Ouabain prevents pathological cardiac hypertrophy and heart failure through activation of phosphoinositide 3 kinase α in mouse

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1 DOI /s RESEARCH Open Access Ouin prevents pthologicl crdic hypertrophy nd hert filure through ctivtion of phosphoinositide 3 kinse α in mouse Jin Wu 1,2, Dxing Li 1,3, Lingling Du 1, Mustf Bldwi 1, Mrjorie E. Gle 1, Amir Askri 1 nd Lijun Liu 1 Astrct Bckground: Use of low doses of digitlis to prevent the development of hert filure ws dvocted decdes go, ut conflicting results of erly niml studies dissuded further reserch on this issue. Recent discoveries of digitlis effects on cell signl pthwys prompted us to reexmine the possiility of this prophylctic ction of digitlis. The specific im of the present study ws to determine if suinotropic doses of in would prevent pressure overlodinduced crdic remodeling in the mouse y ctivting phosphoinositide 3-kinse α (PI3Kα). Results: Studies were done on n existing trnsgenic mouse deficient in crdic PI3Kα () ut with norml crdic contrctility, control mouse (), nd on cultured dult crdiomyocytes. In myocytes, ut not in myocytes, in ctivted PI3Kα nd Akt, nd cused cell growth. This occurred t low in concentrtions tht did not ctivte the EGFR-Src/Rs/Rf/ERK cscde. nd mice were sujected to trnsverse ortic constriction () for 8 weeks. A suinotropic dose of in (5 µg/kg/dy) ws constntly dministrted y osmotic mini-pumps for the first 4 weeks. All mice were monitored y echocrdiogrphy throughout. Ouin erly tretment ttenuted -induced crdic hypertrophy nd firosis, nd improved crdic function in -operted mice ut not in -operted mice. downregulted α2-isoform of N + /K + -ATPse ut not its α1-isoform in herts, nd in tretment prevented the downregultion of α2-isoform. -induced reduction of α2-isoform did not occur in herts. clusions: Our results show tht () sfe doses of in prevent or dely crdic remodeling of pressure overloded mouse hert; nd () these prophylctic effects re due to in inding to α2-isoform resulting in the selective ctivtion of PI3Kα. Our findings lso suggest tht potentil prophylctic use of digitlis for prevention of hert filure in mn deserves serious considertion. Keywords: N + /K + -ATPse, PI3 kinse, Crdic hypertrophy, Hert filure, Ouin, Digitlis Bckground In 1933, Christin [1] dvocted the prophylctic use of digitlis to retrd crdic enlrgement in ptients with hert disese ut without hert filure. In 1965, Willims nd Brunwld [2] presented the first experimentl Correspondence: Lijun.Liu@utoledo.edu 1 Deprtment of Biochemistry nd Cncer Biology, College of Medicine nd Life Sciences, University of Toledo, 3 Arlington Ave., MS 11, Toledo, OH 43614, USA Full list of uthor informtion is ville t the end of the rticle support for this proposl showing tht rts sujected to suprrenl ortic constriction nd treted with dily nontoxic doses of digitoxin prior nd following ortic constriction, exhiited less myocrdil hypertrophy nd lower incidence of ftl hert filure thn rts sujected to ortic constriction ut not treted with digitoxin. Though numer of studies tht followed [3 5] either confirmed or questioned the ove findings nd conclusions, the issue of whether or not digitlis hs prophylctic effect on the growth of the overloded herts seems 215 Wu et l. This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( cretivecommons.org/licenses/y/4./), which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( zero//) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Pge 2 of 15 to hve disppered from the susequent literture. Perhps the question ws rised hed of its time. We hve now reexmined the issue in the light of the recently pprecited effects of digitlis drugs on cell signling pthwys. Here, we present the results of initil studies using fresh experimentl pproches to test for the possile prophylctic effect of digitlis on the hert tht is sujected to pressure overlod. Digitlis drugs (such s digoxin, digitoxin, nd in) re highly specific inhiitors of the N + /K + -ATPse of the plsm memrne of most of higher eukryotic cells [6, 7]. This enzyme (the sodium pump) ctlyzes the coupled ctive trnsport of N + nd K +, mintins resting memrne potentil, regultes cell volume, nd enles the N + -coupled trnsports of multitude of nutrients nd other ions cross the cell memrne. N + / K + -ATPse hs two suunits (α nd β) tht re essentil for ion pumping, nd third suunit ( FXYD protein) tht regultes function [6, 7]. There re multiple isoforms of ech suunit with tissue nd species specificities, nd vritions mong the digitlis sensitivities of the isoforms [6 8]. In the hert, digitlis concentrtions tht prtilly inhiit N + /K + -ATPse to cuse modest increse in intrcellulr N + re sufficient to ffect the roust N + / C 2+ exchnger of the myocyte plsm memrne, cusing significnt increses in intrcellulr C 2+ nd crdic contrctility [9, 1]. This positive inotropic effect hs long een ssumed y mny to e the sis of the clssicl use of digitlis drugs for the tretment of hert filure [9, 1]. In more recent yers, it hs een relized tht in ddition to its vitl ion trnsport function, N + /K + -ATPse my lso ct s signl trnsducer. Through digitlisinduced trnsient communictions with neighoring memrne proteins, the digitlis-inhiited N + / K + -ATPse ctivtes multiple cell signling pthwys, leding to highly cell-specific downstrem consequences [11]. In our erly studies on the signling function of the sodium pump in crdic myocytes, we noted the pprent prdox tht digitlis drugs which hd long een used to tret the hypertrophied filing hert, cused hypertrophy of the cultured crdic myocytes [12]. Susequently, we found tht this drug-induced hypertrophy is due to the ctivtion of PI3K/Akt/mTOR pthwy, nd tht digitlis tretment of the cultured myocytes ctivtes PI3Kα ut not PI3Kγ [13]. More recently, we lso showed tht in-induced ctivtion of PI3Kα nd the resulting hypertrophy re independent of in s positive inotropic effect [14]. These findings, coupled with the extensive prior reserch of others [15] indicting the ssocition of PI3Kα with physiologicl crdic hypertrophy, nd tht of PI3Kγ with pthologicl hypertrophy, led us to suspect tht digitlis-induced hypertrophy my indeed e kin to physiologicl hypertrophy [13, 14]. And since there is mple evidence to suggest tht selective ctivtion of PI3Kα not only induces physiologicl hypertrophy ut my lso prevent the detrimentl effects of the stimuli tht cuse pthologicl hypertrophy [15], we hypothesized tht the forgotten prophylctic effect of digitlis is due to the selective ctivtion of crdic PI3Kα. Here, we report the testing of this hypothesis in the mouse. We used in s prototypic digitlis drug; trnsverse ortic constriction () to induce pressure-overlod nd pthologicl hypertrophy; nd we compred the effects of in tretment on the development nd the consequences of in the wild type mouse nd geneticlly modified mouse from which crdic PI3Kα ws deleted ut exhiited norml crdic contrctility nd histology [16]. Our findings supported the tested hypothesis. Results Comprison of hert size nd function in norml mice nd those deficient in PI3Kα In the present studies, which were designed to test our hypothesis on the prophylctic ction of in, we wished to use the mouse generted y Luo et l. [16] with the muscle specific deletion of p85α regultory suunit nd germ line deletion of p85β regultory suunit of PI3Kα. Becuse this mouse hs not een widely used since its genertion, we deemed it necessry to confirm their min findings on the chrcteristics of this mouse. Our results summrized elow re consistent with nd reinforce those of Luo et l. [16]: (1) In the mouse, reltive to Cre mice (trol mice), protein levels of p85α nd p11α of PI3Kα were gretly reduced in the lystes of the whole hert or the isolted crdiomyocytes, wheres the levels of p11γ of PI3Kγ remined unchnged (Fig. 1); (2) in crdiomyocyte lystes of the mouse, reltive to those of the control, the sl level of PI3Kα ctivity ws gretly reduced, while the ctivity of PI3Kγ ws unchnged (Fig. 1). The remining 1 % of the PI3Kα ctivity in the lyste of the myocytes is most likely due to myocytes tht hve escped gene deletion s noted efore [16], nd s it hs een estlished in cses of mny other crdic-specific deletions [14, 17]; (3) when intct crdiomyoyctes isolted from the nd the control mice were similrly exposed to insulin, the expected ctivtion of Akt ws noted in the control myocytes, ut gretly reduced in the myocytes (Fig. 1c). The serine/threonine kinse Akt, lso known s protein kinse B (PKB), is downstrem component of the signling cscdes tht re dependent on PI3 K ctivtion t the insulin or other growth fctor receptors [15, 16]; (4) The herts of the mice were smller thn those of control (Fig. 1d;

3 Pge 3 of 15 p85α p11α p11γ GAPDH hert p85α p11α p11γ GAPDH crdiomyocyte PI(3)P Origin crdiomyocyte PI3K α PI3K γ PI3K suunits p85 α p11 α p11 γ PI3K suunits p85 α p11 α p11 γ PI3Ks lipid kinse ctivity PI3K α PI3K γ c p-akt Akt d Body Weight (g) Insulin: (min) 1 nm Fig. 1 Chrcteriztion of mouse herts.. Representtive lots of p11α nd p85 suunits of PI3Kα nd p11 γ suunit of PI3Kγ in whole hert lyste (top left) nd in freshly isolted dult mouse crdiomyocytes (top right). GAPDH ws used s loding control. Quntittive dt of expression of PI3 K suunits in nd mouse herts (n = 4) (ottom left) nd crdiomyocytes (n = 7) (ottom right); representtive TLC pltes show PI3Kα nd PI3Kγ lipid kinse ctivities in freshly isolted or crdiomyocytes (top). Quntittive dt of PI3K ctivity in nd crdiomyocytes (n = 4); c insulin (1 nm)- induced Akt signling in nd crdiomyocytes (representtive lots of three experiments) nd d comprison of ody weight, hert weight nd left ventriculr weight in nd mice ( n = 5, n = 7). P < 5, P < 1 vs. Hert Weight (g) Left Ventricle Weight (g) Tle 1), ut crdic contrctility nd function seemed to e norml in the KO herts (Tles 1, 2). Comprison of Ouin s signling effects on crdiomyocytes isolted from norml mice nd those deficient in PI3Kα Adult mouse crdiomyocytes re known to contin the α1 isoform, the α2 isoform, nd the β1 isoform of the N + /K + -ATPse [14, 18]. Since n αβ dimer is the functionl unit to which in inds [7], we immunossyed the lystes of freshly isolted crdiomyocytes of the control nd the mice for α1, α2, nd β1 protein, nd found no significnt differences etween the isoform content in the two mice (Fig. 2). Our previous studies indicting in-induced selective ctivtion of crdic PI3Kα ut not PI3Kγ [13] were done on neontl nd dult crdiomyocytes of rt. To see if similr selective effects of in re noted in dult mouse crdiomyocytes, intct myocytes isolted from control nd mouse herts were exposed to in for 5 min, nd ssyed for PI3Kα nd PI3Kγ ctivity. In control myocytes, in ctivted PI3Kα ut not PI3Kγ ctivity (Fig. 2). Ouin lso cused no significnt ctivtion of PI3Kγ of the myocytes (Fig. 2). sistent with these findings, ininduced ctivtion of Akt during 5 15 min ws oserved in the control myocytes (Fig. 2c), ut not in myocytes (Fig. 2d). When further experiments were done to estimte the minimum dose of in necessry for the rpid ctivtion of Akt in control myocytes, in concentrtion s low s 5 nm cused pronounced ctivtion of Akt fter 5 min tretment (Fig. 2e). Significntly, the ctivtion of ERK 1/2 in the sme control myocytes required much higher in concentrtions (Fig. 2f). In isolted crdiomyocytes, hypertrophy of the terminlly differentited cells my e ssessed y mesuring

4 Pge 4 of 15 Tle 1 Echocrdiogrphic nlysis (seline) of nd mouse herts BW (g) HR (pm) EDA (cm 2 ) ESA (cm 2 ) PWT (cm) SWT (cm) RWT MPI FS LV mss (g) 31.8 ± ± ± 4 88 ± ± 3.14 ± 3.6 ± 2.36 ± 3.45 ± ± 4 KO 28.4 ± 438 ± ± 4 81 ± 4.18 ± 5 87 ± 2 4 ± 2.3 ± 2.45 ± 1.15 ± 5 The nd the Cre controls (12 16 week-old, mle) were sujected to echocrdiogrphy nlysis BW ody weight; HR hert rte; EDA end distolic re; ESA end systolic re; PWT posterior wll thickness; SWT septl wll thickness; RWT reltive wll thickness; MPI myocrdil performnce index; FS frctioning shortening; LV mss left ventriculr mss; n = 21; KO n = 15 p < 5 v.s. Tle 2 Comprison of crdic hemodynmic function (seline) of nd mouse herts BW (g) HR (pm) Ves (LII) Ved (LII) Pes (mmhg) Ped (mmhg) SV (Lll) EF (%) dp/dt (mmhg/s) dp/dt ( mm Hg/s) 23.1 ± ± ± ± ± ± ± ± ± ± 858 KO 21.3 ± ± ± ± ± ± ± ± ± ± 444 Hemodynmic function of nd the Cre controls (12 week-old, mle) were mesured y crdic ctheter s descried in Method. There is no sttisticl significnce etween the nd KO mice BW ody weight; HR hert rte; Ves end systolic volume; Ved end distolic volume; Pes end systolic pressure; Ped end distolic pressure; SV stroke volume; EF ejection frction; +dp/dt the rte of pressure development; dp/dt the rte of relxtion. n=4; KO n=7 the initil rte of protein synthesis [19, 2], nd we hve shown efore tht in-induced ctivtion of PI3Kα/ Akt pthwy leds to increse protein synthesis in isolted mouse dult crdiomyocytes [14]. Compring protein synthesis in the control nd the myocytes, we found tht in (5 μm, 12 h) stimulted hypertrophy significntly in the former ut not in the ltter (Fig. 3). Importntly, endothelin-1 (ET-1) which is known to cuse pthologicl hypertrophy through the ctivtion of PI3Kγ [15], incresed protein synthesis in oth the control nd the myocytes (Fig. 3). Atril ntriuretic peptide (ANP), rin ntriuretic peptide (BNP) nd β-myosin hevy chin (β-mhc) re commonly used crdic hypertrophic mrkers [15]. In greement with our previous findings [14], nd consistent with the present findings of Fig. 3, ET-1 (1 nm, 12 h) stimulted the expression of BNP in oth myocytes, while in hd no significnt effect on BNP expression in either the control or the myocytes (Fig. 3, c). In seprte experiments, we exmined the effects of in, ET-1, nd the comintion of the two on protein synthesis in control crdiomyocytes. The results (Fig. 3d) re consistent with the proposition tht in-induced hypertrophy ntgonizes ET-1-induced hypertrophy t the cellulr level. Assessing the prophylctic effect of in on induced crdic hypertrophy nd dysfunction in norml mice nd those deficient in PI3Kα Supported y the ove findings t the cellulr level, the ims of the following in vivo experiments were to determine if tretment of mice with low doses of in could prevent or dely the known -induced crdic dysfunction nd filure; nd if so, whether or not such in effects were dependent on PI3Kα. - cused chronic chnges were monitored over period of 8 weeks. To ssess in effects, the drug ws infused sucutneously (5 μg/kg/dy) y ALZET osmotic pump for continuous dosing during the first 4 weeks. In these experiments, in concentrtion in circulting lood in hert is dependent on its sorption, distriution, metolism nd excretion. We chose the indicted dosge regimen sed on previous finding of others [21] nd our dt of Fig. 3 in order to keep serum concentrtions of in t low nm rnge (see Discussion). Becuse of previous disgreements on whether or not in ffects lood pressure in rodents [21, 22], it ws necessry to determine the effect of long-term infusion of in on systemic lood pressure in our studies. Tilcuff lood pressure mesurements showed no significnt in effects in shm or groups from the control or the mice (Additionl file 1: Fig. S1). Evidently, in effects on systemic lood pressure re peculir to rts ut not mice s lso noted efore y others [21]. Echocrdiogrphic nlysis showed tht posterior wll thickness, septl wll thickness nd reltive wll thickness incresed during the first 2 weeks fter in the control nd the mice; however, in prevented the -induced increse of wll thickness in the control ut not in the mice (Fig. 4). Echocrdiogrphy lso showed tht left ventriculr chmers egn to dilte fter six weeks of in oth the control nd the mice; the effect eing especilly prominent

5 Pge 5 of 15 NKA suunits NKA-α1 NKA-β1 NKA-α2 GAPDH NKA α1 NKA β1 NKA α2 PI(3)P PI3Ks lipid kinse ctivity PI3K α PI3K γ PI3K γ 2. PI3K α PI3K γ PI3K γ c p-akt Akt Time (min) Rtio of p-akt/akt (reltive vlue) μM μM 1µM 5' 1µM 15' 5 µm 5' 5 µm 15' Rtio of p-akt/akt (reltive vlue) p-akt Akt Time (min) 2. d e f μM 5μM 1 µm 5' 1 µm 15' 5 µm 5' 5 µm 15' Rtio of p-akt/akt nm 5 nm 5 nm 5 µm Rtio of p-erk/erk nm 5 nm 5 nm 5 µm Fig. 2 Comprison of in-induced signling in cultured crdiomyocytes from nd mice.. Representtive lots of N + /K + -ATPse isoforms (top). GAPDH ws used s loding control. Quntittive dt of expression of N + /K + -ATPse isoforms (n = 6) (ottom);. representtive PI(3)P dots show the effect of in (5 μm, 5 min) on PI3Kα nd PI3Kγ lipid kinse ctivities in nd crdiomyocytes, cle () (top). Quntittive dt of PI3K ctivity from the top. (n = 9, P < 5 vs. ); c representtive lots of the effect of in on Akt ctivtion in crdiomyocytes (top). Quntittive dt of Akt ctivtion from the top (n = 6, P < 5 vs. ); d representtive lots of the effect of in on Akt ctivtion in crdiomyocytes (top). Quntittive dt of Akt ctivtion from the top. (n = 6, P < 5 vs. ). e in (5 min) dose- responses on Akt ctivtion in wild-type crdiomyocytes (n = 6, P < 5, P < 1 vs. ) nd f in (5 min) dose- responses on ERK ctivtion in wild-type crdiomyocytes (n = 6, P < 5 vs. ) in the end-systolic dimension (Fig. 5). The erly tretment with in during the first 4 weeks significntly reduced this -induced diltion of left ventricles in the control ut not the mice (Fig. 5). lso reduced contrctile function (Frctionl shortening, FS %) four weeks fter surgery in oth the control nd the mice; nd in pretretment rescued crdic dysfunction in the control ut not the KO mice (Fig. 6, ). Myocrdil Performnce Index (MPI) generted from pulsed wve Doppler imging ws employed to further exmine systolic nd distolic functions. In line with the FS % results, the erly tretment with in prtilly improved crdic function in the control ut not in the mice (Fig. 6c, d). Postmortem nlyses performed fter 8 weeks of showed tht: (1) Ouin pretretment significntly ttenuted the -induced crdic hypertrophy in the control mice ut not the KO mice (Fig. 7); (2) crdic firosis mesured y trichrome stining, which ws evident in ll herts ws significntly reduced y in pretretment in the control ut not the herts (Fig. 7). We lso mesured peri-vsculr firosis in ll groups nd oserved results similr to those of Fig. 7 (dt not shown); (3) the -induced effects on ANP nd BNP mrna expressions noted in oth the control nd the KO mice were ntgonized y in pretretment in the control ut not the herts (Fig. 7c).

6 Pge 6 of 15 3 H-Leucine incorportion ET-1 Gene expression ET-1 ANP BNP β-mhc c Gene expression ET-1 ANP BNP β-mhc [ 3 H]-Phenyllnine incorportion Reltive Chnge ET-1 +ET-1 Fig. 3 Effects of in on hypertrophic growth nd hypertrophic mrker gene expressions in cultured crdiomyocytes of nd mice. Comprison of 12 h tretment of in (5 μm) nd ET-1 (1 nm) on protein synthesis ( 3 H-leucine incorportion) in nd crdiomyocytes (n = 6);, c in nd ET-1 effects on hypertrophic mrkers (fetl gene expression) in nd crdiomyocytes (n = 3) nd d effects of in, ET-1 nd the comintion of the two on protein synthesis ( 3 H-phenyllnine incorportion) (n = 9). P < 5 vs., P < 1 vs., P < 5 vs. d Tken together, the ove in vivo findings provide strong support for the existence of prophylctic effect of in on -induced crdic hypertrophy nd the susequent crdic dysfunction; nd indicte tht this in effect is dependent on the presence of PI3Kα in the hert. Becuse of the longstnding prior findings indicting tht the α2-suunit of the rt crdic N + /K + -ATPse, ut not tht of the α1-suunit, my e trnscriptionlly down-regulted y pressure overlod [23, 24], we lso looked for chnges in the mrna nd protein levels of the α-suunits in the postmortem herts of the experiments of Fig. 7. In the control mouse herts, significntly reduced α2 mrna nd protein levels, ut did not lter the protein level of the α1-suunit (Fig. 8, c). In the control mouse tretment with in for the first 4 weeks, the effect on the protein level of the α2 ws clerly ntgonized y in, though significnt in effect on the α2 mrna ws not detected (Fig. 8, c). sistent with the lck of effect of on the expression of the α1-suunit, in tretment lso did not ffect the expression of α1 protein in the control herts (Fig. 8). In the mouse herts, neither nor with in tretment hd significnt effects on α2 mrna nd α1 nd α2 protein levels reltive to those of shmoperted mice (Fig. 8, d). Tken together, the findings of Fig. 8 clerly indicte the previously unrecognized fct tht PI3Kα is required for the downregultion of the α2 isoform of crdic N + / K + -ATPse. Though previous studies [23, 24] showed tht effects on α2 re trnscriptionl, we showed the possiility tht in my prevent degrdtion or lter the trnsltionl efficiency of α2 isoform of crdic N + / K + -ATPse. Discussion The decisive prt of these studies tht estlish the prophylctic effect of in on pressure overlod-induced hypertrophy re the results of the in vivo experiments on the herts of the control mice nd those deficient in

7 Pge 7 of 15 c Posterior Wll Tickness (PWT) Septl Wll Thickness (SWT) Rettive Wll Thickness (RWT) (w) (w) Posterior Wll Tickness (PWT) Septl Wll Thickness (SWT) (w) (w) (w) (w) Fig. 4 Effects of in on -induced crdic hypertrophy in nd mice. Experiments were done s descried in Methods. Left ventriculr wll thickness ws monitored y echocrdiogrphy efore nd fter 8 weeks of the surgery. Ouin ws infused sucutneously (5 μg/kg/dy) y ALZET osmotic pumps for continuous dosing for the first 4 weeks. Posterior wll thickness (PWT); septl wll thickness (SWT) nd c reltive wll thickness (RWT). n = 6 ~ 7, P < 5 vs. ; P < 5 vs. Rettive Wll Thickness (RWT).6 PI3Kα. These studies, however, would not hve een ttempted hd we not done our initil experiments on the isolted cultured myocytes. It is necessry, therefore, to egin with the discussion of the importnt spects of the in vitro studies. Selective ctivtion of PI3Kα y in nd its effects on the hypertrophy of crdiomyocytes At the cellulr level, crdic hypertrophy is ccompnied y the incresed size of the crdic myocytes most of which hve little or no cpcity to proliferte [25]. In our previous studies on isolted mouse myocytes [26], we showed tht the hypertrophy of these cells s mesured y increse in protein synthesis is stimulted y in through the ctivtion of PI3Kα/Akt cscde, ut we hd no direct evidence tht the in-ctivted PI3K ws the PI3Kα. Our present findings now demonstrte tht only PI3Kα ut not PI3Kγ is ctivted y in (Fig. 2); nd tht only in control myocytes ut not those deficient in PI3Kα, there is in-induced ctivtion of Akt resulting in hypertrophy (Figs. 2c, d, 3c). Our ility to study stimulus-induced hypertrophy in isolted dult crdiomyocytes of mice llowed us to sk if the in-induced selective ctivtion of PI3Kα could ntgonize cellulr hypertrophy induced y selective ctivtor of PI3Kγ such s ET-1. Our experiments showed tht even in these in vitro studies in seemed to reduce ET-1-stimulted hypertrophy (Fig. 2f). In spite of the ovious limittion of these in vitro experiments, their results clerly encourged the conduct of our susequent in vivo studies. The most importnt prt of our findings on the cultured myocytes tht llowed us to proceed to the in vivo experiments ws the dt showing the gret sensitivity of

8 Pge 8 of 15 End Distolic Dimension (EDD) (w) End Distolic Dimension (EDD) Ou (w) End Systolic Dimension (ESD) (w) End Systolic Dimension (ESD) Ou (w) Fig. 5 Effects of in on -induced chmer diltion in nd mice. Experiments were done s descried in Methods. Left ventriculr chmer size ws monitored y echocrdiogrphy efore nd fter 8 weeks of the or shm surgery. Ouin ws infused sucutneously (5 μg/kg/dy) y ALZET osmotic pumps for continuous dosing for the first 4 weeks. End distolic dimension (EDD); end systolic dimension (ESD). n = 6 ~ 7, P < 5 vs. ; P < 5 vs. PI3Kα/Akt pthwy to in concentrtions s low s 5 nm (Fig. 2e). This clerly suggested the fesiility of the more onerous in vivo studies. Previous studies of others [21] hd indicted tht the repeted dily dministrtion of 3 μg/kg of in to mice resulted in extrcellulr levels of 3.3 nm of in-like immune-rective mteril nd no evident toxicity. It hd lso een shown tht in wild-type mouse herts, the first evidence of positive inotropic effect is noted t out 4 nm in [27]. Bsed on this informtion nd our dt of Fig. 2e, we chose to use the dily dose of 5 μg/kg of in in our in vivo experiments (Figs. 4, 5, 6, 7, 8) to ensure tht in regimen is suinotropic nd nontoxic. The results of our in vivo experiments proved tht we hd mde the right choice. In reltion to the very high sensitivity of the PI3Kα/Akt pthwy to in (Fig. 2e), it is importnt to emphsize the contrsting low in sensitivity of the pthwy tht leds to ERK1/2 ctivtion (Fig. 2f). Although our previous studies [11, 13, 14] hd shown tht in dult crdic myocytes two prllel cell signling cscdes; i.e., EGFR-Src/Rs/Rf/ERK nd PI3Kα/Akt re functionlly linked to the in-inhiited N + /K + -ATPse, we hd not tested wide rnge of in concentrtion on these pthwys efore. The present findings (Fig. 2e, f) showing out two orders of mgnitude of difference in the sensitivities of these cscdes in the dult mouse myocytes suggest tht ech of these cscdes is linked to different in inding site. And since the two known α-suunits of these myocytes do indeed hve in sensitivities of out 2 3 orders of mgnitude prt [18, 27], it is resonle to conclude tht the EGFR-Src/ Rs/Rf/ERK cscde is functionlly linked to the insensitive α1-isoform, nd tht the PI3Kα/Akt is functionlly linked to the sensitive α2-isoform. This conclusion lso fits the previously oserved irrelevnce of the EGFR-Src/ Rs/Rf/ERK pthwy to in-induced hypertrophy of the mouse crdiomyocytes [14]. Prevention of pressure overlod induced crdic dysfunction y tretment with nontoxic dose of in The mjor findings of our in vivo studies nd their implictions re rther strightforwrd. In oth the control mice nd those deficient in crdic PI3 Kα, stndrd protocol of produced compensted hypertrophy during the first 4 weeks, followed y decompensted hypertrophy nd filure during the next 4 weeks; nd in tretment during the first 4 weeks clerly ntgonized -induced crdic dysfunction in control mice ut not in those deficient in crdic PI3Kα (Figs. 4, 5, 6,

9 Pge 9 of 15 Frction Shortening (FS, 1%) Week Frction Shortening (FS, 1%) Week + c d Myocrdil Performnce Index (MPI) Week P=6 Myocrdil Performnce Index (MPI) Week + Fig. 6 Effects of in on -induced crdic dysfunction in nd mice. Experiments were done s descried in Methods. Left ventriculr crdic functions were monitored y echocrdiogrphy efore nd fter 8 weeks of the or shm surgery. Ouin ws infused sucutneously (5 μg/kg/dy) y ALZET osmotic pumps for continuous dosing for the first 4 weeks. Frction shortening, FS % in nd KO mice. representtive echocrdiogrphic M-mode views of mouse left ventricles t week 8 fter surgery. c myocrdil performnce index (MPI) in nd KO mice. d representtive Pulsed-Wve Doppler flow used for MPI clcultion. n = 6 ~ 7, P < 5 vs. ; P < 5 vs. 7). In short, our in vivo studies greed with the findings of Willims nd Brunwld [2] regrding the prophylctic effect of digitlis on the hypertrophy of the pressure overloded hert. The mjor difference etween the two studies done nerly hlf century prt is tht they used rts nd digitoxin ut we hve used mice nd in. Our studies provide welth of new informtion on the mechnistic ses of the ove prophylctic effect of in. The most importnt eing tht the sence of this prophylctic effect in herts deficient in PI3 Kα estlishes the hypothesis tht it is the selective ctivtion of this lipid kinse isoform tht opposes the detrimentl effects of the vrious cell signling cscdes tht re ctivted y pressure overlod on the hert [13, 15]. Another importnt mechnistic issue reveled y our findings is the identifiction of the α2 isoform of N + / K + -ATPse s the likely prtner of the PI3Kα for exerting nd regulting in s prophylctic effect on the overloded hert. It is the responsiveness of the PI3Kα/Akt pthwy to low nm in concentrtions tht clerly implictes the α2-isoform in functionl interction with PI3Kα resulting in prevention of the hypertrophy. In ddition, since pressure-overlod down-regultes the α2 isoform nd in reverses this (Fig. 8, c), it seems tht there is second distinct role of in-inhiited α2-isoform: Sving the α2-isoform from disppernce due to overlod-induced trnscriptionl downregultion. The fct tht in herts deficient in PI3Kα neither the overlod-induced downregultion of the α2-isoform, nor the in-induced reversl of this re oserved (Fig. 8, d) clerly indicte tht PI3Kα must lso e involved in the trnscriptionl downregultion of the crdic α2 isoform of N + /K + -ATPse. The mechnism of this novel ction of PI3Kα remins to e studied. A scheme summrizing the ove conclusions is presented in Fig. 9. It is pproprite to note tht other recent evidence hs lso implicted the crdic α2 isoform of N + /K + - ATPse in regultion of crdic hypertrophy. There is n extensive history of reserch suggesting the specil role of the digitlis-sensitive ut quntittively minor α2 isoform in the regultion of intrcellulr C 2+ nd N + of the rodent myocyte [18]; nd there is the more recent evidence [28] showing tht in trnsgenic mice with crdic-specific overexpression of α2 isoform, ut not in mice with overexpression of α1 isoform, -induced hypertrophy nd remodeling re ttenuted. While

10 Pge 1 of 15 + LV weight/body weight (mg/g) Trichome Stining (%) Trichome Stining (%) c mrna LV weight/body Weight (mg/g) ANP + BNP + + ANP BNP Fig. 7 Effects of in on -induced hert weight, crdic firosis nd pthologicl hypertrophic mrkers fter 8 weeks of surgery. Representtive hert imges (left) nd quntittive hert weights (right); representtive imges (left) nd quntittive dt (right) on crdic firosis y trichrome stining (n = 5) nd c fetl genes ANP, BNP mrna expression in nd herts. n = 6 ~ 7, P < 5 vs. ; P < 5 vs. mrna thorough exmintion of the reltion of our findings to those of Correll et l. [28] is outside the scope of this Discussion, we suggest tht the findings of the two lortories re supportive of ech other in emphsizing the need for further studies on how the α2 isoform regultes -induced pthologicl hypertrophy on the one hnd, nd in-induced ctivtion of PI3Kα on the other. Our results indicting tht in the control mouse the prophylctic effect of nontoxic in concentrtions on the pressure overloded hert is due to the drug s interction with the α2 isoform of crdic N + /K + - ATPse is lso significnt when we consider the possile relevnce of the present niml studies to the therpeutic use of digitlis in mn. Becuse ll crdic α isoforms of N + /K + -ATPse in the humn hert hve high digitlis sensitivities similr to the α2-isoform of the rodent hert [8], it is resonle to suspect tht low nm lood levels of in or nother digitlis drug my lso exert similr prophylctic effects in mn without disturing intrcellulr ion concentrtion. Whether only the humn α2-isoform or ll three humn crdic isoforms respond to suinotropic in concentrtions through the ctivtion of PI3Kα remins to e investigted. Regrding the potentil relevnce of the present findings to the clinicl use of digitlis, our findings lso suggest the following possiility: Since numerous reexmintions of the Digitlis Investigtion Group tril [29, 3] hve indicted the eneficil effects of lower serum levels of digoxin ( nm) resulting from the dosge used in the tril, it my e tht such therpeutic enefits were induced through the selective ctivtions of PI3Kα nd the signling cscdes tht re linked to this lipid kinse rther thn y sfe levels of digoxin-induced positive inotropy. Though it is most commonly ssumed tht ny eneficil effect of digitlis in the tretment of hert filure is due to the drug s positive inotropic ction, it is pproprite to recll tht there hs never een consensus on this issue, especilly in studies on mn [3 32]. clusion This study estlishes tht in norml mice low nd sfe doses of in, prototypic digitlis drug, prevent or dely crdic dysfunction nd filure tht re cused y

11 Pge 11 of 15 GAPDH + GAPDH NKA suunits (Reltive Vlue) c NKA 1 NKA 2 NKA suunits (Reltive Vlue) d NKA 1 NKA 2 NKA 2 mrna NKA 2 mrna + + Fig. 8 Effects of in on -induced chnges on N + /K + -ATPse (NKA) isoforms in nd KO herts fter 8 weeks of surgery. Representtive lots (top) nd quntittive dt (ottom) of protein expression of NKA α1 nd α2 in herts; representtive lots (top) nd quntittive dt (ottom) of protein expression of NKA α1 nd α2 in herts; c mrna expression of NKA α2 in herts nd d mrna expression of NKA α2 in KO herts. n = 6 ~ 7, P < 5 vs., P < 5 vs. pressure overlod on the hert. Our findings lso show tht these in effects re exerted through its inding to the α2-isoform of the mouse crdic N + /K + -ATPse, nd the resulting ctivtion of PI3Kα/Akt cell signling cscdes. Also clerly indicted y our results is tht the known downregultion of the crdic α2-isoform y pressure overlod is prevented y in-induced ctivtion of PI3Kα. In conjunction with welth of ville informtion on the clinicl use of digitlis drugs in mn, the present findings lso suggest the need for further studies on the potentil use of these drugs for the prevention of hert filure s dvocted nerly century go. Methods Animl Adult mle mice of 2 6 months of ge were used for this study. PI3K p85α loxp/loxp p85β / mice were gifts from Luo et l. [16]. Strited muscle cretine kinse (mck)- Cre trnsgenic mice (control mice; lso referred to s mice) were purchsed from the Jckson Lortory (Stock 6475). PI3K p85α muscle-specific knockout p85β glol knockout mice (p85α mko p85β / mice, referred to s mice) were crossred y PI3K p85α loxp/loxp p85β / mice with mck-cre mice. All mice were housed in pthogen-free conditions nd mintined with 12 h drk/light cycle with free ccess to food nd wter. Animl cre nd experiments were done following the guidelines of NIH guide for the cre nd use of lortory nimls nd the Institutionl Animl Cre nd Use Committee of the University of Toledo. Adult mouse crdiomyocyte isoltion nd culture week old mle mice were used for isoltion nd culture of crdiomyocytes s previously descried [14, 26]. Mice were heprinized (5 U/Kg) nd nesthetized with Ketmine (2 mg/kg.w.)/xylzine (1 mg/ kg.w.) vi intrperitonel (i.p.) injection. Approximtely, million vile rod-shped cells were yielded

12 Pge 12 of 15 2 isoform Low [in] + 2- N + /K + -ATPse Inhiited 2-N + /K + -ATPse Pressure Overlod GPCR 1 isoform Inhiited 1-N + /K + -ATPse High [in] + 1-N + /K + -ATPse Inhiited 1-N + /K + -ATPse Akt Physiologicl Hypertrophy MAPKs, Clcineurin, PKC, etc. Pthologicl hypertrophy [N + ] i NCX1 [C 2+ ] i Positive Inotropy EGFR/Src PLC Rs Rf PKC MEK/ERK1/2 Pthologicl hypertrophy Cell deth Fig. 9 Schemtic presenttion of in s different growth-relted effects on α1 nd α2 isoforms of N + /K + -ATPse in the mouse crdiomyocytes. α2-isoform: ctivtion y su-inotropic in concentrtions (< 4 nm) of the signling cscdes tht led to physiologicl hypertrophy, nd lock -induced pthologicl hypertrophy nd α1-isoform: induction of positive inotropy y higher concentrtions of in, nd ctivtion of signling cscdes tht leds to pthologicl hypertrophy nd cell deth. See Discussion from one hert. Crdiomyocytes were seeded to the culture dishes (pre-coted with 1 μg/ml lminin) in Modified Egle s Medium, supplemented with 2 mm ATP, 2 mm glutmine, 1 % fetl ovine serum (FBS), 1 mm 2,3-utnedione monoxime (BDM), 1 U/ml penicillin nd 1 μg/ml streptomycin for 2 h in 2 % CO 2 humidified incutor nd then cultured overnight in medium in which FBS ws replced y.1 % Bovine Serum Alumin. This medium ws then chnged to fresh one tht excluded BDM 3 min efore the strt of the indicted experiments. SDS PAGE/western lot nlysis This ws done s descried efore [13, 33]. Briefly, protein ws extrcted from cultured crdiomoycytes or homogenized crdic ventriculr tissue in RIPA uffer. Protein extrcts (1-8 µg) were comined with Lemmli loding uffer contining 5 % 2-mercptoethnol, oiled for 5 min, nd size frctionted y sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS PAGE). When extrcts were to e sujected to SDS-PAGE for the detection of N + /K + -ATPse suunits, they were incuted t 37 C for 15 3 min insted of eing oiled. Proteins were trnsferred to Polyvinylidene Difluoride memrnes. Memrnes were incuted for 1 h with 5 % nonft dried milk in Tris-Buffered Sline Tween-2 uffer nd then incuted overnight t 4 C with primry ntiodies. After incution, memrnes were wshed, incuted with peroxidse-conjugted secondry ntiody, nd nlyzed using ECL (Perkin-Elmer Life Sciences). Primry ntiodies were from BD Trnsduction Lortories: nti-pi3 K p11α, nti-pi3 K p85; Cell Signling Technology: rit nti-phospho 473-Akt, nti- Akt; Developmentl Studies Hyridom Bnk, University of Iow (Iow City, IA): N + /K + -ATPse α1 (α6f); ABR: N + /K + -ATPse α2; Millipore: N + /K + -ATPse β1. Anti-PI3 K p11γ, GAPDH, secondry ntiodies got nti-rit IgG-horserdish peroxidse (HRP), nd got nti-mouse IgG-HRP were purchsed from Snt Cruz Biotechnology. PI3 K lipid kinse ssy This ws conducted s previously descried [13, 14, 33]. Briefly, cells were lysed in RIPA uffer with inhiitors. Equl mount of protein in ech smple were incuted with either nti-pi3 K p85α ntiody (6 195, EMD Millipore), or nti-pi3 K p11γ ntiody (sc-7177, Snt Cruz) overnight t 4 C, followed y incution with Protein A grose eds for 3 h t 4 C. And then, the immune complex ws wshed four times with uffer (1 mm NCl, 1 mm N 3 VO 4, nd 2 mm HEPES, ph 7.5) nd resuspended in 4 µl of uffer (18 mm NCl nd 2 mm HEPES, ph 7.5). PI3 K ctivity in the immunoprecipittes ws ssyed directly on the eds

13 Pge 13 of 15 y stndrd procedure with PI.6 mg/ml (Avnti Polr Lipids, Alster, AL, USA) nd [γ- 32 P] ATP (25 μm) used s sustrtes. The rections were performed t room temperture nd stopped fter 1 min y ddition of 8 µl of 1 M HCl. The lipids were extrcted with 16 µl of chloroform methnol (1:1), spotted on thin-lyer chromtogrphy plte, nd seprted with chloroform-cetone-methnol-glcil cetic cid-h 2 O (4:15:13:12:8). The rection product [γ- 32 P] PI(3)P ws seprted from the origin on the TLC plte nd ws exposed to storge phosphor screen. The screen ws scnned y Typhoon Trio phosphorimger (GE Helthcre, USA) nd the top PI(3)P dots were quntified y Imge J softwre. Protein synthesis ssy Protein synthesis ws mesured using [ 3 H]-Leucine or [ 3 H]-Phenyllnine incorportion ssy s previously descried [14, 33]. Protein synthesis ssy ws used s indiction of myocyte hypertrophy [14]. Rel time reverse trnscription polymerse chin rection (RT PCR) This ws conducted s previously descried [14]. Totl RNAs were extrcted from crdiomyocytes nd hert tissues with RNesy plus mini kit (Qigen, 74134) nd RNesy Firous Tissue Kit (Qigen, 7474) respectively ccording to the mnufcturer s instructions. Highcpcity cdna reverse trnscription kit (AB pplied Biosystems) ws used for RT-PCR. Rel-time PCR ws performed y the SYBR Green method with n Applied Biosystems 75 Fst Rel-Time PCR System. All smples were run in triplicte. In ll cses, glycerldehyde- 3-phosphte dehydrogense (GAPDH) gene ws used for dt stndrdiztion nd normliztion. Gene expression levels nd fold chnge comprisons were ssessed using the ΔCt (cycle threshold) nd ΔΔCt, respectively. Primers (forwrd nd reverse, 5 3 ) were ANP: TCG TCT TGG CCT TTT GGC T nd TCC AGG TGG TCT AGC AGG TTC T; BNP: AAG TCC TAG CCA GTC TCC AGA nd GAG CTG TCT CTG GGC CAT TTC; β-mhc: ATG TGC CGG ACC TTG GAA G nd CCT CGG GTT AGC TGA GAG ATC A; NKA α1: ATC TGA GCC CAA ACA CCT GCT AGT nd AAG CGT CCT TCA GCT CTT CAT CCA; NKA α2: ACA ACC AGA TCC ATG AGG CTG ACA nd TTG AGC AGA GCT GAC GAA GCA; nd GAPDH: CAT GGC CTT CCG TGT TCC TA nd CCT GCT TCA CCA CCT TCT TGA T. surgery nd osmotic mini pump implnttion Microsurgicl procedure of pressure overlod hypertrophy induced through ws performed y modifiction of previously descried procedures [34, 35]. Briefly, 12 week-old mle mice were nesthetized with isoflurne 5 % nd connected to rodent ventiltor. The chest cvity ws entered through sternum ove the third ri, nd ortic constriction ws performed y tying 6 silk suture (SP114, LOOK suture) ginst 27 guge needle for mice or 25 guge needle for KO mice, resulting in similr degrees of hypertrophy in oth groups (Additionl file 1: Fig. S2). operted mice underwent the sme opertion except for tying the suture loosely. We noted tht ortic constriction y 27-guge needle on KO mice cused 1 % mortlity ut ll mice survived in shm group. For pin relief, sucutneous injection of uprenorphine (5.1 mg/kg) ws dministered for ech mouse. Ouin-filled osmotic mini-pumps (Alzet, 24) were implnted underneth the dorsl skin 1 dy fter the surgery under nesthesi y 2 3 % isoflurne. Ouin (5 μg/kg/dy) ws continuously infused for 4 weeks. All mice were euthnized t the end of 8 weeks fter surgery. Body weight nd hert weight were recorded. One prt of ventriculr free wll ws immeditely fixed for histologicl study; the rest of left ventricles were snp-frozen in liquid nitrogen nd stored in 8 C for further nlyses. Mice were sujected to euthnsi y dministrtion of ketmine (2 mg/kg.w.) nd xylzine (1 mg/kg.w.) i.p. t the end of the experiment. Echocrdiogrphy Left ventriculr function nd geometry ws ssessed s previously descried [36]. Briefly, mice underwent echocrdiogrphic ssessment using n ACUSON Sequoi C512 Ultrsound System (Siemens) with 15-MHz liner rry trnsducer. The mice were nesthetized with isoflurne (2 %) in 1 % oxygen in n nesthesi chmer. Anesthesi ws mintined ( %) y msk. Anesthetized mice were weighed, de-hired, trnsferred to heting pd, nd plced in supine position. The prsternl long xis ws used to otin 2-D nd 2-D guided M-mode imges for the ssessment of LV end systolic nd distolic res, dimeters, wll thicknesses nd systolic function. Trnsmitrl Doppler flow ws trced in prsternl long xis view for mesurement of left ventriculr isovolumetric contrction time (IVCT), isovolumetric relxtion time (IVRT) nd ejection time (ET). Myocrdil Performnce Index (MPI), which reflects oth crdic distolic nd systolic function, ws clculted y the following eqution: MPI = (IVCT + IVRT)/ET. Ctheteriztion of mouse left ventricles The procedure ws conducted under stereomicroscope ccording to the pulished protocol [37]. Mice were heprinized (5 U/kg) nd nesthetized with Ketmine (5 15 mg/kg) nd Xylzine ( mg/ kg) vi intrperitonel (i.p.) injection. Mice were plced

14 Pge 14 of 15 on the surgicl pltform nd kept wrm t 37 C. MPVS UltrTM Pressure Volume (P V) system (Millr Instruments, Inc.) nd PowerL 8/3 (ADInstruments, Inc.) were turned on for stiliztion 3 min prior to the recording. The mouse Pressure Volume conductnce ctheter (SPR-839, Millr Instruments, Inc.) ws dvnced into the left ventricle through right crotid rtery. After stiliztion of the signl for 1 15 min, the pressure nd volume signls (P V loops) were cquired nd recorded y PowerL 8/3 (ADInstruments, Inc.) nd MPVS UltrTM Pressure Volume (P V) system (Millr Instruments, Inc.). Rel-time P V loop nd other crdic functions (e.g. HR, dp/dt, etc.) were monitored nd dt nlysis ws conducted with Lchrt 7 (ADInstruments, Inc.) softwre. Msson trichrome stining Left ventricle ws fixed in 1 % uffered formlin conducted y the stndrd Msson s trichrome stining procedure. The stining ws shown s nuclei (lck), cytoplsm, muscle fiers (red) nd collgen (lue). Imges were tken y the Olympus IX51 inverted microscope connected to SPOT Insight 2. Mp Cmer under 2 ojective lens. Seven-fourteen pictures were tken from ech hert smple. The percentge of lue re in ech hert section ws quntified y Imge J softwre (Ntionl Institutes of Helth, USA). Dt nlysis All dt re presented s men ± stndrd error. Sttisticl nlyses were performed y using GrphPd Prism 5. softwre (L Joll, CA, USA). The Student s t test ws used to compre two groups. One-wy nlysis of vrince (ANOVA), followed y the Bonferroni s post hoc test ws used to compre multiple groups. Other method ws listed in the figure legend. Differences were considered sttisticlly significnt t p < 5. Additionl file Additionl file 1: Figure S1. Effect of in infusion on systemic lood pressure in nd mice. Mice were grouped s shm, shm+ (5 μg/kg/dy.w.), or + (5 μg/kg/dy.w.). Ouinfilled osmotic mini-pumps (Alzet, 24) were implnted underneth the dorsl skin one dy fter the surgery. Ouin (5 μg/kg/dy) ws continuously infused for 4 weeks. All mice were euthnized t the end of 8 weeks fter surgery. Blood pressure ws mesured y til-cuff volume-pressure recording (VPR) every two weeks. A. : shm (n=7), shm+ (n = 6), (n = 7), + (n = 6); B. : shm (n = 5), shm+ (n = 4), (n = 4), + (n = 6). Figure S2. Comprison of -induced crdic hypertrophy in nd mice. Experiments were done s descried in Methods. Left ventriculr wll thickness ws monitored y echocrdiogrphy efore nd fter eight weeks of the surgery. A. Reltive wll thickness (RWT). n = 6~7, P < 5 v.s. ; B. Comprison of -induced hypertrophy etween nd mice. There is no difference etween two groups y repeted mesures ANOVA. P < 5. Authors contriutions JW, AA nd LL were involved in the conception nd design of the study; JW, DL, crried out the cellulr nd moleculr studies; MG performed PI3K ssy nd crried out the mouse reeding nd mintennce; JW, LD, MB crried out the mouse surgeries, crdic ctheteriztion nd echocrdiogrphy; JW, DL nlyzed dt nd prepred figures nd tles; JW, AA, nd LL interpreted results of experiments nd drfted the mnuscript; AA, nd LL edited nd revised the mnuscript. All uthors red nd pproved the finl mnuscript. Author detils 1 Deprtment of Biochemistry nd Cncer Biology, College of Medicine nd Life Sciences, University of Toledo, 3 Arlington Ave., MS 11, Toledo, OH 43614, USA. 2 Center for Crniofcil Moleculr Biology, University of Southern Cliforni, Los Angeles, CA, USA. 3 Stte Key Lortory of Te Plnt Biology nd Utiliztion, Anhui Agriculturl University, Hefei, People s Repulic of Chin. Acknowledgements We thnk Dr. Eric E. Morgn for invlule ssistnce with echocrdiogrphic nlyses. This study ws supported y NIH grnt PO1 HL36573 (A.A) nd University of Toledo, College of Medicine nd Life Sciences ridge fund (L.L.). Competing interests The uthors declre tht they hve no competing interests. Received: 21 Septemer 215 Accepted: 3 Novemer 215 References 1. Christin HA. The use of digitlis other thn in the tretment of crdic decompenstion. J Amer Me Assoc. 1933;1: Willims JF Jr, Brunwld E. Studies on digitlis. XI. Effects of digitoxin on the development of crdic hypertrophy in the rt sujected to ortic constriction. Am J Crdiol. 1965;16(4): Cutillett AF, Rudnik M, Arcill RA, Strue R. Effect of prophylctic digitliztion on the development of myocrdil hypertrophy. Am J Physiol. 1977;233(5):H Turto H. Collgen metolism in experimentl crdic hypertrophy in the rt nd the effect of digitoxin tretment. Crdiovsc Res. 1977;11(4): Overeck HW. Elevted rteril pressure, vsculr wll wterlogging, nd impired crdic growth in rts chroniclly receiving digoxin. 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