ARTICLE cro
␣ Subcellular compartmentalization of proximal G q-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes Received for publication, February 6, 2018, and in revised form, March 13, 2018 Published, Papers in Press, April 2, 2018, DOI 10.1074/jbc.RA118.002283 Erika F. Dahl‡, Steven C. Wu§, Chastity L. Healy§, Brian A. Harsch¶, Gregory C. Shearer¶, and Timothy D. O’Connell§1 From the Departments of ‡Pharmacology and §Integrative Biology and Physiology, University of Minnesota, Minneapolis, Minnesota 55455 and the ¶Department of Nutritional Sciences, Pennsylvania State University, University Park, Pennsylvania 16802 Edited by Henrik G. Dohlman
␣ ␣ G protein–coupled receptors that signal through G q (Gq G protein–coupled receptors that signal through G q (Gq ␣ ␣ 2 receptors), such as 1-adrenergic receptors ( 1-ARs) or angio- receptors) share a common proximal signaling pathway Downloaded from tensin receptors, share a common proximal signaling pathway through the activation of phospholipase C1 (PLC1), which   that activates phospholipase C 1 (PLC 1), which cleaves phos- cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to pro-
phatidylinositol 4,5-bisphosphate (PIP2) to produce inositol duce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol
1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these com- (DAG) (1). However, in any cell, simultaneous activation of
mon proximal signaling mechanisms, Gq receptors produce dis- multiple proximal Gq-receptor signaling events would preclude http://www.jbc.org/ tinct physiological responses, yet the mechanistic basis for this the cell’s ability to process different signals and produce a unique outcome. Despite this commonality, each G receptor remains unclear. In the heart, Gq receptors are thought to q induce myocyte hypertrophy through a mechanism termed mediates distinct physiologic processes, but how this specificity excitation–transcription coupling, which provides a mechanis- is achieved is unclear in many cases. Compartmentalization of tic basis for compartmentalization of calcium required for con- signaling provides one mechanism through which a cell could traction versus IP -dependent intranuclear calcium required for handle a multitude of potentially overlapping signals. Cardiac at University of Minnesota Libraries on June 8, 2018 3 myocytes offer several examples of compartmentalized recep- hypertrophy. Here, we identified subcellular compartmental- tor signaling (e.g. the ability to discriminate -adrenergic ization of G -receptor signaling as a mechanistic basis for q (-AR)-G -induced calcium signals that augment contractility unique G receptor–induced hypertrophic phenotypes in car- s q from G receptor–mediated calcium signals that induce hyper- diac myocytes. We show that ␣ -ARs co-localize with PLC1 q 1 trophy) (2). In this case, inositol-sensitive calcium release ␣ and PIP2 at the nuclear membrane. Further, nuclear 1-ARs occurs in the nucleus based on the localization of the inositol  induced intranuclear PLC 1 activity, leading to histone trisphosphate receptor to the inner nuclear membrane. How- deacetylase 5 (HDAC5) export and a robust transcriptional ever, this ultimately raises questions about how Gq receptors, response (i.e. significant up- or down-regulation of 806 traditionally thought to localize to the cell surface, might acti- genes). Conversely, we found that angiotensin receptors vate a nuclear calcium signal to regulate hypertrophy. localize to the sarcolemma and induce sarcolemmal PLC1 ␣ In cardiac myocytes, Gq receptors, including 1-adrenergic, activity, but fail to promote HDAC5 nuclear export, while ␣ endothelin, and angiotensin receptors ( 1-AR, ET-R, and producing a transcriptional response that is mostly a subset AT-R, respectively), regulate vital signaling pathways control- ␣ of 1-AR–induced transcription. In summary, these results ling hypertrophy, cell survival, and inotropy that impact heart
link Gq-receptor compartmentalization in cardiac myocytes failure (HF) (3). Early studies in cell and animal models estab-
to unique hypertrophic transcription. They suggest a new lished the long-held convention that Gq signaling is maladap- model of excitation–transcription coupling in adult cardiac tive and exacerbates HF (4–6). However, several studies have
myocytes that accounts for differential Gq-receptor localiza- challenged the convention that Gq-receptor signaling univer-
tion and better explains distinct physiological functions of Gq sally worsens HF. Clinical studies indicate that antagonists tar- receptors. geting Gq receptors in human HF do not uniformly improve HF
2 The abbreviations used are: Gq receptor, Gq-coupled G-protein–coupled ␣ ␣ receptor; 1-AR, 1-adrenergic receptor; AMVM, adult mouse ventricular This work was supported by start-up funds from the University of Minnesota myocytes; AngII, angiotensin II; AT-R, angiotensin receptor type 1 and 2; (Minneapolis, MN) (to T. D. O.) and start-up funds from Pennsylvania State -AR, -adrenergic receptor; DAG, diacylglycerol; ET-R, endothelin recep-
University (University Park, PA) (to G. C. S.). The authors declare that they tor; HF, heart failure; HDAC, histone deacetylase; IP, inositol phosphate; IP2, have no conflicts of interest with the contents of this article. inositol diphosphate; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3 receptor; This article contains Tables S1–S4 and supporting information. NLS, nuclear localization sequence; OCT3, organic cation transporter 3; PE,  The RNA-Seq data set is available at ArrayExpress under accession number phenylephrine; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC 1, phos- E-MTAB-6770. pholipase C1; QDot, quantum dot; TAMRA, tetramethylrhodamine; 1 To whom correspondence should be addressed: Dept. of Integrative Biol- 2-APB, 2-aminoethoxydiphenyl borate; PC1 and PC2, principle component ogy and Physiology, University of Minnesota, 2231 6th St. SE, Cancer and 1 and 2, respectively; PHD, pleckstrin homology domain; ANOVA, analysis Cardiovascular Research Bldg., 3-141, Minneapolis, MN 55455. Tel.: 612- of variance; FPKM, fragments per kilobase of exon per million reads 625-6750; Fax: 612-301-1543; E-mail: [email protected]. mapped.
8734 J. Biol. Chem. (2018) 293(23) 8734–8749 © 2018 Dahl et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Compartmentalized myocyte Gq-receptor signaling
outcomes. Although AT-R antagonists are standard of care (7), these compartmentalized proximal signals induce differential ␣ 1-AR antagonists worsen HF (3). In mice, 8-fold cardiac myo- activation of nuclear hypertrophic signaling pathways to pro- cyte-specific overexpression of Gq induces cardiac myocyte cell duce unique hypertrophic transcriptomes. Finally, these data death and HF (6). However, Gq levels are increased only 2-fold suggest an entirely new model of excitation–transcription cou- in human HF (8, 9), which in mice produces no obvious pheno- pling that accounts for differential localization of Gq receptors ␣ type (6). Our laboratory previously demonstrated that 1-ARs and better explains the distinct physiologic function of Gq are cardioprotective, as defined by their ability to initiate adapt- receptors in adult cardiac myocytes. ive hypertrophy, survival signaling, and positive inotropy ␣ (reviewed in Ref. 3). The finding that 1-ARs are cardioprotec- Results ␣ tive agrees with the clinical data indicating that 1-antagonists ␣ 1-ARs localize to the nuclei and AT-Rs localize to the worsen HF. Collectively, these data suggest that Gq receptors sarcolemma in adult cardiac myocytes ␣ are functionally unique, some protective, like 1-ARs, and oth- ers maladaptive, like AT-Rs. Here, we sought to define the subcellular localization of ␣ How can these apparently divergent data on cardiac G -re- 1-ARs and AT-Rs in adult cardiac myocytes (Figs. 1 and 2). q ␣ ceptor function be reconciled? A potentially important clue is Previously, we demonstrated that endogenous 1-ARs localize ␣ to the nucleus in adult mouse ventricular myocytes (AMVM) our finding that cardiac 1-ARs primarily localize to and signal Downloaded from at the nucleus unlike other G receptors (reviewed in Refs. 3 and (11, 12, 15). However, reagents typically employed to localize q ␣ 10). Using fluorescent ligands and binding assays in fraction- receptors are generally unreliable, especially 1-AR subtype– ated adult cardiac myocytes, we previously demonstrated that specific antibodies, which lack specificity (16), or fluorescent ␣ ligands, which are no longer commercially available, but none- endogenous 1-ARs localize primarily to the nucleus. We also found that ␣ -ARs contain nuclear localization sequences theless had suboptimal binding kinetics (12). To overcome 1 ␣ http://www.jbc.org/ ␣ these shortcomings, we developed a novel 1-AR ligand (NLSs), and that whereas reconstitution of WT 1-ARs in ␣ -knockout cardiac myocytes restores ␣ -signaling, reconsti- composed of 2-piperazinyl-4-amino-6,7-dimethoxyquinazo- 1 1 ␣ ␣ line, the common pharmacophore of 1-AR antagonists such as tution of 1-NLS mutant receptors does not, demonstrating a ␣ terazosin and doxazosin, attached to a PEG linker with a termi- requirement for 1-nuclear localization (11). Further, we ␣ nal biotin moiety fused to a streptavidin-coated fluorescent showed that 1-ARs activate intranuclear signaling in adult car- diac myocytes based on our observations that the ␣ -agonist quantum dot (QDot) with an emission wavelength of 565 nm at University of Minnesota Libraries on June 8, 2018 1 ␣ phenylephrine activates protein kinase C in isolated nuclei and (Fig. 1A)(17). We validated the 1-QDot-565 by infecting WT ␣ ␣ that blockade of nuclear export inhibits ␣ -mediated signaling AMVM with a GFP-labeled 1A-AR ( 1A-GFP), incubating 1 ␣ for contractile function (11). Finally, we found that organic cat- infected myocytes with the 1-QDot, and co-localizing the GFP ion transporter 3 (OCT3) mediates rapid and specific cate- and QDot fluorescent signals as an indication of receptor bind- cholamine uptake in cardiac myocytes to facilitate ␣ -signaling, ing (Fig. 1B, nuclei indicated with white arrows). In AMVM 1 ␣ ␣ and others have demonstrated that OCT3 knockout mice phe- expressing 1A-GFP, the 1-QDot-565 fluorescent signal co- nocopy the small heart phenotype seen in ␣ -AR knockout ani- localized with the GFP fluorescent signal at the nucleus, and 1 ␣ mals (12, 13). In total, we identified an entirely novel model for pretreatment with the 1-AR antagonist prazosin diminished ␣ QDot fluorescence to nearly undetectable levels, demonstrat- nuclear 1-cardioprotective signaling in cardiac myocytes dis- tinct from the classical model of maladaptive G -receptor ing specificity (Fig. 1C). We employed the same method as q ␣ signaling. above in uninfected WT AMVM, and the 1-QDot-565 bound to endogenous ␣ -ARs at the nuclei in WT AMVM in the Based on these findings, we hypothesized that unique Gq- 1 receptor function is dictated by receptor localization. To test absence of prazosin but was blocked by pretreatment with pra- this hypothesis, we examined the relationship between subcel- zosin, replicating our previous findings (11, 12, 15)(Fig. 1, D ␣ lular compartmentalization of proximal G -receptor signaling and E; quantified in F). Further, the 1-QDot-565 bound q ␣ and the activation of hypertrophic signaling pathways in adult endogenous 1-ARs in nuclei isolated from WT AMVM (Fig. cardiac myocytes. The current model of Gq-receptor hyper- 1G), and this signal could be blocked by prazosin (Fig. 1H), ␣ trophic signaling is largely based on ET-R signaling and sug- indicating the presence of 1-ARs at the nuclear membrane, gests that sarcolemmal Gq receptors produce IP3-dependent again replicating our previous findings (11, 12, 15). Therefore, ␣ intranuclear calcium release, activation of calmodulin kinase, the 1-QDot-565 fluorescent ligand identified endogenous ␣ phosphorylation and nuclear export of histone deacetylases 1-ARs at the nucleus in AMVM and outperformed prior fluo- ␣ (HDACs), and derepression of transcription (2, 14). This model rescent 1-ligands by improving kinetics (30 min) at lower con- is notable for explaining how cytosolic calcium transients centrations (25 nM)(12). ␣ required for contraction are segregated from IP3-dependent Similar to 1-ARs, the lack of validated antibodies for AT-Rs nuclear calcium signals required for hypertrophic signaling. led us to employ another fluorescent ligand to define the local- However, it is not entirely clear how this model might reconcile ization of endogenous AT-Rs in AMVM. In this case, we used the data suggesting that Gq receptors induce unique physiology angiotensin II (AngII), the endogenous ligand of AT-Rs, labeled ␣ or our model of nuclear 1-cardioprotective signaling. Here, we with the red fluorophore tetramethylrhodamine (TAMRA). ␣ report for the first time that in adult cardiac myocytes, 1-ARs We incubated AMVM with AngII-TAMRA in the absence or and AT-Rs localize to and activate PLC1 in unique subcellular presence of unlabeled AngII to demonstrate the specificity of compartments. Further, we demonstrate for the first time that AngII-TAMRA (Fig. 2, A–C). In AMVM, AngII-TAMRA pro-
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duced a distinct localization in confocal sections from the myo- ity (Fig. 2C). To clarify receptor localization, slices from the top cyte surface (Fig. 2B, left), whereas AngII-TAMRA showed and middle of the cell were deconvolved, and 3D surface plots much less specific binding in confocal sections from the middle were created that identified AngII-TAMRA signal predomi- of the myocyte (Fig. 2A, left). Pretreatment with unlabeled nantly at the myocyte surface (Fig. 2, A versus B, center and AngII abolished the AngII-TAMRA signal, indicating specific- right; arrows indicate localization). Finally, a volume rendering Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
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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 M, 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 (ϫ60 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 http://www.jbc.org/ demonstrate receptor density. Scale bar,10m. C, AngII-TAMRA is specific for AT-Rs in AMVM. WT AMVM were pretreated with an excess of unlabeled AngII (50 M, 2 min, 37 °C) and then incubated with AngII-TAMRA (1 M, 5 min, 37 °C). Signal was undetectable, indicating specificity. Scale bar,10m. D, volume plot of AngII-TAMRA–treated AMVM showing surface localization. 3D volume plot was created using FIJI.
␣ was created from a confocal stack of a myocyte labeled with indicate that endogenous 1-ARs and AT-Rs localize to differ- AngII-TAMRA, demonstrating that the majority of the AngII- ent subcellular compartments in adult cardiac myocytes. at University of Minnesota Libraries on June 8, 2018 TAMRA signal localized to the myocyte surface (Fig. 2D).  Previously, a small population of AT-Rs was identified at the PLC 1 and its substrate PIP2 localize to the nuclei in adult nucleus in AMVM based on immunochemical detection and cardiac myocytes  functional assays, although the overall physiologic significance Proximal Gq-receptor signaling involves activation of PLC 1 of this population of AT-Rs is still unclear (18). Further, this and cleavage of PIP2 into IP3 and DAG. Whereas it is generally  population of nuclear AT-Rs, which is activated by intracrine accepted that Gq receptors activate PLC 1 to hydrolyze PIP2 at AngII, represents only a fraction of total AT-Rs (18). Our the sarcolemma (20), it is not clear whether this occurs at the results with AngII-TAMRA indicate that the majority of nucleus in AMVM. Although Gq receptors localize to the ␣ ligand-accessible AT-Rs exist at the sarcolemma, and failure to nucleus in AMVM, including 1-ARs (Fig. 1)(11, 12, 15) as well detect nuclear AT-Rs here is probably because neither AngII as small populations of AT-Rs (18) and ET-Rs (21), it is not  nor AT-R antagonists readily cross the sarcolemma (19). In certain that either PLC 1orPIP2 localizes to the nucleus (22). summary, AngII-TAMRA identified endogenous AT-Rs on the In other cells, PIP2 hydrolysis is observed in the nuclear matrix ␣ sarcolemma, and combined with results from 1-QDot-565 but is reported to be independent of Gq-receptor signaling (23). ␣ ␣ ␦ identification of endogenous 1-ARs at the nucleus, the data However, nuclear 1-ARs activate intranuclear PKC in nuclei
␣ ␣ ␣ Figure 1. 1-ARs localize to the nuclei in adult cardiac myocytes. A, synthesis of 1-AR-QDot-565. The structural backbone of the piperazole class of 1-AR antagonists, 2-piperazinyl-4-amino-6,7-dimethoxyquinazoline, was attached to biotinylated PEG (N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylami- ␣ ␣ no)ethane). The resulting 1-antagonist-PEG–biotinylated molecule was then coupled to streptavidin-conjugated QDot-565 to make 1-QDot-565. B, valida- ␣ ␣ ␣ tion of 1-QDot-565. To define the specificity of the 1-QDot-565, WT AMVM were infected with an adenovirus expressing 1A-GFP and then treated with 25 ␣ ␣ nM 1-AR-QDot-565. Cells were fixed and imaged by confocal microscopy. A representative myocyte is shown, and the channels are as follows: 1A-GFP signal ␣ (green), QDot-565 signal (yellow), and co-localization were determined in post-processing (white). Scale bar,10 m. C, specificity of 1-QDot-565. Myocytes ␣ ␣ ␣ expressing 1A-GFP were pretreated with an excess of unlabeled prazosin (5 M) and then treated with 25 nM 1-AR-QDot-565. Shown are 1A-GFP signal ␣ (green), QDot-565 signal (yellow), and colocalization determined in post-processing (white). Scale bar,10 m. D, 1-ARs localize to the nuclei in AMVM. ␣ ϫ 1-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,10m. E, specificity ␣ ␣ of 1-QDot-565 in WT AMVM. To demonstrate the specificity of the 1-QDot-565, WT AMVM were pretreated with an excess of prazosin (5 M, 30 min, 37 °C) and ␣ ␣ then incubated with 1-AR-QDot-565 (25 nM, 30 min, 37 °C). One representative myocyte is shown. Scale bar,10 m. F, quantification of 1-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 Ϯ S.E. (error bars). Data were analyzed by one-way ANOVA, and p Ͻ 0.05 was considered ␣ ϭ ␣ ϩ ϭ ϭ significant: 1-QDot 565 (n 12 myocytes from four hearts); 1-QDot 565 prazosin (n 3 myocytes from two hearts); QDot 565 alone (n 4 myocytes from ␣ ␣ one heart). Scale bar,10 m. G, 1-QDot-565 labels nuclei isolated from AMVM. Nuclei were isolated from WT AMVM and incubated with 1-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,10m. H, ␣ ␣ specificity of 1-QDot-565 in nuclei isolated from AMVM. To demonstrate the specificity of the 1-QDot-565, nuclei from WT AMVM were pretreated with an ␣ excess of prazosin (5 M, 15 min, 37 °C) and then incubated with 1-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,10m.
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Figure 3. PLC1 localizes to the nuclear membrane in adult cardiac myocytes. A, visualization of siRNA-mediated PLC1 protein knockdown. N38 cells (embryonic mouse hypothalamic cells) were transfected with 80 pmol of either PLC1 siRNA or scrambled siRNA using Lipofectamine RNAiMAX for 72 h at 37 °C. Cells were fixed and stained with a primary antibody against PLC1. Secondary antibody was conjugated to Alexa Fluor 594. Cells were imaged using confocal microscopy (ϫ20). Scale bar,10m. B, quantification of siRNA-mediated PLC1 protein knockdown. Fluorescence intensity was measured using FIJI (n ϭ 2 cultures; four optical areas were measured for scramble, and eight optical areas were measured for PLC1 siRNA). Data are represented as mean Ϯ S.E. (error bars). Data were analyzed by paired student’s t test, and p Ͻ 0.05 was considered significant. C, siRNA-mediated PLC1 mRNA knockdown. N38 cells were transfected as in A. After 72 h, RNA was harvested using an RNAEasy kit, and PLC1 mRNA levels were measured by RT-PCR. Results of densitometry analysis of PLC1 mRNA levels are shown in D. D, quantification of siRNA-mediated PLC1 mRNA knockdown. The ratio of glyceraldehyde-3-phosphate dehydrogenase to PLC1 was quantified using densitometry (n ϭ 3 cultures). Data are represented as mean Ϯ S.E. E, endogenous PLC1 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 PLC1. Secondary antibody was conjugated to Alexa Fluor 488. Myocytes were imaged by confocal microscopy (ϫ60 oil immersion). Arrows, nuclear membrane. Images were cropped (white lines indicate cropped area) and horizontally aligned. Two representative images are shown. Scale bar,10m.
isolated from AMVM (11), suggesting production of DAG from tified in D), and knockdown of PLC protein was visualized by a yet to be defined nuclear phosphatidylinositol species. Fur- decrease in staining of the PLC1 antibody (Fig. 3A; quantified ther, both AT-Rs and ET-Rs induce intranuclear signaling de- in B). Finally, we stained WT AMVM with the PLC1 antibody, pendent on the production of inositol phosphates in AMVM and our results indicate that PLC1 localizes to the sarco- (14, 18). lemma, T-tubules, and nuclear envelope (Fig. 3E, nuclei indi-
Based on prior demonstrations of Gq-receptor localization cated with white arrows).  and signaling at the nucleus, we sought to clarify whether PIP2, the substrate for PLC 1, localizes to the sarcolemma in  PLC 1 is localized to the nucleus in adult cardiac myocytes. AMVM (22, 26, 27), yet identifying a population of nuclear PIP2  First, we sought to validate potential PLC 1 antibodies. Prefer- has been elusive because either 1) PIP2 is not present at the  ably, we would employ AMVM from PLC 1 knockout mice, nucleus or 2) the methods used to detect PIP2 have been insuf- but these mice exhibit spontaneous seizures and high mortality ficient. To address this conundrum, we isolated AMVM nuclei around 3 weeks of age (24). Alternatively, we attempted to use using differential centrifugation and analyzed samples by MS to  siRNA technology to knock down PLC 1 in AMVM but were determine whether PIP2 is present in nuclear membranes in  unable to achieve significant PLC 1 mRNA knockdown by AMVM. To detect the presence of PIP2 species, we ran two 40 h, probably due to low turnover of PLC1 in cultured blanks and two AMVM nuclear samples, adding one of each Ј Ј AMVM. Subsequently, we attempted to validate potential with commercially available 36:2 PIP2, PIP2(4 ,5 )(18:1(9Z)/18: PLC1 antibodies using siRNA technology in the N38 embry- 1(9Z)). Using electrospray ionization-MS scan with precursor onic mouse hypothalamic cell line due to PLC1 enrichment in ion m/z 281 (carboxylated anion of oleic acid; C18:1), we were  the brain (25). To assess PLC 1 knockdown, N38 cells were able identify PIP2 36:2 species in all of the samples except for the transfected with either PLC1 siRNA or scramble siRNA for blank (Fig. 4A, left). Spectra of the peak showing a parent ion 72 h. We observed knockdown of PLC1 mRNA (Fig. 3C; quan- m/z 1021 (Fig. 4A, right) confirmed the identity of the commer-
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Ј Ј 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 Ј Ј 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.
␣  cially available 36:2 phosphatidylinositol 4,5-bisphosphate in AT-Rs, but not 1-ARs, activate PLC 1 at the sarcolemma in
the PIP2-added blank sample, with ion fragments at m/z 259, adult cardiac myocytes 281, 339, and 419 corresponding to the inositol phosphate (IP), Based on the observed distinct subcellular compartmental- the oleic acid backbone, inositol diphosphate (IP ), and inositol ␣ 2 ization of 1-ARs and AT-Rs (Figs. 1 and 2), we reasoned that triphosphate (IP3) components of the fragmented 36:2 PIP2. AT-Rs would activate proximal signaling at the sarcolemma Similar to previous PIP analyses (28), we used negative ion ␣ 2 and 1-ARs at the nuclei. To test this, we measured the com- MS/MS to analyze the product ions of m/z 1045 (38:4 PIP ), the  2 partmentalization of Gq-receptor activation of PLC 1 with the most abundant PIP2 species in the nuclear fraction sample PLC1 activity sensor GFP-C1-PLC␦-PH (GFP-PHD; Fig. 5A). without the PIP2 addition (Fig. 4B). Ions at m/z 259, 283, 303, GFP-PHD is composed of GFP fused to the N terminus of the 339, and 419 correspond to IP, carboxylate anion fatty acyl pleckstrin homology domain of PLC␦1, which preferentially chains of stearic acid, the arachidonic acid backbone, IP2, and binds PIP2 over other membrane phosphatidylinositols in vitro IP3, all portions of the fragmented 38:4 PIP2 spectrum. Our (29). In general, GFP-PHD associates with PIP2 in membranes in  results indicate that PIP2 localizes to membranes within the the basal state, and upon Gq-receptor stimulation, PLC 1 hydro- nuclei of AMVM, which to our knowledge is the first such dem- lyzes PIP2, and as PIP2 is depleted, GFP-PHD dissociates from the onstration. In total, our findings reveal that PLC1 and its sub- membrane, as illustrated in Fig. 5B. In AMVM expressing GFP-
strate, PIP2, are found in the same subcellular compartments as PHD, the probe localized to the sarcolemma and T-tubules in the ␣ both the 1-AR (nuclear) and AT-R (sarcolemma), suggesting basal state, in agreement with previous reports (22). More impor- ␣ the potential for compartmentalized Gq-receptor signaling in tantly, the 1-agonist phenylephrine (PE), in the absence or pres- adult cardiac myocytes. ence of prazosin, produced no change in the localization of GFP-
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PHD compared with vehicle (Fig. 5C; quantified in D). Conversely, (Fig. 5E; quantified in F). These results demonstrate that AT-Rs, ␣  AngII induced a marked dissociation of GFP-PHD from the mem- but not 1-ARs, activate PLC 1 at the sarcolemma in adult cardiac brane compared with vehicle, which was blocked by the nonselec- myocytes, consistent with the subcellular compartmentalization tive AT-R antagonist losartan, indicating a receptor-specific effect of each receptor. Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
8740 J. Biol. Chem. (2018) 293(23) 8734–8749 Compartmentalized myocyte Gq-receptor signaling ␣  1-ARs, but not AT-Rs, activate PLC 1 at the nuclear envelope icant, export of HDAC5 at 30 min (Fig. 7A; quantified in B). By in adult cardiac myocytes 1 h, PE significantly induced HDAC5 nuclear export, whereas Interestingly, GFP-PHD was not detected at the nuclear AngII did not (Fig. 7C; quantified in D), consistent with previ- membrane in the basal state (Fig. 5). We suggest there are two ous reports for PE (30). To determine whether PE-induced HDAC5 nuclear export was IP -dependent, AMVM expressing explanations for this observation: 1) PIP2 is not present in the 3 HDAC5-GFP were pretreated with the IP R inhibitor 2-amino- nuclear membrane, despite our identification of PIP2 in nuclear 3 membranes (Fig. 4), or 2) GFP-PHD, which lacks an NLS, is ethoxydiphenyl borate (2-APB). Pretreatment with 2-APB abolished the PE-mediated HDAC5 nuclear export (Fig. 7E; unable to target the nucleus to bind nuclear PIP2. To clarify this and additionally determine whether G receptor–mediated quantified in F). Taken together, these results indicate that q ␣ activation of PLC1 possibly occurs at the nucleus, we inserted 1-ARs, but not AT-Rs, activate IP3-dependent HDAC5 an NLS sequence at the N terminus of GFP-PHD to create NLS- nuclear export in AMVM. GFP-PHD (Fig. 6A ). Insertion of an NLS promoted nuclear ␣ 1-ARs and AT-Rs induce unique transcriptomes in adult localization of GFP-PHD (Fig. 6, C and E), suggesting that PIP2 is found in the nucleus, in agreement with detection of nuclear cardiac myocytes ␣ PIP2 by MS (Fig. 4). NLS-GFP-PHD associates with PIP2 in the Physiologically, 1-ARs and AT-Rs have diametrically op-
␣ Downloaded from nuclear membrane in the basal state, and upon nuclear Gq- posed effects on the heart. 1-ARs induce physiologic hypertro-  receptor stimulation, PLC 1 hydrolyzes PIP2, and as PIP2 is phy, survival signaling, and positive inotropy and are not asso- depleted, NLS-GFP-PHD dissociates from the nuclear mem- ciated with fibrosis (3). AT-Rs induce pathologic hypertrophy, brane and moves into the nucleoplasm, as illustrated in Fig. 6B. myocyte cell death, and negative inotropy and are pro-fibrotic Using the same experimental conditions as our experiments (31, 32). We hypothesized that these differences in the physio- ␣ ␣ with GPF-PHD, the 1-agonist PE induced a marked dissocia- logic function of cardiac 1-ARs and AT-Rs would be revealed http://www.jbc.org/ tion of NLS-GFP-PHD from the nuclear membrane, which was in their transcriptomes and reflective of their distinct subcellu- ␣ blocked by prazosin (Fig. 6C; quantified in D). Conversely, lar localization. To evaluate 1-AR and AT-R transcriptomes, AngII, in the absence or presence of losartan, produced little we treated mice with vehicle, PE (30 mg/kg/day), or AngII (0.5 change in the localization of NLS-GFP-PHD (Fig. 6E; quanti- mg/kg/day) continually for 3 days using osmotic minipumps, at ␣ fied in F). These results demonstrate that 1-ARs, but not which point we isolated cardiac myocytes and performed AT-Rs, primarily activate PLC1 at the nuclear membrane in RNA-Seq. The doses of PE and AngII that we used are known to at University of Minnesota Libraries on June 8, 2018 AMVM. The combined results from experiments using GFP- induce hypertrophy without a concomitant increase in blood ␣ PHD and NLS-GFP-PHD indicate that 1-AR– and AT-R– pressure (33–36). Consistent with our HDAC5 results and mediated proximal signaling is confined to distinct subcellular with the fact that HDAC5 activation relieves transcriptional ␣ compartments, consistent with receptor localization. repression, 1-ARs induced a larger transcriptional response, with a total of 806 genes changed (increased or decreased) ␣ 1-ARs, but not AT-Rs, induce IP3-dependent nuclear export of 1.7-fold versus vehicle, whereas AT-Rs induced a much HDAC5 in adult cardiac myocytes smaller response, with only 173 genes changed 1.7-fold ver-
Gq receptors are thought to induce hypertrophy through a sus vehicle. 1.7-fold expression over vehicle was used as the mechanism known as excitation–transcription coupling (2). threshold because adult cardiac myocytes are postmitotic
Conventionally, sarcolemmal Gq receptor–mediated produc- and generally do not induce large transcriptional responses. ␣ tion of IP3 elicits intranuclear calcium release from the nuclear Interestingly, between 1-ARs and AT-Rs, 155 genes were ␣ envelope, activation of calmodulin kinase type II, and phosphor- changed by both agonists, indicating that 1-ARs induced ylation and nuclear export of HDAC5 (2). Nuclear compart- 651 unique genes, whereas AT-Rs induced only 18 unique mentalization of IP3-dependent calcium release allows myo- genes (Fig. 8A). These results suggest that the AT-R tran- ␣ cytes to distinguish calcium required for contraction from scriptome is largely a subset of the 1-AR transcriptome in calcium required for transcriptional signaling. Here, we exam- cardiac myocytes. ined whether differentially localized Gq receptors would have To parse the RNA-Seq results further, we initially attempted the same effect on HDAC5 export. In AMVM expressing to utilize the Ingenuity Pathway Analysis (IPA) software, but HDAC5-GFP, PE but not AngII induced a moderate, but signif- the majority of the database is derived from oncogenic studies
␣  Figure 5. AT-Rs, but not 1-ARS, activate PLC 1 at the sarcolemma in adult cardiac myocytes. A, schematic representation of GFP-PHD. GFP-PHD consists ␦ ␦ of the pleckstrin homology domain of phospholipase C attached to the C terminus of GFP. The PH domain of PLC has high affinity for PIP2, and in the basal  state, GFP-PHD binds to membrane PIP2, and cleavage of PIP2 by PLC 1 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 PLC 1(Activated), PIP2 is cleaved, ␣  and the probe moves into the cytoplasm. C and E, 1-ARs (C) do not activate PLC 1 at the sarcolemma, but AT-Rs do (E). WT AMVM expressing GFP-PHD were treated with vehicle (left)orPE(20M, 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 M, 30-min pretreatment, 37 °C, middle and right panels, respec- tively). 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 m. D and F, quantification of sarcolemmal PLC 1 activity downstream of 1-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 2, and p Ͻ 0.05 was considered significant. Data are represented as mean Ϯ S.E. (error bars): vehicle (n ϭ 30 myocytes from seven hearts); PE (n ϭ 24 myocytes from four hearts); AngII (n ϭ 29 myocytes from four hearts). ns, not significant.
J. Biol. Chem. (2018) 293(23) 8734–8749 8741 Compartmentalized myocyte Gq-receptor signaling and lacks cardiac myocyte specific pathways. Thus, we derived two into gene ontologies corresponding to Gq-receptor biology: our own analysis and sorted genes that were regulated by hypertrophy, survival signaling, inotropy, and fibrosis (Fig. 8B ␣ ␣ 1-ARs alone, AT-Rs alone, or were in common between the and Tables S1–S4). 1-ARs most robustly altered genes in all Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
8742 J. Biol. Chem. (2018) 293(23) 8734–8749 Compartmentalized myocyte Gq-receptor signaling
categories as compared with common genes and AT-R–only transcription (Fig. 9A). Whereas the current model of ␣ genes. Whereas it is surprising that 1-ARs altered more excitation–transcription coupling suggests that Gq receptors fibrotic genes than AT-Rs, these genes are not classically asso- induce IP3 production at the sarcolemma leading to activation ciated with alterations in the extracellular matrix leading to of IP3-dependent calcium release at the nucleus to induce fibrosis (Table S4). Finally, principle component analysis was HDAC5 export and promote gene transcription, it fails to performed to determine the degree of difference between vehi- explain how Gq receptors might produce unique physiological cle-, AngII-, and PE-treated samples (Fig. 8C). Principle com- function in cardiac myocytes. ponent 1 (PC1) accounted for 66% of the variance and aligned Both ET-Rs and insulin-like growth factor receptors con- ␣ with the 1-AR transcriptomes, whereas PC2 accounted for form to the traditional excitation–transcription model (2, 14, 22% of the variance and aligned with the AT-R transcriptome. ␣ 37). Our data indicate that 1-ARs might support this model as The AngII-treated samples also closely grouped with the vehi- well, but interestingly, AT-Rs do not. Although we observed cle-treated samples, indicating that AT-Rs do not induce a AT-R–mediated activation of PLC1, we failed to detect highly distinct transcriptome from control, consistent with our AT-R–mediated nuclear export of HDAC5 and found a much gene ontology results (Fig. 8, A and B) The top 25 genes deter- smaller transcriptional response. One interpretation of this mining PC1 and PC2 are presented in Fig. 8D. Taken together, result is that close proximity to the nucleus is required for G ␣ q 1-ARs robustly activate transcription in adult cardiac myo- Downloaded from receptor–mediated activation of IP3-dependent hypertrophic cytes, whereas AT-Rs minimally activate transcription. These ␣ signaling. In support of this interpretation, 1-ARs localize to results identify distinct differences in the transcriptomes the inner nuclear membrane (11), and ET-Rs and insulin-like ␣ induced by 1-ARs and AT-Rs that align with their distinct growth factor receptors localize to the bottom of T-tubules in subcellular localization and activation of proximal signaling to close apposition to the nucleus in adult cardiac myocytes (37, produce differential activation of nuclear hypertrophic signal- 38). Further, the failure of AT-Rs to induce nuclear export of http://www.jbc.org/ ing pathways. HDAC5 suggests that AT-R–mediated activation of PLC1at
Discussion the sarcolemma either fails to generate enough IP3 to reach the nucleus or that IP is degraded before it reaches the nucleus. Here, we identified an entirely novel mechanistic explana- 3 The potential degradation of IP3 before reaching the nucleus tion for unique Gq-receptor function in adult cardiac myocytes
might be analogous to the compartmentalization of cAMP sig- at University of Minnesota Libraries on June 8, 2018 predicated upon subcellular compartmentalization of proximal naling in cardiac myocytes (39). Gq-receptor signaling and propose a novel model of excitation– ␣ Here, we propose a new model of excitation–transcription transcription coupling. We found that 1-ARs localize to the nucleus and induce intranuclear activation of PLC1, stimulate coupling that is based on compartmentalization of Gq receptors that explains distinct physiologic function of Gq receptors in IP3-dependent nuclear export of HDAC5, and activate a robust and unique transcriptome associated with hypertrophic, sur- adult cardiac myocytes. Our model suggests that Gq receptor– vival, inotropic, and (anti-)fibrotic gene programs. Conversely, induced IP3 production is compartmentalized and that IP3 pro- we observed that AT-Rs primarily localize to and activate duced inside the nucleus (or possibly in close proximity to the PLC1 at the sarcolemma but have little effect on nuclear nucleus) induces HDAC5 export to promote gene transcrip- export of HDAC5 and induce a small transcriptome that is a tion, whereas IP3 produced at a distance from the nucleus (at ␣ the sarcolemma) has a different and smaller effect on transcrip- subset of the 1-transcriptome. More importantly, these find- tional regulation (Fig. 9B). In summary, we suggest that G - ings are consistent with our hypothesis that Gq-receptor local- q ization dictates function by showing that compartmentaliza- receptor compartmentalization has a large influence on the transcriptomes induced by differentially localized G receptors, tion of proximal Gq-signaling is correlated with phenotypic q outcome in adult cardiac myocytes. illustrating the fundamental physiologic importance of Gq-re-
The excitation–transcription model of Gq receptor– ceptor compartmentalization. ␣ mediated hypertrophic signaling in adult cardiac myocytes With regard to our model of nuclear 1-induced HDAC5 proposes that sarcolemmal Gq receptors induce IP3 production export, previous work indicated that HDAC5 export down- ␣ and IP3-sensitive intranuclear calcium release from perinuclear stream of 1-ARs occurs mainly through activation of protein calcium stores to activate calmodulin kinase, phosphorylate kinase D, and our results do not exclude activation of this sig- and induce nuclear export of HDAC5, and thereby activate naling cascade (40). Nevertheless, in agreement with our
␣  Figure 6. 1-ARs and, to a lesser extent, AT-Rs activate PLC 1 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 PLC 1(Activated), PIP2 is cleaved, and the probe ␣  moves into the nucleoplasm. C and E, 1-ARs (C) activate PLC 1 at the nuclear membrane, but AT-Rs do not (E). WT AMVM expressing NLS-GFP-PHD were treated with either vehicle (left panels)orPE(20M, 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 M, 30-min pretreatment, 37 °C; middle and right panels,  ␣ respectively). Arrows indicate nuclear membrane activation of PLC 1by 1-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,10m. D and F, quantification of nuclear  ␣ PLC 1 activity downstream of 1-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 2, and p Ͻ 0.05 was considered significant. Data are represented as mean Ϯ S.E. (error bars): vehicle (n ϭ 30 myocytes from seven hearts); PE (n ϭ 14 myocytes from four hearts); AngII (n ϭ 16 myocytes from four hearts). ns, not significant.
J. Biol. Chem. (2018) 293(23) 8734–8749 8743 Compartmentalized myocyte Gq-receptor signaling Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
␣ ␣ Figure 7. 1-ARS induce HDAC5 export, whereas AT-Rs do not. A and C, 1-ARs activate HDAC5 nuclear export, whereas AT-Rs do not. WT AMVM expressing HDAC5-GFP were treated with either vehicle (left panels), 20 M PE (middle panels; insets of zoomed-in nuclei), or 100 nM AngII (right panels) for either 30 min (A) ␣ or1h(C)at37°C.Scale bar,10 m. B and D, quantification of HDAC5 export induced by 1-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)and1h(D). Data are represented as mean Ϯ S.E. (error bars) Data were analyzed by one-way ANOVA, and p Ͻ 0.05 was considered significant: vehicle (n ϭ 12 myocytes from six ϭ ϭ 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 M, 30 min at 37 °C) before treatment with PE (1 h at 37 °C; middle). Scale bar,10m. F, quantification Ϯ 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 Ͻ 0.05 was considered significant: vehicle (n ϭ 12 myocytes from six hearts); 2-APB (n ϭ 12 myocytes from six hearts); PE (n ϭ 12 myocytes from six hearts); PE ϩ 2-ABP (n ϭ 12 myocytes from six hearts).
␣ results, Luo et al. (41) demonstrated that 1-ARs activate IP3- The consensus view of cardiac Gq-receptor function has been dependent nuclear calcium transients in cardiac myocytes, con- that Gq receptors mediate pathologic remodeling, promoting ␣ sistent with our finding that 1-ARs induce IP3-sensitive maladaptive hypertrophy, myocyte cell death, and negative ino- HDAC nuclear export. At this time, the discrepancy between tropic responses (4). However, in clinical trials of hypertension these findings is not entirely clear. (Antihypertensive and Lipid-Lowering Treatment to Prevent
8744 J. Biol. Chem. (2018) 293(23) 8734–8749 Compartmentalized myocyte Gq-receptor signaling Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
␣ ␣ Figure 8. 1-ARs robustly activate transcriptional responses in adult cardiac myocytes whereas AT-ARs do not. A, Venn diagram of 1-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 1.7ϫ absolute -fold change and false discovery rate–corrected p Ͻ 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 1-AR– and AT-R–induced transcriptomes. Gq 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-B, 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 1-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.
␣ Heart Attack Trial (ALLHAT)) and HF (Vasodilator Heart Fail- of 1-ARs stands in stark contrast to the consensus view of ␣ ure Trial (V-HeFT)), 1-AR antagonists worsened outcomes maladaptive Gq-receptor function. Here, our data suggest that ␣ (42, 43). Further, our studies indicate that 1-ARs are cardio- compartmentalization of Gq receptors could explain differ- protective, promoting adaptive or physiologic hypertrophy, ences in Gq-receptor function. We demonstrated that although ␣  prevention of cell death, and positive inotropic effects identify- both 1-ARs and AT-Rs activate PLC 1, they do so in different ␣ ing a mechanistic basis for the negative results of 1-AR antag- subcellular compartments, which has a profound effect on acti- onists in clinical trials (3). In short, the cardioprotective nature vation of intranuclear hypertrophic signaling and transcrip-
J. Biol. Chem. (2018) 293(23) 8734–8749 8745 Compartmentalized myocyte Gq-receptor signaling Downloaded from http://www.jbc.org/ at University of Minnesota Libraries on June 8, 2018
Figure 9. Novel model of excitation–transcription coupling in adult cardiac myocytes. A, conventional model of excitation–transcription coupling in adult ␣  cardiac myocytes. All G q-receptors localize to the sarcolemma in adult cardiac myocytes. Upon ligand binding, sarcolemmal PLC 1 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 IP3Ronthe 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. 1-ARs (nuclear) and AT-Rs (sarcolemmal) are differentially localized in adult cardiac myocytes ␣  ␣ (Figs. 1 and 2). Upon ligand binding to either 1-ARs or AT-Rs, PLC 1 is activated either at the nucleus ( 1-AR; Fig. 6) or at the sarcolemma (AT-R, Fig. 5).  ␣  AT-R–induced PLC 1 activation at the sarcolemma fails to induce HDAC5 nuclear export, whereas 1-AR–induced PLC 1 activation at the nucleus induces ␣ ␣ HDAC5 nuclear export. Furthermore, 1-AR–induced HDAC5 nuclear export is IP3-dependent (blocked by 2-APB) (Fig. 7). Consistent with 1-induced, IP3-de- ␣ pendent HDAC5 nuclear export, 1-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 1-AR–induced transcriptional responses (potentially suggesting a different non-HDAC dependent mechanism of tran- scription) (Fig. 8).
tional activation. Therefore, we suggest that nuclear Gq-recep- AT-Rs localize to the nucleus as well (18, 21), although their ␣ tor signaling, typified by 1-ARs, is cardioprotective. Aside physiologic significance is unclear. Conversely, we observed ␣ from 1-ARs, a small population of functional ET-Rs and AT-Rs primarily at the sarcolemma, but not in close proximity
8746 J. Biol. Chem. (2018) 293(23) 8734–8749 Compartmentalized myocyte Gq-receptor signaling
ϫ 10 ϫ 8 to the nucleus, which might suggest that Gq-receptor signaling 5.9 10 ); GFP-PHD, 100 (titer: 2.4 10 ); NLS-GFP-PHD at the sarcolemma is pathologic, which is supported by studies (titer: 1.4 ϫ 1010); and HDAC5-GFP (titer: 6.1 ϫ 1015). ␣ ␣ with AT-Rs (31, 32). In support of this concept, adenoviral Synthesis of 1-QDot-565—The synthesis of the 1-QDot- mediated expression of the PLC1b at the sarcolemma induces 565 was performed as described (17) with a few modifications contractile dysfunction (44). In summary, our hypothetical that are described in the supporting information. ␣  model of compartmentalized Gq-receptor signaling, where Localization of 1-ARs, AT-Rs, and PLC 1 in AMVM— ␣ nuclear Gq-receptor signaling is cardioprotective, suggests a Methods used to define the localization of 1-ARs, AT-Rs, and  more nuanced view of Gq-receptor function in cardiac PLC 1 are described in the supporting information. myocytes. Identification of nuclear PIP2—PIP2 extraction was carried The concept of GPCR compartmentalization in cardiac myo- out as described (52) with some modifications. The electros- cytes is not without precedence. In co-cultures of sympathetic pray mass spectrum of each sample was analyzed on a hybrid  ganglionic neurons and neonatal rat cardiac myocytes, 1-ARs quadrupole triple ion trap mass spectrometer (Triple TOF localize to regions of axonal contact rich in SAP97, AKAP97, 5600, AB Sciex Instrument). All scans were performed in neg-  catenins, and cadherins, whereas 2-ARs are excluded from ative ionization mode and a mass-to-charge (m/z) range from  Ј Ј these domains (45). In adult cardiac myocytes, 1-ARs are dis- 50 to 1200. PIP2(4 ,5 )(18:1(9Z)/18:1(9Z)) was obtained from  tributed over the entire sarcolemma, whereas 2-ARs are Avanti Polar Lipids. Downloaded from restricted deep within T-tubules (46). Finally, platelet-derived Quantification and statistical analysis growth factor receptors, which also signal through Gs, localize to caveolae in cardiac myocytes and do not induce an inotropic The methods for the analysis of phospholipase C1 activity, response (47). These examples demonstrate that cardiac myo- HDAC5 nuclear export, and hypertrophic transcriptional pro- cytes compartmentalize G -mediated GPCR signaling, analo-
s files adenoviruses are described in the supporting information. http://www.jbc.org/ gous to our findings with Gq receptors. In conclusion, our find- Details and methods used for statistical analysis can be found in ings support a model of compartmentalized Gq-receptor the supporting information. signaling in adult cardiac myocytes and suggest a revision of the classic model of excitation–transcription coupling. Our new Author contributions—E. F. D., S. C. W., G. C. S., and T. D. O. concep- model, largely based on our identification of cardioprotective tualization; E. F. D., C. L. H., B. A. H., and T. D. O. data curation;
␣ at University of Minnesota Libraries on June 8, 2018 nuclear 1-AR signaling, provides a plausible mechanistic basis E. F. D., B. A. H., and T. D. O. formal analysis; E. F. D. validation; to explain the unique function of cardiac Gq receptors. Addi- E. F. D., S. C. W., C. L. H., B. A. H., and T. D. O. investigation; E. F. D. tionally, this model suggests a reexamination of the classic par- visualization; E. F. D., S. C. W., C. L. H., B. A. H., and T. D. O. method- ology; E. F. D. and T. D. O. writing-original draft; S. C. W., C. L. H., and adigm of maladaptive Gq signaling in cardiac myocytes in favor T. D. O. writing-review and editing; C. L. H. and G. C. S. project admin- of a more nuanced view of compartmentalized Gq-receptor signaling. istration; G. C. S. resources; T. D. O. and G. C. S. funding acquisition.
Experimental procedures Acknowledgments—We thank Dr. Timothy McKinsey (University of Experimental models: Mice Colorado) for the generous gift of the HDAC5 virus, Dr. Alessandro Bartolomucci (University of Minnesota) for the generous gift of N38 In this study, male and female C57BL/6J mice (10–15 weeks cells, Christy Long for her effort in making the GFP-PHD virus, Dr. Jop of age) were used for primary adult cardiac myocytes. Male Van Berlo for assistance in conceptualizing this project, and the Uni- FVB/NJ mice (10–11 weeks of age) were used for infusion of versity of Minnesota Genomics Core. agonist to measure Gq receptor–induced hypertrophic tran- scriptional responses. All animals were sourced from Jackson Laboratories. The use of all animals in this study conformed to References the United States Public Health Service Guide for Care and Use 1. Kamato, D., Mitra, P., Davis, F., Osman, N., Chaplin, R., Cabot, P. J., Afroz, of Laboratory Animals and was approved by the University of R., Thomas, W., Zheng, W., Kaur, H., Brimble, M., and Little, P. J. (2017) Gaq proteins: molecular pharmacology and therapeutic potential. Cell. Minnesota institutional animal care and use committee. Mol. Life Sci. 74, 1379–1390 CrossRef Medline 2. Wu, X., Zhang, T., Bossuyt, J., Li, X., McKinsey, T. A., Dedman, J. R., Method details Olson, E. N., Chen, J., Brown, J. H., and Bers, D. M. (2006) Local InsP3-de- Isolation and culture of AMVM—This was carried out as de- pendent perinuclear Ca2ϩ signaling in cardiac myocyte excitation-tran- scribed previously (48). scription coupling. J. Clin. Invest. 116, 675–682 CrossRef Medline Isolation and labeling of nuclei from AMVM—This was car- 3. O’Connell, T. D., Jensen, B. C., Baker, A. J., and Simpson, P. C. (2014) Cardiac ␣1-adrenergic receptors: novel aspects of expression, signaling ried out as described previously (49). Labeling is described in mechanisms, physiologic function, and clinical importance. Pharmacol. the supporting information. Rev. 66, 308–333 Medline ␣ Adenoviral production—The 1A-GFP adenovirus was 4. Dorn, G. W., 2nd, and Brown, J. H. (1999) Gq signaling in cardiac adapta- described previously (50). Production of the GFP-C1-PLC␦-PH tion and maladaptation. Trends Cardiovasc. Med. 9, 26–34 CrossRef (GFP-PHD) and NLS-GFP-PHD adenoviruses are described in Medline 5. Adams, J. W., Sakata, Y., Davis, M. G., Sah, V. P., Wang, Y., Liggett, S. B., the supporting information. The HDAC5-GFP adenovirus was ␣ Chien, K. R., Brown, J. H., and Dorn, G. W., 2nd (1998) Enhanced G q a gift from Dr. Timothy McKinsey (51). For all experiments, signaling: a common pathway mediates cardiac hypertrophy and apopto- cultured AMVM were counted and infected with adenoviruses tic heart failure. Proc. Natl. Acad. Sci. U.S.A. 95, 10140–10145 CrossRef ␣ at the following multiplicity of infection: 1A-GFP, 1000 (titer: Medline
J. Biol. Chem. (2018) 293(23) 8734–8749 8747 Compartmentalized myocyte Gq-receptor signaling
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J. Biol. Chem. (2018) 293(23) 8734–8749 8749 SUPPORTING INFORMATION
EXPANDED METHODS
Contact for Reagent and Resource Sharing
Information and requests for resources and reagents should be directed to and will be fulfilled by the Lead
Contact, Timothy O’Connell ([email protected]).
Experimental Model and Subject Details
Mouse Lines
In this study, male and female C57BL/6J mice (10-15 weeks of age) were used as the source of primary adult cardiac myocytes. Male and female C57BL/6J mice were included due to no historical evidence indicating differences in receptor (α1-AR and AT-R) density or signaling cascades. Male FVB/NJ (10-11 weeks of age) mice were used for infusion of agonist to measure Gq-receptor induced hypertrophic transcriptional responses. Only male FVB/NJ mice were used for the Gq-receptor induced hypertrophic transcriptional responses due to historical evidence indicating a more robust hypertrophic response by male mice. All FVB/NJ mice were randomized to treatment groups (vehicle (n=6), AngII (n=4), or PE
(n=4)). For implantation of mini-osmotic pumps, mice were anesthetized under isoflurane (3% for induction, 1.5% for maintenance) and animals were treated with buprenorphrine (1 mg/kg) for post- surgical analgesia. After implantation of osmotic pumps, animals were housed singly for the duration of the study to ensure safety of the incision site. All animals were treatment naïve and housed under 12-hour light/dark cycle and fed standard chow. The use of all animals in this study conformed to the PHS Guide for Care and Use of Laboratory Animals and was approved by the University of Minnesota Institutional
Animal Care and Use Committee. The University of Minnesota provides AAALAC-accredited services and resources for using animals in research, including animal ordering, care, facilities, housing, and training for researchers and lab personnel.
Cell Lines Cell lines used in this study include, N38 embryonic mouse hypothalamus cells to validate the PLCβ1 antibody, and HEK293 cells used for adenoviral amplification. Both cell lines were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were incubated at 5% CO2 and 37°C. No information on sex or cell authentication information is available for either cell line.
Method Details
Isolation and Culture of AMVM:
As we have described previously (1), adult mouse ventricular myocytes were isolated from wild-type male and female C57BL/6J mice between 10 and 15 weeks of age. Briefly, mice were anesthetized with isoflurane (3% for induction, 1.5% for maintenance), injected with heparin (100 IU/mL), the pleural cavity was opened and the heart removed, cannulated on a retrograde perfusion apparatus, and perfused with collagenase type II to dissociate ventricular myocytes. Isolated cardiac myocytes were plated at a density of 50 rod-shaped myocytes per square millimeter on laminin-coated culture dishes. Myocytes were cultured in MEM with Hank’s Balanced Salt Solution, 1 mg/ml bovine serum albumin, 10 mM 2,3- butanedione monoxime, and 100 U/ml Penicillin in a 2% CO2 incubator at 37°C. All reagents were purchased from Sigma Aldrich unless otherwise specified. Full details of the buffers, enzymes, cell culture medium, all procedures for isolation and culturing of AMVM and a detailed diagram of the perfusion apparatus were described in detail previously (1). Based on our previous experience, myocyte isolation are ~95% pure, but we cannot exclude the possibility of minimal contamination with fibroblasts and endothelial cells (1).
Following plating, myocytes were counted at a magnification of 20X to determine cell viability.
Adenovirus was added based on previously determined multiplicity of infections. Infected myocytes were incubated in a 2% CO2 incubator at 37°C for 40 hours. If myocytes were left uninfected (α1-AR QDot 565, AngII-TAMRA, and PLCβ1 immunocytochemistry experiments), myocytes were incubated at in a
2% CO2 incubator at 37°C for 24 hours before beginning experiments.
Isolation of Nuclei from AMVM
Immediately following isolation, myocytes were allowed to settle by gravity before aspiration of supernatant. Myocytes were washed in Buffer A (10 mM K-HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM activated Na3VO4, 1 mM DTT, 50 U/mL DNase, 10 µL/mL protease inhibitor cocktail) and centrifuged at 25 x g for 2 minutes. Supernatant was aspirated and the myocytes were resuspended in
Buffer A. Myocytes were incubated on ice for 15 minutes to allow for swelling, and subsequently homogenized for 20 strokes using a dounce homogenizer. The homogenate was centrifuged for 2 minutes at 25 x g. The supernatant was collected and the pellet was resuspended in Buffer A. The homogenization and centrifugation steps were repeated, and the pellet was resuspended in Buffer A. The resuspended pellet was then sonicated and the homogenate was centrifuged at 2,000 x g for 15 minutes to collect nuclei. 1.95M sucrose was added to the pellet and layered on top of 1.95M sucrose in an Ultra-Clear
Beckman Coulter 14 x 89 mm centrifuge tube. The supernatant containing the myocyte sarcolemmal fraction and cytosolic fractions was added to a separate Ultra-Clear Beckman Coulter 14 x 89 mm centrifuge tube. The nuclei (tube 1) and myocyte sarcolemmal/cytosolic fractions (tube 2) were centrifuged at 100,000 x g for 90 minutes at 4°C. The nuclear pellet from tube 1 was resuspended in
Buffer C (20 mM Na-HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 25% Glycerol, 0.5 mM
DTT, 0.5 mM PMSF, 10 µL/mL protease inhibitor cocktail). The supernatant from tube 2 was collected as the cytosolic fraction and the pellet from the tube 2 was resuspended in Buffer C for the myocyte sarcolemmal membrane fraction.
Adenoviral Production
α1A-GFP: As previously described (2), the α1A-GFP fusion protein was constructed with GFP fused to the C-terminus of the human α1A-AR (NM000680) cDNA. The α1A-AR cDNA was amplified with primers designed to remove the stop codon and insert Bgl II and Mlu I restriction sites 5’ and 3’, respectively. The resulting PCR product was cloned into pCR2.1-TOPO (Invitrogen), then subcloned into the Bgl II-Mlu I restriction sites in the multi-cloning site of the humanized pGFP2-N3 vector with GFP at the C-terminus. To generate adenovirus, the α1A-GFP was amplified by PCR with primers designed to insert Pme I and Xba I restriction sites at the 5’ and 3’ ends, respectively. The resulting PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen), then subcloned into the Pme I-Xba I restriction sites in the Ad5CMV K-NpA vector under control of the CMV promoter.
GFP-PHD and NLS-GFP PHD: The GFP-C1-PLCdelta-PH (GFP-PHD) plasmid was obtained from
Addgene. To construct NLS-GFP-PHD, the nuclear localization sequence (NLS) from the classical monopartite NLS of the SV40 large T-antigen (3) was added to the C-terminus of GFP-PHD using mini- gene technology. The sequence of the monopartite NLS is: GCA TGT GTC CAA AGA AAA AGC GCA
AGG TAA. Integrated DNA Technologies synthesized the mini gene that included monopartite NLS flanked by an Age I and Xho I restriction sites. pShuttle CMV GFP-PHD was digested with Age I and
Xho I and the resulting fragment was replaced with the AgeI-monopartite NLS-Xho I mini-gene using ligation.
HDAC5-GFP: The HDAC5-GFP adenovirus was a gift from Timothy McKinsey, Ph.D. (4).
Viral amplification and purification: All viruses, except the α1A-GFP as described above, were produced using the AdEasy Adenoviral Vector System (Agilent). Briefly, chosen fusion proteins were cloned into the pShuttle-CMV vector. Positive clones were recombined into bacterial cell line BJ5183 using electroporation. DNA was isolated from positive clones and transfected into HEK293 cells using
Effectene transfection reagent. Virus was amplified for three rounds of replication. Viruses were purified using OptiPrep gradients and ultracentrifugation (35,000 x g for 18 hours). Viruses were titered using
Adeno-X Rapid Titer Kit per manufacturer’s instructions. All constructs were sequence verified in the
University of Minnesota sequencing core. For experiments, cultured AMVM were counted at 20X and infected with adenoviruses at the following multiplicity of infection α1A-GFP: 1000 (titer: 5.9 x 1010);
GFP-PHD 100 (titer: 2.4 x 108); NLS-GFP-PHD (titer: 1.4 x 1010) and HDAC5-GFP (titer: 6.1 x 1015).
Synthesis of α1-QDot-565
The synthesis of α1-QDot-565 is a two-step process in which the pharmacophore of piperazinyl α1AR antagonists is first attached to biotinylated PEG, and the resulting α1-PEG-biotin product is then joined to a streptavidin coated QDot to create a novel fluorescent α1AR antagonist. First, 2-Piperazinyl-4-amino-
6,7-dimethoxyquinazoline (MW = 289.33) was combined with Poly(ethylene glycol) (N- hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (average Mn 3,800) at a molecular weight ratio of 1:2 in an excess of anhydrous dimethyl sulfoxide in a round bottom flask under a blanket of argon. The mixture was constantly stirred and heated at 40°C for 40 hours. The reaction mixture was then added to an excess of HPLC grade diethyl ether, filtered, and speed vacuumed overnight. The dried precipitate was resuspended in water and subsequently lyophilized for 48 hours. The final product, 2-
Piperazinyl-4-amino-6,7-dimethoxyquinazoline-Poly(ethylene glycol)-biotin, was then stored under a blanket of argon at -20°C. Second, 2-Piperazinyl-4-amino-6,7-dimethoxyquinazoline-Poly(ethylene glycol)-biotin was resuspended in water and added to streptavidin conjugated QDot 565 at a ratio of 20:1.
The mixture was rocked at 4°C for 30 minutes in a dark tube to make the final product, α1-QDot 565.
Localization of α1-ARs in AMVM
To define the localization of α1-ARs in adult cardiac myocytes, cultured AMVM plated on coverslips were incubated with 25 nM α1-QDot 565 for 30 minutes at 37°C in the presence and absence of 5 µM prazosin. Myocytes were fixed in 4% paraformaldehyde and mounted in Fluoromount G. Confocal images were captured on an Olympus FluoView FV1000 IX2 Inverted Confocal Microscope at 60X magnification (oil immersion). To detect α1-ARs in the nuclear membrane, myocyte nuclei were isolated, and resuspended in buffer N (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4-7H2O, 10 mM Na-HEPES, 4.6 mM NaHCO3, 30 mM Taurine, 10 mM 2, 3-Butanedione monoxime, 5.5 mM Glucose) (1). Nuclei were subsequently incubated with 25 nM α1-QDot 565 for 15 minutes at 37°C in the absence or presence of 5 µM prazosin. Nuclei were further stained with 1 µM
DRAQ5 (ThermoFisher Scientific) Nuclei were mounted in Fluoromount G and imaged with confocal microscopy as above.
Localization of AT-Rs in AMVM
To define the localization of AT-Rs in adult cardiac myocytes, cultured AMVM plated on coverslips were incubated with 1 µM Angiotensin II-TAMRA (AngII-TAMRA) in the presence and absence of 5 µM unlabeled AngII for 5 minutes at 37°C. Slides were prepared exactly as described for α1-QDot 565.
Confocal images were captured on an Olympus FluoView FV1000 IX2 Inverted Confocal Microscope at
60X magnification (oil immersion). Images were deconvolved for 10 cycles using AutoQuant X3. 3D surface plots were created in FIJI using the 3D surface plot plugin. 3D images were created in FIJI using the volume viewer plugin.
Localization of NCX1 in AMVM
To define the localization of NCX1 in adult cardiac myocytes, cultured AMVM plated on coverslips were fixed and stained with anti NCX1 (1:200 dilution) followed by the appropriate AlexaFluor 488 secondary antibody (1:500 dilution). Cells were imaged by confocal microscopy.
Localization of Phospholipase C β1 in AMVM
The PLCβ1 antibody was validated by measuring PLCβ1 mRNA levels and PLCβ1 protein levels by immunocytochemistry following siRNA-mediated knockdown of PLCβ1 in N38 mouse embryonic hypothalamus cells (generous gift from the lab of Dr. Alessandro Bartolomucci). N38 cells were plated on glass coverslips at a density of 0.5 x 106 in 35 mm dishes. 24 hours after plating, cells were transfected with 80 pmol of either PLCβ1 Gene Solution siRNA or Silencer Negative Control siRNA using
Lipofectamine RNAiMAX reagent, following manufacturer’s instructions. Cells were incubated at 37°C for 72 hours. A portion of cells from each treatment (PLCβ1 siRNA and negative control siRNA) were harvested for quantitative reverse transcriptase PCR analysis using the RNeasy Fibrous Tissue Mini Kit, following manufacturer’s instructions. cDNA was synthesized from 2 µg of total RNA using SuperScript
III First-Strand Synthesis System for RT-PCR. PCR primer sequences are as follows: PLCβ1 forward
GCC CCT GGA GAT TCT GGA GT and PLCβ1 reverse GGG AGA CTT GAG GTT CAC CTT T. The second portion of cells were fixed and stained with anti PLCβ1-C terminal (1:100 dilution) followed by the appropriate AlexaFluor488 secondary antibody (1:500 dilution). Cells were imaged by confocal microscopy. Adult cardiac myocytes were fixed and stained using the exact dilution of primary antibody as N38 cells. AF488 (1:500 dilution) was used as the secondary antibody.
Identification of Nuclear PIP2
PIP2 extraction was carried out as described by(5) with some modifications. Briefly, 20 µL of the nuclear fraction was pipetted into 1.7 mL Eppendorf tubes with 225 µL of cold methanol added to the samples.
After briefly vortexing the samples, 750 µL of cold methyl-tert-butyl ether was added to the samples followed by brief vortex and shaking for 6 minutes at 4°C. 250 µL of HPLC grade water was then added to the samples and briefly vortexed and the phases were separated by centrifugation at 14,000 rpm for 2 minutes. 350 µL of the upper organic phase was collected and placed in 1.5 mL Eppendorf tubes and dried in a speed vac. The samples were resuspended with 100 µL of methanol/toluene (9:1 v/v), vortexed briefly, and centrifuged at 14,000 rpm for 2 minutes to remove any debris. 50 µL of the samples was transferred to 250 µL autosampler vials. The samples’ electrospray mass spectra were 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 at an infusion rate of 5 µl/min. PIP2[4’,5’](18:1(9Z)/18:1(9Z)) was obtained from Avanti Polar Lipids.
Quantification and Statistical Analysis
Analysis of Phospholipase C β1 Activity
To measure PLCβ1 activity in adult cardiac myocytes, cultured AMVM plated on coverslips were infected with either 100 MOI GFP-PHD or 500 MOI NLS-GFP-PHD for 40 hours at 37°C. Myocytes were treated with either vehicle, PE (20 µM) in the absence and presence of prazosin (1 mM), or AngII
(100 nM) in the absence or presence of losartan (5 µM) for 5 minutes at 37°C. Pretreatment with antagonists (prazosin or losartan) was carried out for 30 minutes at 37°C before agonist (PE or AngII) treatment. Myocytes were fixed and mounted exactly as described for α1-QDot 565. Confocal images were captured on an Olympus FluoView FV1000 IX2 Inverted Confocal Microscope at 60X magnification (oil immersion). 3D surface plots were created in FIJI using the 3D surface plot plugin.
Analysis of HDAC5 Nuclear Export
To measure Gq-receptor induced HDAC5 nuclear export, cultured AMVM plated on coverslips were infected with 5,000 MOI HDAC5-GFP for 40 hours at 37°C. Myocytes were treated with either vehicle,
PE (20 µM), or AngII (100 nM) for either 30 minutes or 1 hour at 37°C. Pretreatment with 2-APB (2 µM) was carried out for 30 minutes at 37°C prior to PE treatment. Myocytes were fixed and mounted exactly as described for α1-QDot 565. Confocal images were captured on an Olympus FluoView FV1000 IX2
Inverted Confocal Microscope at 60X magnification (oil immersion).
Analysis of Hypertrophic Transcriptional Profiles
To analyze Gq-receptor hypertrophic transcriptional profiles, 10 to 11 week old FVB/NJ mice were dosed at 30 mg/kg/day for PE (n=4), 0.5 mg/kg/day for AngII (n=4), or vehicle (n=6) using implanted micro- osmotic pumps for 72 hours. The doses of PE and AngII are known to induce hypertrophy without a concomitant increase in blood pressure (6-9) Total RNA was harvested from isolated myocytes using RNeasy Fibrous Tissue Mini Kit, following manufacturer’s instructions. Using total RNA as input, rRNA was removed via oligo-dT purification to enrich for mRNA, followed by random-primed reverse- transcription, second-strand cDNA synthesis, ligation of indexed adaptors for Illumina sequencing, and subsequent library creation from the resulting double-stranded DNA. 125 base pair paired end stranded
RNA Sequencing was performed by the University of Minnesota Genomics Core using the Illumina
HiSeq 2500 resulting in 10-20 million reads per sample.
Statistical Analysis
All data plotting and statistical analysis was performed using Prism 6 software. All information on statistical parameters can be found in figure legends. Images of α1-QDot 565 (α1-QDot 565, α1-QDot
565 + prazosin, QDot 565 alone (not shown)) were quantified using FIJI analyze particle plug-in. Data were analyzed by One-Way ANOVA and P<0.05 was considered significant. PLCβ1 immunofluorescent intensity was quantified on whole images using FIJI. Data were analyzed by student’s t-test and P<0.05 was considered significant. PLCβ1 mRNA levels were quantified by densitometry using ImageLab (Bio-
Rad). Data were analyzed by student’s t-test and P<0.05 was considered significant. To quantify activation of PLCβ1, AMVM expressing GFP-PHD or NLS-GFP-PHD were exposed to agonist or vehicle, and classified as responders (movement of probe off the membrane) and non-responders (no apparent movement of probe) by another investigator blinded to treatment group. Data were analyzed by
Chi-Square and P<0.05 was considered significant. HDAC5-GFP fluorescent intensity was measured for whole myocytes and nuclei using FIJI. Nuclear fluorescence was subtracted from whole cell fluorescence to calculate cytosolic fluorescence. Ratio of cytosolic to nuclear fluorescence was plotted. Data were analyzed by One-Way ANOVA and P<0.05 was considered significant.
For RNAseq analysis, 126bp FastQ paired-end reads (n=11.6 Million per sample) were trimmed using Trimmomatic (v 0.33) enabled with the optional “-q” option; 3bp sliding-window trimming from 3’ end requiring minimum Q30. Quality control checks on raw sequence data for each sample were performed with FastQC. Read mapping was performed via Bowtie (v2.2.4.0) using the UCSC mouse genome (mm10) as reference. Gene quantification was done via Cuffquant for FPKM values and Feature
Counts for raw read counts. Differentially expressed genes were identified using the edgeR (negative binomial) feature in CLCGWB (Qiagen) using raw read counts. We filtered the generated list based on a minimum 1.7X Absolute Fold Change and FDR corrected p < 0.05. The filtered gene list was then used for further analysis of hypertrophic, survival-signaling, inotropic, and fibrotic gene ontologies. Search criterion were defined by commonly associated terms used in concordance with hypertrophy, survival- signaling, inotropy, and fibrosis, respectively. Search terms for hypertrophy were NF-kappa B, 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, matrix. All genes are represented with Entrez gene names (https://www.ncbi.nlm.nih.gov/gene). Prcomp was used to perform principal component analysis on a file containing normalized FPKM values across 14 samples with 24,345 genes across 3 groups. Ggplot was used to draw the PCA graph and stat_ellipse was used to delineate the 95% confidence intervals.
KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-phospholipase C beta 1 antibody- C-terminal abcam Cat#ab185724 Anti-NCX1 abcam Cat#ab2869 AlexaFluor 594 goat anti-rabbit IgG Invitrogen Cat#A11012 AlexaFluor 488 goat anti-rabbit IgG Invitrogen Cat#A11008
Bacterial and Virus Strains BJ5183 electroporation competent cells Agilent Technologies Cat#200154 AD5-CMV K-NpA ViraQuest Inc. N/A Chemicals, Peptides, and Recombinant Proteins Collagenase Worthington Cat#LS004176 Biochemical Corporation Laminin BD Bioscience Cat#354232 Effectene Qiagen Cat# 301425 OptiPrep Accurate Chemical Cat# AN1114542 2-Piperazinyl-4-amino-6,7-dimethoxyquinazoline Toronto Research Cat#P480500 Chemicals Poly(ethylene glycol) (N-hydroxysuccinimide 5- Sigma Aldrich Cat#757799 pentanoate) ether 2-(biotinylamino)ethane QDot 565 Steptavidin Conjugate ThermoFisher Cat#Q10133MP Scientific Prazosin Hydochloride Sigma Aldrich Cat# P7791 DRAQ5 Fluorescent Probe Solution ThermoFisher Cat# 62251 Scientific (R)-(-)-Phenylephrine hydrochloride Sigma Aldrich Cat#P6126 Fluoromount G Electron Microscopy Cat#17984-25 Sciences Angiotensin II-TAMRA AnaSpec Cat#AS-61181 Human Angiotensin II Sigma Aldrich Cat#A9525 Losartan potassium salt Tocris Bioscience Cat#3798/50 Lipofectamine RNAiMAX reagent Invitrogen Cat#13778100 2-APB Sigma Aldrich Cat#D9754 PIP2[4’,5’](18:1(9Z)/18:1(9Z)) Avanti Polar Lipids Cat# 850155 Critical Commercial Assays Adeno-X Rapid Titer Kit Takara Cat#632250 RNeasy Fibrous Tissue Mini Kit Qiagen Cat# 74704 SuperScript III First-Strand Synthesis System for RT- Invitrogen Cat# 18080051 PCR Deposited Data RNA Sequencing Data http://dx.doi.org/10.17 N/A 632/pbw8ww4k55.1 Experimental Models: Cell Lines Human embryonic kidney cells (HEK293) ATCC Cat#CRL-1573 Mouse embryonic hypothalamic cells (N38) CELLutions Cat#CLU118 Biosystems Inc Experimental Models: Organisms/Strains C57BL/6J Jackson Labs Cat#000664 FVB/NJ Jackson Labs Cat#001800
Oligonucleotides PLCβ1 forward and reverse primers Integrated DNA N/A Technologies NLS-GFP-PHD Mini Gene Integrated DNA N/A Technologies Silencer Negative Control siRNA Ambion Cat#AM4636 PLCβ1 Gene Solution siRNA Qiagen Cat#GS18795 Recombinant DNA pGFP2-N3 vector BioSignal Packard Discontinued GFP-C1-PLCdelta-PH Addgene plasmid #21179 Software and Algorithms Prism 6 GraphPad N/A AutoQuant X3 Media Cybernetics N/A
SUPPLEMENTAL TABLES Supplementary Table 1. Hypertrophy Phenotype Genes
Gene Ontology AngII PE Common Category Immune Response 0 19 5 Increase 0 19 5 - Cd14, Cd44, Cd84, Clec4n, Fcgr2b, Il6, Inhba, Fcer1g, Fcgr1, Fcgr3, H2- Lgals3, Tgfb2 DMb1, H2-DMb2, Havcr2, Il1b, Ncf4,Slc11a1, Tgfb3, Tlr1, Tnfrsf11a, Tnfsf11, Tnfsf14, Trem2 Decrease 0 0 0 - - - Kinase & Phosphatase 0 6 4 Activity Increase 0 6 3 - Btk, Ikbke, Pak1, Pi16, Cdkn1a, Nckap1l, Pik3ap1, Rcan1 Ptk2b Decrease 0 0 1 - - Akap5 Transcription Regulation 0 7 1 Increase 0 6 1 - Bcl3, Col14a1, Igfbp2, Il7r, Spi1 Nupr1, Zc3h12d Decrease 0 1 0 - Nlrc5 - G-protein Coupled 0 6 0 Receptors & Activity
Increase 0 5 0 - Frzb, Htr2b, Rasgrp4, Rrad, - Sfrp1 Decrease 0 1 0 - Adra1a - Growth Factor 0 4 2 Increase 0 4 2 - Cgref1, Ctgf, Hbegf, Igf1 Csf2rb, Fhl1 Decrease 0 0 0 - - -
Supplementary Table 1. Molecular function of Hypertrophic Genes induced by α1ARs, ATRs, or in common. As described in Figure 8, genes were sorted into Gq-receptor functional subclasses of hypertrophy, survival signaling, inotropy, and fibrosis using non-overlapping search terms unique to Gq- receptor biologic functions. Search terms for hypertrophy were: NF-kappa B, GATA4, MEF2, NFAT, ANF, TGF, hypertrophy, myosin heavy chain, MHC, c-myc, c-fos, cell growth, natriuretic. Hypertrophic genes were further sorted by molecular function and listed in the table, with the number of genes increased or decreased indicated along with the specific genes in each category.
Supplementary Table 2. Survival Signaling Phenotype Genes
Gene Ontology AngII PE Only Common Category Only Immune Response 0 18 12 Increase 0 18 12 - Ccl12, Ccl4, Ccl6, Ccr1, Ccl2, Ccl9, Ccl22, Cd44, Cd84, Gdf15, Gdf6, Ccr2, Cd24a, Crlf1, Il1b, Il33, Lgals1, Lif, Tgfb3, Il6, Il11, Il1rn, Tnfrsf11a, Tnfrsf11b, Inhba, Socs3, Tnfrsf23, Tnfsf11, Tnfsf14 Tgfb2 Decrease 0 0 0 - - - Kinase & Phosphatase 0 20 7 Activity Increase 0 15 6 - Dram1, Dusp26, Havcr2, Hck, Cdkn1a, Hsb1, Hk3, Ikbke, Pak1, Pak3, Mylk2, Nckap1l, Pak6, Peg10, Pik3ap1, Ptn, Ptk2b, Snai1 Ptpn6Ptprc, Sphk1 Decrease 0 5 1 - Dusp4, Ephb1, Map4k2, Ptprr, Kit Prkcg G-protein Coupled 0 16 0 Receptors & Activity
Increase 0 14 0 - Ccl7, Ccl8, Ccr5, Cdh1, - Cx3cr1, Frzb, Gpnmb, Htr2b, Lpar3, Mpz, Ncf2, Ntsr2, Rgs14, Sfrp1 Decrease 0 2 0 - Adra1a, Brinp1 - Transcription Regulation 2 6 4 Increase 1 5 3 Atf3 Creb3l1, Irf5, Isl1, Myc, Ankrd2, Plac8, Twist1 Spi1 Decrease 1 1 1 Sox4 Sox7 Mycn Enzyme 0 11 0 Increase 0 10 0 Supplementary Table 2. Molecular function of Survival Signaling Genes induced by α1ARs, ATRs, or in common. As described in Figure 8, genes were sorted into Gq-receptor functional subclasses of hypertrophy, survival signaling, inotropy, and fibrosis using non-overlapping search terms unique to Gq- receptor biologic functions. Search terms for survival signaling were: ERK, MAPK, apoptotic, apoptosis, survival, cell death. Survival signaling genes were further sorted by molecular function and listed in the table, with the number of genes increased or decreased indicated along with the specific genes in each category.
Supplementary Table 3. Inotropy Phenotype Genes
Gene AngII PE Common Ontology Category Kinase & 0 23 7 Phosphatase Activity Increase 0 20 5 - Bmp1, Camk2n2, Ccl4, Ccr1, Acp5, Fgr, Mylk2, Cnn1, Coro1a, Dbn1, Dpysl3, Nckap1l, Ptk2b Fbn1, Fkbp10, Hck, Myo1g, Pacsin1, Pak1, Pak3, Pak6, Plek, Ptpn6, Ptprc, Sphk1 Decrease 0 3 2 - Dgkh, Mylk4, Prkcg Akap5, Kit G-protein 0 19 3 Coupled Receptors & Activity Increase 0 15 2 - Ackr1, Adgre1, Cdh1, Dock2, Lrp8, Ptafr Fpr2, Fstl1Htr2b, Lpar3, Rab27b, Rac2, Rhou, Sfrp1, Tgm2, Tnfsf11, Was Decrease 0 4 1 - Adra1a, Celsr3, Gpr17, Tbxa2r Apln Immune 0 12 6 Response Increase 0 12 6 - Alox5ap, C3ar1, C5ar1, Ccl12, Ccl2, Cd24a, Crlf1, Ccr5, Cd84,Cxcl13, Gpr35, Il1b, Sele, Selp, Tgfb2 Ncf1, Panx1, Runx1 Decrease 0 0 0 - - - Ion Channel 0 13 4 Increase 0 9 4 - Atp1a3, Clca3a, Fzd2, Itih4, Hcn1, Ltbp2, Sypl2, Kcnab2, Kcnn4, Slc8a2, Trem2, Syt12 Trpm2 Decrease 0 4 0 - Ano10, Cacna1s, Clcn1, Cracr2a -
Supplementary Table 3. Molecular function of Inotropy Genes induced by α1ARs, AT-Rs, or in common. As described in Figure 8, genes were sorted into Gq-receptor functional subclasses of hypertrophy, survival signaling, inotropy, and fibrosis using non-overlapping search terms unique to Gq- receptor biologic functions. Search terms for inotropy were: calcium, contraction, sarcoplasmic reticulum, troponin, myosin, actin, ryanodine, protein kinase C, and sarcomere. Inotropy genes were further sorted by molecular function and listed in the table, with the number of genes increased or decreased indicated along with the specific genes in each category.
Supplementary Table 4. Fibrosis Phenotype Genes
Gene Ontology Category AngII PE Common Extracellular Matrix 1 27 11 Modulation Increase 1 27 11 Mmp9 Acan, Adamts2, Col11a1, Adam8, Col12a1, Col14a1, Adamts4, Col16a1, Col18a1, Col1a1, Col6a1, Col6a2, Eln, Col1a2, Emilin1, Fbln2, Fbn1, Col3a1, Fbn2, Frem1, Itga11, Col5a1, Itgb3, Mmp12, Mmp14, Col5a2, Mmp3, Mmp19, Mmp8, Mrc2, Spp1, Thbs1, P4ha3, Pcolce, Postn, Thbs4 Serpinh1, Vacn Decrease 0 0 0 - - - Immune Response 0 6 7 Increase 0 6 7 - Bgn, Ccdc80, Cd44, Ccl2, Cxcl5, Tgfb3, Tnfsf11b Il17ra, Il6, Itgb2, Lgals3, Tgfb2 Decrease 0 0 0 - - - Kinase & Phosphatase 0 7 2 Activity Increase 0 6 2 - Ccnb1, Cnn1, Dusp26, Cdkn1a, Ptk2b Fam20c, Pak1, Pak3 Decrease 0 1 0 - Ptprz1 - Transcription Regulation 0 7 0 Increase 0 5 0 - Bcl3, Creb3l1, Myc, Nupr1, - Sh3pxd2b Decrease 0 2 0 - Dach1, Tcf15 - Peptidase 0 4 3 Increase 0 4 3
Supplementary Table 4. Molecular function of Fibrosis Genes induced by α1ARs, AT-Rs, or in common. As described in Figure 8, genes were sorted into Gq-receptor functional subclasses of hypertrophy, survival signaling, inotropy, and fibrosis using non-overlapping search terms unique to Gq- receptor biologic functions. Search terms for fibrosis were: collagen, matrix metalloprotease, fibrosis, fibroblast, extracellular matrix, matrix. Fibrosis genes were further sorted by molecular function and listed in the table, with the number of genes increased or decreased indicated along with the specific genes in each category.
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