (GFP-PHD) and NLS-GFP-PHD adenoviruses are described in the supporting information. The HDAC5-GFP adenovirus was a gift from Dr. Timothy McKinsey (51). For all experiments, cultured AMVM were counted and infected with adenoviruses at the following multiplicity of infection: α₁A-GFP, 1000 (titer: 5.9 × 10¹⁰); GFP-PHD, 100 (titer: 2.4 × 10⁸); NLS-GFP-PHD (titer: 1.4 × 10¹⁰); and HDAC5-GFP (titer: 6.1 × 10¹⁵).

Synthesis of α₁-QDot-565

The synthesis of the α₁-QDot-565 was performed as described (17) with a few modifications that are described in the supporting information.

Localization of α₁-ARs, AT-Rs, and PLCβ1 in AMVM

Methods used to define the localization of α₁-ARs, AT-Rs, and PLCβ1 are described in the supporting information.

Identification of nuclear PIP₂

PIP₂ extraction was carried out as described (52) with some modifications. The electrospray mass spectrum of each sample was analyzed on a hybrid quadrupole triple ion trap mass spectrometer (Triple TOF 5600, AB Sciex Instrument). All scans were performed in negative ionization mode and a mass-to-charge (m/z) range from 50 to 1200. PIP₂(4′,5′)(18:1(9Z)/18:1(9Z)) was obtained from Avanti Polar Lipids.

Quantification and statistical analysis

The methods for the analysis of phospholipase Cβ1 activity, HDAC5 nuclear export, and hypertrophic transcriptional profiles adenoviruses are described in the supporting information. Details and methods used for statistical analysis can be found in the supporting information.

Author contributions

E. F. D., S. C. W., G. C. S., and T. D. O. conceptualization; E. F. D., C. L. H., B. A. H., and T. D. O. data curation; E. F. D., B. A. H., and T. D. O. formal analysis; E. F. D. validation; E. F. D., S. C. W., C. L. H., B. A. H., and T. D. O. investigation; E. F. D. visualization; E. F. D., S. C. W., C. L. H., B. A. H., and T. D. O. methodology; E. F. D. and T. D. O. writing-original draft; S. C. W., C. L. H., and T. D. O. writing-review and editing; C. L. H. and G. C. S. project administration; G. C. S. resources; T. D. O. and G. C. S. funding acquisition.