RESEARCH PAPER b-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via camp and PKAbph_

Transcription

1 British Journl of Phrmcology (21), 16, The Authors Journl compiltion 21 The British Phrmcologicl Society All rights reserved /1 RESEARCH PAPER b-adrenoceptor stimultion potentites insulin-stimulted PKB phosphoryltion in rt crdiomyocytes vi camp nd PKAbph_ Jorid T Stuenæs 1, Astrid Bolling 1, Ad Ingvldsen 1, Cmill Rommundstd 1,2, Emin Sudr 1,3, Fng-Chin Lin 1,4, Yu-Ching Li 1,4 nd Jørgen Jensen 1,4 1 Deprtment of Physiology, Ntionl Institute of Occuptionl Helth, Oslo, Norwy, 2 School of Phrmcy, University of Oslo, Oslo, Norwy, 3 Institute Vinc, Lbortory of Rdiobiology nd Moleculr Genetics, Belgrde, Serbi, nd 4 Deprtment of Physicl Performnce, Norwegin School of Sport Sciences, Oslo, Norwy Bckground nd purpose: Genetic pproches hve documented protein kinse B (PKB) s pivotl regultor of hert function. Insulin strongly ctivtes PKB, wheres drenline is not considered mjor physiologicl regultor of PKB in hert. In skeletl muscles, however, drenline potentites insulin-stimulted PKB ctivtion without hving effect in the bsence of insulin. The purpose of the present study ws to investigte the interction between insulin nd b-drenergic stimultion in regultion of PKB phosphoryltion. Experimentl pproch: Crdiomyocytes were isolted from dult rts by collgense, nd incubted with insulin, isoprenline, nd other compounds. Protein phosphoryltion ws evluted by Western blot nd phospho-specific ntibodies. Key results: Isoprenline incresed insulin-stimulted PKB Ser 473 nd Thr 38 phosphoryltion more thn threefold in crdiomyocytes. Isoprenline lone did not increse PKB phosphoryltion. Isoprenline lso incresed insulin-stimulted GSK-3b Ser 9 phosphoryltion pproximtely twofold, supporting tht PKB phosphoryltion incresed kinse ctivity. Dobutmine (b 1- gonist) incresed insulin-stimulted PKB phosphoryltion s effectively s isoprenline (more thn threefold), wheres slbutmol (b 2-gonist) only potentited insulin-stimulted PKB phosphoryltion by pproximtely 8%. Dobutmine, but not slbutmol, incresed phospholmbn Ser 16 phosphoryltion nd glycogen phosphorylse ctivtion (PKA-medited effects). Furthermore, the camp nlogue tht ctivtes PKA (dibutyryl-camp nd N 6 -benzoyl-camp) incresed insulin-stimulted PKB phosphoryltion by more thn threefold without effect lone. The Epc-specific ctivtor 8-(4-chlorophenylthio)-2 -O-methylcAMP (7) incresed insulin-stimulted PKB phosphoryltion by pproximtely 5%. Db-cAMP nd N 6 -benzoyl-camp, but not 7, incresed phospholmbn Ser 16 phosphoryltion. Conclusions nd implictions: b-drenoceptors re strong regultors of PKB phosphoryltion vi camp nd PKA when insulin is present. We hypothesize tht PKB medites importnt signlling in the hert during b-drenergic receptors stimultion. British Journl of Phrmcology (21) 16, ; doi:1.1111/j x; published online 23 Mrch 21 Keywords: Hert; Akt; GSK-3; phosphoryltion; phosphtidylinositol 3-kinse; phoshodiesterse; phospholmbn; hypertrophy; roliprm; ERK Abbrevitions: DNA-PK, DNA-dependent protein kinse; Epc, exchnge protein directly ctivted by camp; ERK, extrcellulr signl-regulted kinse; GSK-3, glycogen synthse kinse-3; IGF-1, insulin like growth fctor-1; makap, muscle-specific A-kinse nchoring protein; MEK, mitogen-ctivted protein kinse kinse; mtorc2, mmmlin trget of rpmyosin (mtor) complex-2; PDK1, phosphoinositide dependent kinse-1; PTEN, phosphtse nd tensin homolog deleted on chromosome 1 Introduction Genetic pproches hve provided evidence tht protein kinse B (PKB or Akt) is n importnt regultor of norml Correspondence: J Jensen, Deprtment of Physiology, Ntionl Institute of Occuptionl Helth, P. O. Box 8149 Dep., N-33, Oslo, Norwy. E-mil: jorgen.jensen@stmi.no Received 3 Mrch 29; revised 5 October 29; ccepted 23 December 29 hert functions nd dys-regultion cuses crdic disese (Debosch et l., 26). Deletion of PKB decreses hert size (Debosch et l., 26) wheres overexpression of constitutively ctivted PKB increses hert size nd cuses dilted myopthy (Condorelli et l., 22; Mtsui et l., 22). However, PKB hs beneficil effects nd protects the herts during ischemi reperfusion (Mtsui et l., 22). Growth fctors like insulin nd insulin-like growth fctor-1 (IGF-1) ctivtes PKB in the hert (Chesley et l., 2; Beuloye et l., 21). Insulin ctivtes PKB vi PI 3-kinse (Shepherd et l., 1998; Shepherd,

2 camp-pka signlling regultes PKB in hert JT Stuenæs et l ) nd phosphoryltion of PKB t Thr 38 nd t Ser 473 (Vnhesebroeck nd Alessi, 2). PDK1 phosphoryltes PKB Thr 38 (Vnhesebroeck nd Alessi, 2). PKB Ser 473 is phosphorylted by mtorc2 complex in insulin-sensitive tissues (Srbssov et l., 25), but DNA-PK, PKCbII nd integrinlinked kinse hve been reported to phosphorylte PKB Ser 473, t lest in some cell types (Persd et l., 21; Kwkmi et l., 24; Huston et l., 28). Activted PKB phosphoryltes glycogen synthse kinse (GSK)-3 (GSK-3 Ser 21 nd GSK-3b Ser 9 ) nd GSK-3 is, like PKB, point of integrtion of hypertrophic signlling in the hert (Sugden et l., 28). Genetic mnipultions of regultors of PKB ctivity, like PDK1, PTEN nd PI 3-kinse, hve provided further evidence tht regultion of PKB signlling is essentil for norml hert growth nd function (Shioi et l., 2; Schwrtzbuer nd Robbins, 21; Crckower et l., 22; Mor et l., 23; Byscs et l., 26; Heineke nd Molkentin, 26). b-adrenoceptors regulte centrl physiologicl functions in the hert (Ruehr et l., 24; Zheng et l., 25) nd defective camp regultion promotes hert filure (Lehnrt et l., 25). The signlling pthwys for b 1- nd b 2-drenoceptors differ (Steinberg, 1999; Pvoine nd Defer, 25; Zheng et l., 25). Stimultion of b 1-drenergic receptors increses concentrtion of camp tht ctivtes PKA leding to phosphoryltion of phospholmbn (PLB), rynodine receptor, phosphorylse kinse (which ctivtes glycogen phosphorylse) nd numerous other proteins (Drgo nd Colyer, 1994; Steinberg, 1999). In contrst, stimultion of b 2-drenoceptors is not thought to increse PLB Ser 16 phosphoryltion or to ctivte glycogen phosphorylse (Kuschel et l., 1999; Jo et l., 22), lthough smll increse in PLB Ser 16 phosphoryltion hs been observed (Brtel et l., 23). Although b 2-drenergic receptors ctivtes denylyl cyclse, the produced camp is rpidly broken down (Xing et l., 25) nd b 2-drenoceptors medite dditionl signlling vi PI 3-kinse, extrcellulr signl-regulted kinse (ERK) nd phospholipse A 2 (Steinberg, 24; Pvoine nd Defer, 25). Interestingly, prolonged stimultion of b 1-drenoceptors cuses poptosis in crdiomyocytes wheres b 2-drenoceptors my improve cell survivl fter hypoxi (Zhu et l., 21; Zhu et l., 23). The g-isoform of clss 1 PI 3-kinse (PI3Kg) is ctivted by G protein-coupled receptors (Wymnn nd Mrone, 25) nd b-drenoceptors hve been reported to medite effects vi PI 3-kinse nd PKB in hert cells (Chesley et l., 2; Oudit et l., 23; Tseng et l., 25). b 2-Adrenoceptors ctivte PI3Kg vi G i-coupled G bg but PKB ctivtion is much less thn during IGF-1 stimultion (Chesley et l., 2). Recently, camp hs lso been reported to medite effect vi exchnge protein directly ctivted by camp (Epc) (De rooij et l., 1998; Jensen, 27) nd it hs been reported tht Epc medited hypertrophy in neontl crdiomyocytes (Morel et l., 25; Métrich et l., 28). Stimultion of b 2-drenoceptors does not increse camp concentrtion throughout the cell nd comprtmentlized signlling llows ctivtion of specific pools of PKA (Steinberg, 24). A huge number of phosphodiesterses limit the spred of camp nd comprtmentlize b 2-drenergic signlling (Fischmeister et l., 26). The PDE4 fmily is coded by four genes comprising ~2 members (Hously et l., 25), nd the PDE4 subfmily is involved in estblishing comprtmentlized b 2-drenergic signlling in crdiomyocytes (Xing et l., 25). ERK phosphoryltes most PDE4 isoforms, nd ERK-medited phosphoryltion decreses phosphodiesterse ctivity of the long isoforms wheres ctivity of most of the short PDE4 isoforms increses (Mckenzie et l., 2; Hously et l., 25). The roles of ERK nd PDE4 for b 2-drenergic signlling hve not chieved much ttention in crdiomyocytes from dult rts. Recently, we reported complex interction between b-drenoceptors nd insulin signlling in skeletl muscles. While b-drenoceptor stimultion did not ctivte PKB in the bsence of insulin, b-drenoceptor stimultion strongly potentited insulin-stimulted PKB phosphoryltion nd ctivity, nd camp mimicked the effect of b-drenoceptor stimultion (Brennesvik et l., 25; Jensen et l., 28). The purpose of the present study ws to test the hypothesis tht stimultion of b-drenoceptors potentites insulin-stimulted PKB phosphoryltion in crdiomyocytes vi camp nd PKA. Methods Chemicls nd ntibodies Collgense 2 nd DNAse were from Worthington (Lkewood, NJ, USA) nd Nturl Mouse Lminin from Invitrogen (Crlsbd, CA, USA). Isoprenline, dobutmine, slbutmol, PD98,59, dibutyryl-camp (db-camp; #D627), roliprm, drenline, nordrenline nd wortmnnin were from Sigm (St. Louis, MO, USA). N 6 -Benzoyl-cAMP (N 6 -camp; B9), 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (7; C41) nd 8-Bromodenosine-3, 5 -cyclic monophosphorothiote, Rp-isomer (Rp-8-Br-cAMPS; B1) were from BIOLOG Life Science Institute (Bremen, Germny). Insulin (Actrpid) ws from Novo Nordisk (Bgsværd, Denmrk). Anti-nti-GSK-3 (#5-412) nd nti-mouse HRP-conjugte ntibodies (#12-349) were from Upstte (Lke Plcid, NY, USA). Anti-phospho- Akt Ser 473 (#9271), nti-phospho-akt Thr 38 (#9275), ntiphospho-gsk-3/b Ser 21 /Ser 9 (#9331) nd nti-rbbit HRPlinked ntibodies (#774) were from Cell Signling Technology (Dnvers, MA, USA). Anti-PLB (#A1-14) nd nti-phospho-plb Ser 16 (A1-12) were from Bdrill (Leeds, UK). ECL (RPN216) ws from Amershm Phrmci (Buckinghmshire, UK) nd ECL (WBKLS5) from Millipore (Bedford, MA, USA). Other chemicls were stndrd nlyticl grdes from Merck (Drmstdt, Germny), Sigm (St. Louis, MO, USA) nd Bio-Rd (Hercules, CA, USA). Animls Mle Wistr rts were obtined from B&K Universl (Nittedl, Norwy) nd cclimtized in our lbortory niml fcilities for 2 weeks with free ccess to food nd tp wter before the experiment. Experiments were pproved nd conducted in conformity with lws nd regultions controlling experiments nd procedures for niml reserch in Norwy nd the Europen Convention for the Protection of Vertebrte Animls used in Experimentl nd Other Scientific Purposes. Hert cell isoltion nd incubtion Herts from dult mle rts (45 g) were quickly removed under pentobrbitone nesthesi (.8 ml pentobrbitone British Journl of Phrmcology (21)

3 camp-pka signlling regultes PKB in hert 118 JT Stuenæs et l 5 mg ml -1 i.p.) nd plced in ice-cold sline. The herts were, still immersed in sline, connected to the Lngendorff perfusion pprtus through cnnul inserted into the ort nd crdiomyocytes were isolted by procedure modified fter Stokke et l. (Stokke et l. 1996). The herts were initilly perfused for ~1 min with C 2+ -free Joklik S-MEM medium (Invitrogen, ) dded 24 mm NHCO 3, 1.2 mm MgSO 4, 1 mm DL-crnitine, ph 7.4 (buffer A). Perfusion ws continued for 25 min t 37 C with buffer A dded 2 U ml -1 collgense 2 nd.1% BSA t flow rte of 6 7 ml min -1 with recircultion. Perfusion buffers were gssed with 95% O 2/5% CO 2. In some of the initil experiments trypsin (63 U ml -1, Sigm) ws dded. The effect of isoprenline on insulin-stimulted PKB phosphoryltion ws similr in crdiomyocytes isolted with nd without trypsin nd dt re pooled. The herts were removed from the cnnule nd the ventriculr tissue cut nd torn into smll frgments in 3 ml buffer A dded.5 mm CCl 2 nd 1% BSA. Cogulted blood nd visible connective tissue were removed, nd the suspension ws incubted for 1 min, shking (1 stroke per minute), t 37 C nd gently gssed. The hert tissue ws trnsferred to glss tube nd centrifuged (2 g, ~2 s). The pellet ws resuspended in 3 ml with buffer A dded 2 U ml -1 collgense 2,.1% BSA nd DNse I (.6 U ml -1 ), nd hert cells were llowed to dissocite for 15 2 min (shking: 1 stroke per minute, 37 C nd gently gssed). Centrifugtion ws repeted nd the crdiomyocyte pellet resuspended in ~3 ml buffer A dded.5 mm CCl 2 nd 1% BSA, nd llowed to rest t 37 C for 5 min without gittion. The cell suspension ws filtered through nylon mesh (size ~25 mm), centrifuged s bove. The finl pellet contining the crdiomyocytes ws resuspended in culture medium (Medium 199 with.2% BSA, 2 mm DL-crnitine, 5 mm cretine, 5 mm turine, 1 mu ml -1 insulin, M 5-triiodo-D-thyronine, 1 IU ml -1 penicillin nd 1 IU ml -1 streptomycin; ~15 ml per hert). After 1 3 min t 37 C equilibrted with 95% O 2/5% CO 2, crdiomyocytes were plted in 9.5 cm 2 TC dishes (Corning, NY, USA) coted with lminin. After 2 h in incubtor (37 C, 5% CO 2) in 2 ml culture medium the culture medium ws chnged to remove non-ttched cells nd cell debris. After chnge of medium, nerly ll cells ttched to lminin were rod-shped crdiomyocytes. The crdiomyocytes were incubted overnight for experiments the following dy. Protein content within experiments ws rther similr in ech well. In different experiment, men protein content per well vried between 4 nd 8 mg. For lminin coting, dishes were treted with 5 ml 1mg ml -1 lminin dissolved in Medium 199 for 1 h. Prior to experiments, crdiomyocytes were preincubted for 2 h in 2 ml buffer contining 12 mm NCl, 3.3 mm KCl, 1.2 mm KH 2PO 4, 24 mm NHCO 3,.8 mm MgSO 4, 1 mm CCl 2,.1% BSA, 5.5 mm D-glucose, ph 7.4 (PB). In experiments, crdiomyocytes were incubted for 15 min in buffer s bove with or without insulin (1 mu ml -1 ), isoprenline (1-6 M), dobutmine (1-6 M) or slbutmol (1-6 M). In experiments with camp nlogues nd MEK inhibitor (PD98,59), substnces were dded fter 9 min of preincubtion. Crdiomyocytes were therefore incubted for 3 min with PD98,59 (5 mm) or.5 mm of the camp nlogues (db-camp, N 6 -benzoyl camp or 7) prior to the 15 min incubtion with insulin (1 mu ml -1 ) unless otherwise stted in legends. In experiments with the PDE4 inhibitor roliprm (1 mm) the inhibitor ws dded fter 15 min of preincubtion. Crdiomyocytes were therefore incubted with roliprm for 15 min before insulin (1 mu ml -1 ) nd slbutmol (1-6 M) were dded for 15 min. PD98,59 nd roliprm were dissolved in DMSO; in these experiments,.1% DMSO ws included in wells. Western blot nlysis Crdiomyocytes were scrped off the wells in 25 or 35 ml well -1 of ice-cold lysis buffer contining 1 mm NPO 4 buffer ph 7.2, 15 mm NCl, 2 mm EDTA, 5 mm NF, 1% Nonidet P-4, 1% sodium deoxycholte,.1% SDS (w/v),.2 mm N 3VO 4 nd 2 ml ml -1 of protese inhibitor cocktil (P834; Sigm). The lystes were trnsferred to microtubes nd rotted for 45 min t 4 C. After centrifugtion (11 6 g; 15 min; 4 C) protein concentrtion ws determined in the superntnt (DC Protein Assy, Bio-Rd, Hercules, CA, USA) with protein stndrd from Sigm (P8119). Lystes were diluted to equl concentrtion on experimentl dys (1 mg ml -1 ) nd stored t -7 C. For Western blot, lystes were prepred with Lemmli buffer nd proteins (~15 mg) were seprted by electrophoresis (Mini-PROTEAN 3 # from Bio-Rd) in 1% SDS- PAGE. A 15% SDS-PAGE ws run for nti-plb nd nti-plb Ser 16 probing. Proteins were trnsferred from gel into PVDF membrne (Immobilon-P.45 mm, #IPVH1 from Millipore) for 1 h t.25 A with ice-continer in trnsfer buffer (25 mm Tris, 192 mm glycine nd 1% methnol). Membrnes were wshed 3 1 min in PBS-T (8 mm NA 2HPO 4, 2 mm NH 2PO 4, 1 mm NCl nd.1% Tween-2, ph 7.4) nd incubted (blocked) in 5% dried non-ft skim milk solved in PBS-T for 2 h t room temperture to minimize nonspecific binding. All wshing nd incubtion were done with gentle shking. Membrnes were wshed 2 3 s in PBS-T before incubtion overnight t 4 C with primry ntibodies diluted in PBS-T with 3% BSA (w/v). Dilutions of primry ntibodies vried from 1:5 to 1:4. After 6 1 min wsh, membrnes were incubted t room temperture for 1 h with secondry ntibody diluted in PBS-T with 1% BSA (w/v). Dilutions of secondry ntibodies vried from 1:2 to 1:4. After 6 1 min wsh, membrnes were incubted in ECL. Signls were detected from enhnced chemiluminescence fter exposed to film (Kodk X-OMAT UV Plus Film or FUJI RX Ct. nr , Tokyo, Jpn). The films were scnned, nd by using densitometry the signls were quntified (Scion Imge, Scion Corportion). Glycogen phosphorylse ctivity Crdiomyocytes were scrped off the wells in 35 ml homogenizing buffer [5 mm MES, 1 mm NF, 5 mm EDTA nd 1 mm 2-mercptoethnol (ph 6.1)]. The cell suspension ws minced in MixerMill (Retsch, Hn, Germny) for 2 3 s nd centrifuged (3 g; 1 min; 4 C) nd the superntnt frozen t -7 C. Glycogen phosphorylse ctivity ws mesured in reverse direction with 48 mm glucose 1-phosphte British Journl of Phrmcology (21)

4 camp-pka signlling regultes PKB in hert JT Stuenæs et l 119 nd.5 mci ml -1 [ 14 C(U)]-glucose 1-phosphte (PerkinElmer, Shelton, CT, USA) by the filter pper method s described previously (Gilboe et l., 1972; Frnch et l., 1999). Totl phosphorylse ctivity ws determined in the presence of 3 mm 5 -AMP in the ssy buffer, phosphorylse ctivity in the bsence of AMP, nd percentge of phosphorylse in the -form ws clculted. Totl protein concentrtion in the superntnt ws determined (DC Protein Assy) using Protein Stndrd (P8119, Sigm) s reference. Sttistics Dt re presented s men SE. Anlysis of vrince ws performed to investigte differences nd Fishers lest significnt difference ws used s post hoc test to compre different tretments. P <.5 ws considered s significnt. Results Protein kinse B Ser 473 nd Thr 38 phosphoryltion ws not detectble in crdiomyocytes incubted in buffer without hormones. As expected, insulin incresed phosphoryltion of PKB t both Ser 473 nd Thr 38 (Figure 1). Isoprenline did not stimulte PKB Ser 473 or Thr 38 phosphoryltion when present lone. Interestingly, isoprenline incresed insulin-stimulted PKB Ser 473 nd Thr 38 phosphoryltion by more thn threefold (Figure 1A nd B). Insulin incresed phosphoryltion of GSK-3b t Ser 9 (PKB phosphoryltion site) nd combintion of insulin nd isoprenline incresed GSK-3b Ser 9 phosphoryltion further supporting tht PKB ctivity ws incresed (Figure 1C). Dose response curve for insulin-stimulted PKB Thr 38 phosphoryltion in the presence nd bsence of isoprenline showed tht isoprenline lso hd strong effect t physiologicl concentrtions of insulin (Figure 1F). Dose response curve for isoprenline-medited PKB Ser 473 phosphoryltion showed tht high physiologicl concentrtions of isoprenline were required to see effect on insulin-stimulted PKB phosphoryltion (Figure 1G). PLB Ser 16 phosphoryltion (PKA phosphoryltion site) ws not detectble in bsl condition nd during insulin stimultion. Isoprenline incresed PLB Ser 16 phosphoryltion nd insulin did not influence isoprenline-stimulted PLB Ser 16 phosphoryltion (Figure 1). About 2% of glycogen phosphorylse ws in -form in bsl condition, nd isoprenline incresed glycogen phosphorylse ctivtion to bout 55% (Tble 1). Insulin decresed isoprenline-medited glycogen phosphorylse ctivtion (Tble 1) nd tended to decrese bsl glycogen phosphorylse ctivtion (P =.91). Wortmnnin completely blocked PKB phosphoryltion in crdiomyocytes stimulted with insulin lone nd combintion of insulin nd isoprenline (Figure 2). In prllel with the inhibited PKB phosphoryltion, wortmnnin lso completely blocked GSK-3b Ser 9 phosphoryltion stimulted by insulin nd isoprenline (Figure 2). The b 1-gonist dobutmine nd the b 2-gonist slbutmol were used to get indictions of whether isoprenline medited its effect vi b 1-orb 2-drenergic receptors. Dobutmine (b 1- gonist) incresed insulin-stimulted PKB phosphoryltion t both Thr 38 nd Ser 473 to similr level s isoprenline did (Figure 3A nd B). Slbutmol (b 2-gonist) lso incresed insulin-stimulted PKB phosphoryltion but ws fr less potent thn isoprenline nd dobutmine (Figure 3). Neither dobutmine nor slbutmol incresed PKB phosphoryltion in the bsence of insulin. GSK-3b Ser 9 phosphoryltion ws high in crdiomyocytes incubted with insulin nd dobutmine supporting tht PKB phosphoryltion incresed ctivity (Figure 3C). Slbutmol did not significntly ctivte glycogen phosphorylse or phosphorylte PLB t Ser 16 s expected (Tble 1; Figure 3D). Dobutmine, on the other hnd, incresed glycogen phosphorylse ctivtion nd PLB Ser 16 phosphoryltion (Tble 1; Figure 3D). Adrenline nd nordrenline incresed insulin-stimulted PKB Ser 473, PKB Thr 38 nd GSK-3b Ser 9 phosphoryltion in similr mnner s isoprenline (Figure 3E). Moreover, drenline nd nordrenline incresed PLB Ser 16 phosphoryltion (Figure 3E). In the bsence of insulin, drenline nd nordrenline did not influence PKB or GSK-3b phosphoryltion (Figure 3E). The camp nlogue db-camp mimicked the effect of isoprenline on PKB phosphoryltion. Alone, db-camp did not increse PKB phosphoryltion, but db-camp potentited insulin-stimulted PKB Ser 473 nd Thr 38 phosphoryltion (Figure 4A nd B). GSK-3b Ser 9 phosphoryltion ws lso higher in crdiomyocytes incubted with db-camp nd insulin compred with crdiomyocytes incubted with insulin lone (Figure 4C). N 6 -camp, PKA-selective camp nlogue, lso incresed insulin-stimulted PKB Ser 473 nd Thr 38 phosphoryltion pproximtely fourfold without hving effect lone (Figure 4A nd B). N 6 -camp (.5 mm) incresed glycogen phosphorylse ctivtion (Tble 1) nd cused PLB Ser 16 phosphoryltion (Figure 4D). The Epcspecific camp nlogue 7 incresed insulin-stimulted PKB phosphoryltion by bout 8% (Figure 4). The Epc ctivtor (.5 mm) did not ctivte glycogen phosphorylse (Tble 1) or stimulte PLB Ser 16 phosphoryltion (Figure 4D) supporting tht PKA ws not ctivted. Inhibition of PKA with Rp-8-Br-cAMPS (.5 mm) reduced glycogen phosphorylse ctivtion in crdiomyocytes incubted with M isoprenline from % to % (P <.5; n = 4 in both groups). Rp-8-Br-cAMPS lso reduced PKB Ser 473 phosphoryltion in crdiomyocytes incubted with insulin nd isoprenline (Figure 5) wheres Rp-8- Br-cAMPS did not influence insulin-stimulted PKB Ser 473 phosphoryltion (Figure 5). PDE4 is required for b 2-drenoceptor subtype-specific signlling in crdiomyocytes (Xing et l., 25). Furthermore, lrge complex consisting of PDE4, ERK, MEK, makap, Epc nd PKA hs been reported to exist in neontl crdiomyocytes (Dodge-kfk et l., 25). In the present study, we used roliprm (PDE4 inhibitor) nd PD98,59 (MEK inhibitor) to test the hypothesis tht ERK-medited PDE4 phosphoryltion reduced the bility of b 2-drenoceptor stimultion to increse insulin-stimulted PKB phosphoryltion. Roliprm incresed PKB phosphoryltion when crdiomyocytes were incubted with both insulin nd slbutmol (Figure 6A nd B). Roliprm lso incresed GSK-3b Ser 9 phosphoryltion in hert cells incubted with both slbutmol nd insulin (Figure 6C). Inhibition of PDE4 by roliprm did not increse PLB Ser 16 phosphoryltion in bsl condition but incresed PLB Ser 16 phosphoryltion when slbutmol ws present British Journl of Phrmcology (21)

5 camp-pka signlling regultes PKB in hert 12 JT Stuenæs et l A B PKB Ser 473 phosphoryltion Isoprenline Insulin C PKB Thr 38 phosphoryltion Isoprenline Insulin D b GSK-3β Ser 9 phosphoryltion Isoprenline Insulin E G PLB Ser 16 phosphoryltion (% of Iso) F Isoprenline Insulin PKB Thr 38 PKB Totl Iso (1 6 M) Insulin (µu ml 1 ) 3 6 Insulin 5 Insulin + Iso PKB Thr 38 phosphoryltion (% of insulin 1. µu ml 1 ) 1 3 1, 1, Insulin (µu ml 1 ) British Journl of Phrmcology (21)

6 camp-pka signlling regultes PKB in hert JT Stuenæs et l 121 Figure 1 Effect of isoprenline nd insulin on phosphoryltion of protein kinse B (PKB), glycogen synthse kinse (GSK)-3b nd phospholmbn (PLB) in isolted crdiomyocytes. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h prior to 15 min incubtion with or without isoprenline (1-6 M) in the bsence or presence of insulin (1 mu ml -1 ). Crdiomyocytes were prepred for Western blot s described in Methods. (A) Effect of isoprenline nd insulin on PKB Ser 473 phosphoryltion. Grph shows mens of quntified blots with insulin s 1%; dt re from three different experiments; n = 6 9 in ech group; representtive blot is shown in (E). (B) Effect of isoprenline nd insulin on PKB Thr 38 phosphoryltion. Grph shows mens of quntified blots with insulin s 1%; dt re from three different experiments; n = 6 7 in ech group. (C) Effect of isoprenline nd insulin on phosphoryltion GSK-3b Ser 9 phosphoryltion. Grph shows mens of quntified blots with insulin s 1%; dt re from three different experiments; n = 6 7 in ech group. (D) Effect of isoprenline nd insulin on PLB Ser 16 phosphoryltion. Grph shows mens of quntified blots with isoprenline s 1%; dt re from four different experiments; n = 8 9 in ech group. (E) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9, PLB Ser 16 phosphoryltion nd totl GSK-3b nd totl PLB in different tretments groups. (F) Dose response curve for insulin-stimulted PKB Ser 38 phosphoryltion in the bsence (open circles) nd presence of 1-6 M isoprenline (filled squres); symbols re mens of quntified blots (with 1 mu ml -1 of insulin s 1%) from three different experiments; n = 9 for insulin 1 mu ml -1 nd n = 6 for other symbols. (G) Dose response curve for isoprenline-medited PKB Ser 473 phosphoryltion in the presence of 1 mu ml -1 insulin; symbols re mens of quntified blots (with insulin s 1%) from four different experiments; n = 19 for insulin nd n = 9 12 for other symbols. Significntly higher thn insulin. Tble 1 Glycogen phosphorylse ctivtion in crdiomyocytes fter exposure to b-drenergic receptor gonists or camp nlogues Glycogen phosphorylse (% -form) (-) Insulin (+) Insulin Control (22) (12) Isoprenline (21) b (6) Dobutmine ,b (12) Slbutmol (6) N 6 -camp (8) (6) Crdiomyocytes were incubted overnight in medium nd preincubted for 2 h in buffer without ny hormones prior to 15 min exposure to isoprenline (1-6 M), dobutmine (1-6 M) slbutmol (1-6 M) nd insulin (1 mu ml -1 ). Crdiomyocytes were exposed to.5 mm of the camp nlogues for 45 min. Adrenline nd nordrenline incresed glycogen phosphorylse % to nd respectively (n = 4 in both groups; P <.5 compred with control). Dt re men SEM. Number of smples in prentheses. Significntly higher thn control nd slbutmol. b Significntly lower thn isoprenline without insulin. 7, 8-(4-chlorophenylthio)-2 -O-methyl-Cmp; N 6 -camp, N 6 -benzoyl-camp. Figure 2 Wortmnnin blocks phosphoryltion of protein kinse B (PKB) nd glycogen synthse kinse (GSK)-3b stimulted by insulin lone nd in combintion with isoprenline. Representtive blots for PKB Ser 473, PKB Thr 38 nd GSK-3b Ser 9 phosphoryltion in crdiomyocytes incubted with insulin (1 mu ml -1 ), isoprenline (1-6 M) nd wortmnnin s indicted. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h with 1 mm wortmnnin dded fter 15 min. After preincubtion (nd 15 min incubtion with wortmnnin) insulin nd isoprenline ws dded for 15 min. (Figure 6D). Roliprm did not influence bsl or insulinstimulted PKB phosphoryltion. Inhibition of MEK by PD98,59 incresed PKB Ser 473, PKB Thr 38 nd GSK-3 Ser 9 phosphoryltion in crdiomyocytes when both insulin nd slbutmol were present (Figure 7A C). PD98,59 lso incresed slbutmol-medited PLB Ser 16 phosphoryltion (Figure 7D) supporting tht PKA becme ctivted. Bsl nd insulin-stimulted PKB Ser 473 phosphoryltion ws not influenced by PD98,59. Discussion A novel nd importnt finding in the present study ws tht stimultion of b-drenergic receptors incresed insulinstimulted PKB phosphoryltion in crdiomyocytes. Interestingly, stimultion of b 1-drenergic receptors incresed insulinstimulted PKB phosphoryltion (>3%) much more thn stimultion of b 2-drenergic receptor (~8%). Furthermore, the camp nlogues ctivting PKA (db-camp nd N 6 -camp) incresed insulin-stimulted PKB s much s isoprenline. In ddition, the Epc-specific camp nlogue 7 incresed insulin-stimulted PKB phosphoryltion lthough to lesser extent. Our findings couples for the first time b-drenoceptorcamp-pka signlling to stimultion of PKB phosphoryltion in crdiomyocytes nd indictes the need to reconsider the role of b-drenergic receptor in the regultion of PKB. In prticulr, studies of b-drenoceptor signlling in hert need to tke into ccount the potentil synergistic crosstlk with other hormones. Insulin is considered strong ctivtor of PKB (Bertrnd et l., 28) nd it is therefore notble tht stimultion of b-drenoceptors incresed insulin-stimulted PKB Ser 473 nd Thr 38 phosphoryltion by three- to fourfold. Isoprenline lso incresed insulin-stimulted GSK-3b Ser 9 phosphoryltion supporting tht PKB ctivity ws incresed, nd our dt suggest tht b-drenoceptors re powerful regultors of PKB when insulin is present. PKB hs centrl role for regultion of crdic function nd it hs been shown tht overexpression of constitutively ctivted PKB increses hert size nd cuses dilted myopthy (Condorelli et l., 22; Mtsui et l., 22) wheres deletion of PKB decreses hert size (Debosch et l., 26). Furthermore, PKB protects the herts during ischemi reperfusion (Mtsui et l., 22) nd regultes British Journl of Phrmcology (21)

7 camp-pka signlling regultes PKB in hert 122 JT Stuenæs et l A B PKB Ser 473 phosphoryltion ,b,b PKB Thr 38 phosphoryltion ,b,b Insulin Slbutmol Dobutmine Isoprenline C Insulin Slbutmol Dobutmine Isoprenline D GSK-3β Ser 9 phosphoryltion ,b,b Insulin Slbutmol Dobutmine Isoprenline E British Journl of Phrmcology (21)

8 camp-pka signlling regultes PKB in hert JT Stuenæs et l 123 Figure 3 Effect of dobutmine (b 1-gonist), slbutmol (b 2-gonist), isoprenline, drenline nd nordrenline on insulin-stimulted protein kinse B (PKB), glycogen synthse kinse (GSK)-3b nd phospholmbn (PLB) phosphoryltion. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h prior to 15 min incubtion with slbutmol (1-6 M), dobutmine (1-6 M), isoprenline (1-6 M), drenline (1-6 M) nd nordrenline (1-6 M) in the bsence or presence of insulin (1 mu ml -1 ). Grphs show mens of quntified blots with insulin s 1%; representtive blots re shown in (D). (A) Effect of dobutmine, slbutmol nd isoprenline on insulin-stimulted PKB Ser 473 phosphoryltion. Dt re from five different experiments; n = 2 for insulin nd n = 1 14 for other groups. (B) Effect of dobutmine, slbutmol nd isoprenline on insulin-stimulted PKB Thr 38 phosphoryltion. Dt re from five different experiments; n = 18 for insulin nd n = 8 11 for other groups. (C) Effect of dobutmine, slbutmol nd isoprenline on insulin-stimulted GSK-3b Ser 9 phosphoryltion. Dt re from five different experiments; n = 17 for insulin nd n = 8 11 for other groups. (D) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9 nd PLB Ser 16 phosphoryltion nd totl PKB in different tretments groups. (E) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9 nd PLB Ser 16 phosphoryltion in crdiomyocytes incubted with drenline (1-6 M) nd nordrenline (1-6 M) lone or in combintion with insulin. Significntly higher thn insulin; b Significntly higher thn insulin + slbutmol. A B PKB Ser 473 phosphoryltion (% of insulin) * * * *,c,b Insulin db-camp N6-cAMP PKB Thr 38 phosphoryltion (% of insulin) * * * *,c,b Insulin db-camp N6-cAMP C D GSK-3β Ser 9 phosphoryltion (% of insulin) * * * *,c,b Insulin db-camp N6-cAMP Figure 4 Effect of camp nlogues on phosphoryltion of protein kinse B (PKB), glycogen synthse kinse (GSK)-3b nd phospholmbn (PLB). After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h with camp nlogues (.5 mm) dded fter 9 min. After preincubtion (nd 3 min incubtion with camp nlogues) insulin ws dded for 15 min. Grphs show mens of quntified blots with insulin s 1%; representtive blots re shown in (D). (A) Effect of camp nlogues on PKB Ser 473 phosphoryltion in the bsence or presence of 1 mu ml -1 insulin. Dt re from five different experiments; n = 19 for insulin nd n = 1 for other groups. (B) Effect of camp nlogues on PKB Thr 38 phosphoryltion in the bsence or presence of 1 mu ml -1 insulin. Dt re from five different experiments; n = 19 for insulin nd n = 1 for other groups. (C) Effect of camp nlogues on GSK-3b Ser 9 phosphoryltion in the bsence or presence of 1 mu ml -1 insulin. Dt re from five different experiments; n = 27 for insulin nd n = 1 for other groups. (D) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9 nd PLB Ser 16 phosphoryltion nd totl PKB in different tretments groups. Significntly higher thn insulin; b Significntly higher thn insulin + 7; c Significntly higher thn insulin + N 6 -camp. *Significntly lower thn insulin. 7, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP; db-camp, dibutyryl-camp; N 6 -camp, N 6 -benzoyl-camp. British Journl of Phrmcology (21)

9 camp-pka signlling regultes PKB in hert 124 JT Stuenæs et l Figure 5 The protein kinse A (PKA) inhibitor Rp-8-Br-cAMPS reduces PKB Ser 473 phosphoryltion in crdiomyocytes incubted with insulin nd isoprenline. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer for 3 h with Rp-8-Br-cAMPS (.5 mm) nd without ny hormones. After preincubtion insulin (1 mu ml -1 ) nd isoprenline (3 1-9 M) ws dded for 15 min. Dt re from three different experiments; n = 6 7 for ll groups. Representtive blots for PKB Ser 473 nd totl PKB re shown bove the grph. Significntly higher thn insulin; b Significntly lower thn insulin + isoprenline. other functions in the hert (Shiojim nd Wlsh, 26). Although we minly used GSK-3 Ser 9 phosphoryltion s redout of PKB ctivity, it is worth to note tht GSK-3 per se is considered s key hypertrophic signlling molecule in the hert (Sugden et l., 28). Tken together, our dt show tht stimultion of b-drenoceptors, in the presence of insulin, strongly increses phosphoryltion of the two hypertrophic signlling molecules PKB nd GSK-3. Isoprenline lone did not significntly increse PKB phosphoryltion in crdiomyocytes from dult rts. Previously, b-drenergic stimultion hs been reported to increse PKB Ser 473 phosphoryltion in neontl crdiomyocytes (Chesley et l., 2; Morisco et l., 25) nd in H9c2 crdiomyocytes (Yno et l., 27). However, b-drenoceptor-medited PKB ctivtion is low compred with IGF (Chesley et l., 2) nd b-drenoceptors re normlly not considered s n importnt regultor of PKB in the hert (Shiojim nd Wlsh, 26). Indeed, in vivo injection of isoprenline hs lso shown to increse PKB phosphoryltion in dult rts (Tseng et l., 25) nd mice (Oudit et l., 23); in vivo insulin will lwys be present nd it is possible tht the effect of isoprenline represents potentition of insulin ction. Leblis et l. (Leblis et l. 24) hve lso reported tht stimultion of b 1-drenoceptors increse PI 3-kinse ctivity, but dt on PKB were not reported. Even though these dt seem to contrdict the finding tht b-drenoceptor stimultion did not increse PKB phosphoryltion in the bsence of insulin, we hve previously reported similr effect of b-drenergic stimultion on PKB phosphoryltion in skeletl muscles: drenline hd no effect in the bsence of insulin but potentited insulinstimulted PKB phosphoryltion nd ctivity (Brennesvik et l., 25; Jensen et l., 28). In the present study, drenline nd nordrenline incresed insulin-stimulted PKB phosphoryltion s efficiently s isoprenline, supporting physiologicl role of the sympthodrenl system in the regultion of PKB in the hert. Stimultion of b 1-drenoceptors incresed insulinstimulted PKB phosphoryltion much more thn stimultion of b 2-drenoceptors. This ws not expected since b 2-drenoceptor stimultion, previously hs been coupled to PKB ctivtion (Chesley et l., 2; Oudit et l., 23; Tseng et l., 25), but not b 1-drenoceptor stimultion. It is well documented tht b 1- nd b 2-drenoceptors hve different signlling mechnism in crdiomyocytes from dult rts (Steinberg, 1999; Pvoine nd Defer, 25; Zheng et l., 25). In greement with these studies, we found tht stimultion of b 1-drenergic receptors gonist dobutmine incresed glycogen phosphorylse ctivtion nd PLB Ser 16 phosphoryltion wheres slbutmol did not significntly ctivte glycogen phosphorylse nd phosphoryltes PLB Ser 16 (Kuschel et l., 1999; Jo et l., 22). The physiologicl role of b 1- nd b 2-drenoceptors lso differs nd it hs been reported tht stimultion of b 1-drenoceptors cuses poptosis wheres stimultion of b 2-drenoceptors prevents poptosis (Communl et l., 1999; Zhu et l., 21; Zhu et l., 23). Becuse ctivtion of PKB prevents poptosis, it ws exciting tht ddition of insulin mde b 1-drenoceptor stimultion to powerful ctivtor of PKB phosphoryltion. Furthermore, b 1-drenoceptors stimultes hypertrophy much more thn b 2-drenoceptors, nd our results rise the possibility tht b 1-drenoceptors medite hypertrophic signlling vi PKB nd GSK-3. We hypothesized tht b-drenoceptors potentited insulinstimulted PKB phosphoryltion vi camp; the hypothesis ws supported by the fct tht db-camp incresed insulinstimulted PKB phosphoryltion. To further dissect the signlling pthwy we used the PKA-specific camp nlogues N 6 -camp, which strongly incresed insulin-stimulted PKB phosphoryltion without hving effect lone nd therefore mimicked the effect of isoprenline on PKB phosphoryltion. Moreover, the camp nlogue Rp-8-Br-cAMPS, which prevents PKA ctivtion, reduced PKB Ser 473 phosphoryltion in crdiomyocytes incubted with insulin nd isoprenline. These dt show for the first time tht camp increses PKB phosphoryltion vi PKA in hert. Both PKB nd camp signlling hs previously been reported to medite dilted crdiomyopthy (Lehnrt et l., 25; Shiojim nd Wlsh, 26). Our findings now couples camp nd PKA signlling to phosphoryltion of PKB, nd links the two hypertrophic signlling pthwys in hert tht were previously regrded s independent. The Epc-specific camp nlogue 7 lso incresed insulin-stimulted PKB phosphoryltion in hert, but less thn db-camp nd N 6 -camp. The fct tht 7 did not stimulte PLB Ser 16 phosphoryltion or ctivte glycogen phosphorylse supports tht the effect ws specific for Epc, nd our dt suggest tht both PKA nd Epc increse insulinstimulted PKB phosphoryltion. In skeletl muscles, we reported tht 7 incresed insulin-stimulted PKB phosphoryltion (Brennesvik et l., 25), nd suggested tht the effect ws medited vi Epc only becuse the PKA inhibitor H89 further incresed the drenline-medited potentition of British Journl of Phrmcology (21)

10 camp-pka signlling regultes PKB in hert JT Stuenæs et l 125 A PKB Ser 473 phosphoryltion C Insulin Slbutmol Roliprm b B PKB Thr 38 phosphoryltion D Insulin Slbutmol Roliprm b GSK-3β Ser 9 phosphoryltion Insulin Slbutmol Roliprm b Figure 6 Roliprm increses phosphoryltion of protein kinse B (PKB), glycogen synthse kinse (GSK)-3b nd phospholmbn (PLB) in crdiomyocytes incubted with slbutmol nd insulin. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h with 1 mm roliprm dded fter 15 min. After preincubtion (nd 15 min incubtion with 1 mm roliprm) insulin (1 mu ml -1 ) nd slbutmol (1-6 M) were dded for 15 min. Grph shows mens of quntified blots with insulin s 1%; representtive blot is shown in (D). (A) Effect of roliprm on insulin-stimulted PKB Ser 473 phosphoryltion in the bsence or presence of slbutmol. Dt re from four different experiments; n = 16 for insulin nd n = 7 15 for other groups. (B) Effect of roliprm on insulin-stimulted PKB Thr 38 phosphoryltion in the bsence or presence of slbutmol. Dt re from six different experiments; n = 22 for insulin nd n = 9 12 for other groups. (C) Effect of roliprm on insulin-stimulted GSK-3b Ser 9 phosphoryltion in the bsence or presence of slbutmol. Dt re from six different experiments; n = 24 for insulin nd n = 9 12 for other groups. (D) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9 nd PLB Ser 16 phosphoryltion nd totl GSK-3b nd PLB in different tretments groups. Significntly higher thn insulin; b Significntly higher thn insulin + slbutmol. insulin-stimulted PKB phosphoryltion. However, H89 is rther unspecific inhibitor for PKA (Lochner nd Moolmn, 26), nd we hve lter seen tht N 6 -camp increses insulinstimulted PKB phosphoryltion in soleus muscles (J. Jensen, unpublished). Therefore, we suspect tht H89 influences PKB phosphoryltion vi mechnisms independent of PKA nd there re no contrdictions between the studies. Our dt suggest tht ctivtion of both PKA nd Epc cn increse insulin-stimulted PKB phosphoryltion in crdiomyocytes from dult rts. However, Epc seems much less effective thn PKA. The mechnism for PKA-medited PKB phosphoryltion is not obvious, but requires PI 3-kinse ctivtion s wortmnnin completely blocked PKB nd GSK-3 phosphoryltion in crdiomyocytes stimulted with insulin nd isoprenline. A possibility is tht b-drenoceptor stimultion incresed insulin-stimulted PI 3-kinse ctivity, but this does not occur in skeletl muscles where stimultion of b-drenoceptors lso potentited insulin-stimulted PKB nd GSK-3 phosphoryltion (Brennesvik et l., 25; Jensen et l., 27; Jensen et l., 28). Therefore, it is lso likely tht stimultion of b-drenoceptors regulte other components in the PI 3-kinse signlling pthwy such s PTEN, PDK1, mtorc2 or the phosphtse tht dephosphoryltes PKB; this ide is supported by recent study showing tht inhibition of PKA ttenuted PDGF-medited PIP 3 ccumultion independent of PI 3-kinse ctivity in fibroblsts (Deming et l., 28). It is lso possible tht b-drenoceptor British Journl of Phrmcology (21)

11 camp-pka signlling regultes PKB in hert 126 JT Stuenæs et l A B PKB Ser 473 phosphoryltion Insulin Slbutmol PD 98, C b PKB Thr 38 phosphoryltion Insulin Slbutmol PD 98, D b GSK-3β Ser 9 phosphoryltion Insulin Slbutmol PD 98, b Figure 7 The MEK inhibitor PD98,59 increses phosphoryltion of protein kinse B (PKB), glycogen synthse kinse (GSK)-3b nd phospholmbn (PLB) in crdiomyocytes incubted with slbutmol nd insulin. After n overnight incubtion in medium, crdiomyocytes were preincubted in buffer without ny hormones for 2 h with 5 mm PD98,59 dded fter 9 min. After preincubtion (nd 3 min incubtion with 5 mm PD98,59) insulin (1 mu ml -1 ) nd slbutmol (1-6 M) were dded for 15 min. Grph shows mens of quntified blots with insulin s 1%; representtive blot is shown in (D). (A) Effect of PD98,59 on insulin-stimulted PKB Ser 473 phosphoryltion in the bsence or presence of slbutmol. Dt re from three different experiments; n = 1 for insulin nd n = 4 6 for other groups. (B) Effect of PD98,59 on insulin-stimulted PKB Thr 38 phosphoryltion in the bsence or presence of slbutmol. Dt re from five different experiments; n = 16 for insulin nd n = 7 1 for other groups. (C) Effect of PD98,59 on insulin-stimulted GSK-3b Ser 9 phosphoryltion in the bsence or presence of slbutmol. Dt re from four different experiments; n = 14 for insulin nd n = 6 8 for other groups. (D) Representtive blots showing PKB Ser 473, PKB Thr 38, GSK-3b Ser 9 nd PLB Ser 16 phosphoryltion nd totl PKB nd PLB in different tretments groups. Significntly higher thn insulin; b Significntly higher thn insulin + slbutmol. stimultion ctivtes other kinses, like DNA-PK, PKCb2 or integrin-linked kinse, which hve been reported to phosphorylte PKB Ser 473 in some cell types (Persd et l., 21; Kwkmi et l., 24; Huston et l., 28). Although the present study does clrify the mechnisms by which b-drenoceptor stimultion increses insulin-stimulted PKB phosphoryltion in crdiomyocytes, our dt show tht the PI 3-kinse ctivtion is involved. PDE4 is required for b 2-drenoceptor subtype-specific signlling in crdiomyocytes (Xing et l., 25). ERK phosphoryltes most PDE4 isoforms nd phosphoryltion decreses phosphodiesterse ctivity of the long isoforms wheres ctivity of most of the short isoforms increses (Mckenzie et l., 2; Hously et l., 25). In the present study, we used roliprm (PDE4 inhibitor) nd PD98,59 (MEK inhibitor) to test the hypothesis ERK-medited PDE4 phosphoryltion reduced the bility of b 2-drenoceptor stimultion to increse insulin-stimulted PKB phosphoryltion. Interestingly, inhibition of PDE4 (roliprm) or MEK (PD98,59) incresed the effect of slbutmol on insulin-stimulted PKB phosphoryltion. Roliprm nd PD98,59 lso incresed PLB Ser 16 when slbutmol ws present supporting tht PKA becme ctivted. These dt lso support tht stimultion of b 2-drenoceptors ctivte denylyl cyclse in dult rt crdiomyocytes, but the produced camp is broken down immeditely by PDE4 in process tht requires ERK ctivtion. Unfortuntely, the present study does not determine which isoforms of ERK nd PDE4 tht medite this effect in dult crdiomyocytes. Comprtmentlized b 2-drenergic signlling involves PDE4 in lrge protein complexes (Billie nd British Journl of Phrmcology (21)

12 camp-pka signlling regultes PKB in hert JT Stuenæs et l 127 Hously, 25) nd Dodge-Kfk et l. recently reported lrge complex consisting of PDE4, ERK, MEK, makap, Epc nd PKA in neontl crdiomyocytes (Dodge-kfk et l., 25). The dt in the present study support tht ERKmedited phosphoryltion of PDE4 contribute to comprtmentliztion of b 2-drenoceptor signlling in dult crdiomyocytes nd reduce PKA-medited potenttion of insulin-stimulted PKB phosphoryltion. A cler limittion of the present study is tht we do not describe complete signlling pthwy for the increse in insulin-stimulted PKB phosphoryltion during stimultion of b-drenoceptors. However, the present study for the first time report tht camp-medited PKA ctivtion increses PKB phosphoryltion in crdiomyocytes. It is lso limittion tht we do not report physiologicl role of b-drenoceptormedited potenttion of insulin-stimulted PKB phosphoryltion. However, the fct tht isoprenline-medited PKB phosphoryltion depends on insulin in crdiomyocytes highlights tht insulin should be included in studies with b-drenoceptor gonist, nd it would be prticulr interesting to investigte whether stimultion of b 1-drenoceptors still cuses poptosis in crdiomyocytes when insulin is present. The present study lso opens new perspectives. Defective regultion of both PKB nd camp signlling hve been coupled to crdic hypertrophy (Zheng et l., 25), nd the crosstlk between camp-pka nd PKB now couples these two pthwys. camp signlling hs, to the best of our knowledge, not been coupled to PI3Kg ctivtion, nd it is therefore ppeling to speculte tht camp-medited PKB phosphoryltion my involves clss I A PI 3-kinse. Crckower et l. (Crckower et l. 22) reported tht crdic specific deletion of PTEN induced crdic hypertrophy vi PI3K nd elevted PKB phosphoryltion, nd it is possible tht combined ctivtion of insulin nd b-drenoceptor signlling my induce crdic hypertrophy vi this signlling pthwy. Metbolic syndrome is ssocited with incresed sympthetic nervous ctivity nd hyperinsulinemic nd it my be ttrctive to hypothesize tht this interction between insulin nd b-drenoceptor signlling contributes to the incresed risk for development of hert filure in type 2 dibetes (Ashrfin et l., 27). Moreover, s PKB ctivtion improves recovery from ischemi reperfusion (Mtsui et l., 21), it would lso be relevnt to investigte whether combintion of insulin nd b-drenoceptor will improve recovery from ischemi. The crosstlk mechnisms between insulin nd b-drenoceptors in the regultion of PKB should be fully chrcterized in the hert, s they my represent novel trgets for tretment of crdic diseses. In conclusion, b-drenoceptors re powerful regultors of PKB phosphoryltion in the presence of insulin. The potentition of insulin-stimulted PKB phosphoryltion occurs vi camp nd PKA, nd stimultion of b 1-drenergic receptors incresed insulin-stimulted PKB phosphoryltion much more thn b 2-drenergic receptor stimultion. Stimultion of b 2-drenoceptors ctivtes denylyl cyclse but co-ctivtion of PDE4 prevents camp ccumultion nd PKA ctivtion in dult rt crdiomyocytes. Moreover, the crosstlk between insulin nd b-drenergic receptors in crdiomyocytes show tht it is importnt to study b-drenergic signlling in the presence of insulin or other growth fctors. Acknowledgements We thnk professors Peter R Shepherd (University of Aucklnd, NZ) nd Bjørn S Skålhegg (University of Oslo) for comments to the mnuscript. The study ws supported by Novo Nordisk Foundtion nd the Europen Commission vi COST BM62. Conflicts of interest The uthors declre no conflicts of interest. References Ashrfin H, Frenneux MP, Opie LH (27). Metbolic mechnisms in hert filure. Circultion 116: Billie GS, Hously MD (25). Arrestin times for comprtmentlised camp signlling nd phosphodiesterse-4 enzymes. Curr Opin Cell Biol 17: Brtel S, Kruse EG, Wllukt G, Krczewski P (23). New insights into b 2-drenoceptor signling in the dult rt hert. Crdiovsc Res 57: Byscs JR, Skmoto K, Armit L, Arthur JS, Alessi DR (26). Evlution of pproches to generte PDK1 tissue specific knock-in mice. J Biol Chem 281: Beuloye C, Bertrnd L, Kruse U, Mrsin AS, Dresselers T, Vnstpel F et l. (21). No-flow ischemi inhibits insulin signling in hert by decresing intrcellulr ph. Circ Res 88: Bertrnd L, Hormn S, Beuloye C, Vnoverschelde JL (28). Insulin signlling in the hert. Crdiovsc Res 79: Brennesvik EO, Ktori C, Ruzzin J, Jebens E, Shepherd PR, Jensen J (25). Adrenline potentites insulin-stimulted PKB ctivtion vi camp nd Epc: implictions for cross tlk between insulin nd drenline. Cell Signl 17: Chesley A, Lundberg MS, Asi T, Xio RP, Ohtni S, Lktt EG et l. (2). The b 2-drenergic receptor delivers n ntipoptotic signl to crdic myocytes through G i-dependent coupling to phosphtidylinositol 3 -kinse. Circ Res 87: Communl C, Singh K, Swyer DB, Colucci WS (1999). Opposing effects of b 1- nd b 2-drenergic receptors on crdic myocyte poptosis: role of pertussis toxin-sensitive G protein. Circultion 1: Condorelli G, Drusco A, Stssi G, Bellcos A, Roncrti R, Iccrino G et l. (22). Akt induces enhnced myocrdil contrctility nd cell size in vivo in trnsgenic mice. Proc Ntl Acd Sci USA 99: Crckower MA, Oudit GY, Kozierdzki I, Sro R, Sun H, Sski T et l. (22). Regultion of myocrdil contrctility nd cell size by distinct PI3K-PTEN signling pthwys. Cell 11: Debosch B, Treskov I, Lupu TS, Weinheimer C, Kovcs A, Courtois M et l. (26). Akt1 is required for physiologicl crdic growth. Circultion 113: Deming PB, Cmpbell SL, Bldor LC, Howe AK (28). Protein kinse A regultes 3-phosphtidylinositide dynmics during plteletderived growth fctor-induced membrne ruffling nd chemotxis. J Biol Chem 283: Dodge-Kfk KL, Soughyer J, Pre GC, Crlisle Michel JJ, Lngeberg LK, Kpiloff MS et l. (25). The protein kinse A nchoring protein makap coordintes two integrted camp effector pthwys. Nture 437: Drgo GA, Colyer J (1994). Discrimintion between two sites of phosphoryltion on djcent mino cids by phosphoryltion British Journl of Phrmcology (21)