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Originally published In Press as doi:10.1074/jbc.M608495200 on December 17, 2006

J. Biol. Chem., Vol. 282, Issue 8, 5488-5495, February 23, 2007
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Epac-mediated Activation of Phospholipase C{epsilon} Plays a Critical Role in beta-Adrenergic Receptor-dependent Enhancement of Ca2+ Mobilization in Cardiac Myocytes*

Emily A. Oestreich{ddagger}, Huan Wang§, Sundeep Malik{ddagger}, Katherine A. Kaproth-Joslin, Burns C. Blaxall||, Grant G. Kelley**, Robert T. Dirksen{ddagger}, and Alan V. Smrcka{ddagger}1

From the {ddagger}Departments of Pharmacology and Physiology and the ||Cardiovascular Research Institute, University of Rochester School of Medicine, Rochester, New York 14642, §Systems Biology Division, The Wistar Institute, Philadelphia, Pennsylvania, 19104, and Departments of Pharmacology and **Medicine, SUNY Upstate Medical University, Syracuse, New York 13210

Received for publication, September 5, 2006 , and in revised form, December 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently we demonstrated that PLC{epsilon} plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC{epsilon} regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca2+ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC{epsilon}-/- cardiac myocytes but rescued by transduction with PLC{epsilon}, indicating that Epac is upstream of PLC{epsilon}. Furthermore, transduction of PLC{epsilon}+/+ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLC{epsilon} pathway. We also show that Epac/PLC{epsilon}-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC{epsilon} that enhances intracellular Ca2+ release in cardiac myocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of adrenergic receptors by either neurohumoral or systemic release of the catecholamines epinephrine and norepinephrine produces acute increases in cardiac contractility during stress and exercise to increase cardiac output and oxygen delivery to tissues. Much of the increase in cardiac output is due to the direct stimulation of the beta adrenergic receptor (betaAR)2 in cardiac myocytes (1, 2). Activation of betaAR activates Gs and adenylyl cyclase resulting in the production of cAMP and subsequent activation of protein kinase A, which phosphorylates key components of the calcium handling and contractile machinery.

Analysis of a phospholipase C (PLC){epsilon} knock-out mouse model (PLC{epsilon}-/-) generated in our laboratory indicates that PLC{epsilon} contributes to betaAR-dependent regulation of cardiac function (3). PLC{epsilon}-/- mice exhibit significantly decreased left ventricular developed pressure in response to acute stimulation with the betaAR agonist, isoproterenol. Isolated myocytes from PLC{epsilon}-/- mice exhibit decreased isoproterenol-dependent enhancement of electrically evoked Ca2+ release in the absence of effects on betaAR density or cAMP generation. These data implicate PLC{epsilon} as a novel component of betaAR regulation of Ca2+ release, which had not previously been described in the heart. However, the pathway linking betaAR stimulation to PLC{epsilon} activation is unknown.

PLC-mediated phosphatidyl-4,5-bisphosphate hydrolysis resulting in intracellular Ca2+ release and protein kinase C activation is an integral signaling component of many physiological processes in a variety of tissues, including the cardiovascular system (4, 5). PLC{epsilon} is a recently identified PLC isoform expressed in the heart as well as other tissues (6–8). A particularly unique feature of this enzyme is direct regulation of its phosphatidyl-4,5-bisphosphate hydrolysis activity by Ras and Ras family members including Rap (6, 8–11). In addition, PLC{epsilon} has guanine nucleotide exchange factor activity and can activate small G proteins such as Rap1 (12). Thus, PLC{epsilon} is uniquely positioned to respond to multiple receptor inputs in the heart and coordinate both PLC and guanine nucleotide exchange factor activities that could be important for cardiac function.

Here we have demonstrated for the first time a new pathway involving PLC{epsilon} and the cAMP-responsive Rap guanine nucleotide exchange factor, Epac, in adult ventricular cardiac myocytes directly downstream of betaAR-dependent cAMP generation and upstream of CICR. Epac has recently been demonstrated to mediate cAMP responses in a number of systems (13) and has been shown to activate PLC{epsilon} in a Rap-dependent manner (14). We show here that cAMP-stimulated Epac/Rap1-dependent activation of PLC{epsilon} acts in concert with the classical cAMP-dependent activation of PKA to facilitate betaAR-mediated enhancement of intracellular Ca2+ release from the sarcoplasmic reticulum in cardiac myocytes. This novel pathway introduces a new level of regulation of cardiac function by the betaAR system and is likely to have implications for the treatment of cardiac disease.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Adult Ventricular Cardiac Myocytes—Adult ventricular cardiac myocytes (AVMs) were isolated from 4-month-old wild-type or PLC{epsilon}-/- mice as previously described (3). Mice were anesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight) by intraperitoneal injection. For all experiments, cells were plated at a density of 10,000 cells/35-mm dish on laminin (BD Biosciences)-coated coverslips in minimum essential medium supplemented with 2 mM L-glutamine, 2.5% fetal bovine serum (Hyclone), and 1% penicillin/streptomycin with the addition of 1 µM Blebbistatin (Sigma) to prevent spontaneous contraction.

Measurement of Electrically Evoked Ca2+ Transients in Single Ventricular Myocytes—Cells were loaded with 2 µM Indo-1/AM (Molecular Probes) in plating medium and then transferred to Ringer solution (145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes). Individual cells were excited at 350 nm, and fluorescence emission at 405 and 485 nm was collected at 100 points/s and monitored as fluorescence ratio (405:485). Following an initial 20-s equilibration in control Ringer's solution or Ringer's solution containing 1 µM isoproterenol (Sigma) or other activators or inhibitors, myocytes were electrically stimulated (20 ms, 8 V, 0.5–1 Hz, 60 s) in the presence or absence of agonist/inhibitors using an extracellular electrode placed close to the cell of interest. Ca2+ transient magnitude was assessed from the change in 405/485 ratio averaged across five consecutive electrical pulses/cell. For each experiment, data were collected from 5–15 cells in the absence of agonist stimulation to assess the magnitude and kinetics of naïve electrically stimulated Ca2+ transients. The kinetics of the Ca2+ removal following evoked release was estimated by fitting a standard single exponential to the decay phase of the Ca2+ transient.

Pharmacological Inhibition of PLC Activity in PLC{epsilon}+/+ and PLC{epsilon}-/- Ventricular Myocytes—After data collection in the absence of betaAR stimulation, cells were perfused with 1 µM isoproterenol (Sigma) for 20 s followed by a 60-s train of electrical stimuli in the presence of isoproterenol at a pace of 1 Hz. Data were collected for 5–15 cells in the presence of isoproterenol alone. 2 µM U73122 [GenBank] [1-(6-[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]-amino/hexyl)-1H-pyrroledione] or U73343 [GenBank] [1-(6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]-hexyl-2,5-pyrrolidine-dione], an inactive analog of U73122 [GenBank] as control (Sigma), was added to the bath with constant perfusion along with 1 µM isoproterenol perfusion for 20 s followed by 60 s of electrical stimuli, and data were analyzed once a maximal response was reached for 5–15 cells. Once maximal Ca2+ transient amplitude was reached, no decrease in amplitude over time was observed during the course of the measurements.

Pharmacological Manipulation of Epac and PKA—For Epac activation, after data were collected in the absence of agonist, cells were constantly perfused with 10 µM cpTOME (Calbiochem) for 20 s followed by a 60-s train of electrical stimuli at 1 Hz. The change in 405/485 ratio was determined for 10–20 cells/animal. For PKA activation, cells were pretreated for 30 min with 300 µM 6-Bnz-cAMP (Axxora, LLC San Diego, CA) during Indo-1/AM loading and data were then collected as previously described. 300 µM 6-Bnz-cAMP was present throughout the experiment. For PKA inhibition, cells were treated with 10 µM H89 (Calbiochem) alone prior to perfusion with 1 µM isoproterenol. H89 was present throughout perfusion. For inhibition of PKA-specific responses, cells were pretreated with 10 µM H89 plus 300 µM 6-Bnz-cAMP for 30 min followed by constant perfusion with 300 µM 6-Bnz-cAMP. Alternatively, cells were pretreated for 30 min with a combination of 350 µM Rp-cAMPs and 8-Br-Rp-cAMPs (Axxora) to assess PKA inhibition. Data were collected as previously.

Transduction of Cardiac Myocytes with Adenovirus—Adenoviruses were prepared using the AdEasy system with the cytomegalovirus promoter used to drive expression of either yellow fluorescent protein (YFP) or FLAG-PLC{epsilon}. Adult AVMs were isolated and adhered to laminin-coated coverslips for 2 h preinfection. The plating medium was then removed and replaced with fresh medium containing 300 m.o.i. of either YFP control, PLC{epsilon}, or RapGAP1 adenovirus. RapGAP1 adenovirus was kindly provided by Dr. Patrick Casey, Duke University (15). After 2 h, the virus was removed, and fresh medium was added to the cells. PLC{epsilon} expression was not detectable by direct Western analysis at 24 h but was detectable at 48 h. PCR analysis described below was used to detect PLC{epsilon} expression at 24 h. Because endogenous PLC{epsilon} was comparable with adenovirally expressed PLC{epsilon} at 24 h (based on reverse transcription (RT)-PCR analysis), experiments were performed 24 h post-infection to minimize PLC{epsilon} overexpression and more closely mimic endogenous expression levels. For Rap dominant negative inhibition, myocytes were infected with an adenovirus-expressing RapGAP1 driven by a cytomegalovirus promoter. Cells were analyzed 48 h post-infection to maximize both RapGAP1 overexpression and Rap inhibition. Indo-1/AM fluorescence ratio (405/485) was assessed as previously ±1 µM isoproterenol or ±10 µM cpTOME. Myocytes were electrically stimulated at 0.5 Hz in all experiments with adenovirally infected AVMs.

Immunoblotting—For Epac and Rap1 expression level comparisons, tissue lysates were prepared from hearts excised from 4-month-old wild-type and PLC{epsilon}-/- mice. To assess expression of proteins by adenovirus and expression levels of PKA subunits, AVM cell lysates were prepared. Proteins were resolved by standard SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) for blotting. Epac rabbit polyclonal antibody (used at 1:1000) was from Upstate%20Biotechnology">Upstate Biotechnology, Rap1 and Rap1GAP rabbit polyclonal antibodies (used at 1:200) were from Santa Cruz Biotechnology, and PKA mouse monoclonal antibodies (used at 1:1000) were from BD Transduction Laboratories. PLC{epsilon} rabbit polyclonal antibody was kindly provided by Dr. Grant Kelley, SUNY Upstate Medical University (used at 1:3000). G protein beta-subunit rabbit polyclonal antibody, B600 (16), was used at 1:40,000.

RT-PCR—For detection of PLC{epsilon} mRNA in PLC{epsilon}+/+, PLC{epsilon}-/-, and PLC{epsilon}-/- AVMs transduced with FLAG-PLC{epsilon}-expressing adenovirus (300 m.o.i., 10,000 cells), RNA was prepared using the RNAeasy minikit (Qiagen, Inc., Valencia, CA) following the recommendations of the manufacturer. The Superscript III RT-PCR kit (Invitrogen) was used with 150 ng of total RNA template for RT-PCR reactions with the mouse PLC{epsilon} primers 5'-ACCCTGCGGTAAATGTTCTG-3' and 5'-ATGTGAATTCCGTGCTACCC-3' to give a predicted 300-bp product. GAPDH primers 5'-CAACGGGAAGCCCATCACCAT-3' and 5'-CCTTGGCAGCACCAGTGGATGC-3' yielded a predicted 350-bp product. RT was done for 30 min at 42 °C followed by incubation at 94 °C for 2 min. The PCR parameters were denaturation at 94 °C for 30 s, annealing at 45 °C for 45 s., and extension at 72 °C. The number of PCR cycles was 23, 26, and 30 for GAPDH and 30, 32, and 35 for PLC{epsilon}.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We recently reported a significant decrease in beta-adrenergic receptor-dependent enhancement of electrically evoked Ca2+ release in ventricular myocytes isolated from PLC{epsilon}-/- mice (3) and hypothesized that PLC{epsilon} plays a direct role in the betaAR-signaling cascade. PLC activation as a result of betaAR stimulation in the heart has not been previously described. A potential pathway from the betaAR to PLC{epsilon} demonstrated in human embryonic kidney 293 fibroblasts involves stimulation of GS, elevation of cAMP, activation of Epac, increased guanine nucleotide exchange on Rap, and subsequent PLC{epsilon} activation (14). To investigate whether this pathway operates in cardiac myocytes, we utilized cpTOME, a cAMP analog with preferential specificity for Epac activation (17). In freshly isolated AVMs, electrically evoked Ca2+ transients were assessed in the presence and absence of 10 µM cpTOME. Surprisingly, cpTOME treatment alone increased the magnitude of electrically stimulated Ca2+ transients ~2-fold (Fig. 1, A and B) in PLC{epsilon}+/+ myocytes. Strikingly, however, cpTOME did not effect electrically evoked Ca2+ release in PLC{epsilon}-/- myocytes (Fig. 1, A and B), supporting the idea that Epac acts upstream of PLC{epsilon}. Western blotting confirmed that Epac and Rap expression are not significantly altered in heart tissue from PLC{epsilon}-/- mice (Fig. 1C).

To provide further evidence for involvement of PLC{epsilon} in Epac regulation of CICR, PLC{epsilon} was re-introduced into PLC{epsilon}-/- myocytes by adenoviral transduction. PLC{epsilon} mRNA was detected by semiquantitative RT-PCR at levels similar to PLC{epsilon}+/+ AVMs after 24 h of adenoviral infection (Fig. 1D), but at this point, adenovirally expressed PLC{epsilon} protein was not reliably detectable by immunoblotting. In general, this 250-kDa protein does not express to high levels even with adenoviral transduction. PLC{epsilon} protein was detectable at 48 h (Fig. 1E), demonstrating that PLC{epsilon} protein is expressed by this virus in AVMs. We chose to assay myocyte function 24 h post-infection to more closely mimic low endogenous levels of PLC{epsilon} expression. The cpTOME-induced increase in Ca2+ transient amplitude was completely rescued by re-introduction of PLC{epsilon} into PLC{epsilon}-/- myocytes (Fig. 1F). Together, these data indicate that an Epac-PLC{epsilon} pathway exists in cardiac myocytes to regulate CICR that is activated by cAMP downstream of betaAR stimulation.


Figure 1
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  		FIGURE 1.
Epac-mediated increase in Ca2+ transient amplitude requires PLC{epsilon}. A, representative electrically evoked (8 V, 20 ms, 1 Hz) Ca2+ transients (405/485 nm) from single PLC{epsilon}+/+ and PLC{epsilon}-/- cardiac myocytes loaded with Indo-1/AM. Black arrows indicate the addition of 10 µM cpTOME. B, average (±S.E.) peak Ca2+ transient amplitude ({Delta}405/485 nm) for PLC{epsilon}+/+ and PLC{epsilon}-/- myocytes in the absence and presence of 10 µM cpTOME. Data are pooled from 30–60 cells for each treatment obtained from three separate pairs of PLC{epsilon}+/+ and PLC{epsilon}-/- mice. ***, p < 0.001, one-way ANOVA, Bonferroni post-test. C, Epac and Rap1 levels in PLC{epsilon}-/- and PLC{epsilon}+/+ hearts. 5 µgof cytosolic protein from total heart extracts were analyzed by Western blotting. G proteinbeta-subunit was analyzed as a loading control. D, RT-PCR using 150 ng of total RNA isolated from AVMs 24 h post-transduction with a PLC{epsilon} adenovirus (300 m.o.i.). E, at 48 h post-transduction, PLC{epsilon}-/- myocyte extracts were assessed by Western blotting for PLC{epsilon} protein. Western blots were also assessed for muscle-specific A kinase-anchoring protein (mAKAP) expression to confirm equivalent protein loading. F, PLC{epsilon}-/- myocytes transduced with either YFP control or PLC{epsilon} adenovirus (300 m.o.i.). After 24 h of PLC{epsilon} expression, cells were treated with or without 10 µM cpTOME. ***, p < 0.001, one-way ANOVA, Bonferroni post-test.

 
Because the primary function of Epac is to act as a guanine nucleotide exchange factor for Rap GTPases and Rap is a known activator of PLC{epsilon}, we hypothesized that Rap activation is required for maximal enhancement of Ca2+ release following betaAR stimulation. To implicate Rap in the betaAR pathway, we transduced wild-type AVMs with an adenovirus-encoding expression of RapGAP1 (Fig. 2A), a protein that accelerates GTP hydrolysis on activated Rap1, returning Rap1 to its GDP-bound inactive state (18). Expression of RapGAP1 has been used to inhibit Rap1 and implicate Rap function in a number of systems (15, 19, 20). Wild-type myocytes expressing RapGAP1 had 40% decreased responsiveness to isoproterenol stimulation compared with myocytes transduced with a control adenovirus encoding YFP (Fig. 2B). The resulting level of isoproterenol responsiveness is comparable with that observed for PLC{epsilon}-/- myocytes (3) (see Fig. 4B). PLC{epsilon}-/- myocytes expressing RapGAP1 showed no further decrease in isoproterenol responsiveness (data not shown), supporting a specific role for Rap-mediated PLC{epsilon} activation downstream of betaAR stimulation. As expected, RapGAP1 expression almost completely suppressed the response to cpTOME in wild-type AVMs (Fig. 2C). Overall, these data indicate Rap activity is required for betaAR-mediated activation of PLC{epsilon} and places the Epac-Rap-PLC{epsilon} pathway downstream of betaAR stimulation.

The data in Figs. 1 and 2 provide strong evidence that the Epac/Rap/PLC{epsilon} pathway contributes to betaAR signaling in the heart. Classically, betaAR signaling in cardiac myocytes has been thought to rely on PKA activation. To assess relative contributions of PKA and Epac signaling to betaAR responses, we treated isolated myocytes from either wild-type or PLC{epsilon}-/- mice with a selective activator of PKA, 6-Bnz-cAMP (17). 6-Bnz-cAMP treatment enhanced electrically evoked Ca2+ release as expected; however, the increase was significantly lower than that produced by isoproterenol (Fig. 3A). To further confirm the idea that a portion of the isoproterenol response is PKA-independent, AVMs were treated with H89, a PKA inhibitor that does not affect the Epac pathway (Fig. 3A) (17). H89 treatment partially inhibited isoproterenol-dependent enhancement of evoked Ca2+ release but completely blocked 6-Bnz-cAMP-dependent enhancement. This indicates that H89 completely inhibits the PKA pathway but only partially inhibits the isoproterenol-dependent pathway, supporting the idea that a signaling pathway in addition to PKA activation is required to support full isoproterenol responsiveness. Importantly, H89 does not significantly inhibit the response to cpTOME and thus does not inhibit the Epac-PLC{epsilon} pathway. We also examined whether Epac and PKA pathways could additively reconstitute the maximal isoproterenol response. PLC{epsilon}+/+ AVMs were treated with 6-Bnz-cAMP alone to activate PKA, cpTOME alone to activate Epac, or both to stimulate both pathways concomitantly. Both 6-Bnz-cAMP and cpTOME treatment alone significantly enhanced electrically evoked Ca2+ release. As expected in both cases, this enhancement was less than that of isoproterenol treatment. However, co-treatment of AVMs with 6-Bnz-cAMP and cpTOME resulted in an additive enhancement of CICR equivalent to that of isoproterenol treatment (Fig. 3B).

Based on these data, we hypothesized that the Epac/Rap/PLC{epsilon} pathway acts in parallel and independent of the canonical PKA pathway following betaAR stimulation in the heart. If this is true, the PKA pathway downstream of cAMP accumulation should be unaffected in PLC{epsilon}-/- myocytes. To ensure that PKA activity was intact in PLC{epsilon}-/- myocytes, freshly isolated myocytes were treated with 6-Bnz-cAMP to assess the effects on electrically evoked Ca2+ transient amplitude. As expected, 6-Bnz-cAMP produced a 2.4-fold increase in Ca2+ transient amplitude in PLC{epsilon}-/- myocytes (Fig. 3C). Importantly, this level of responsiveness was equivalent to both maximal isoproterenol responses observed in PLC{epsilon}-/- myocytes (Fig. 3C) and 6-Bnz-cAMP stimulation in PLC{epsilon}+/+ myocytes (Fig. 3D). H89 completely blocked both isoproterenol-dependent and 6Bnz-cAMP-dependent enhancement of Ca2+ transients in myocytes isolated from PLC{epsilon}-/- mice (Fig. 3E). A second PKA inhibitor combination of Rp-cAMPs and 8-Br-Rp-cAMPs also completely inhibited both isoproterenol-dependent and 6-Bnz-cAMP-dependent enhancement of Ca2+ transients in PLC{epsilon}-/- AVMs (17). Finally, expression of the PKA regulatory and catalytic subunits is not significantly altered in hearts isolated from PLC{epsilon}-/- mice (Fig. 3F). Together, these data indicate that the PKA pathway is intact in AVMs from PLC{epsilon}-/- mice and is responsible for the remainder of the isoproterenol-dependent enhancement of evoked Ca2+ release when the Epac/PLC{epsilon} pathway has been ablated in PLC{epsilon}-/- animals.


Figure 2
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  		FIGURE 2.
Epac-mediated Rap1 activation is required for PLC{epsilon}-dependent enhancement of electrically evoked Ca2+ release. A, PLC{epsilon}-/- myocytes transduced with either YFP control or RapGAP1 adenovirus (300 m.o.i.). After 48 h, the myocytes were lysed directly in SDS-PAGE sample lysis buffer, and 10 µg of protein was analyzed for RapGAP1 expression by Western blotting. B, Ca2+ transient amplitude ({Delta}405/485) for YFP- or RAPGAP1-expressing myocytes in the absence or presence of 1 µM isoproterenol. Data are pooled from 30–60 cells for each treatment obtained from three separate pairs of PLC{epsilon}+/+ and PLC{epsilon}-/- mice. **, p < 0.01 YFP versus RapGAP1 with isoproterenol (ISO); one-way ANOVA, Bonferroni post-test. C, Ca2+ transient amplitude ({Delta}405/485) for YFP- or RapGAP1-expressing myocytes in the presence or absence of 10 µM cpTOME. Data are pooled from 30–60 total cells for each treatment obtained from three separate pairs of PLC{epsilon}+/+ and PLC{epsilon}-/- mice. ***, p < 0.001 YFP versus RapGAP1 with isoproterenol, one-way ANOVA, Bonferroni post-test.

 


Figure 3
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  		FIGURE 3.
Relative roles of PKA and Epac in regulation of myocyte CICR. A, PKA inhibition with H89 only partially blocks isoproterenol (ISO)-enhanced Ca2+ release, and PKA activation only partially increases Ca2+ release in PLC{epsilon}+/+ myocytes. Ca2+ release ({Delta}405/485 nm) was measured following activation of betaAR (1 µM isoproterenol), PKA (300 µM 6-Bnz-cAMP, 30 min pre-incubation during Indo-1/AM loading), or Epac (10 µM cpTOME). PKA was inhibited with H89 (2 µM). Results are the average (±S.E.); ***, p < 0.001; **, p < 0.01; nd, no difference one-way ANOVA, Bonferroni post-test. H89 had no effect on basal Ca2+ transients. B, effects of co-treatment with 6-Bnz-cAMP (300 µM, 30min) and 10 µM cpTOME on electrically evoked Ca2+ release in PLC{epsilon}+/+ myocytes are additive and equivalent to treatment with 1 µM isoproterenol. Data are pooled from 30–60 total cells for each treatment obtained from 3 PLC{epsilon}+/+ mice. Results are average (±S.E.); ***, p < 0.001 isoproterenol treatment versus cpTOME or 6-Bnz-cAMP treatment alone; nd, no difference, isoproterenol versus 6-Bnz-cAMP/cpTOME co-treatment and 6-Bnz-cAMP versus cpTOME treatment, one-way ANOVA, Bonferroni post-test. C, effects of 6-Bnz-cAMP (300 µM, 30 min) and isoproterenol (1 µM) on average (±S.E.) peak electrically evoked Ca2+ release in PLC{epsilon}-/- myocytes. D, PKA activation with 6-Bnz-cAMP produced equivalent levels of enhanced CICR in PLC{epsilon}-/- or PLC{epsilon}+/+ myocytes. Both cell types were treated with 6-Bnz-cAMP as described above. Results are average (±S.E.). E, Ca2+ transient amplitudes from PLC{epsilon}-/- mice treated with 1 µM isoproterenol, 300 µM 6-Bnz-cAMP, 10 µM H89, 350 µM Rp-cAMPs, and 8-Br-Rp-cAMPs. H89, Rp-cAMPs, and 8-Br-Rp-cAMPs had no effect on base line Ca2+ transients. Results are average (±S.E.). ***, p < 0.001, one-way ANOVA. F, cytosolic extracts (5 µg) from hearts isolated from PLC{epsilon}-/- and PLC{epsilon}+/+ mice were analyzed for expression of the regulatory and catalytic subunits of PKA. G protein beta-subunit was assessed as a loading control.

 
To further confirm a role for acute PLC{epsilon} activity in betaAR regulation of CICR, AVMs from PLC{epsilon}+/+ mice were treated with U73122 [GenBank] , an inhibitor of multiple PLC isoforms in intact cells (21, 22). Acute treatment with U73122 [GenBank] (but not the inactive control U73343 [GenBank] ) decreased isoproterenol stimulation of evoked Ca2+ transients by 50% (Fig. 4A and 4B). U73122 [GenBank] did not affect either base line (data not shown) or isoproterenol stimulation of electrically evoked Ca2+ release in PLC{epsilon}-/- myocytes, indicating that, in these experiments, the effects of U73122 [GenBank] were mediated specifically by the inhibition of PLC{epsilon} (Fig. 4B). These data also indicate that the observed effects on betaAR signaling are specific to PLC{epsilon} with no contribution from other PLC isoforms present in cardiac myocytes. Because U73122 [GenBank] blocks PLC-dependent inositol 1,4,5-trisphosphate (IP3) production and Ca2+ mobilization in cells, the data support a direct role for PLC{epsilon}-phosphatidyl-4,5-bisphosphate hydrolytic activity in betaAR-dependent enhancement of CICR.


Figure 4
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  		FIGURE 4.
Acute PLC{epsilon} activity is involved in betaAR regulation of CICR. A, representative electrically evoked (8 V, 20 ms, 1 Hz) Ca2+ transients (405/485 nm) from single PLC{epsilon}+/+ and PLC{epsilon}-/- cardiac myocytes loaded with Indo-1/AM. Black bars indicate the addition of 1 µM isoproterenol and 2 µM U73122. B, average (±S.E.) peak electrically evoked Ca2+ release represented as the change in fluorescence ratio ({Delta}405/485 nm). Data are representative of three preparations (n = 3 each of PLC{epsilon}+/+ and PLC{epsilon}-/- mice), 10–20 cells/treatment/animal. **, p < 0.01; *, p < 0.05; one-way ANOVA, Bonferroni post-test. U73122 did not affect base line or evoke release in the absence of 1 µM isoproterenol. C, average (±S.E.) peak Ca2+ transient amplitude following treatment with or without 1 µM isoproterenol in PLC{epsilon}-/- cardiac myocytes transduced with either YFP or PLC{epsilon} (300 m.o.i.) for 24 h. Data were pooled from 20–40 cells for each treatment from n = 3 PLC{epsilon}-/- mice. Isoproterenol-stimulated YFP versus isoproterenol-stimulated PLC{epsilon} are shown. ***, p < 0.001; one-way ANOVA, Bonferroni post-test.

 
To further support these observations, we transduced PLC{epsilon}-/- AVMs with PLC{epsilon} adenovirus. Isoproterenol-stimulated increases in Ca2+ transient amplitude analyzed 24 h post-infection were enhanced ~3-fold over basal in the PLC{epsilon}-expressing myocytes, similar to that observed for PLC{epsilon}+/+ myocytes, compared with only a 1.5–2-fold over increase in YFP-expressing PLC{epsilon}-/- myocytes (Fig. 4C). The rescue of full isoproterenol responsiveness by re-introduction of PLC{epsilon} in AVMs of PLC{epsilon}-/- mice provides compelling evidence that specific deletion of PLC{epsilon} was responsible for the observed reduction in betaAR regulation of CICR.


Figure 5
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  		FIGURE 5.
Analysis of SR Ca2+ load and Ca2+ clearance in PLC{epsilon}+/+ and PLC{epsilon}-/- mice. A, SRCa2+ content was assessed from the peak Ca2+ release in response to a maximal concentration (10 mM) of caffeine. Data are from 10–15 cells/animal for 20 animals of each genotype. No significant difference was observed between AVMs obtained from PLC{epsilon}+/+ and PLC{epsilon}-/- mice. B, isoproterenol-induced increase in SR Ca2+ load was not significantly different in AVMs obtained from PLC{epsilon}+/+ and PLC{epsilon}-/- mice. Data are presented as caffeine release + isoproterenol/caffeine release - isoproterenol). C, Ca2+ clearance in AVMs obtained from PLC{epsilon}+/+ and PLC{epsilon}-/- mice was determined by fitting a single exponential function (colored lines) to the decay phase of electrically evoked Ca2+ transients in both the presence (upper traces) and absence (lower traces) of isoproterenol. D, average (±S.E.) values for Ca2+ transient decay time constants ({tau}) were pooled from five transients/cell, 10–15 cells/animal from 20 animals of each genotype. Isoproterenol treatment significantly accelerated the time constant of Ca2+ transient decay in AVMs obtained from both PLC{epsilon}+/+ and PLC{epsilon}-/- mice (isoproterenol-stimulated versus control; ***, p < 0.001, one-way ANOVA, Bonferroni post-test). However, the time constants of Ca2+ transient decay were not significantly different between PLC{epsilon}+/+ and PLC{epsilon}-/- mice in either the presence or absence of isoproterenol (one-way ANOVA, Bonferroni post-test).

 
PLC{epsilon}-dependent changes in betaAR-mediated stimulation of electrically evoked Ca2+ release could potentially result from PLC{epsilon} activity enhancing SR Ca2+ uptake leading to an increase in SR Ca2+ load. To test whether PLC{epsilon} activity acting downstream of betaAR activation alters SR Ca2+ load, we assessed releasable SR Ca2+ content by applying caffeine (10 mM) to maximally activate the ryanodine receptor. Peak caffeine-induced Ca2+ release was not different between PLC{epsilon}+/+ and PLC{epsilon}-/- animals (Fig. 5A). betaAR stimulation increases SR Ca2+ load by enhancing SR Ca2+ re-uptake following phosphorylation and subsequent disassociation of phospholamban from sarcoendoplasmic reticulum Ca2+-ATPases. As expected, isoproterenol treatment enhanced SR Ca2+ load, and this increase was similar in AVMs obtained from both PLC{epsilon}+/+ and PLC{epsilon}-/- mice (Fig. 5B). Thus, betaAR-mediated enhancement of SR Ca2+ load is not altered by PLC{epsilon} deletion. We also assessed the aggregate activity of rapid Ca2+ clearance mechanisms during excitation contraction coupling (e.g. sodium/calcium exchange and sarcoendoplasmic reticulum Ca2+-ATPase re-uptake) by fitting a single exponential to the time course of Ca2+ transient decay under both base line conditions and following application of isoproterenol (Fig. 5C). Although betaAR stimulation significantly accelerated the rate of Ca2+ transient decay, no significant difference was observed between PLC{epsilon}+/+ and PLC{epsilon}-/- myocytes (Fig. 5D). The data in Fig. 5 demonstrate that betaAR-mediated enhancement of SR Ca2+ load and myoplasmic Ca2+ clearance are not significantly altered by PLC{epsilon} ablation, indicating that PLC{epsilon} regulates the Ca2+ trigger and or release process.


Figure 6
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  		FIGURE 6.
Model showing betaAR stimulation of CICR in the heart involves cAMP activation of two parallel and independent signaling pathways (PKA and Epac-Rap1-PLC{epsilon}). Agents used in this study to identify this pathway are indicated as stimulatory (blue arrows) or inhibitory (red perpendicular bars).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
betaARs enable the heart to respond to stress by increasing SR Ca2+ release and thus cardiac contractility. A role for PKA-dependent phosphorylation and functional regulation of cardiac Ca2+ handling proteins including L-type Ca2+ channels, ryanodine receptors, and phospholamban is well documented (23, 24). Since its discovery in 1998, Epac has been shown to also contribute to cAMP signaling in many processes once thought to depend entirely on PKA phosphorylation events (13, 25). Our data demonstrate for the first time that cAMP-dependent activation of Epac, in addition to PKA activation, is required for maximal betaAR stimulation of CICR in the myocardium. The data support a model where an Epac-Rap-PLC{epsilon} pathway acts downstream of cAMP and parallel to the PKA pathway during betaAR stimulation (Fig. 6). It is likely that the decreased contractile function observed in PLC{epsilon} null mice in response to betaAR stimulation (3) arises from the loss of PLC{epsilon}-dependent enhancement of Ca2+ release from the SR, but this needs to be further investigated.

Although this study has characterized the upstream signaling pathway that links betaAR stimulation to PLC{epsilon} activation in cardiac myocytes, the downstream targets of PLC{epsilon} activity in betaAR-mediated enhancement of CICR are unknown. Acute inhibition of PLC{epsilon} activity with U73122 [GenBank] (Fig. 4) implicates PLC{epsilon}-dependent phosphatidyl-4,5-bisphosphate hydrolysis activity, as opposed to its Rap-guanine nucleotide exchange factor activity, in the betaAR response. PLC{epsilon} hydrolytic activity, as with all PLC enzymes, leads to the production of IP3 and diacylglycerol. Knock-out of the type 2 IP3 receptor, the predominant IP3 receptor found in cardiac myocytes, does not affect betaAR responsiveness in isolated atrial cardiac myocytes (26). Based on this and other data, it appears unlikely that IP3 generation and subsequent Ca2+ release from IP3 receptors is responsible for the observed PLC{epsilon}-mediated increase in CICR in cardiac myocytes, although it has recently been shown that local IP3 release can regulate cardiac hypertrophy (27). Diacylglycerol-dependent protein kinase C activation is a more likely candidate for influencing CICR. Multiple protein kinase C isoforms are expressed in the heart, and protein kinase C stimulation exerts both positive and negative inotropic effects, depending on which isoform is stimulated (28). Potential targets of protein kinase C include proteins that regulate Ca2+ handling (L-type Ca2+ channel, ryanodine receptor, phospholamban) or action potential duration (K+ channels) (Fig. 6). As for PKA, there are multiple potential downstream targets of PLC{epsilon} that need to be thoroughly investigated before a comprehensive understanding of how PLC{epsilon} activity contributes to CICR and cardiac contractility can be fully appreciated.

Defining the betaAR-mediated upstream regulation of PLC{epsilon} in cardiac myocytes is one important step in ongoing efforts to elucidate the mechanism of PLC{epsilon} action in cardiac function. The pathway outlined here is clearly important for maximal betaAR regulation of cardiac function. Mice deficient in PLC{epsilon} also exhibit an increased susceptibility to cardiac hypertrophy and fibrosis (3), hallmarks of heart failure progression. However, the precise role PLC{epsilon} signaling plays in enhancement of CICR and PLC{epsilon}-dependent protection from hypertrophy may be mediated by separate downstream signaling pathways. A recent report suggests a role for Epac and Rap in the suppression of cardiac hypertrophy in an ERK5-dependent manner (29). On the other hand, other investigators suggest that chronic activation of Epac in neonatal cardiac myocyte culture promotes hypertrophy (30). Because sympathetic regulation of cardiac function is critical for the normal response of the heart to stress, and perturbation of beta-adrenergic signaling is one of the major contributors to progressive heart failure, determining specific roles of the Epac-Rap-PLC{epsilon} signaling pathway in normal and pathological cardiac function could be of important therapeutic value to heart failure patients.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM053536 (to A. V. S.), DK56294 (to G. G. K.), and AR44657 (to R. T. D.), an American Heart Association Scientist Development grant (to B. C. B.), and Oral Cellular and Molecular Biology Training Grant T32 DE07202-15 (to E. A. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine, 601 Elmwood Ave., Box 711, Rochester, NY 14642. Tel.: 585-275-0892; Fax: 585-273-2652; E-mail: alan_smrcka{at}urmc.rochester.edu.

2 The abbreviations used are: CICR, Ca2+-induced Ca2+ release; PLC, phospholipase C; cpTOME, 8–4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate; PKA, protein kinase A; betaAR, beta-adrenergic receptor; 6-Bnz-cAMP, N6-benzoyladenosine-3',5'-cyclic monophosphate; Rp-cAMPs, adenosine-3',5'-cyclic monophosphorothioate Rp-isomer; 8-Br-Rp-cAMPs, 8-bromoadenosine-3',5'-cyclic monophosphorothioate Rp-isomer; YFP, yellow fluorescent protein; AVM, adult ventricular cardiac myocyte; m.o.i., multiplicity of infection; RT, reverse transcription; IP3, inositol 1,4,5-trisphosphate; ANOVA, analysis of variance; SR, sarcoplasmic reticulum. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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