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. 2009 Jan 16;284(3):1514-22.
doi: 10.1074/jbc.M806994200. Epub 2008 Oct 27.

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II

Emily A Oestreich  1 , Sundeep Malik, Sanjeewa A Goonasekera, Burns C Blaxall, Grant G Kelley, Robert T Dirksen, Alan V Smrcka
Affiliations

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II

Emily A Oestreich et al. J Biol Chem. .

Abstract

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.

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Figures

FIGURE 1.
FIGURE 1.
Epac-mediated enhancement of CICR requires both PLCε hydrolytic and RapGEF activities. A, domain structure of PLCε (CDC25 GEF, Ras family small GTPase guanine nucleotide exchange factor domain; PH, pleckstrin homology; EF, EF-hand Ca2+-binding domain, X and Y, PIP2 hydrolysis catalytic domain; C2, Ca2+-dependent lipid-binding domain; RA1 and RA2, Ras association domains). ΔCDC25(677–772), GEF deletion mutant, no RapGEF activity; H1460L, catalytic domain point mutation, lacks PIP2 hydrolysis activity; K2150E, RA2 domain point mutation, eliminates stimulation of PLC activity by Ras and Rap1. B, PLCε–/– cardiac myocytes were transduced with YFP, PLCε wild type, or PLCε domain mutant adenoviruses. 24 h post-transduction, equal expression of PLCε mRNA was demonstrated by reverse transcriptase-PCR (lower). C, cardiac myocytes were transduced with wild type (WT) and mutant PLCε viruses and protein expression was measured after 48 h by Western blotting. D and E, average (±S.E.) peak Ca2+ transient amplitude (Δ405/485) for naïve PLCε–/– myocytes and 24 h post-transduction with YFP control, PLCε wild type, or PLCε domain mutant adenovirus (300 multiplicity of infection) with or without 1 μm isoproterenol (D) or 10 μm cpTOME (E). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 2.
FIGURE 2.
PLCε is required for βAR-mediated Rap activation in cardiac myocytes. Hearts from PLCε+/+ or PLCε–/– mice were cannulated through the aorta and perfused with or without 1 μm isoproterenol for 10 min. 1 mg of heart lysate was incubated with either GST-tagged RalGDS-RBD for assaying activated Rap or GST-tagged Raf1-RBD for activated Ras. Gβ1 was used as a gel loading control. Data are representative of 3 experiments showing similar results.
FIGURE 3.
FIGURE 3.
Pharmacological inhibition of PKC attenuates βAR enhancement of CICR. A, IP3R inhibition with 2-APB (20 μm, 5 min pre-treatment) does not inhibit βAR-dependent increases in Ca2+ transient amplitude (Δ405/485). Ca2+ transients were measured in the absence and presence of 1 μm isoproterenol. Data are pooled from 10 to 15 cells per treatment condition. Results are average (±S.E.). B and C, PKC inhibition (1 μm BIM, 5 min pre-treatment) significantly blunts (B) isoproterenol- and (C) cpTOME-induced increases in Ca2+-transient amplitude in PLCε+/+, but not PLCε–/– cardiac myocytes. BIM did not affect naïve Ca2+ transient amplitude. Data were pooled from 20–40 cells for each treatment condition from n = 3 PLCε+/+ and n = 2 PLCε–/– mice. Results are average (±S.E.); ***, p < 0.001; #, p < 0.001 for PLCε–/– response compared with PLCε+/+; ns is not significant; one-way ANOVA, Bonferroni post test.
FIGURE 4.
FIGURE 4.
PLCε-dependent enhancement of CICR requires specific activation of PKCε. A, left, PKCε translocates to the membrane fraction following treatment with 1 μm isoproterenol (30 s) or 10 μm cpTOME (3 min) in PLCε+/+ but not PLCε–/– cardiac myocytes. PKCα does not translocate to the membrane in response to βAR stimulation. PMA treatment (500 nm, 10 min) was used as a positive control for PKC translocation. 3 μg of cardiac myocyte membrane fractions was analyzed for PKC isoform translocation. Gβ subunit was used as a loading control. Right, densitometric quantitation of PKCε membrane translocation from cells isolated from 5 PLCε+/+ and 3 PLCε–/– mice. Data are represented as a percentage of maximal translocation evoked by PMA treatment. **, p < 0.01; ***, p < 0.001; ns, not significant as compared with nontreated PLCε+/+ cells. One-way ANOVA, Bonferroni post-test. B, left, PLCε+/+ cardiac myocytes were transfected with PKCε-specific siRNA or a CY3-labeled negative control siRNA. PKCε protein levels are nearly completely knocked down in cardiac myocytes transfected with PKCε-specific siRNA relative to negative control siRNA at 36 h post-transfection. Lower left, PKCα protein levels are not significantly affected by PKCε siRNA 36 h post-transfection. Right, densitometric quantitation of PKCε protein expression from myocytes transfected with either PKCε siRNA or Cy3-labeled negative control siRNA pooled from three separate experiments. C, knockdown of PKCε significantly decreases isoproterenol-induced enhancement of CICR and completely eliminates cpTOME responsiveness in PLCε+/+ cardiac myocytes. Data are pooled Ca2+ transient amplitudes (Δ404/485) from 20 to 40 cells per condition, n = 3 mice. Results are average (±S.E.); ***, p < 0.001, one-way ANOVA, Bonferroni post-test.
FIGURE 5.
FIGURE 5.
PKC-dependent activation of CamKII is required for PLCε-mediated enhancement of CICR. CamKII inhibition (1 μm KN-93, 30 min pretreatment) significantly blunts (A) Iso and (B) cpTOME (10 μm) enhancement of electrically evoked Ca2+ transient amplitude in AVM from PLCε+/+, but not PLCε–/– mice. Pretreatment with KN92, an inactive analogue of KN-93, does not affect Iso responsiveness. Data were pooled from 5 to 15 cells for each treatment condition from 3 PLCε+/+ and 3 PLCε–/– mice. Results are average (±S.E.); ***, p < 0.001; ns, not significant; one-way ANOVA, Bonferroni post test. C, phosphorylation of CamKII at Thr286 was measured by Western blotting of extracts of AVM treated with Iso (1 μm), cpTOME (10 μm), and BIM (1 μm) as indicated (n = 5 animals). Right panel, pooled data from densitometric quantitation. Results are average (±S.E.); *, p < 0.05; one-way ANOVA, Bonferroni post test. D, phosphorylation of RyR2 Ser2815 and PLB Thr17 was measured by Western blotting of extracts of AVM treated with cpTOME (10 μm) or BIM (1 μm) as indicated (n = 3 animals each). Right panel, pooled data from densitometric quantitation and normalized relative to untreated cells. Results are average (±S.E.); **, p < 0.01; ***, p < 0.001; one-way ANOVA, Bonferroni post test.
FIGURE 6.
FIGURE 6.
PLCε ablation significantly reduces isoproterenol stimulation of depolarization-induced intracellular calcium transients without altering L-type Ca2+ current density. Perforated patch clamp experiments were used to simultaneously monitor depolarization-induced L-type Ca2+ currents (A and B) and intracellular Ca2+ transients (C and D) in the absence (closed symbols) and presence (open symbols) of βAR-stimulation with 1 μm isoproterenol in AVM from PLCε+/+ (circles) and PLCε–/– (squares) mice. A, average (±S.E.) voltage dependence of L-type Ca2+ current density. B, average (±S.E.) fold stimulation of maximal L-type Ca2+ conductance in AVM from 5 PLCε–/– and 5 PLCε+/+ mice. C, average (±S.E.) voltage dependence of intracellular Ca2+ transients. D, average (±S.E.) fold stimulation of peak intracellular Ca2+ transient (measured at –30 mV) in AVM from 5 PLCε–/– and 5 PLCε+/+ mice. *, p < 0.05, t test.
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