Phospholipase Cϵ Scaffolds to Muscle-specific A Kinase Anchoring Protein (mAKAPβ) and Integrates Multiple Hypertrophic Stimuli in Cardiac Myocytes*

To define a role for phospholipase Cϵ (PLCϵ) signaling in cardiac myocyte hypertrophic growth, PLCϵ protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCϵ depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCϵ depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCϵ catalytic activity was required for hypertrophy development, yet PLCϵ depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCϵ was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCϵ-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCϵ complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCϵ integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy.

Pathologic hypertrophic growth of the myocardium, a process associated with the development of heart failure, occurs in response to various stimuli including pressure overload and chronic increases in circulating hormones that place long term stress on the heart. Many of these hormones act on receptors in cardiac myocytes including adrenergic and endothelin receptors to initiate hypertrophic signaling cascades (1)(2)(3). The exact participants in these hypertrophic signaling cascades have been the subject of intense investigation. One common signaling mechanism closely coupled to receptor activation is activation of phosphatidylinositol-specific phospholipase C (PLC) 3 (4). A role for PLC activity in regulation of hypertrophic signaling has been suggested because G q , a potent stimulator of hypertrophy (5), directly stimulates PLC␤ as a major regulatory pathway (6). Additionally, growth factors such as insulin-like growth factor-1 (IGF-1) that stimulate physiological hypertrophy activate PLC␥ (7,8). Recently, it has been reported that a splice variant of PLC␤, PLC␤1b, is required for hypertrophy downstream of ␣1-adrenergic receptor stimulation and suggested to be a major mediator of G q -dependent cardiac hypertrophy (9).
The PLC family consists of 12 isoforms regulated by diverse upstream signal inputs, all of which hydrolyze phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol trisphosphate (IP 3 ) (10,11). Although containing a common PLC catalytic core domain, PLC␤, PLC␥, PLC␦, and PLC⑀ isoforms are very different in terms of overall domain structure and regulatory mechanisms, indicating that they are unlikely to play overlapping roles in the cells. PLC⑀ is unique in that it is directly regulated by various small GTPases, including Ras, Rho, and Rap (12)(13)(14)(15)(16), and G␤␥ subunits, but not G␣ q . PLC⑀ has been shown to signal downstream of G protein-coupled receptors, including ␤-adrenergic receptors (␤ARs), lysophosphatidic acid and sphingosine 1-phophate receptors (EdgRs), proteaseactivated receptors (13,17), and growth factor receptor tyrosine kinases, including PDGF receptors (15) and EGF receptors (13). Thus, PLC⑀ is ideally positioned to integrate multiple signaling inputs in various cell types including cardiac myocytes.
Recent studies of PLC⑀ Ϫ/Ϫ mice revealed alterations in ␤ARdependent regulation of cardiac ionotropy and hypertrophy (18). These mice had no hypertrophy at baseline but developed greater hypertrophy than wild type animals in response to chronic isoproterenol treatment. Thus, PLC⑀ appeared to protect mice from stimulus-induced cardiac hypertrophy. In wild type mice, the levels of PLC⑀ mRNA and protein increase during pressure overload or chronic isoproterenol (Iso) treatment. PLC⑀ RNA levels are elevated in biopsies from human heart failure samples. To explore a mechanistic role for PLC⑀ in hypertrophy, we utilized an siRNA protocol in an established cellular model of cardiac hypertrophy, the neonatal rat ventric-ular myocyte (NRVM) (19). Surprisingly, in this cell model, we found that PLC⑀ appears to be required for development of cardiac hypertrophy downstream of multiple hypertrophic stimuli, rather than protective.
We also examined the subcellular scaffolding of PLC⑀ that might lead to generation of IP 3 and DAG at specific cellular sites critical for activation of hypertrophic transcriptional mechanisms. A common family of scaffolding molecules in the heart is the protein kinase A anchoring protein (AKAP) family (20,21). One member of this family that is involved in cytokineand adrenergic-induced hypertrophy is the alternatively spliced isoform of muscle AKAP (mAKAP␤) expressed exclusively in striated myocytes (22). mAKAP␤ binds multiple proteins besides protein kinase A (PKA) including exchange factor activated by cAMP (Epac), adenylate cyclase 5 (23), cAMP-phosphodiesterase 4D3 (PDE4D3), extracellular signal-regulated kinase 5 (ERK5) (22), protein phosphatase 2A (PP2A) (21), and calcineurin (24). mAKAP␤ localizes to the nuclear envelope of myocytes, where it is targeted by nesprin-1␣ (25). It has also been suggested that mAKAP␤ scaffolds PKA to ryanodine receptor type 2 (RyR2) in the sarcoplasmic reticulum (SR) (26), but this is controversial. Here we demonstrate that PLC⑀ binds directly to mAKAP␤ and that the scaffolding by mAKAP␤ is important for the ability of PLC⑀ to regulate cardiomyocyte hypertrophy. This suggests that scaffolding of PLC⑀ to the nuclear envelope by mAKAP␤ regulates expression of hypertrophic genes.
Adenoviral Constructs-Short interfering hairpin RNAs (siRNAs) were designed for targeting mouse, rat, or human PLC⑀: siRNAPLC⑀ 5, 5ЈGCCAAATATTCCTACAGCA; RANsiRNA, ACTGTCACAAGTACCTACA (scrambled siRNAPLC⑀ 5). siRNA sequences were subcloned into pShuttle-CMV as in (28), and recombinant viruses were generated following the manufacturer's protocol for the Q-biogene AdEasy adenoviral vector system. Adenoviruses expressing PLC⑀-RA1 and mAKAP-SR1 were prepared by PCR amplification of the appropriate cDNA fragments and were subcloned into a shuttle vector, under the control of the mouse cytomegalovirus promoter, which also expresses the YFP protein under the control of a separate mouse cytomegalovirus promoter. The shuttle vector was recombined with the parent vector in HEK293 cells to generate adenovirus expressing the PLC⑀-RA1 or mAKAP-SR1 domain. Recombined virus was amplified in HEK293 cells and purified by CsCl gradient centrifugation. Viral titers were calculated by an immunoreactivity spot assay (29).
Glutathione S-Transferase (GST) Fusion Proteins-GST fusion protein constructs were prepared as described previously (23). BL21 DE3 cells containing pGEX-4T2 plasmids with the indicated regions of mAKAP fused to the C terminus of GST were grown to an optical density (600 nm) of 0.6 followed by induction with 100 mol/liter isopropyl-1-thio-␤-D-galactopyranoside for 16 h at 18°C in 1 liter of each. Cells were harvested by centrifugation followed by lysis in 30 ml of lysis buffer (10 mmol/liter Tris-Cl, pH 7.5, 150 mmol/liter NaCl, 2 mmol/liter EDTA, and 10 mmol/liter ␤-mercaptoethanol) ϩ protease inhibitors: 133 M phenylmethylsulfonyl fluoride, 21 g/ml 1-chloro-3-tosylamido-7-amino-2-heptanone and L-1tosylamido-2-phenylethyl chloromethyl ketone, 0.5 g/ml aprotinin, 0.2 g/ml leupeptin, 1 g/ml pepstatin A, 42 g/ml p-tosyl-L-arginine methyl ester, 10 g/ml soybean trypsin inhibitor, by probe sonication. Nonidet P-40 detergent was added to a final concentration of 1% (v/v), and the samples were incubated on ice for 30 min. Lysates were cleared of insoluble material by centrifugation at 100,000 ϫ g for 30 min. Soluble lysates were incubated with 500 l of glutathione agarose beads (Thermo Fisher) for 2 h at 4°C. Beads were harvested by centrifugation and washed four times in 20 ml of lysis buffer. Beads bound to GST fragments were analyzed directly by SDS-PAGE and staining with Coomassie Blue as well as by an Amido Black protein assay and stored at 4°C.
For GST-mAKAP fragment pulldowns, 2 g of GST fusion protein bound to 25 l of beads was mixed with 50 ng of purified PLC⑀ in 500 l of pulldown buffer (10 mmol/liter Tris-Cl, pH 7.5, 150 mmol/liter NaCl, 2 mmol/liter EDTAϩ). Mixtures were rotated for 2 h at 4°C. Beads were harvested by centrifugation at 16,000 ϫ g at 4°C for 2 min. After supernatant removal, beads were washed three times with 500 l of pulldown buffer. Beads were boiled in 30 l of sample buffer, and 10 l was loaded on a 7.5% (w/v) SDS-polyacrylamide gel for resolving and Western blotting.
HEK Cell Culture and Transfection-5 ϫ 10 5 HEK293 cells cultured in Dulbecco's modified Eagle's medium (DMEM) ϩ 10% (v/v) FBS were plated on poly-D-lysine-coated 6-well plates the day before transfection. 2 g of DNA was transfected using Lipofectamine Plus reagent (Invitrogen). Proteins were expressed for 48 h prior to harvesting for immunoprecipitation.
Neonatal Cardiac Myocyte Culture, Adenoviral Infection, and Hormone Treatment-NRVMs were isolated and cultured from hearts from 2-3-day-old Sprague-Dawley rats as has been previously described (19) with modifications. Briefly, hearts were excised, and ventricles were separated and rinsed in Hanks' balanced salt solution prior to digestion with three rounds of collagenase type II (Worthington) in Hanks' balanced salt solution without Ca 2ϩ and Mg 2ϩ . Cells were collected by centrifugation and resuspended in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mmol/liter glutamine, and 2 g/ml vitamin B-12. Non-myocytes were removed by preplating of the cells for 1 h at 37°C. The NRVMs were cultured in DMEM as described above containing 10 mol/liter cytosine arabinoside. 10 mol/ liter cytosine arabinoside was applied for the first 3 days. After overnight incubation, FBS was reduced to 1% (v/v) followed by DMEM lacking serum for at least 24 h. After NRVMs were plated for 6 -8 h, adenovirus expressing PLC⑀ siRNA 5, random siRNA, PLC⑀, PLC⑀(H1460L), PLC⑀-RA1, mAKAP-SR1, or YFP as control was added (multiplicity of infection 50) for 4 -6 h followed by washing. NRVMs were then cultured in serumfree DMEM medium containing 0.1ϫ insulin-transferrin-selenium. After adenovirus infection for at least 2 days and serum starvation for at least 24 h, 10 nmol/liter ET-1, 10 mol/liter Iso, 10 mol/liter norepinephrine (NE), or 10 nmol/liter IGF-1 was added for 48 h to induce hypertrophy.
[ 3 H]Leucine Incorporation-NRVMs were plated in 24-well 0.2% (w/v) gelatin-coated plates at a density of 2 ϫ 10 5 cells/ml and 500 l/well. [ 3 H]Leucine (1 Ci/ml) was used to label newly translated proteins for 48 h in the absence or presence of hypertrophic agonist as described above. Cells were washed twice with ice-cold PBS and incubated in 5% (w/v) trichloroacetic acid for 30 min at 4°C to precipitate the protein. Precipitates were washed twice and solubilized in 0.4 N NaOH for 2 h. Trichloroacetic acid (TCA) precipitable radioactivity was measured by scintillation counting. For analysis, data were pooled from 2-3 independent experiments performed in triplicate.
Measurement of PLC Activity in NRVMs-NRVMs were plated in 24-well 0.2% (w/v) gelatin-coated plates at a density of 2 ϫ 10 5 cells/ml and 500 l/well. After adenovirus infection for 2 days, NRVMs were labeled for 2 days with [ 3 H]inositol (20 Ci/ml) in inositol-free DMEM supplemented as above. LiCl was added to a final concentration of 100 mM for 10 min at 37°C. Then agonist was added for 30 min at 37°C. Total inositol phosphates (IPs) produced were measured as in Ref. 30.
Measurement of ANF Levels by Real Time PCR-NRVMs were plated in 6-well 0.2% gelatin-coated plates at a density of 5.0 ϫ 10 5 cells/ml and 500 l/well. For real time PCR, total RNA was extracted from NRVM using the RNeasy mini kit (Qiagen). 300 ng was reverse-transcribed with reverse primers to GAPDH and atrial natriuretic factor (ANF) using SuperScript III reverse transcriptase (Invitrogen). Real time PCR was performed on this product on the BioRadCFX96 real time system with a C 1000 thermal cycler using the iQ SYBR Green supermix (Bio-Rad).
Isolation of Nuclei from NRVMs-The protocol was as previously reported except that the fraction containing SR, plasma membrane, and Golgi bodies was not further purified (31).
Statistical Analysis-For the majority of the [ 3 H]leucine incorporation, cell area, real time PCR, and inositol phosphate measurements, significance was calculated by dividing the mean ligand-stimulated response by the unstimulated response for either random or PLC⑀ siRNA treatment to determine fold activation. The difference in fold activation for random siRNA versus PLC⑀ siRNA was analyzed for statistical significance using a one-tailed unpaired Student's t test. Selected data in Fig. 3A were analyzed by one-way analysis of variance with Bonferroni's post test.

PLC⑀ Knockdown in NRVMs Inhibits
Endothelin-1-dependent Hypertrophy-We previously established an siRNA protocol for PLC⑀-specific knockdown in Rat-1 fibroblasts. This siRNA treatment did not affect expression of other PLC isoforms or expression of G proteins, and several siRNA sequences produced similar biological effects (28). We selected the most effective siRNA sequence from those tested (siRNAPLC⑀ 5) that knocked down PLC⑀ by 97% in Rat-1 fibroblasts and created an adenovirus for infection of NRVMs. Supplemental Fig.  1A shows RT-PCR analysis of PLC⑀ RNA extracted from NRVMs infected for 3 and 6 days. PLC⑀ mRNA was effectively depleted after 3 days and remained low after 6 days of viral infection. PLC⑀ protein was also depleted after 3 days of viral infection (supplemental Fig. 1B), whereas the levels of other PLC isoforms were unaffected by the siRNA treatment (supplemental Fig. 1C).
First, we examined the role of PLC⑀ in ET-1-dependent cardiac hypertrophy. ET-1 treatment for 48 h caused a significant increase in three markers of hypertrophy: [ 3 H]leucine incorporation (Fig. 1A), cell area (Fig. 1, B and C), and ANF mRNA (Fig.  1D) in random siRNA infected cells, indicating stimulation of the hypertrophic response. Surprisingly, PLC⑀ siRNA blocked ET-1-dependent increases in all hypertrophic markers (Fig. 1,  A-D), indicating a requirement for PLC⑀ for ET-1-dependent hypertrophy in NRVMs. To show that this result was not due to a nonspecific effect of the PLC⑀ siRNA, we co-expressed PLC⑀ with either a LacZ control or PLC⑀ with a non-coding mutation that provides resistance to the siRNA (PLC⑀ (res)) to replace the endogenously depleted PLC⑀ protein with exogenously expressed PLC⑀. Leucine incorporation was monitored as a measure of hypertrophy (supplemental Fig. 1D). As in Fig. 1A, PLC⑀ siRNA suppressed ET-1-induced [ 3 H]leucine incorporation, but co-expression with PLC⑀ (Res) prevented this effect, demonstrating rescue of the hypertrophic response upon re-expression of PLC⑀ in the PLC siRNA background.
Overall, suppression of the hypertrophic response to ET-1 by PLC⑀ depletion was unexpected because it is the opposite of what was observed in the PLC⑀ Ϫ/Ϫ mice where the loss of PLC⑀ appeared to increase hypertrophy. Regardless of this apparent contradiction, this result was also unexpected because ET-1 is thought to stimulate phosphatidyl inositol hydrolysis through a G q -dependent mechanism that would not be expected to directly involve PLC⑀. ET A receptors can also signal via G 12/13 -Rho-, G i /␤␥-, and G s -dependent pathways, and it is possible that PLC⑀ is regulated through one of these signaling mechanisms. It was noted that PLC⑀ knockdown alone increased cell area in the absence of stimulation by ET-1, but this appears to be unrelated to the hypertrophic response because the other markers of hypertrophy, ANF mRNA levels and protein synthe-sis, are not significantly altered by PLC⑀ siRNA treatment in the absence of stimulation by agonist.
PLC⑀ Is a Central Participant in Ligand-dependent Hypertrophic Responses in NRVMs-PLC⑀ has the potential to be regulated by multiple upstream signaling mechanisms. We have extensively characterized a role in cAMP-Epac-and Rap-dependent signaling, and PLC⑀ can respond to multiple G protein-coupled receptor-dependent and receptor tyrosine kinasedependent pathways through Ras, Rho, G␤␥, or other potential regulatory molecules (13,16,17,32). For this reason, we examined the effects of PLC⑀ knockdown on stimulation of hypertrophy by agonists that signal through distinct cellular mechanisms. Iso likely works through a cAMP-dependent mechanism. NE activates G q -dependent signaling via stimulation of ␣1-adrenergic receptors in neonatal myocytes (33). IGF-1 signals via tyrosine phosphorylation and couples to PLC␥ (8). Again, PLC⑀ knockdown almost completely inhibited ligand-dependent hypertrophy by all of these agonists ( Fig. 2 and supplemental Fig. 2), indicating a central role of PLC⑀ in hypertrophic signaling by diverse hormonal regulatory mechanisms.
To determine whether PLC⑀ overexpression can promote hypertrophy in NRVMs, cells were infected with an adenovirus expressing PLC⑀ under the control of a mouse cytomegalovirus promoter, and [ 3 H]leucine incorporation was measured. PLC⑀ expression alone significantly increased leucine incorporation (Fig. 3A), indicating that increasing PLC⑀ expression was sufficient to increase hypertrophy. ET-1 was able to further increase  [ 3 H]leucine incorporation in the context of PLC⑀ overexpression. To determine whether PLC⑀ catalytic activity was required for the PLC⑀-dependent increase in hypertrophy, we expressed PLC⑀ with a point mutation in the catalytic domain that eliminates PLC hydrolytic activity (PLC⑀(H1460L)) and measured hypertrophy. Expression of PLC⑀(H1460L) did not stimulate hypertrophy when compared with the YFP control. Expression of PLC⑀(H1460L) also suppressed hypertrophy stimulated by ET-1, likely acting in a dominant negative manner (Fig. 3A). Adenoviral expression of PLC⑀ and PLC⑀(H1460L) gives equal levels of PLC⑀ protein in ventricular myocytes (not shown). These data indicate that the products of PLC⑀ catalytic activity are important for the hypertrophic response. To examine the role of PLC⑀ in receptor-driven phosphatidylinositol 4,5-bisphosphate hydrolysis, we knocked down PLC⑀ with siRNA and examined ET-1-and NE-stimulated IP production. PLC⑀ knockdown had no effect on the ability of these agonists to stimulate total IP production (Fig. 3,  B and C). We propose that PLC⑀ activity does not significantly contribute to global cellular IP production either because it is expressed at very low levels, because its localization is restricted, or both. Possible mechanisms for subcellular localization of activity are discussed in depth below. Overall, these data suggest that PLC⑀ is necessary for hypertrophy driven by multiple neurohumoral stimuli and that PLC⑀ activity is sufficient to drive hypertrophic responses.

mAKAP Binds PLC⑀ in Transfected Cells and in the Heart-
Because the data suggest that PLC⑀ may be acting locally to produce its physiological effects and because one of the regulators of PLC⑀, Epac, scaffolds to mAKAP␤, we tested whether PLC⑀ could interact with mAKAP␤. Myc-mAKAP was co-expressed with PLC⑀ in HEK293 cells followed by immunoprecipitation of mAKAP and immunoblotting for either PLC⑀ or mAKAP (Fig. 4A). PLC⑀ was found in Myc-mAKAP immunoprecipitates only when both proteins were expressed. No PLC⑀ immunoreactivity was detected if either PLC⑀ or Myc-mAKAP was absent. To determine whether mAKAP␤ and PLC⑀ associate in cardiac tissue, mouse heart lysates were prepared from PLC⑀ ϩ/ϩ and PLC⑀ Ϫ/Ϫ mice followed by PLC⑀ immunopre-  cipitation and immunoblotting for mAKAP␤ (Fig. 4B). Lysates from PLC⑀ Ϫ/Ϫ mouse hearts were used as controls to confirm that bands observed from the immunoprecipitation were the result of association with immunoprecipitated PLC⑀ and not nonspecific immunoprecipitation. mAKAP␤ immunoreactivity was detected in immunoprecipitates from PLC⑀ ϩ/ϩ heart lysates but not in immunoprecipitates from PLC⑀ Ϫ/Ϫ hearts, indicating that mAKAP␤ associates with PLC⑀ in native cardiac tissue. PLC⑀ also co-precipitated with mAKAP␤ when mouse heart extracts were immunoprecipitated with mAKAP␤ antisera (Fig. 4C, lane 3).
Direct Binding of PLC⑀ to mAKAP␤ and Mapping of the Binding Site on the Scaffold-To test for a direct interaction between PLC⑀ and mAKAP and to map the PLC⑀ binding regions on mAKAP, GST fusion proteins corresponding to different domains of the scaffold were purified (Fig. 5A) and tested for binding to purified full-length PLC⑀. First, fragments of mAKAP indicated in Fig. 5A were fused to GST, purified, and tested for binding to purified full-length PLC⑀. A fragment corresponding to mAKAP 586 -1286 consistently bound specifically to PLC⑀, demonstrating direct binding of PLC⑀ to mAKAP and localization to this region of mAKAP (Fig. 5B). The 586 -1286 domain contains three spectrin repeat-like domains, the third of which is involved in interactions with nesprin-1␣ that localizes mAKAP␤ to the nuclear envelope in cardiac myocytes (25). To further dissect these domains, fragments comprising each of the different repeats were fused to GST, purified, and tested for PLC⑀ binding. Of these fragments, GST-615-915 binds the strongest, with fragment GST-1065-1240 also showing some binding (Fig. 5C). The 615-915 fragment of mAKAP contains the first of the three spectrin repeat-like structures (amino acids 772-882). Spectrin repeat-like domains contain two conserved tryptophans (Trp-793 and Trp-867 in the mAKAP first spectrin repeat-like domain) that are important for thermodynamic stability. Mutation of analogous Trp residues to proline in the third spectrin repeat-like domain disrupted interactions between mAKAP and nesprin-1␣. Trp-793 was mutated to proline in the mAKAP spectrin repeat-like domain 1, and binding of this mutant protein fused to GST to purified PLC⑀ was examined (Fig. 5D). This mutation strongly decreased binding of PLC⑀ to this domain, confirming that PLC⑀ binds to the first spectrin repeat-like domain in mAKAP.
Identification of PLC⑀ Functional Domains Required for mAKAP Interactions-To identify specific domains in PLC⑀ that mediate mAKAP binding, PLC⑀ truncated at the C terminus (⌬CT) to delete the Ras association (RA) domains or PLC⑀ with a GEF domain deletion (⌬GEF) was co-expressed with Myc-mAKAP in HEK293 cells (Fig. 6A). The ⌬CT mutant was unable to interact with mAKAP in co-immunoprecipitation assays, whereas the interaction was unaffected by the ⌬GEF mutation (Fig. 6B). Next, either the PLC⑀-RA1 or the PLC⑀-RA2 domains were expressed with Myc-mAKAP followed by Myc-mAKAP immunoprecipitation. Both PLC⑀-RA1 and PLC⑀-RA2 independently bound to co-expressed mAKAP (Fig.  6C). These data indicate that the RA domains are necessary and sufficient for binding of PLC⑀ to mAKAP. Given that the RA domains are important for Rap and Ras interactions, we determined whether activated RapG12V or RasG12V altered PLC⑀-mAKAP binding in transfected cells. Expression of activated Rap or Ras did not affect PLC⑀-mAKAP co-immunoprecipitation (data not shown).
Disruption of mAKAP-PLC⑀ Binding by Expression of PLC⑀ RA1 or mAKAP-SR1-To determine whether expression of PLC⑀-RA1 or mAKAP-SR1 could compete for PLC⑀ binding to mAKAP, mAKAP and PLC⑀ were co-expressed in HEK293 cells, plus or minus co-expression of PLC⑀-RA1 or mAKAP-SR1 domains. Myc-mAKAP protein complexes were immunoprecipitated with Myc antibodies and immunoblotted for PLC⑀. PLC⑀ co-immunoprecipitated with mAKAP, but expression of either PLC⑀-RA1 or mAKAP-SR1 prevented mAKAP-PLC⑀ co-immunoprecipitation (Fig. 7A). To test whether PLC⑀-RA1 or mAKAP-SR1 can disrupt endogenous mAKAP-PLC⑀ signaling complexes in NRVMs, NRVMs were infected with adenoviruses expressing either domain followed by immunoprecipitation of mAKAP and immunoblotting for PLC⑀. PLC⑀ co-immunoprecipitated with mAKAP when YFP was expressed as a control (Fig. 7B), although the complex was difficult to detect. In general, endogenous PLC⑀ is difficult to detect and has to be enriched by direct IP from most tissues to enable adequate detection by immunoblotting. Nevertheless, expression of either PLC⑀-RA1 or mAKAP-SR1 in NRVMs significantly reduced the association of PLC⑀ with mAKAP immunoprecipitates (Fig. 7B), indicating that expression of either of these domains disrupts endogenous mAKAP␤-PLC⑀ complexes in NRVMs.
To confirm the location of endogenous mAKAP␤ in NRVMs, isolated myocytes were fixed and stained with mAKAP antibody (data not shown). As reported previously, there was a very strong perinuclear staining of mAKAP (25). Reliable detection of PLC⑀ by immunocytochemistry was not possible, likely due to its low abundance coupled with nonspecific background staining with PLC⑀ antibodies. Given the interaction with mAKAP␤ and the predominant perinuclear localization of mAKAP, it is likely that PLC⑀ interaction with FIGURE 4. PLC⑀ interacts with mAKAP in transfected HEK293 cells and in native mouse heart tissue. A, HEK293 cells were transfected with Myc-mAKAP alone, PLC⑀ alone, or PLC⑀ with Myc-mAKAP. Cells were lysed and immunoprecipitated (IP) with an anti-Myc specific antibody. Lysates were Western blotted (IB) for either PLC⑀ or Myc. B, Nonidet P40 soluble lysates were prepared from hearts isolated from PLC⑀ ϩ/ϩ mice, or PLC⑀ Ϫ/Ϫ mice as a control, immunoprecipitated with anti PLC⑀ antibodies, and immunoblotted for mAKAP␤. C, Nonidet P40 lysates from PLCe ϩ/ϩ mice as in B were immunoprecipitated with either anti-mAKAP or PLC⑀ antibodies and immunoblotted as indicated. Lysates were directly blotted with mAKAP␤ antisera as loading controls. Each experiment was repeated at least 3 times with similar results. JULY 1, 2011 • VOLUME 286 • NUMBER 26 mAKAP␤ localizes some of the PLC⑀ pool to the perinuclear region of the cell. To confirm the nuclear localization of PLC⑀, NRVMs were fractionated to isolate nuclei and immunoblotted for PLC⑀. PLC⑀ was significantly enriched in the nuclear fraction when compared with whole cell NRVM lysate, consistent with association with nuclear-bound mAKAP. Nuclear enrichment was confirmed by measuring the enrichment of the nuclear protein LAP-2 and the mAKAP␤. Both proteins were greatly enriched relative to the whole cell NRVM lysates. To confirm that the nuclei were free of SR, the lysates were probed for calsequestrin, which was enriched in an SR fraction but was absent from the nuclear fraction. mAKAP␤ spectrin repeat domain 3, but not repeats 1 or 2, has been shown to mediate interactions with nesprin-1␣, which localizes mAKAP to the perinuclear region (25) of cardiac myocytes. Nevertheless, because PLC⑀ interacts with spectrin repeat domain 1, we tested whether expression of either PLC⑀-RA1 or mAKAP-SR1 disrupted perinuclear localization of mAKAP␤ in NRVMs. Neither domain altered the localization of mAKAP␤ as assessed by immunocytochemistry of NRVMs  (supplemental Fig. 3), indicating that these domains effectively disrupt endogenous mAKAP␤-PLC⑀ interactions without influencing mAKAP␤ localization to the nuclear envelope. Thus, expression of PLC⑀-RA1 or mAKAP-SR1 likely disrupts PLC⑀ localization at the nuclear envelope, while leaving the mAKAP␤ scaffold itself undisturbed.

Phospholipase C⑀ Regulation of Hypertrophy
Importance of PLC⑀ Scaffolding to mAKAP in Cardiomyocyte Function-We have shown that PLC⑀ is an important central mediator of hypertrophy and that it is scaffolded to mAKAP␤, which we have also shown to be important for cardiac myocyte hypertrophy (16). To establish the functional relevance of mAKAP␤-PLC⑀ complexes, we tested whether mAKAP␤-PLC⑀ binding was required for the hypertrophy of NRVMs. NRVMs were infected with an adenovirus expressing either PLC⑀-RA1 or mAKAP-SR1 to disrupt PLC⑀-mAKAP binding followed by treatment with 10 nM ET-1 for 48 h. Expression of either PLC⑀-RA1 or mAKAP-SR1 significantly blunted ET-1dependent increases in [ 3 H]leucine incorporation and ANF mRNA expression (Fig. 8, A and B). PLC⑀-RA1 is related to Ras binding domains but does not bind Ras or Rap (34). Nevertheless, to be sure that PLC⑀-RA1 was not inhibiting hypertrophy by blocking Ras or Rap signaling, we measured IGF-1-stimulated ERK phosphorylation, in the presence or absence of PLC⑀-RA1 expression. The IGF-1-stimulated increase in ERK1/2 phosphorylation in NRVMs was not affected by expression of PLC⑀-RA1 (data not shown), indicating that these proteins do not globally interfere with Ras signaling. That two independent approaches to disrupting mAKAP␤-PLC⑀ complexes, using two different binding domains from two different proteins, both significantly inhibited ET-1-dependent hypertrophy strongly suggests that scaffolding of PLC⑀ to mAKAP␤ is required for hypertrophic responses mediated by ET-1. PLC⑀-RA1 expression in NRVMs also significantly inhibited [ 3 H]leucine incorporation in response to Iso, NE, and IGF-1, indicating a general role for nuclear scaffolding of PLC⑀ (supplemental Fig. 4).

DISCUSSION
Here we demonstrate that PLC⑀ plays a central role in mediating hypertrophy downstream of multiple agonists and that subcellular scaffolding of PLC⑀ to mAKAP␤, likely at the nuclear envelope, is critical for this process. Mechanistically, the upstream pathways that regulate PLC⑀ for these hypertrophic responses remain to be defined in NRVMs. Possible mechanisms for activation include G 12/13 -dependent Rho pathways, cAMP-Epac pathways, and Ras pathways. ET A receptors can activate G 12/13 , IGF-1 receptors can activate Ras, and ␤AR can activate cAMP-Epac-Rap, all of which are potential PLC⑀ regulators in NRVMs. ␣1-Adrenergic receptors signal primarily through G q , which would not activate PLC⑀, but G q activation can lead to activation of Rho and Ras pathways that could regulate PLC⑀ activity. That PLC⑀ can respond to such diverse signals ideally positions it to be a central integrator of multiple hormonal signals that impinge on the cardiac myocytes during physiologic and pathological stimulation.  Regardless of the mechanism for activation, the catalytic activity of PLC⑀ is required for hypertrophy because expression of a catalytically inactive form of PLC⑀ does not stimulate hypertrophy and blocks development of ET-1-dependent hypertrophy. PLC activity results in the production of two second messengers, DAG and IP 3 , and roles for both of these molecules have been suggested in hypertrophy. Nevertheless, the data show that PLC⑀ is not responsible for the majority of global IP 3 generation downstream of ET A or ␣1AR. An attractive hypothesis to explain this is that PLC⑀ scaffolded to mAKAP␤ at the nuclear envelope generates IP 3 and DAG locally to regulate nuclear Ca 2ϩ or PKC signals without significantly contributing to the pool of total IPs. In ventricular myocytes, IP 3 receptors are localized primarily to the nuclear envelope and release a pool of nuclear calcium distinct from that involved in calcium-induced calcium release (35,36). This nuclear calcium pool has been implicated in regulating hypertrophic gene expression through two pathways: (i) activation of nuclear CamKII leading to phosphorylation of histone deacetylase 5 (HDAC5) and derepression of myocyte enhancer factor (MEF)-dependent transcription; and (ii) activation of calcineurin-dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) leading to increased nuclear factor of activated T-celldependent transcription. Additionally, PKC activation has been implicated in hypertrophy, and PKC-PKD-dependent phosphorylation of HDAC leads to export of HDAC5 from the nucleus (37). PLC⑀ could be involved in the local generation of either IP 3 or DAG at the nuclear envelope necessary for these processes.
The PLC isoform responsible for the majority of IP 3 production downstream of G q -coupled receptors has been suggested to be PLC␤ because the major known target of G␣ q is PLC␤ (6,38), although G␣ q can also regulate Rho activation through direct stimulation of RhoGEFs (39). Data directly implicating PLC␤ in cardiac hypertrophy have been lacking until recently. These new data show that PLC␤1b overexpression increases NRVM hypertrophy and that inhibition of PLC␤1b sarcolemmal membrane association can inhibit ␣1AR-dependent hypertrophy (9). Our published results in astrocytes (17) and fibroblasts (28) indicate that surprisingly, many G q -coupled receptors generate significant accumulation of total IPs through PLC⑀, suggesting the possibility that some portion of hypertrophy driven through G q -linked receptors could be downstream of PLC⑀ activation. Nevertheless, the bulk of IP 3 generation in NRVMs does not appear to involve PLC⑀ and is likely PLC␤-driven, suggesting that PLC⑀-dependent local phosphatidylinositol 4,5-bisphosphate hydrolysis, distinct from the bulk IP pool, is required for hypertrophic responses. It is possible that hypertrophy in response to ET-1 and NE is both PLC␤1b-dependent and PLC⑀-dependent and that two distinct pools of IP 3 and/or DAG are required for the hypertrophic response.
We previously reported that PLC⑀ activity is important for CamKII-dependent phosphorylation of Ryr2 and regulation of cardiac Ca 2ϩ cycling (41). Based on this observation, we predict that mAKAP␤ scaffolded PLC⑀ represents a pool of PLC⑀ that is distinct from a separately scaffolded pool that regulates RyR2. mAKAP␤ has been reported to bind to RyR2 (26), but this is at odds with the observation that the majority of mAKAP␤ is found at the nuclear envelope, whereas the bulk of RyR2 protein is in the SR. Nevertheless, it remains possible that a small pool of mAKAP␤ can scaffold PLC⑀ to Ryr2. This hypothesis remains to be investigated.
Our data demonstrating a requirement for PLC⑀ in hormone-regulated hypertrophic responses contrast with our previously reported in vivo observation that global PLC⑀ Ϫ/Ϫ mice are more sensitive to the development of hypertrophy in response to chronic Iso treatment (18). A possible explanation for the difference in the cellular and in vivo data is that in the context of development of the PLC⑀ Ϫ/Ϫ mice, compensatory pathways were up-regulated that sensitized mice to hypertrophy. In particular, we found that CamKII phosphorylation is basally increased in PLC⑀ Ϫ/Ϫ mice (supplemental Fig. 5), which could lead to enhanced sensitivity to stress-induced hypertrophy because CamKII activation has been shown to be prohypertrophic (40). CamKII activation is not elevated in PLC⑀ siRNA-treated myocytes (data not shown). It is not unusual for compensatory pathways to be up-or down-regulated in global knock-out mice where the gene product is lost from birth. This suggests that our previous conclusion from global PLC⑀ Ϫ/Ϫ mice suggesting a protective role for PLC⑀ was incorrect. Analysis of mice with cardiac myocyte-specific, acute PLC⑀ knockdown would help to resolve this discrepancy.
Overall, these data demonstrate that PLC⑀ plays a central role in mediating hypertrophy in response to multiple neurohumoral stimuli and that scaffolding to mAKAP␤ is critical for this. The human heart is exposed to multiple hormonal inputs during the development of heart failure. PLC⑀ represents a potentially interesting therapeutic target as a central integrator of these inputs, possibly allowing for more efficacious treatment of heart failure. Because PLC⑀ plays an important role in the CNS, pancreas, and kidney as well as other tissues, targeting PLC⑀ activity directly might not be desirable. Development of an approach that could specifically interfere with PLC⑀-mAKAP␤ interactions could provide a more selective approach to targeting PLC⑀ and its role in hypertrophy.