Phospholipase C epsilon scaffolds to muscle-specific A kinase anchoring protein (mAKAPbeta) and integrates multiple hypertrophic stimuli in cardiac myocytes

Abstract

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.

FIGURE 1.

PLCϵ is required for ET-1-dependent cardiomyocyte hypertrophy. NRVMs were infected with PLCϵ-siRNA or random siRNA (Ran siRNA) adenoviruses followed by stimulation with 10 nmol/liter ET-1 as described under “Experimental Procedures.” A, [³H]leucine incorporation was measured as described under “Experimental Procedures.” Data are combined results from four separate preparations of myocytes with each experiment performed in triplicate. Con, control. B, NRVMs were fixed and stained with α-actinin antisera as described under “Experimental Procedures.” Scale bar is 100 μm. C, NRVM surface areas were calculated using the NIH ImageJ software from over 400 myocytes for each condition. Data were pooled from 2 independent experiments. D, ANF mRNA was measured by real time PCR and normalized to GAPDH as described under “Experimental Procedures.” Data are combined from two different preparations of cells. Error bars indicate S.E. *, p < 0.05, ***, p < 0.001.

FIGURE 2.

PLCϵ mediates cardiomyocyte hypertrophy downstream of multiple signaling pathways. Cells were treated as in Fig. 1 except with 10 μmol/liter Iso (A), 10 nmol/liter IGF-1 (B), or 10 μmol/liter NE (C). [³H]Leucine incorporation and ANF mRNA were measured as in Fig. 1. Data are pooled from three preparations of NRVMs for leucine incorporation performed in triplicate or two preparations of NRVMs for ANF measurement. Error bars indicate S.E. **, p < 0.01, ***, p < 0.001. Con, control; Ran siRNA, random siRNA.

FIGURE 3.

PLCϵ hydrolytic activity promotes hypertrophy but does not contribute significantly to global IP₃ production in NRVMs. A, NRVMs were infected with adenoviruses expressing either PLCϵ or catalytically inactive PLCϵ(H1460L) and treated with or without ET-1 for 48 h. [³H]Leucine incorporation was measured as described under “Experimental Procedures.” Data are pooled results from 3 separate experiments performed in triplicate, ***, p < 0.001. Error bars indicate S.E. CON, control. B and C, total inositol phosphates were measured after a 30-min treatment of NRVMs with or without (CON) 100 nm ET-1 (B) or 100 μmol/liter NE (C). Data are combined results from 2 separate experiments performed in triplicate. N.S., not significant; RansiRNA, random siRNA.

FIGURE 4.

PLCϵ interacts with mAKAP in transfected HEK293 cells and in native mouse heart tissue. A, HEK293 cells were transfected with Myc-mAKAP alone, PLCϵ alone, or PLCϵ with Myc-mAKAP. Cells were lysed and immunoprecipitated (IP) with an anti-Myc specific antibody. Lysates were Western blotted (IB) for either PLCϵ or Myc. B, Nonidet P40 soluble lysates were prepared from hearts isolated from PLCϵ^+/+ mice, or PLCϵ^−/− mice as a control, immunoprecipitated with anti PLCϵ antibodies, and immunoblotted for mAKAPβ. C, Nonidet P40 lysates from PLCe^+/+ mice as in B were immunoprecipitated with either anti-mAKAP or PLCϵ antibodies and immunoblotted as indicated. Lysates were directly blotted with mAKAPβ antisera as loading controls. Each experiment was repeated at least 3 times with similar results.

FIGURE 5.

Direct binding of PLCϵ to mAKAP subdomains. A, diagram of mAKAP showing defined domains for binding PDK1, AC5, RyR2, and PDE4D3, and below, showing the regions that were cloned as GST fusion proteins. B, GST fusion proteins corresponding to the larger fragments shown in A were tested for binding to purified PLCϵ. The top panel is a Western blot (IB) for PLCϵ after pulldown with the indicated GST fragments. The bottom panel is a Coomassie Blue-stained gel of the purified GST fusion proteins. C, GST fusion proteins corresponding to the smaller subfragments of the 586–1286 binding domain defined in B were tested for binding to purified PLCϵ. The top panel is a Western blot for PLCϵ after pulldown with the indicated GST fragments. The bottom panel is a Coomassie Blue-stained gel of the purified GST fusion proteins. D, the 615–915 fragment defined in C was mutated by site-directed mutagenesis (W793P), purified, and tested for binding to purified PLCϵ. The top panel is a Western blot for PLCϵ after pulldown with the indicated GST fragments. The bottom panel is a Coomassie Blue-stained gel of the purified GST fusion proteins. Each experiment was repeated at least 3 times with similar results.

FIGURE 6.

The RA domains of PLCϵ bind to mAKAP. A, domain structure of PLCϵ showing the CDC25 guanine nucleotide exchange factor domain (CDC25 GEF), a putative pleckstrin homology domain (PH), the EF hand domain, the X and Y catalytic domain, the C2 lipid binding domain, and the RA homology domains. Only RA2 binds Ras. B, Myc-mAKAP alone or with the indicated PLCϵ constructs was cotransfected into HEK293 cells, and Myc-mAKAP was immunoprecipitated (IP) followed by immunoblotting (IB) with anti-PLCϵ antibody (top panel) or Myc antibody as a control (bottom panel). C, Myc-mAKAP was cotransfected with either PLCϵ-RA1 or PLCϵ-RA2 domains. Cells were lysed and immunoprecipitated with anti-Myc antibodies and immunoblotted with the antibodies indicated on the right. Each experiment was repeated at least 3 times with similar results.

FIGURE 7.

The PLCϵ-RA1 and mAKAP-SR1 domains compete for mAKAP-PLCϵ interactions. A, PLCϵ and Myc-mAKAP were cotransfected (Transfect.) into HEK393 cells in the presence or absence of PLCϵ-RA1 or mAKAP-SR1 domains. Myc-mAKAP was immunoprecipitated (IP) and Western blotted (IB) for PLCϵ (top panel) or Myc (bottom panel). Each experiment was repeated at least 3 times with similar results. B, NRVMs were infected with adenoviruses expressing YFP, PLCϵ-RA1, or mAKAP-SR1 for 48 h. Lysates were prepared (1 mg of protein, PLCϵ-RA1-infected cells, or 2 mg of protein, mAKAP-SR1-infected cells), and mAKAPβ was immunoprecipitated. Immunoprecipitates were immunoblotted for either PLCϵ or mAKAPβ. This experiment was repeated twice. C, NRVMs were fractionated to isolate nuclei and 10 μg of protein was analyzed by Western blotting. SR/PM, sarcoplasmic reticulum/plasma membrane.

FIGURE 8.

PLCϵ-RA1 or mAKAP-SR1 expression in neonatal cardiac myocytes inhibits development of cardiac hypertrophy in response to ET-1 treatment. Cells infected with adenovirus expressing either PLCϵ-RA1 or YFP (A) or mAKAP-SR1 or YFP (B) were treated with or without (CON) 10 nmol/liter ET-1 for 48 h followed by measurement of [³H]leucine incorporation or ANF mRNA. Data were normalized to YFP with no ET-1 treatment (CON). Data are the combined results from 3 separate experiments performed in triplicate for leucine incorporation or 2 separate experiments for ANF mRNA. Error bars indicate S.E. *, p < 0.05, **, p < 0.01, ***, p < 0.001.