Epac-mediated Activation of Phospholipase Cɛ Plays a Critical Role in β-Adrenergic Receptor-dependent Enhancement of Ca2+ Mobilization in Cardiac Myocytes

Abstract

Recently we demonstrated that PLCɛ plays an important role in β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of βAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLCɛ 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 PLCɛ-/- cardiac myocytes but rescued by transduction with PLCɛ, indicating that Epac is upstream of PLCɛ. Furthermore, transduction of PLCɛ+/+ 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 βAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLCɛ pathway. We also show that Epac/PLCɛ-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent βAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLCɛ that enhances intracellular Ca2+ release in cardiac myocytes. Recently we demonstrated that PLCɛ plays an important role in β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of βAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLCɛ 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 PLCɛ-/- cardiac myocytes but rescued by transduction with PLCɛ, indicating that Epac is upstream of PLCɛ. Furthermore, transduction of PLCɛ+/+ 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 βAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLCɛ pathway. We also show that Epac/PLCɛ-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent βAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLCɛ that enhances intracellular Ca2+ release in cardiac myocytes. 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 β adrenergic receptor (βAR) 2The 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; βAR, β-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. in cardiac myocytes (1Brodde O.E. Michel M.C. Pharmacol. Rev. 1999; 51: 651-690PubMed Google Scholar, 2Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (795) Google Scholar). Activation of βAR 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)ɛ knock-out mouse model (PLCɛ-/-) generated in our laboratory indicates that PLCɛ contributes to βAR-dependent regulation of cardiac function (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google Scholar). PLCɛ-/- mice exhibit significantly decreased left ventricular developed pressure in response to acute stimulation with the βAR agonist, isoproterenol. Isolated myocytes from PLCɛ-/- mice exhibit decreased isoproterenol-dependent enhancement of electrically evoked Ca2+ release in the absence of effects on βAR density or cAMP generation. These data implicate PLCɛ as a novel component of βAR regulation of Ca2+ release, which had not previously been described in the heart. However, the pathway linking βAR stimulation to PLCɛ 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 (4Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1243) Google Scholar, 5Hwang J.I. Oh Y.S. Shin K.J. Kim H. Ryu S.H. Suh P.G. Biochem. J. 2005; 389: 181-186Crossref PubMed Scopus (96) Google Scholar). PLCɛ is a recently identified PLC isoform expressed in the heart as well as other tissues (6Kelley G.G. Reks S.E. Ondrako J.M. Smrcka A.V. EMBO J. 2001; 20: 743-754Crossref PubMed Scopus (310) Google Scholar, 7Lopez I. Mak E.C. Ding J. Hamm H.E. Lomasney J.W. J. Biol. Chem. 2001; 276: 2758-2765Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 8Shibatohge M. Kariya K. Liao Y. Hu C.D. Watari Y. Goshima M. Shima F. Kataoka T. J. Biol. Chem. 1998; 273: 6218-6222Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). 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 (6Kelley G.G. Reks S.E. Ondrako J.M. Smrcka A.V. EMBO J. 2001; 20: 743-754Crossref PubMed Scopus (310) Google Scholar, 8Shibatohge M. Kariya K. Liao Y. Hu C.D. Watari Y. Goshima M. Shima F. Kataoka T. J. Biol. Chem. 1998; 273: 6218-6222Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 9Kelley G.G. Reks S.E. Smrcka A.V. Biochem. J. 2004; 378: 129-139Crossref PubMed Scopus (92) Google Scholar, 10Seifert J.P. Wing M.R. Snyder J.T. Gershburg S. Sondek J. Harden T.K. J. Biol. Chem. 2004; 279: 47992-47997Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Wing M.R. Snyder J.T. Sondek J. Harden T.K. J. Biol. Chem. 2003; 278: 41253-41258Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition, PLCɛ has guanine nucleotide exchange factor activity and can activate small G proteins such as Rap1 (12Jin T.G. Satoh T. Liao Y. Song C. Gao X. Kariya K. Hu C.D. Kataoka T. J. Biol. Chem. 2001; 276: 30301-30307Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, PLCɛ 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 PLCɛ and the cAMP-responsive Rap guanine nucleotide exchange factor, Epac, in adult ventricular cardiac myocytes directly downstream of βAR-dependent cAMP generation and upstream of CICR. Epac has recently been demonstrated to mediate cAMP responses in a number of systems (13Bos J.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 733-738Crossref PubMed Scopus (423) Google Scholar) and has been shown to activate PLCɛ in a Rap-dependent manner (14Schmidt M. Evellin S. Weernink P.A. von Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (285) Google Scholar). We show here that cAMP-stimulated Epac/Rap1-dependent activation of PLCɛ acts in concert with the classical cAMP-dependent activation of PKA to facilitate βAR-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 βAR system and is likely to have implications for the treatment of cardiac disease. Isolation of Adult Ventricular Cardiac Myocytes—Adult ventricular cardiac myocytes (AVMs) were isolated from 4-month-old wild-type or PLCɛ-/- mice as previously described (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google Scholar). 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 naiöve 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 PLCɛ+/+ and PLCɛ-/- Ventricular Myocytes—After data collection in the absence of βAR 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 [1-(6-[17β-3-methoxyestra-1,3,5(10)-triene-17-yl]-amino/hexyl)-1H-pyrroledione] or U73343 [1-(6-[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]-hexyl-2,5-pyrrolidine-dione], an inactive analog of U73122 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 using the system with the used to of either yellow fluorescent protein or Adult were isolated and to coverslips for 2 The plating medium was then and with medium containing 300 of either PLCɛ, or was by K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). 2 the was and medium was added to the cells. PLCɛ was not by direct analysis at but was at analysis described was used to PLCɛ at endogenous PLCɛ was with expressed PLCɛ at on reverse were to PLCɛ and endogenous For Rap inhibition, myocytes were with an by a were analyzed to both and Rap inhibition. Indo-1/AM fluorescence ratio was assessed as previously μm isoproterenol or μm Myocytes were electrically stimulated at in all with Epac and Rap1 level were from from 4-month-old wild-type and PLCɛ-/- To assess of proteins by and of PKA cell were were by standard and transferred to for Epac at was from Rap1 and at were from and PKA mouse at were from Transduction PLCɛ was by at G protein J. Smrcka A.V. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google was used at of PLCɛ in and PLCɛ-/- with m.o.i., 10,000 was using the CA) following the of the The was used with of for with the mouse PLCɛ and to a and a was for 30 min at followed by at for 2 The were at for 30 at for and at The number of was and 30 for and and for PLCɛ. We recently reported a decrease in β-adrenergic enhancement of electrically evoked Ca2+ release in ventricular myocytes isolated from PLCɛ-/- mice (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google Scholar) and that PLCɛ plays a direct role in the βAR-signaling PLC activation as a of βAR stimulation in the heart has not been previously described. A pathway from the βAR to PLCɛ demonstrated in stimulation of of activation of Epac, increased guanine nucleotide exchange on Rap, and subsequent PLCɛ activation (14Schmidt M. Evellin S. Weernink P.A. von Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (285) Google Scholar). To this pathway in cardiac we cpTOME, a cAMP analog with for Epac activation F. J. S. F. C. J.L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In isolated electrically evoked Ca2+ were assessed in the presence and absence of 10 μm cpTOME treatment alone increased the magnitude of electrically stimulated Ca2+ A and in PLCɛ+/+ myocytes. cpTOME not electrically evoked Ca2+ release in PLCɛ-/- myocytes A and the that Epac acts upstream of PLCɛ. that Epac and Rap are not significantly in heart from PLCɛ-/- mice To for of PLCɛ in Epac regulation of CICR, PLCɛ was PLCɛ-/- myocytes by PLCɛ was by at to PLCɛ+/+ after of but at this expressed PLCɛ protein was not by In this protein not to with PLCɛ protein was at that PLCɛ protein is expressed by this in We to function to endogenous of PLCɛ The increase in Ca2+ transient amplitude was rescued by of PLCɛ PLCɛ-/- myocytes data that an pathway in cardiac myocytes to CICR that is by cAMP downstream of βAR the function of Epac is to as a guanine nucleotide exchange factor for Rap and Rap is a of PLCɛ, we that Rap activation is for maximal enhancement of Ca2+ release following βAR To implicate Rap in the βAR we wild-type with an of a protein that GTP hydrolysis on Rap1 to its inactive F. M. S. PubMed Scopus Google Scholar). of has been used to Rap1 and implicate Rap function in a number of systems K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, E.A. Y. M. C.D. J.L. J. Cell Biol. PubMed Scopus Google Scholar). myocytes had decreased to isoproterenol stimulation with myocytes with a control The resulting level of isoproterenol is with that observed for PLCɛ-/- myocytes (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google Scholar) PLCɛ-/- myocytes no decrease in isoproterenol not a role for PLCɛ activation downstream of βAR the response to cpTOME in wild-type data Rap activity is for βAR-mediated activation of PLCɛ and the pathway downstream of βAR PLCɛ activity is in βAR regulation of CICR. A, electrically evoked V, 20 ms, 1 Ca2+ from single PLCɛ+/+ and PLCɛ-/- cardiac myocytes loaded with the addition of 1 μm isoproterenol and 2 μm electrically evoked Ca2+ release as the change in fluorescence ratio Data are of each of PLCɛ+/+ and PLCɛ-/- 10–20 ANOVA, U73122 not or release in the absence of 1 μm isoproterenol. Ca2+ transient amplitude following treatment with or 1 μm isoproterenol in PLCɛ-/- cardiac myocytes with either or PLCɛ for Data were from cells for each treatment from PLCɛ-/- PLCɛ are ANOVA, The data in 1 and 2 that the pathway contributes to βAR signaling in the heart. βAR signaling in cardiac myocytes has been to on PKA To assess of PKA and Epac signaling to βAR responses, we treated isolated myocytes from either wild-type or PLCɛ-/- mice with a of PKA, 6-Bnz-cAMP F. J. S. F. C. J.L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). 6-Bnz-cAMP treatment electrically evoked Ca2+ release as the increase was significantly that by isoproterenol To the that a of the isoproterenol response is were treated with a PKA that not the Epac pathway F. J. S. F. C. J.L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). H89 treatment inhibited isoproterenol-dependent enhancement of evoked Ca2+ release but This indicates that H89 the PKA pathway but the isoproterenol-dependent the that a signaling pathway in addition to PKA activation is to isoproterenol H89 not significantly the response to cpTOME and not the pathway. We also Epac and PKA could the maximal isoproterenol PLCɛ+/+ were treated with 6-Bnz-cAMP alone to activate PKA, cpTOME alone to activate Epac, or both to both 6-Bnz-cAMP and cpTOME treatment alone significantly electrically evoked Ca2+ in both this enhancement was that of isoproterenol However, of with 6-Bnz-cAMP and cpTOME in an enhancement of CICR to that of isoproterenol treatment on we that the pathway acts in and independent of the PKA pathway following βAR stimulation in the heart. this is the PKA pathway downstream of cAMP be in PLCɛ-/- myocytes. To that PKA activity was in PLCɛ-/- isolated myocytes were treated with 6-Bnz-cAMP to assess the effects on electrically evoked Ca2+ transient 6-Bnz-cAMP a increase in Ca2+ transient amplitude in PLCɛ-/- myocytes this level of was to both maximal isoproterenol responses observed in PLCɛ-/- myocytes and 6-Bnz-cAMP stimulation in PLCɛ+/+ myocytes H89 both isoproterenol-dependent and enhancement of Ca2+ in myocytes isolated from PLCɛ-/- mice A PKA combination of Rp-cAMPs and 8-Br-Rp-cAMPs also inhibited both isoproterenol-dependent and enhancement of Ca2+ in PLCɛ-/- F. J. S. F. C. J.L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). of the PKA and is not significantly in isolated from PLCɛ-/- mice data that the PKA pathway is in from PLCɛ-/- mice and is for the of the isoproterenol-dependent enhancement of evoked Ca2+ release the Epac/PLCɛ pathway has been in PLCɛ-/- To a role for acute PLCɛ activity in βAR regulation of CICR, from PLCɛ+/+ mice were treated with an of multiple PLC in cells J. Biol. Chem. Full Text PDF PubMed Google Scholar, Mol. Pharmacol. 2004; PubMed Scopus Google Scholar). treatment with U73122 not the inactive control decreased isoproterenol stimulation of evoked Ca2+ by and U73122 not either not or isoproterenol stimulation of electrically evoked Ca2+ release in PLCɛ-/- indicating in experiments, the effects of U73122 were mediated by the inhibition of PLCɛ These data also that the observed effects on βAR signaling are to PLCɛ with no from other PLC present in cardiac myocytes. U73122 inositol production and Ca2+ in the data a direct role for activity in βAR-dependent enhancement of CICR. To we PLCɛ-/- with PLCɛ increases in Ca2+ transient amplitude analyzed were over in the to that observed for PLCɛ+/+ with a over increase in PLCɛ-/- myocytes The of isoproterenol by of PLCɛ in of PLCɛ-/- mice that of PLCɛ was for the observed in βAR regulation of CICR. in βAR-mediated stimulation of electrically evoked Ca2+ release could from PLCɛ activity Ca2+ to an increase in Ca2+ To PLCɛ activity downstream of βAR activation Ca2+ we assessed Ca2+ by to activate the Ca2+ release was not PLCɛ+/+ and PLCɛ-/- βAR stimulation increases Ca2+ by Ca2+ following and subsequent of from reticulum isoproterenol treatment Ca2+ and this increase was in from both PLCɛ+/+ and PLCɛ-/- mice Thus, βAR-mediated enhancement of Ca2+ is not by PLCɛ We also assessed the activity of Ca2+ clearance during exchange and reticulum by fitting a single exponential to the time course of Ca2+ transient decay both and following of isoproterenol βAR stimulation significantly the of Ca2+ transient no was observed PLCɛ+/+ and PLCɛ-/- myocytes The data in 5 demonstrate that βAR-mediated enhancement of Ca2+ and Ca2+ clearance are not significantly by PLCɛ indicating that PLCɛ regulates the Ca2+ and or release the heart to respond to stress by Ca2+ release and cardiac A role for and regulation of cardiac Ca2+ handling proteins including Ca2+ and is well Nat. 2004; PubMed Scopus Google Scholar, Nature. 2002; 415: PubMed Scopus Google Scholar). its in Epac has been shown to also to cAMP signaling in many processes once to on PKA (13Bos J.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 733-738Crossref PubMed Scopus (423) Google Scholar, J. J.L. Nature. 1998; PubMed Scopus Google Scholar). data demonstrate for the first time that cAMP-dependent activation of Epac, in addition to PKA activation, is for maximal βAR stimulation of CICR in the The data a model an pathway acts downstream of cAMP and to the PKA pathway during βAR stimulation is likely that the decreased contractile function observed in PLCɛ mice in response to βAR stimulation (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google Scholar) from the of enhancement of Ca2+ release from the SR, but this to be this has the upstream signaling pathway that βAR stimulation to PLCɛ activation in cardiac the downstream of PLCɛ activity in βAR-mediated enhancement of CICR are unknown. inhibition of PLCɛ activity with U73122 phosphatidyl-4,5-bisphosphate hydrolysis as to its nucleotide exchange factor in the βAR PLCɛ as with all PLC to the production of and of the 2 the receptor in cardiac not βAR in isolated cardiac myocytes X. A.V. F. J. Circ. Res. 2005; PubMed Scopus Google Scholar). on this and other that generation and subsequent Ca2+ release from receptors is for the observed increase in CICR in cardiac has recently been shown that release can cardiac X. T. J. X. T.A. J. J. PubMed Scopus Google Scholar). protein kinase C activation is a likely for CICR. protein kinase C are expressed in the and protein kinase C stimulation both and on which isoform is stimulated T. J. 2005; PubMed Scopus Google Scholar). of protein kinase C proteins that Ca2+ handling Ca2+ or for PKA, are multiple downstream of PLCɛ that to be a of PLCɛ activity contributes to CICR and cardiac contractility can be the βAR-mediated upstream regulation of PLCɛ in cardiac myocytes is important in to the of PLCɛ in cardiac function. The pathway here is important for maximal βAR regulation of cardiac function. Mice in PLCɛ also exhibit an increased to cardiac and (3Wang H. Oestreich E.A. Maekawa N. Bullard T.A. Vikstrom K.L. Dirksen R.T. Kelley G.G. Blaxall B.C. Smrcka A.V. Circ. Res. 2005; 97: 1305-1313Crossref PubMed Scopus (106) Google of heart However, the role PLCɛ signaling plays in enhancement of CICR and from be mediated by downstream signaling A a role for Epac and Rap in the of cardiac in an manner K.L. J. Michel Nature. 2005; PubMed Scopus Google Scholar). the other other that activation of Epac in cardiac M. F. F. Circ. Res. 2005; 97: PubMed Scopus Google Scholar). regulation of cardiac function is for the response of the heart to and of β-adrenergic signaling is of the to heart of the signaling pathway in and cardiac function could be of important to heart