Essential Opposite Roles of ERK and Akt Signaling in ...28°C and maintained under a 14:10-h...

1521-0103/357/2/345–356$25.00 http://dx.doi.org/10.1124/jpet.115.230763THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:345–356, May 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Essential Opposite Roles of ERK and Akt Signaling in CardiacSteroid-Induced Increase in Heart Contractility s

Nahum Buzaglo, Haim Rosen, Hagit Cohen Ben Ami, Adi Inbal, and David LichtsteinDepartment of Medical Neurobiology (N.B., H.C. B.A, A.I., D.L.) and Department of Microbiology and Molecular Genetics (H.R.),Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received November 16, 2016; accepted February 16, 2016

ABSTRACTInteraction of cardiac steroids (CS) with the Na1, K1-ATPaseelicits, in addition to inhibition of the enzyme’s activity, theactivation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized thatthe activities of these pathways are involved in CS-inducedincrease in heart contractility. This hypothesis was tested usingin vivo andexvivowild type (WT) andsarcoplasmic reticulumCa(21)atpase1a-deficient zebrafish (accordion, acc mutant) experimentalmodel. Heart contractility was measured in vivo and in primarycardiomyocytes in WT zebrafish larvae and acc mutant. Ca21

transients were determined ex vivo in adult zebrafish hearts.CS dose dependently augmented the force of contraction of larvaeheart muscle and cardiomyocytes and increased Ca21 transientsin WT but not in acc mutant. CS in vivo increased the phosphor-ylation rate of ERK and Akt in the adult zebrafish heart of the

two strains. Pretreatment of WT zebrafish larvae or cardiomyo-cytes with specific MAPK inhibitors completely abolished theCS-induced increase in contractility. On the contrary, pre-treatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivowithout affecting CS-induced Ca21 transients. Furthermore,pretreatment of the acc mutant larvae or cardiomyocytes withAkt inhibitor restored the CS-induced increase in heart con-tractility also without affecting Ca21 transients. These resultssupport the notion that the activity of MAPK pathway isobligatory for CS-induced increases in heart muscle contrac-tility. Akt activity, on the other hand, plays a negative role, viaCa21 independent mechanisms, in CS action. These findingspoint to novel potential pharmacological intervention to in-crease CS efficacy.

IntroductionCardiac steroids (CS), such as ouabain, digoxin, and bufalin,

extracted from various plants and toad skin, are used toincrease the force of contraction of heart muscle and regulateits rhythm in heart failure and arrythmogenic patients,respectively (Kanji and Maclean, 2012; Ambrosy et al.,2014). Nevertheless, the therapeutic window for CS is ex-tremely small. Whereas about 1 nM digoxin is consideredbeneficial, significant signs of toxicity are observed already at3 nM (Kanji andMaclean, 2012). The advantage of using CS ina clinical setting is still debatable. A comprehensiveDIG study(Rekha Garg et al., 1997) showed that digoxin did not reduceoverall mortality, but rather the rate of hospitalization, bothoverall and for worsening heart failure. Recent studies,however, have shown that heart failure in patients treatedwith digoxin was associated with lower all-cause mortalityand hospitalization than in patients in the placebo group,

advocating the use of this drug, despite its small therapeuticindex (Gheorghiade et al., 2013; vanVeldhuisen et al., 2013). Acomprehensive understanding of the mechanisms involved inCS-induced effects on heart contractility may lead to newpharmacological tools for the improvement of CS use.The only established receptor for CS is the ubiquitous plasma

membrane sodium- potassium-dependent adenosine triphos-phatase (Na1, K1-ATPase). The Na1, K1-ATPase belongs tothe P-typeATPase family and transportsNa1 out of cell andK1

into cell against their electrochemical gradients, using the freeenergy obtained from ATP hydrolysis (Toyoshima et al., 2011).This transporter plays a crucial role in maintaining the Na1

and K1 gradients across the plasma membrane. Consequently,its activity is a major determinant in regulating cell volume, aswell as cytoplasmic pHandCa12 via theNa1/H1 exchanger andthe Na1/Ca1 exchanger, respectively(Kaplan, 2002). The bind-ing of CS to a specific site located in the extracellular loop of thea subunit of Na1, K1-ATPase causes the inhibition of ATPhydrolysis and ion transport by the pump, reducing Na1 andK1 gradients across the plasma membrane and, as a result,affecting numerous cell functions (Lingrel, 2010). These effectsof CS on ionic gradients are the common explanation for themechanism underlying the CS-induced increase in the force of

This work was supported by grants from the Ministry of Trade and Industry,[NOFAR 032-5398], Israel and the Szolts Foundation, The Hebrew Universityof Jerusalem, and the Walter and Greta Chair in Heart Studies (to D.L).

dx.doi.org/10.1124/jpet.115.230763.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: acc, zebrafish accordion mutant; Akt, protein kinase B; ANOVA, analysis of variance; CO, cardiac output; CS, cardiac steroids; EF,ejection fraction; ERK, extracellular signal-regulated kinases; FAC, fractional area change; hpf, hours postfertilization; LA, long axis; MAPK, mitogen-activated protein kinases; Na1, K1-ATPase, sodium-potassium-dependent adenosine triphosphatase; PD98059, 2’-Amino-3’-methoxyflavone; SA,short axis; SERCA, sarcoplasmic reticulum Ca(21) atpase; Src, Src tyrosine kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene; WT, wild type.

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contractionofheartmuscle.That is, inhibition ofNa1,K1-ATPaseby CS causes an increase in intracellular Na1, which, in turn,attenuates theNa1/Ca21exchangeactivity, resulting in increasedintracellular Ca21 concentration and consequently greater con-tractility. The reversion of the intracellularCa21 to its basal levelsdepends on its sequestration in the sarcoplasmic reticulum andmitochondria, mainly by the sarcoplasmic reticulum Ca(21)atpase (SERCA) enzymes (Kranias and Hajjar, 2012).Studies in the past decade have demonstrated that in

addition to pumping ions, the Na1, K1-ATPase is engaged inthe assembly of multiple protein complexes into functionalmicrodomains that transmit signals into the cell (Xie and Cai,2003). The interaction of CS, at nanomolar of subnanomolarconcentrations, with Na1, K1-ATPase activates signal trans-duction cascades of the Src-kinase/MAP-kinase and PI3K1A/PDK/Akt pathways in different cell types, including cardio-myocytes, smooth muscle, neuronal, and epithelial cells(Khundmiri et al., 2007; Wu et al., 2013). This CS-inducedsignal transduction activation was shown to be involved inseveral physiologic processes, including the regulation of geneexpression, cell viability, differentiation, and smooth musclecontraction (Xie and Cai, 2003).The involvement ofMAPandAkt kinases, proteins upstream

to the contractile machinery, in the regulation of musclecontractility was demonstrated in several experimental sys-tems. For example, ERK inhibition was found to abrogatesustained contraction and normalized angiotensin II effects inspontaneously hypertensive rats (Touyz et al., 1999). Similarly,Akt, which is a crucial factor in the regulation of heart musclehypertrophy, participates in intracellular Ca21 homeostasis(Chaanine and Hajjar, 2011), and its activation improvedcontractile function in failing mouse heart (Condorelli et al.,2002). The involvement of CS-induced activation of intracellu-lar signaling in their positive inotropic effect in the heart wasaddressed only by Tian et al. (2001) who showed that inhibitionof Src or ERK abolished the ouabain-induced increase inintracellular Ca21 and contractility in rat cardiac myocytes.To address the possible role of MAPK and Akt activities in

CS-induced increases in heart contractility and the involve-ment of intracellular Ca21 in these effects ex vivo and in vivousing appropriate mutant, we tested the influence of CS inzebrafish larvae and in zebrafish isolated heart and primarycardiomyocytes under different experimental conditions. In thetwo experimental systems, inhibition of Src or ERK abolishedthe CS-induced increase in heart contractility. On the contrary,Akt inhibition augmented CS-induced heart positive inotropywithout affecting basal or CS-induced Ca21 transients. Theseresults demonstrate that CS-activated signaling cascadesregulate CS-induced increase in contractility by mechanismssome ofwhich are independent of changes in intracellularCa21.

Materials and MethodsAquaculture. Experiments were performed on wild-type (WT, AB

strain) zebrafish (Danio rerio). The fish were maintained in accor-dance with the principles established by the National Institutes ofHealth. TheHebrewUniversity Animal CareCommittee approved theuse of the animals and the experimental protocols used in this study(Approval #MD-11-12979-2). The fish were kept in small aquaria at28°C and maintained under a 14:10-h light:dark cycle. The zebrafishlarvae were maintained in the presence of 0.1% methylene blue(M9140, Sigma-Aldrich Inc, Israel) at 28°C. All measurements weretaken in embryos at 72 hours postfertilization (hpf).

Pharmacological manipulations. Ouabain, bufalin, digoxin,acetylcholine, carbachol, protein phosphatase 2, and U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene) werepurchased from Sigma-Aldrich Inc. The compounds were dissolvedin egg water (0.3 g Instant Ocean Salt in RO H2O) to a final selectedconcentration. The larvae were transferred to this solution in aminimal volume and after 60- to 90-minute incubation at 28°C, thedrug of choice, dissolved in E3 medium, was added. At selected timeintervals at 28°C, the larvae were transferred to filming/anesthetizingmedium.

Filming process. Zebrafish larvae heart imaging was performedusing an Olympus CKX41 (Tokyo, Japan) upright microscope with�10 or �20 magnification and integrated incandescent illumination.A FastCam imi-tech (Gyeonggi-do, Korea) high-speed digital camerawith 640�480 pixel grayscale image sensor was mounted on themicroscope, using ImCam software (IMI Technology, Co. Ltd,Gyeonggi-do, South Korea) for high-speed video recording. The larvaewere anesthetized by placing them in 0.1% SeaKem LE Agarose(BMA, Rockland, ME) containing 15 mM ethyl-3-aminobenzoatemethanesulfonate salt (Sigma-Aldrich Inc.). Each larva in 0.5 mlfilming/anesthetizing medium was transferred to a 96-well tissueculture plate at room temperature, and sequential images of the heartwere obtained with the larvae positioned on their side at 80 fps during10 seconds with a shutter speed of 0.016 second.

Quantification of heart contractility. Physiologic parametersof cardiovascular performance in the zebrafish larvae were evaluatedas previously described by Shin et al. (2010). Image analysisapplications ImageJ (National Institutes of Health, Bethesda, MD)were used, allowing delineation of the endomyocardial border at theend of systole or end of diastole to define the ventricular area.Sequential still frames were analyzed to capture ventricular end-systole and end-diastole images. These areas were used to calculatefractional area change (FAC), according to the equation: FAC 5 [enddiastolic area (EDA) - end systolic area (ESA)]/end diastolic area*100(Fig. 1, B and C). The ejection fraction (EF) was determined by anindependent estimate of ventricular volume. This was achieved byplacing scan lines across the midventricular short axis (SA) and longaxis (LA) at the end of systole and diastole (Fig. 1. D and E). Theseparameters were used to quantify ventricular volume using thevolume equation for an ellipsoid: Vol5 4/3*3.14*LA*SA

2. A minimumof five sequential pairs of systolic and diastolic cycles were measuredand analyzed in 9–12 larvae for each treatment. Each of the presentedexperiments was repeated at least three times, with identical results.

Measurements of Ca21 transients. Spontaneous Ca21 tran-sients were measured in isolated hearts from adult zebrafish. Thezebrafish were stunned by a blow to the head, and the hearts wereremoved quickly and placed in Dulbecco’s modified Eagle’s mediumcontaining 10% fetal bovine serum at room temperature. A total of 3–5hearts were placed in a small Petri dish containing 100 ml Krebs-Ringer solution (in mM: 119 NaCl, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, 1.3MgCl2, 20 HEPES, and 11 D-glucose) with 0.01 mM Fura-2 AM(Biotium, Inc., Hayward, CA). After incubation for 10 minutes (37°C,5% CO2), 150 ml of Krebs-Ringer solution were added to the Petridishes, whichwas incubated for an additional 30minutes. The dyewasremoved from the solution by two incubations (10 minutes each, roomtemperature) inDulbecco’smodifiedEagle’smedium-fetal bovine serumsolution. Intracellular Ca21 transients were measured in stabilizedmedium containing 1% low-melt agarose in Krebs-Ringer solution. Thehearts underwent alternating excitation at340 and380nmwith 510nmemission, using a PTI fluorimetric system (Photon Technology In-ternational, Madison, WI) as previously described(Cohen et al., 2007).The Ca21 levels are presented as the ratio of 340/380 nm fluorescenceemission.

Isolation of zebrafish ventricular myocytes. Adult (4–12months old) ventricular myocytes were obtained by enzymatic disso-ciation. The zebrafish were stunned by a blow to the head and thebrainwas pithed. The heartwas quickly removed and placed in a smallPetri dish containing 10 ml isolation solution (in mM): 100 NaCl, 10

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KCl, 1.2 KH2PO4, 4MgSO4, 50 taurine, 20 glucose, and 10HEPES, pH6.9. The ventricle was cut free from the bulbus and atrium undera binocular. Ventricles from 3 fish were incubated for 45 minutes at32°C in a solution containing perfusion buffer (in mM: 150 NaCl, 5.4KCl, 1.5 MgSO4, 0.4 NaH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES,pH 7.7), Collagenases II and IV (Gibco, Grand Island, NY, 5 mg/mleach) and additional CaCl2 (2.012 mM final concentration) wereadded. After Eppendorf centrifugation (1 minute, 250 g at roomtemperature) the precipitated cells were suspended in 1 ml perfusionbuffer for 30 minutes at room temperature before use. Spontaneouscontraction was observed in about 10% of the cells in the preparation.

Measurement of cardiomyocyte contractility. Cells weretransferred to a chamber with a quartz base and examined using aninverted epifluorescence microscope (Nikon Diaphot 200, Tokyo,Japan). The myocytes were field-stimulated (0.4 Hz, 70 V, squarewaves), and contractions were measured using a video motion edgedetector (Crescent Electronics, Sandy, UT) at the rate of 5 Hz, aspreviously described (Cohen et al., 2007). Cardiomyocyte performancewas calculated as the percentage of resting cell length. The slopes ofcontraction and relaxation (1dL/dt and 2dL/dt) were calculated fromthe linear portions of the changes in contractility. Nine cells per eachgroup were measured. Each of the experiments was repeated at leastthree times with identical results.

Heart dissection and protein extraction from adult zebra-fish. Adult zebrafish (∼12 months old) were transferred to swimmingmedium containing different concentrations of CS. At various timepoints (5–30 minutes) the zebrafish were transferred to dissectionmedia, and the hearts were immediately removed and transferred toRIPA lysis buffer (Sigma-Aldrich) and protease inhibitor cocktail at a1:100 dilution. The tissue was homogenized in an ultrasonic homog-enizer (Microson,NewYork,NY), and aliquots of the homogenatewerestored at 270°C until used.

Western blotting. Protein dilution and separation on SDS-PAGEelectrophoresis and their transfer to a polyvinylidene fluoride mem-branewere carried out as previously described (Goldstein et al., 2006).The membranes were incubated for 1 hour at room temperature withone of the specific antibodies against Phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204), Rabbit mAb #4370 (Cell signaling). or Phospho-Akt(Ser473) (193H12), Rabbit mAb #4058 (Cell signaling) at a 1:1000dilution in TBS containing 0.1% Tween. The wash with TBS contain-ing 0.1% Tween and exposure to horseradish peroxidase-conjugatedsecondary goat anti-rabbit IgG antibody (1:50,000) and membranestripping prior to exposure to a different antibody were performed aspreviously described (Goldstein et al., 2006). Detectionwas carried outwith the aid of a Luminata Crescendo Western HRP Substrates(Jackson Immuno Research Labs, West Grove, PA), according to the

Fig. 1. In vivo heart contractility measurementsand their validation. (A) Schematic of zebrafishlarva at 72 hpf. The single atrium and ventriclethat lie anteriorly on the ventral surface of the fishis marked by an arrow. Video microscopy wasperformed at 80 fps. The heart is shown in highmagnification at the end of systole (B andD) and atthe end of diastole (C and E). Image analysis toolsallowed the delineation of the endomyocardialborder (polygon) at the end of systole or diastole todefine the ventricular area. FAC and EF weremeasured as described in the Materials andMethods. The effect of adrenergic and cholinergicagonists on zebrafish heart contractility weredetermined by placing zebrafish larvae in stan-dard swimming medium (E3) containing 1 and10 mM carbachol, for 60 minutes or 1 and 10 mMof adrenalin for 90 minutes (F and G). The larvaewere then anesthetized and images of the heartwere obtained and analyzed as described above. Aminimum five pairs of systolic and diastolic cycleswere measured and analyzed in 10 larvae for eachtreatment. Zebrafish heart rate and calculatedcardiac output are shown in (Hand I), respectively.*Significantly different from control, P , 0.05(ANOVA with a post hoc Bonferroni-adjustedStudent’s t test).

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manufacturer’s instructions. Preliminary experiments verified thatthe stripping and reblotting procedure did not affect the quantificationof any of the proteins.

Statistics. Differences between experimental and control groupsin the in vivo experiments, quantification of heart contractility, andconsequent Western blots were analyzed using analysis of variance(ANOVA) with a post hoc Bonferroni-adjusted Student’s t test.Statistical differences in Ca21 transients and cardiomyocytes contrac-tility were analyzed by a mixed design (within/between subjects)ANOVA, which was performed using the SPSS program (IBM, NewYork, NY). Mixed design ANOVA is a statistical technique thatexamines differences on a dependent variable (a within-subject vari-able, measured multiple times on the same subject) due to multipleexperimental conditions (the between-subject factor) while controllingtype 1 error (Gueorguieva and Krystal, 2004). This is achieved byexamining how the overall sum of squares in the dependent variable(in our case, the five repeated length measurements of each cell) isrelated to the between-subject effect (in our case, the four experimen-tal conditions, control, kinase inhibitor, ouabain, and both). None ofthe within-subject effects were significant, indicating that the fivelength measurements of each cell were not significantly differentfrom each other neither when collapsed across the experimentalgroups [F(4,27) 5 0.273, P 5 0.936] nor within the experimentalgroups [F(12, 87) 5 0.902, P 5 0.548], supporting the stability ofthe measurements for each cell. The between-subject was significant[F(3,30) 5 16.047, P 5 0.000], indicating that at least in one of theexperimental conditions the length was dissimilar to the others.

ResultsValidation of heart contractility measurements in

zebrafish larvae. To validate the in vivo heart contractilitymeasurements, the effects of adrenergic and cholinergicagonists on the heart contractility of WT zebrafish larvaewere tested. As shown in Fig. 1, treatment of zebrafish larvaewith carbachol decreased the heart force of contraction andrate (Fig. 1, F–I), resulting in a reduction of 10.396 2.2% and16.92 6 3% in cardiac output (CO) (Fig. 1I) at 1 and 10 mM,respectively. Similar results were obtained with acetylcholine(Supplemental Fig. 1). Larvae exposed to adrenalin showedthe opposite effect, i.e., an increase in heart contractility andrate, resulting in an increase of 18.34 6 1.97% and 30.48 62.67% in CO at 1 and 10 mM, respectively (Fig. 1I). Similarresults were obtained with noradrenalin (Supplemental Fig. 1).These results are in complete agreement with the well-established effects of these compounds, demonstrating the capa-bility of the zebrafish experimental system to identify changes inheart contractility and rate under physiologic conditions.CS-induced increase in heart contractility in zebra-

fish larvae. Zebrafish larvae were treated with differentconcentrations of ouabain, digoxin, and bufalin for 90 minutes.The exposure to low concentrations of ouabain (0.05–1 nM) ledto a significant increase in the heart force of contraction in adose-dependent manner. This was manifested by significantincreases in FAC, EF, and CO, with amaximal increase of 3863.68% in CO at 0.2 nM but no change in heart rate (Fig. 2).Similar results were obtained in larvae treated with digoxin orbufalin (Figs. 3 and 4, respectively). Although some diversity inCS effectiveness was apparent in our experimental system,1 nM was chosen for the following experiments. At thisconcentration, the three CS increased significantly the force ofcontraction, without affecting heart rate. Hence, as in manyother species, the heart of zebrafish larvae respondby increasedheart contractility to CS treatment.

The crucial role of intracellular Ca21 in the CS-inducedincrease in heart contractility is well established. Hence, in theinitial experiments wemeasured CS action on the zebrafish accmutant. Thismutant lacks the activity of the SERCA1a isoformexclusively in its muscle cells, inducing slow calcium clearancefrom the cytoplasm to the sarcoplasmic reticulum (Olson et al.,2010). As predicted, exposure of acc larvae to 1 nM ouabain,digoxin, or bufalin for 90 minutes had no effect on heartcontractility (Fig. 5). The same result was obtained at otherCS concentrations, which increased contractility in the WT(Figs. 2, 3, and 4). These results confirm the notion that, as inother species, CS-induced increases in heart contractility inzebrafish larvae largely depend on Ca21 homeostasis.CS-induced ERK and Akt phosphorylation in adult

zebrafish heart in vivo. In recent years several laboratoriesestablished the effects of CS on the phosphorylation of ERKandAkt proteins in different cells and species (Mohammadi et al.,2003; Wu et al., 2013). To test this phenomenon in zebrafish,adult specimens were exposed to 1 mM CS for 5 minutes, afterwhich the hearts were removed and the proteins extracted. Thephosphorylation states of ERK and Akt in the protein extractswere examined byWestern blot analysis. As seen inFig. 6, A–D,the addition of ouabain to the swimming media of WT adultzebrafish resulted in a 1306 17.95%and 100620.06% increasein ERK and Akt phosphorylation in the heart, respectively.Similar results were obtained using digoxin and bufalin (Fig. 6,A–D). In addition,CS stimulatedERKandAkt phosphorylationalso in accmutants in a manner similar to that seen in the WT(Fig. 6, E–H).MAPK inhibitors attenuate CS-induced increases in

heart contractility in vivo. The hypothesis that the MAPKpathway is involved in CS-induced increases in heart contractilitywas tested using pharmacological tools. Zebrafish larvae wereexposed to MAPK inhibitors for 30 minutes, after which CS wereadded and heart contractility was measured 90 minutes later. Asseen in Fig. 7, the inhibitor of Src family kinases, PP2, atconcentrations that did not affect heart function (50 nM),completely abolished the CS-induced increase in contractility(Fig. 7, A and B). Preincubation of the larvae with two differentspecificERKinhibitors,U0126 (1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene monoethanolate) and PD98059(2’-Amino-3’-methoxyflavone), at concentrations that did not affectbasal contractility (1 mM), prevented the CS-induced increase incontractility (Fig. 7, C and D). Control experiments verified thatU0126 and PD98059 inhibit the CS-induced increase in ERKphosphorylation in the adult zebrafish heart (data not shown).Akt inhibitor potentiates CS-induced increase in

heart contractility in vivo. Akt is involved in the PI3K/Akt/mTOR and other signaling pathways and has a key role inmultiple cellular processes such as glucose metabolism,apoptosis, and cell proliferation in the heart and other organs(Xia and Xu, 2015). To test the possible involvement of Aktactivation in CS-induced increases in heart contractility,inhibition of Akt by MK-2206 on zebrafish heart contractilitywas investigated. The exposure of WT zebrafish larvae for2 hours to MK-2206 (10 nM) did not affect heart contractilityparameters. However, this treatment resulted in a doubling ofthe CS-induced increase in heart contractility compared withthe CS effect in the absence of the inhibitor (Fig. 8, A and B).This augmentation of the response to CS on contractility wasapparent in both the FAC and EF determinations and was notaccompanied by any effect on heart rate (Supplemental Fig. 2).

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The effect of Akt inhibitor was also tested in the accmutant. Although, as mentioned above, this mutant was notaffected by CS at any of the tested concentrations (Fig. 5), a

significant increase in heart contractility caused by 1 nMCS was observed in the presence of Akt inhibitor (Fig. 8, Cand D).

Fig. 3. Effect of digoxin on zebrafish heart contractility. The effects of the steroid on FAC (A), EF (B), heart rate (C), and CO (D) are shown. Zebrafish larvaewere placed in standard swimming medium (E3) containing 0.1–1000 nM digoxin for 90 minutes at 28°C. The experimental procedures were performed asdescribed in the legend to Fig. 2. *Significantly higher than the control, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).

Fig. 2. Effect of ouabain on zebrafish heart contractility. Zebrafish larvae were placed in standard swimming medium (E3) containing 0.025–1 nM ouabainfor 90 minutes at 28°C. The larvae were then anesthetized and images of the heart were obtained and analyzed as described in the legend to Fig. 1. Aminimum five pairs of systolic and diastolic cycles weremeasured and analyzed in 12 larvae for each treatment. The effects of the steroid on FAC (A), EF (B),heart rate (C), and CO (D) are shown. *Significantly higher than the control, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).


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ERK and Akt inhibitors affect CS-induced increasesin contractility in primary zebrafish cardiomyocytes.The results presented above on the effects of ERK and Akt

inhibitors on CS-induced increases in heart contractility mayhave resulted from indirect effects of the inhibitors and/or CSon neuronal or endocrine systems, rather than from direct

Fig. 5. Effect of CS on zebrafish acc mutant heart contractility. Zebrafish acc larvae aged 72 hpf were placed in standard swimming medium (E3)containing different concentrations of ouabain (Oua), bufalin (Buf), or digoxin (Dig) for 90 minutes. The effects of the steroid on FAC (A), EF (B), heartrate (C), and CO (D) are shown. The larvae were then anesthetized and images of the heart were obtained and analyzed as described in the legend to Fig.1. A minimum five pairs of systolic and diastolic cycles were measured and analyzed in 10 larvae for each treatment. *Significantly higher than thecontrol, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).

Fig. 4. Effect of bufalin on zebrafish heart contractility. The effects of the steroid on FAC (A), EF (B), heart rate (C), and CO (D) are shown. Zebrafish larvaewere placed in standard swimming medium (E3) containing 0.01–10 nM bufalin for 90 minutes at 28°C. The experimental procedures were performed asdescribed in the legend to Fig. 2. *Significantly higher than the control, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).

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action on the heart. To test this hypothesis, a similar set ofexperiments was performed on isolated adult zebrafish car-diomyocytes. As seen in Fig. 9 and Table 1, treatment of

zebrafish cardiomyocytes with 0.1 nM ouabain resulted in athree- to fourfold increase in contractility with a concomitantincrease in contractility and relaxation rise time.

Fig. 6. Effect of CS on ERK and Aktphosphorylation in adult zebrafish heart.Adult WT zebrafish (A–D) or acc mutants(E–H) were exposed to 1 mM of ouabain,bufalin, or digoxin for 5 minutes in swim-ming medium. The hearts were then re-moved and placed in lysis buffer. The levelsof phospho-ERK and phospho-Akt wereexamined by Western blot (see Materialsand Methods). Total ERK and Akt servedas control. Each bar represents themean6S.E. of three experiments. *Significantlyhigher than the control, P , 0.05 (ANOVAwith a post hoc Bonferroni-adjusted Stu-dent’s t test).

Fig. 7. Effect of Src andERK1/2 inhibitors onCS-induced increase in heart contractility. Zebrafish larvae aged 72 hpfwere placed in standard swimmingmediumcontaining 50 nM protein phosphatase 2 (Src inhibitor) or 1mMU0126 (ERK inhibitor) for 30minutes at 28°C. Next 1 nM ouabain, digoxin, or bufalin were addedto themedium for 90minutes at 28°C.The larvaewere then anesthetized and images of the heartwere obtained andanalyzed as described in the legend toFig. 1.Aminimum five pairs of systolic and diastolic cycles in 12 larvae weremeasured and analyzed for each treatment. (A) FACmeasurements for Src inhibitor with andwithoutCS. (B)EFmeasurements for Src inhibitorwith andwithoutCS. (C) FACmeasurements for ERK inhibitorwith andwithoutCS. (D) EFmeasurements forERK inhibitor with and without CS. *Significantly higher than the control, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).


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Although the addition of 10 nMof ERK inhibitor PD98059 tothe cell medium increased contractility and relaxation risetime, it did not affect the amplitude of cell contraction.However, in the presence of the inhibitor, ouabain-inducedincreases in all contractility parameters were completelyprevented (Fig. 9, A and B, and Table 1). On the contrary,preincubation of cardiomyocytes with 1 nM Akt inhibitor MK-2206, which by itself did not affect any of the contractilityparameters, enhanced the ouabain-induced augmentation incontractility manifested by 113, 183, and 169% increases inamplitude, contraction rise time, and relaxation rise time,respectively (Fig. 9, C andD, andTable 1). These results confirmthe direct action of ouabain and the ERK and Akt inhibitors onthe heart.In addition, similar to the in vivo experiments, 0.1 nM

ouabain or 1 nMMK-2206 did not affect the amplitude of adultaccmutant primary cardiomyocytes contractility, demonstrat-ing the obligatory role of SERCA1a in CS action under normalcondition. However, the addition of ouabain to these cells inthe presence of Akt inhibitor restored ouabain-induced in-crease in muscle contraction manifested by 67% increase inthe contraction amplitude (Fig. 9, E and F, and Table 1). Theseresults support the notion that Akt activity plays a negativeregulatory role in CS action.Akt inhibition effect on CS-induced increases in

contractility is Ca21 independent. The novel finding ofthe potentiation and restoration effects of Akt inhibition onCS-induced increases in heart contractility in WT and accmutants, respectively, may result from changes in CS-inducedCa21 transients or other Ca21 independent mechanisms. To

test the mechanisms involved, basal and CS-induced Ca21

transients were measured in adult WT and acc zebrafishisolated hearts. Spontaneous cardiac rhythm in the accmutant was significantly reduced compared with that in theWT. In the WT zebrafish heart, as expected, the addition of200 mM ouabain to the medium caused a significant increase inamplitude (27%) and in rise slope (61%) and decay slope (86%) oftheCa21 transients (Fig. 10A). Similar changes in theCS-inducedCa21 signals were obtained after preincubation of the heart withAkt inhibitor (Fig. 10B and Table 2), indicating that the ouabaineffects on Ca21 oscillation are independent of Akt activity.Furthermore, the addition of 200 mM ouabain to acc zebrafishheart did not result in any changes in the Ca21 transientsparameters (Fig. 10C and Table 2), showing that SERCA1aactivity is required for CS-induced alterations in Ca21 signals.The addition of 200 mM ouabain to acc zebrafish hearts in thepresence of Akt inhibitor (Fig. 10D and Table 2), a condition thatinduced an increase in heart contractility, also did not cause anychanges in Ca21 transient parameters. These Ca21 measure-ments support the notion that in zebrafish heart Akt inhibitiondoes not change ouabain-induced effects on Ca21 transients.

DiscussionThe zebrafish is a well-established experimental model for

the study of embryonic development. In recent years it hasentered the field of cardiovascular research as an organismoffering distinct advantages for dissecting molecular path-ways of cardiovascular development, regeneration, and func-tion (Leong et al., 2010; Wilkinson et al., 2014). We used

Fig. 8. Effect of Akt inhibitor on CS-induced increase in heart contractility inWT zebrafish and in accmutants. Zebrafish larvae aged 72 hpf were placedin standard swimming medium containing 10 nMMK2206 for 30 minutes at 28°C. Next, 1 nM ouabain, digoxin, or bufalin were added to the medium for90 minutes at 28°C. The larvae were then anesthetized, and images of the heart were obtained and analyzed as described in the legend to Fig. 1. Aminimum five pairs of systolic and diastolic cycles were measured and analyzed in 12 larvae for each treatment. (A) FAC measurements for MK-2206 inWT larvae with or without CS. (B) EFmeasurements for MK-2206 inWT larvae with or without CS. (C) FACmeasurements for MK-2206 in accmutantswith or without CS. (D) EFmeasurements forMK-2206 in accmutants with or without CS. *Significantly higher than the control, P, 0.05, #significantlyhigher than ouabain-treated grope P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).

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zebrafish larvae to address the hypothesis that MAPK andAkt signaling pathways play a role in CS-induced increases inheart contractility. To address this issue, an in vivo cardiacfunction system for zebrafish larvae was established. Theeffects of CS and kinase inhibitors were studied using liveimaging of the fully developed cardiovascular zebrafish lar-vae. The method, originally developed by Shin et al. (2010),was based on the assumption that the heart ventricle has anelliptic shape and construction of the changes in the areabetween systole and diastole (Shin et al., 2010). In the presentstudy, the measurements were improved by determinations ofthe ventricular area using continuous drawings of the polygon

border of the ventricle, yielding accurate FAC and EF of theheart in vivo (Fig. 1, B–E). The systemwas validated by testingthe effects of adrenergic and cholinergic agonists added to theswimming media on zebrafish heart contractility. As expected,adrenalin (1 mM) induced a significant increase in heartcontractility and rate (Fig. 1, F–I). This classic responseresembles that seen in other species. Similarly, cholinergicagonists (i.e., acetylcholine, 1 mM) induced a decrease in heartcontractility and rate (Fig. 1) comparable to its effects in otheranimal models. Hence, this method of cardiac measurementenables efficient determination of key aspects of cardiac func-tion, such as FAC, EF, heart rate, and calculation of CO ofzebrafish larvae, and can be used for in vivo physiologic andpharmacological investigations.The addition of ouabain, digoxin, or bufalin to zebrafish

larvae swimming medium increased dose dependently theforce of contraction of heartmuscle.Whereas 0.1 nMbufalin orouabain produced a significant increase in heart contractility,an about tenfold higher concentration of digoxin was requiredto yield a similar effect (Figs. 2, 3, and 4). This effect and thedifferential potencies are similar to the one seen in mammals,demonstrating the suitability of the zebrafish model forthe study of CS action. CS-induced toxicity, manifested byreduced FAC, EF, or mortality, was observed at concentra-tions 1000 times higher than those required for a significantpositive inotropic effect, indicating a wider dose response forCS in this organism.Inhibition of Na1, K1-ATPase by CS is the recognized

mechanism of action for their ability to increase the force ofcontraction of heart muscle. The exclusiveness of this mech-anism was challenged by the demonstration that the in-teraction of CS with Na1, K1-ATPase elicits the activationof several major signaling cascades, including ERK, Akt, andiNOS (Tian et al., 2001; Gan et al., 2012; Wu et al., 2013).

TABLE 1Contractility parameters of ouabain-induced increase in primarycardiomyocyte contractility in the presence and absence of ERK or AktinhibitorsThe experiments were performed as described in the legend to Fig. 9. Average twitchamplitude, contraction, and relaxation rates were measured in 9 cells in eachgroup.

Amplitude ContractionRate (2dv/dt)Relaxation

Rate (+dv/dt)

Control 0.14 6 0.04 0.63 6 0.16 0.41 6 0.090.1 nM Oua 0.53 6 0.1# 3.65 6 0.38* 2.92 6 0.37*10 nM PD98059 0.13 6 0.01 0.87 6 0.09* 0.78 6 0.14*Oua & PD98059 0.15 6 0.02 1.06 6 0.09 0.79 6 0.08Control 0.13 6 0.01 0.77 6 0.14 0.50 6 0.110.1 nM Oua 0.38 6 0.05* 1.84 6 0.28* 1.64 6 0.23*Oua & MK 0.43 6 0.04*# 3.37 6 0.27*# 2.78 6 0.32*#1 nM MK2066 0.19 6 0.03 0.98 6 0.19 0.87 6 0.20Acc mutantControl 0.13 6 0.01 1.58 6 0.29 0.56 6 0.190.1 nM Oua 0.14 6 0.03 2.43 6 0.84 0.07 6 0.05*Oua & MK 0.22 6 0.02* 3.65 6 0.71* 0.52 6 0.261 nM MK2066 0.15 6 0.02 2.61 6 0.74 0.98 6 0.34

*Significantly higher than control P , 0.05, #significantly higher than ouabain-treated cells (post hoc Bonferroni-adjusted between subjects ANOVA).

Fig. 9. Effect of ERK and Akt inhibitors on CS-induced increase in primary cardiomyocyte contractility. Primary adult zebrafish cardiomyocytes wereprepared as described in Materials and Methods and exposed to ouabain, ERK, or Akt inhibitor or a combination thereof for 20 minutes at roomtemperature. The cardiomyocytes were stimulated at 0.5 Hz, and cell shortening was recorded. Nine cells per each group were measured. Arepresentative twitch of % shortening of cells exposed to ouabain with or without ERK inhibitor (A) or to ouabain with or without Akt inhibitor (C) isshown. Quantification of the data as % of control is shown in (B) and (D). A representative twitch of % shortening of cells from acc mutant exposed toouabain with or without Akt inhibitor is shown in (E), and quantification of the data as % of control of responses is shown in (F). *Significantly higherthan the control, P , 0.05, #significantly higher than ouabain-treated group P , 0.05 (post hoc Bonferroni-adjusted between subjects ANOVA).


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These signaling pathways, once activated byCS, participate innumerous physiologic functions including cell viability, kid-ney and muscle function, and cardiac hypertrophy (Liu et al.,2007; Xie et al., 2013; Wang et al., 2014). Indeed, using an exvivo experimental system, it was shown that inhibition of Srcand ERK attenuated the CS-induced increase in heartcontractility by affecting Ca12 homeostasis (Tian et al., 2001).The effect of CS on ERK and Akt phosphorylation was shown

in many tissue culture cells including rat brain (Yu et al., 2010)and heart (Ceolotto et al., 2003) and opossum kidney proximaltubular cells (Khundmiri et al., 2007). Similarly, the exposure ofadult zebrafish to CS caused an about twofold increase in ERKand Akt phosphorylation in heart tissue in the WT fish and inthe acc mutants (Fig. 6). The protein phosphorylation mayresult in conformational changes after the interaction of CSwith the Na1, K1-ATPase. Alternatively, the phosphorylationmay be the consequence of indirect mechanisms, such aschanges in intracellular Ca12 (Agell et al., 2002) or in muscletension (Xu et al., 1996). The observation that CS did not

increase the heart force of contraction nor Ca21 transients butdid augment ERK and Akt phosphorylation in the accmutantsfavors the firstmechanism. In addition, the observation thatCSdid not induce an increase in heart contractility in the accmutant but did stimulate ERK phosphorylation indicates thatthe CS-inducedMAPKactivation per se is not sufficient to elicitan increase in contractility.The inhibition of MAPK pathway by the addition of Src

kinase orERK inhibitors did not affect the force of contraction ofthe zebrafish larvae hearts or cardiomyocytes. These treat-ments, however, completely abolished the increased contractioncaused by CS in vivo and ex vivo (Fig. 7 and Fig. 9, A and B,respectively). These findings are in accordance with the notionthat MAPK activation is required for the CS-induced increasein heart contractility. A similar conclusion was drawn in aprevious study demonstrating that ERK inhibitors attenuate aCS-induced increase in contractility in rat cardiomyocytes (Tianet al., 2001). The possible mechanism involved in this effectmay be mediated by the established link between ERK

Fig. 10. Effects of ouabain in the presence and absence of Akt inhibitor on Ca2+ transients in zebrafish isolated adult hearts . Hearts isolation and Ca2+

transients measurements were performed as described in Materials and Methods. Representative Ca2+ transients in WT and in acc mutants in controland after 200 mM ouabain administration are shown in (A) and (B), respectively. The effects of ouabain in the presence of Akt inhibitor in wt and accmutant is shown in (C) and (D), respectively. The quantitative analyses of these Ca2+ oscillations are depicted in Table 2.

TABLE 2Effect of ouabain and Akt inhibitor on Ca2+ transients in adult zebrafish heartThe experiments were performed as described in the legend to Fig. 10. Average of Ca2+ transient were measured in 10 isolated zebrafish heart in each group.

Control Ouabain 200 mM MK-2206 (1 nM) Ouabain & MK-2206

Rate (wave/min) 58 6 7.4 52 6 6.8 48 6 4.8 48 6 5Amplitude (Maximal deflection from baseline) 5.28 6 0.32 6.59 6 0.71* 4.8 6 0.13 5.91 6 0.28*#

Area (Area under the transient relative to baseline) 2.52 6 0.28 2.94 6 0.44* 2.81 6 0.19 2.08 6 0.23Rise slope (+D Fluorescence in the linear phase/sec) 4.85 6 1.29 7.84 6 2.02* 1.65 6 0.28* 2.68 6 0.34*#

Decay slope (-D Fluorescence in the linear phase/sec) 10.73 6 7.54 20 6 3.51* 4.65 6 0.54 7.87 6 1.11Acc mutantRate (wave/min) 31 6 5 35 6 8.4 25 6 3 31 6 4.4Amplitude (Maximal deflection from baseline) 1.79 6 0.11 1.67 6 0.15* 1.85 6 0.05 1.76 6 0.05Area (Area under the transient relative to baseline) 2.64 6 0.65 2.54 6 0.56 2.79 6 0.23 2.92 6 0.4Rise slope (+D Fluorescence in the linear phase/sec) 0.48 6 0.07 0.47 6 0.1 0.51 6 0.04 0.56 6 0.05Decay slope (-D Fluorescence in the linear phase/sec) 1.88 6 0.33 1.92 6 0.46 1.72 6 0.22 1.61 6 0.19

*Significantly higher than control P , 0.05; #significantly higher than MK-2206 P , 0.05 (post hoc Bonferroni-adjusted between subjects ANOVA).

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activity and intracellular Ca12 concentration (Agell et al., 2002;Andrikopoulos et al., 2011). The identical dependence of the CS-induced increase in contractility in vivo and in cardiomyocytespoints to the direct effect of the steroid on the zebrafish heart.Previous studies have shown the involvement of Akt in the

regulation of cardiac hypertrophy, proliferation, and contrac-tility (Ballou et al., 2015). These investigations demonstratedthat Akt activation affects Ca12 levels (Das et al., 2014) orgene expression linked to the contractile machinery (Ouditand Penninger, 2009). The final outcome of Akt inhibition onheart contractility is diverse. Akt inhibition reduced intracel-lular Ca12 in cardiomyocytes, resulting in attenuation ofcontractility (Graves et al., 2012). On the other hand, areduction in Akt activity, under stress conditions, potentiatedcontractile protein expression, resulting in improved contrac-tility (Das et al., 2014). In this study, we discovered that theaddition of Akt inhibitor in vivo and ex vivo potentiated theCS-induced increase in contractility (Fig. 8, A and B, and Fig.9, C andD, respectively). Furthermore, the exposure of the accmutants to the Akt inhibitor caused the appearance of CS-induced increase in heart contractility at concentrations thatwere ineffective in its absence (Fig. 8, C and D, and Fig. 9, Eand F). Measurements of changes in CS-induced Ca21 tran-sients in zebrafish heart (Fig. 10, Table 2) revealed that Aktinhibition did not affect the ouabain-induced increase in Ca21

transient amplitude in WT. Similarly, in acc mutant, whereCS induced increase in force of contraction in the presence ofAkt inhibitor, CS did not cause an increase in Ca21 amplitude.Because inhibition of Akt in both the wt and in acc mutantsresulted in a similar phenotype, without concomitant changesin Ca21 transients, we conclude that themechanisms involvedin this effect are not associated with alterations in intracellu-lar Ca21 and may be a consequence of other changes such asgene expression or Ca21 sensitivity.Taking into consideration all the findings, we propose that

CS-induced increases in heart contractility result from twopathways. Inhibition of Na1, K1-ATPase after CS bindingincreases the concentration of intracellular Na1 and, conse-quently, the cytoplasmic Ca12 level and contractility. Concur-rently, CS binding to Na1, K1-ATPase activates intracellularsignaling pathways that regulate contractility: CS-inducedERK phosphorylation, presumably directly or by increasingCa12 turnover, augments the CS-induced effect. On the otherhand, CS-induced Akt phosphorylation, by unknown mecha-nisms that may involve changes in the contractile machineryand/or reduced Ca12 sensitivity, attenuates the CS-inducedeffects. The final outcome of the activation of the signalingpathways emerges from the balance between the two branches.This concept of CS-induced effects suggests that changes inERK and Akt activities by inhibitors or genetic manipulationhave a significant influence on the efficacy and toxicity of theCS.As mentioned in the introduction, the small therapeutic

index of CS precludes their common use in patients with heartfailure. Our study shows that despite the vast phylogeneticdistance, many characteristics of CS action in the zebrafishmodel are identical to those seen in humans. This includes thedosage required for their positive inotropic effect, as well asthe activated signaling pathways and the apparent changes inintracellular Ca21 (Rekha Garg et al., 1997). Hence, it may beinferred from our results that a combined treatment with CSand Akt inhibitors of heart failure patients would increase theCS therapeutic index.

Acknowledgments

We thank Dr. Michal Horowitz for advice and support in cellmotility measurements and Dr. Yuval Kalish for assistance in thestatistical analyses.

Author Contributions

Participated in research design: Buzaglo, Inbal, and Lichtstein.Conducted experiments: Buzaglo and Cohen-Ben Ami.Performed data analysis: Buzaglo, Rosen, and Inbal.Wrote or contributed to the writing of the manuscript: Buzaglo,

Rosen, and Lichtstein.

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