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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.115.230763
J Pharmacol Exp Ther 357:345–356, May 2016
Essential Opposite Roles of ERK and Akt Signaling in Cardiac
Steroid-Induced Increase in Heart Contractility s
Nahum Buzaglo, Haim Rosen, Hagit Cohen Ben Ami, Adi Inbal, and David Lichtstein
Department 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
Introduction
Cardiac steroids (CS), such as ouabain, digoxin, and bufalin,
extracted from various plants and toad skin, are used to
increase the force of contraction of heart muscle and regulate
its rhythm in heart failure and arrythmogenic patients,
respectively (Kanji and Maclean, 2012; Ambrosy et al.,
2014). Nevertheless, the therapeutic window for CS is extremely small. Whereas about 1 nM digoxin is considered
beneficial, significant signs of toxicity are observed already at
3 nM (Kanji and Maclean, 2012). The advantage of using CS in
a clinical setting is still debatable. A comprehensive DIG study
(Rekha Garg et al., 1997) showed that digoxin did not reduce
overall mortality, but rather the rate of hospitalization, both
overall and for worsening heart failure. Recent studies,
however, have shown that heart failure in patients treated
with digoxin was associated with lower all-cause mortality
and hospitalization than in patients in the placebo group,
This work was supported by grants from the Ministry of Trade and Industry,
[NOFAR 032-5398], Israel and the Szolts Foundation, The Hebrew University
of 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.
two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the
CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CSinduced increase in heart contractility both in vivo and ex vivo
without affecting CS-induced Ca21 transients. Furthermore,
pretreatment of the acc mutant larvae or cardiomyocytes with
Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca21 transients. These results
support the notion that the activity of MAPK pathway is
obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via
Ca21 independent mechanisms, in CS action. These findings
point to novel potential pharmacological intervention to increase CS efficacy.
advocating the use of this drug, despite its small therapeutic
index (Gheorghiade et al., 2013; van Veldhuisen et al., 2013). A
comprehensive understanding of the mechanisms involved in
CS-induced effects on heart contractility may lead to new
pharmacological tools for the improvement of CS use.
The only established receptor for CS is the ubiquitous plasma
membrane sodium- potassium-dependent adenosine triphosphatase (Na1, K1-ATPase). The Na1, K1-ATPase belongs to
the P-type ATPase family and transports Na1 out of cell and K1
into cell against their electrochemical gradients, using the free
energy 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, as
well as cytoplasmic pH and Ca12 via the Na1/H1 exchanger and
the Na1/Ca1 exchanger, respectively(Kaplan, 2002). The binding of CS to a specific site located in the extracellular loop of the
a subunit of Na1, K1-ATPase causes the inhibition of ATP
hydrolysis and ion transport by the pump, reducing Na1 and
K1 gradients across the plasma membrane and, as a result,
affecting numerous cell functions (Lingrel, 2010). These effects
of CS on ionic gradients are the common explanation for the
mechanism underlying the CS-induced increase in the force of
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, mitogenactivated 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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ABSTRACT
Interaction of cardiac steroids (CS) with the Na1, K1-ATPase
elicits, in addition to inhibition of the enzyme’s activity, the
activation of intracellular signaling such as extracellular signalregulated (ERK) and protein kinase B (Akt). We hypothesized that
the activities of these pathways are involved in CS-induced
increase in heart contractility. This hypothesis was tested using
in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(21)
atpase1a-deficient zebrafish (accordion, acc mutant) experimental
model. Heart contractility was measured in vivo and in primary
cardiomyocytes 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 larvae
heart muscle and cardiomyocytes and increased Ca21 transients
in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the
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Materials and Methods
Aquaculture. Experiments were performed on wild-type (WT, AB
strain) zebrafish (Danio rerio). The fish were maintained in accordance with the principles established by the National Institutes of
Health. The Hebrew University Animal Care Committee approved the
use of the animals and the experimental protocols used in this study
(Approval #MD-11-12979-2). The fish were kept in small aquaria at
28°C and maintained under a 14:10-h light:dark cycle. The zebrafish
larvae were maintained in the presence of 0.1% methylene blue
(M9140, Sigma-Aldrich Inc, Israel) at 28°C. All measurements were
taken in embryos at 72 hours postfertilization (hpf).
Pharmacological manipulations. Ouabain, bufalin, digoxin,
acetylcholine, carbachol, protein phosphatase 2, and U0126 (1,4diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene) were
purchased from Sigma-Aldrich Inc. The compounds were dissolved
in egg water (0.3 g Instant Ocean Salt in RO H2O) to a final selected
concentration. The larvae were transferred to this solution in a
minimal volume and after 60- to 90-minute incubation at 28°C, the
drug of choice, dissolved in E3 medium, was added. At selected time
intervals at 28°C, the larvae were transferred to filming/anesthetizing
medium.
Filming process. Zebrafish larvae heart imaging was performed
using 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 camera
with 640480 pixel grayscale image sensor was mounted on the
microscope, using ImCam software (IMI Technology, Co. Ltd,
Gyeonggi-do, South Korea) for high-speed video recording. The larvae
were anesthetized by placing them in 0.1% SeaKem LE Agarose
(BMA, Rockland, ME) containing 15 mM ethyl-3-aminobenzoate
methanesulfonate salt (Sigma-Aldrich Inc.). Each larva in 0.5 ml
filming/anesthetizing medium was transferred to a 96-well tissue
culture plate at room temperature, and sequential images of the heart
were obtained with the larvae positioned on their side at 80 fps during
10 seconds with a shutter speed of 0.016 second.
Quantification of heart contractility. Physiologic parameters
of cardiovascular performance in the zebrafish larvae were evaluated
as previously described by Shin et al. (2010). Image analysis
applications ImageJ (National Institutes of Health, Bethesda, MD)
were used, allowing delineation of the endomyocardial border at the
end of systole or end of diastole to define the ventricular area.
Sequential still frames were analyzed to capture ventricular endsystole and end-diastole images. These areas were used to calculate
fractional area change (FAC), according to the equation: FAC 5 [end
diastolic area (EDA) - end systolic area (ESA)]/end diastolic area*100
(Fig. 1, B and C). The ejection fraction (EF) was determined by an
independent estimate of ventricular volume. This was achieved by
placing scan lines across the midventricular short axis (SA) and long
axis (LA) at the end of systole and diastole (Fig. 1. D and E). These
parameters were used to quantify ventricular volume using the
volume equation for an ellipsoid: Vol 5 4/3*3.14*LA*SA2. A minimum
of five sequential pairs of systolic and diastolic cycles were measured
and analyzed in 9–12 larvae for each treatment. Each of the presented
experiments was repeated at least three times, with identical results.
Measurements of Ca21 transients. Spontaneous Ca21 transients were measured in isolated hearts from adult zebrafish. The
zebrafish were stunned by a blow to the head, and the hearts were
removed quickly and placed in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum at room temperature. A total of 3–5
hearts were placed in a small Petri dish containing 100 ml KrebsRinger solution (in mM: 119 NaCl, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, 1.3
MgCl2, 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 Petri
dishes, which was incubated for an additional 30 minutes. The dye was
removed from the solution by two incubations (10 minutes each, room
temperature) in Dulbecco’s modified Eagle’s medium-fetal bovine serum
solution. Intracellular Ca21 transients were measured in stabilized
medium containing 1% low-melt agarose in Krebs-Ringer solution. The
hearts underwent alternating excitation at 340 and 380 nm with 510 nm
emission, using a PTI fluorimetric system (Photon Technology International, Madison, WI) as previously described(Cohen et al., 2007).
The Ca21 levels are presented as the ratio of 340/380 nm fluorescence
emission.
Isolation of zebrafish ventricular myocytes. Adult (4–12
months old) ventricular myocytes were obtained by enzymatic dissociation. The zebrafish were stunned by a blow to the head and the
brain was pithed. The heart was quickly removed and placed in a small
Petri dish containing 10 ml isolation solution (in mM): 100 NaCl, 10
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contraction of heart muscle. That is, inhibition of Na1, K1-ATPase
by CS causes an increase in intracellular Na1, which, in turn,
attenuates the Na1/Ca21 exchange activity, resulting in increased
intracellular Ca21 concentration and consequently greater contractility. The reversion of the intracellular Ca21 to its basal levels
depends on its sequestration in the sarcoplasmic reticulum and
mitochondria, 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 in
the assembly of multiple protein complexes into functional
microdomains that transmit signals into the cell (Xie and Cai,
2003). The interaction of CS, at nanomolar of subnanomolar
concentrations, with Na1, K1-ATPase activates signal transduction cascades of the Src-kinase/MAP-kinase and PI3K1A/
PDK/Akt pathways in different cell types, including cardiomyocytes, smooth muscle, neuronal, and epithelial cells
(Khundmiri et al., 2007; Wu et al., 2013). This CS-induced
signal transduction activation was shown to be involved in
several physiologic processes, including the regulation of gene
expression, cell viability, differentiation, and smooth muscle
contraction (Xie and Cai, 2003).
The involvement of MAP and Akt kinases, proteins upstream
to the contractile machinery, in the regulation of muscle
contractility was demonstrated in several experimental systems. For example, ERK inhibition was found to abrogate
sustained contraction and normalized angiotensin II effects in
spontaneously hypertensive rats (Touyz et al., 1999). Similarly,
Akt, which is a crucial factor in the regulation of heart muscle
hypertrophy, participates in intracellular Ca21 homeostasis
(Chaanine and Hajjar, 2011), and its activation improved
contractile function in failing mouse heart (Condorelli et al.,
2002). The involvement of CS-induced activation of intracellular signaling in their positive inotropic effect in the heart was
addressed only by Tian et al. (2001) who showed that inhibition
of Src or ERK abolished the ouabain-induced increase in
intracellular 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 involvement of intracellular Ca21 in these effects ex vivo and in vivo
using appropriate mutant, we tested the influence of CS in
zebrafish larvae and in zebrafish isolated heart and primary
cardiomyocytes under different experimental conditions. In the
two experimental systems, inhibition of Src or ERK abolished
the CS-induced increase in heart contractility. On the contrary,
Akt inhibition augmented CS-induced heart positive inotropy
without affecting basal or CS-induced Ca21 transients. These
results demonstrate that CS-activated signaling cascades
regulate CS-induced increase in contractility by mechanisms
some of which are independent of changes in intracellular Ca21.
ERK and Akt Signaling in Cardiac Steroid Inotropic Effect
347
KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES, pH
6.9. The ventricle was cut free from the bulbus and atrium under
a binocular. Ventricles from 3 fish were incubated for 45 minutes at
32°C in a solution containing perfusion buffer (in mM: 150 NaCl, 5.4
KCl, 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/ml
each) and additional CaCl2 (2.012 mM final concentration) were
added. After Eppendorf centrifugation (1 minute, 250 g at room
temperature) the precipitated cells were suspended in 1 ml perfusion
buffer for 30 minutes at room temperature before use. Spontaneous
contraction was observed in about 10% of the cells in the preparation.
Measurement of cardiomyocyte contractility. Cells were
transferred to a chamber with a quartz base and examined using an
inverted epifluorescence microscope (Nikon Diaphot 200, Tokyo,
Japan). The myocytes were field-stimulated (0.4 Hz, 70 V, square
waves), and contractions were measured using a video motion edge
detector (Crescent Electronics, Sandy, UT) at the rate of 5 Hz, as
previously described (Cohen et al., 2007). Cardiomyocyte performance
was calculated as the percentage of resting cell length. The slopes of
contraction and relaxation (1dL/dt and 2dL/dt) were calculated from
the linear portions of the changes in contractility. Nine cells per each
group were measured. Each of the experiments was repeated at least
three times with identical results.
Heart dissection and protein extraction from adult zebrafish. Adult zebrafish (∼12 months old) were transferred to swimming
medium containing different concentrations of CS. At various time
points (5–30 minutes) the zebrafish were transferred to dissection
media, and the hearts were immediately removed and transferred to
RIPA lysis buffer (Sigma-Aldrich) and protease inhibitor cocktail at a
1:100 dilution. The tissue was homogenized in an ultrasonic homogenizer (Microson, New York, NY), and aliquots of the homogenate were
stored at 270°C until used.
Western blotting. Protein dilution and separation on SDS-PAGE
electrophoresis and their transfer to a polyvinylidene fluoride membrane were carried out as previously described (Goldstein et al., 2006).
The membranes were incubated for 1 hour at room temperature with
one 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 1:1000
dilution in TBS containing 0.1% Tween. The wash with TBS containing 0.1% Tween and exposure to horseradish peroxidase-conjugated
secondary goat anti-rabbit IgG antibody (1:50,000) and membrane
stripping prior to exposure to a different antibody were performed as
previously described (Goldstein et al., 2006). Detection was carried out
with the aid of a Luminata Crescendo Western HRP Substrates
(Jackson Immuno Research Labs, West Grove, PA), according to the
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Fig. 1. In vivo heart contractility measurements
and their validation. (A) Schematic of zebrafish
larva at 72 hpf. The single atrium and ventricle
that lie anteriorly on the ventral surface of the fish
is marked by an arrow. Video microscopy was
performed at 80 fps. The heart is shown in high
magnification at the end of systole (B and D) and at
the end of diastole (C and E). Image analysis tools
allowed the delineation of the endomyocardial
border (polygon) at the end of systole or diastole to
define the ventricular area. FAC and EF were
measured as described in the Materials and
Methods. The effect of adrenergic and cholinergic
agonists on zebrafish heart contractility were
determined by placing zebrafish larvae in standard swimming medium (E3) containing 1 and
10 mM carbachol, for 60 minutes or 1 and 10 mM
of adrenalin for 90 minutes (F and G). The larvae
were then anesthetized and images of the heart
were obtained and analyzed as described above. A
minimum five pairs of systolic and diastolic cycles
were measured and analyzed in 10 larvae for each
treatment. Zebrafish heart rate and calculated
cardiac output are shown in (H and I), respectively.
*Significantly different from control, P , 0.05
(ANOVA with a post hoc Bonferroni-adjusted
Student’s t test).
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Buzaglo et al.
Results
Validation of heart contractility measurements in
zebrafish larvae. To validate the in vivo heart contractility
measurements, the effects of adrenergic and cholinergic
agonists on the heart contractility of WT zebrafish larvae
were tested. As shown in Fig. 1, treatment of zebrafish larvae
with carbachol decreased the heart force of contraction and
rate (Fig. 1, F–I), resulting in a reduction of 10.39 6 2.2% and
16.92 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 showed
the opposite effect, i.e., an increase in heart contractility and
rate, resulting in an increase of 18.34 6 1.97% and 30.48 6
2.67% in CO at 1 and 10 mM, respectively (Fig. 1I). Similar
results were obtained with noradrenalin (Supplemental Fig. 1).
These results are in complete agreement with the wellestablished effects of these compounds, demonstrating the capability of the zebrafish experimental system to identify changes in
heart contractility and rate under physiologic conditions.
CS-induced increase in heart contractility in zebrafish larvae. Zebrafish larvae were treated with different
concentrations of ouabain, digoxin, and bufalin for 90 minutes.
The exposure to low concentrations of ouabain (0.05–1 nM) led
to a significant increase in the heart force of contraction in a
dose-dependent manner. This was manifested by significant
increases in FAC, EF, and CO, with a maximal increase of 38 6
3.68% in CO at 0.2 nM but no change in heart rate (Fig. 2).
Similar results were obtained in larvae treated with digoxin or
bufalin (Figs. 3 and 4, respectively). Although some diversity in
CS effectiveness was apparent in our experimental system,
1 nM was chosen for the following experiments. At this
concentration, the three CS increased significantly the force of
contraction, without affecting heart rate. Hence, as in many
other species, the heart of zebrafish larvae respond by increased
heart contractility to CS treatment.
The crucial role of intracellular Ca21 in the CS-induced
increase in heart contractility is well established. Hence, in the
initial experiments we measured CS action on the zebrafish acc
mutant. This mutant lacks the activity of the SERCA1a isoform
exclusively in its muscle cells, inducing slow calcium clearance
from 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 heart
contractility (Fig. 5). The same result was obtained at other
CS concentrations, which increased contractility in the WT
(Figs. 2, 3, and 4). These results confirm the notion that, as in
other species, CS-induced increases in heart contractility in
zebrafish larvae largely depend on Ca21 homeostasis.
CS-induced ERK and Akt phosphorylation in adult
zebrafish heart in vivo. In recent years several laboratories
established the effects of CS on the phosphorylation of ERK and
Akt 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, after
which the hearts were removed and the proteins extracted. The
phosphorylation states of ERK and Akt in the protein extracts
were examined by Western blot analysis. As seen in Fig. 6, A–D,
the addition of ouabain to the swimming media of WT adult
zebrafish resulted in a 130 6 17.95% and 100 6 20.06% increase
in ERK and Akt phosphorylation in the heart, respectively.
Similar results were obtained using digoxin and bufalin (Fig. 6,
A–D). In addition, CS stimulated ERK and Akt phosphorylation
also in acc mutants 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 MAPK
pathway is involved in CS-induced increases in heart contractility
was tested using pharmacological tools. Zebrafish larvae were
exposed to MAPK inhibitors for 30 minutes, after which CS were
added and heart contractility was measured 90 minutes later. As
seen in Fig. 7, the inhibitor of Src family kinases, PP2, at
concentrations 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 different
specific ERK inhibitors, U0126 (1,4-Diamino-2,3-dicyano-1,4-bis(oaminophenylmercapto)butadiene monoethanolate) and PD98059
(2’-Amino-3’-methoxyflavone), at concentrations that did not affect
basal contractility (1 mM), prevented the CS-induced increase in
contractility (Fig. 7, C and D). Control experiments verified that
U0126 and PD98059 inhibit the CS-induced increase in ERK
phosphorylation 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 in
multiple 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 Akt
activation in CS-induced increases in heart contractility,
inhibition of Akt by MK-2206 on zebrafish heart contractility
was investigated. The exposure of WT zebrafish larvae for
2 hours to MK-2206 (10 nM) did not affect heart contractility
parameters. However, this treatment resulted in a doubling of
the CS-induced increase in heart contractility compared with
the CS effect in the absence of the inhibitor (Fig. 8, A and B).
This augmentation of the response to CS on contractility was
apparent in both the FAC and EF determinations and was not
accompanied by any effect on heart rate (Supplemental Fig. 2).
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manufacturer’s instructions. Preliminary experiments verified that
the stripping and reblotting procedure did not affect the quantification
of any of the proteins.
Statistics. Differences between experimental and control groups
in the in vivo experiments, quantification of heart contractility, and
consequent 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 contractility were analyzed by a mixed design (within/between subjects)
ANOVA, which was performed using the SPSS program (IBM, New
York, NY). Mixed design ANOVA is a statistical technique that
examines differences on a dependent variable (a within-subject variable, measured multiple times on the same subject) due to multiple
experimental conditions (the between-subject factor) while controlling
type 1 error (Gueorguieva and Krystal, 2004). This is achieved by
examining how the overall sum of squares in the dependent variable
(in our case, the five repeated length measurements of each cell) is
related to the between-subject effect (in our case, the four experimental conditions, control, kinase inhibitor, ouabain, and both). None of
the within-subject effects were significant, indicating that the five
length measurements of each cell were not significantly different
from each other neither when collapsed across the experimental
groups [F(4,27) 5 0.273, P 5 0.936] nor within the experimental
groups [F(12, 87) 5 0.902, P 5 0.548], supporting the stability of
the 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 the
experimental conditions the length was dissimilar to the others.
ERK and Akt Signaling in Cardiac Steroid Inotropic Effect
349
The effect of Akt inhibitor was also tested in the acc
mutant. Although, as mentioned above, this mutant was not
affected by CS at any of the tested concentrations (Fig. 5), a
significant increase in heart contractility caused by 1 nM
CS was observed in the presence of Akt inhibitor (Fig. 8, C
and 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 larvae
were placed in standard swimming medium (E3) containing 0.1–1000 nM digoxin for 90 minutes at 28°C. The experimental procedures were performed as
described 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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Fig. 2. Effect of ouabain on zebrafish heart contractility. Zebrafish larvae were placed in standard swimming medium (E3) containing 0.025–1 nM ouabain
for 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. A
minimum five pairs of systolic and diastolic cycles were measured 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 increases
in contractility in primary zebrafish cardiomyocytes.
The results presented above on the effects of ERK and Akt
inhibitors on CS-induced increases in heart contractility may
have resulted from indirect effects of the inhibitors and/or CS
on 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), heart
rate (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 the
control, P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).
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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 larvae
were placed in standard swimming medium (E3) containing 0.01–10 nM bufalin for 90 minutes at 28°C. The experimental procedures were performed as
described 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).
ERK and Akt Signaling in Cardiac Steroid Inotropic Effect
351
action on the heart. To test this hypothesis, a similar set of
experiments was performed on isolated adult zebrafish cardiomyocytes. As seen in Fig. 9 and Table 1, treatment of
zebrafish cardiomyocytes with 0.1 nM ouabain resulted in a
three- to fourfold increase in contractility with a concomitant
increase in contractility and relaxation rise time.
Fig. 7. Effect of Src and ERK1/2 inhibitors on CS-induced increase in heart contractility. Zebrafish larvae aged 72 hpf were placed in standard swimming medium
containing 50 nM protein phosphatase 2 (Src inhibitor) or 1 mM U0126 (ERK inhibitor) for 30 minutes at 28°C. Next 1 nM ouabain, digoxin, or bufalin were added
to the medium for 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. A
minimum five pairs of systolic and diastolic cycles in 12 larvae were measured and analyzed for each treatment. (A) FAC measurements for Src inhibitor with and
without CS. (B) EF measurements for Src inhibitor with and without CS. (C) FAC measurements for ERK inhibitor with and without CS. (D) EF measurements for
ERK 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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Fig. 6. Effect of CS on ERK and Akt
phosphorylation 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 swimming medium. The hearts were then removed and placed in lysis buffer. The levels
of phospho-ERK and phospho-Akt were
examined by Western blot (see Materials
and Methods). Total ERK and Akt served
as control. Each bar represents the mean 6
S.E. of three experiments. *Significantly
higher than the control, P , 0.05 (ANOVA
with a post hoc Bonferroni-adjusted Student’s t test).
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test the mechanisms involved, basal and CS-induced Ca21
transients were measured in adult WT and acc zebrafish
isolated hearts. Spontaneous cardiac rhythm in the acc
mutant was significantly reduced compared with that in the
WT. In the WT zebrafish heart, as expected, the addition of
200 mM ouabain to the medium caused a significant increase in
amplitude (27%) and in rise slope (61%) and decay slope (86%) of
the Ca21 transients (Fig. 10A). Similar changes in the CS-induced
Ca21 signals were obtained after preincubation of the heart with
Akt inhibitor (Fig. 10B and Table 2), indicating that the ouabain
effects on Ca21 oscillation are independent of Akt activity.
Furthermore, the addition of 200 mM ouabain to acc zebrafish
heart did not result in any changes in the Ca21 transients
parameters (Fig. 10C and Table 2), showing that SERCA1a
activity is required for CS-induced alterations in Ca21 signals.
The addition of 200 mM ouabain to acc zebrafish hearts in the
presence of Akt inhibitor (Fig. 10D and Table 2), a condition that
induced an increase in heart contractility, also did not cause any
changes in Ca21 transient parameters. These Ca21 measurements support the notion that in zebrafish heart Akt inhibition
does not change ouabain-induced effects on Ca21 transients.
Discussion
The zebrafish is a well-established experimental model for
the study of embryonic development. In recent years it has
entered the field of cardiovascular research as an organism
offering distinct advantages for dissecting molecular pathways of cardiovascular development, regeneration, and function (Leong et al., 2010; Wilkinson et al., 2014). We used
Fig. 8. Effect of Akt inhibitor on CS-induced increase in heart contractility in WT zebrafish and in acc mutants. Zebrafish larvae aged 72 hpf were placed
in standard swimming medium containing 10 nM MK2206 for 30 minutes at 28°C. Next, 1 nM ouabain, digoxin, or bufalin were added to the medium for
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. A
minimum five pairs of systolic and diastolic cycles were measured and analyzed in 12 larvae for each treatment. (A) FAC measurements for MK-2206 in
WT larvae with or without CS. (B) EF measurements for MK-2206 in WT larvae with or without CS. (C) FAC measurements for MK-2206 in acc mutants
with or without CS. (D) EF measurements for MK-2206 in acc mutants with or without CS. *Significantly higher than the control, P , 0.05, #significantly
higher than ouabain-treated grope P , 0.05 (ANOVA with a post hoc Bonferroni-adjusted Student’s t test).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Although the addition of 10 nM of ERK inhibitor PD98059 to
the cell medium increased contractility and relaxation rise
time, it did not affect the amplitude of cell contraction.
However, in the presence of the inhibitor, ouabain-induced
increases in all contractility parameters were completely
prevented (Fig. 9, A and B, and Table 1). On the contrary,
preincubation of cardiomyocytes with 1 nM Akt inhibitor MK2206, which by itself did not affect any of the contractility
parameters, enhanced the ouabain-induced augmentation in
contractility manifested by 113, 183, and 169% increases in
amplitude, contraction rise time, and relaxation rise time,
respectively (Fig. 9, C and D, and Table 1). These results confirm
the direct action of ouabain and the ERK and Akt inhibitors on
the heart.
In addition, similar to the in vivo experiments, 0.1 nM
ouabain or 1 nM MK-2206 did not affect the amplitude of adult
acc mutant primary cardiomyocytes contractility, demonstrating the obligatory role of SERCA1a in CS action under normal
condition. However, the addition of ouabain to these cells in
the presence of Akt inhibitor restored ouabain-induced increase in muscle contraction manifested by 67% increase in
the contraction amplitude (Fig. 9, E and F, and Table 1). These
results support the notion that Akt activity plays a negative
regulatory role in CS action.
Akt inhibition effect on CS-induced increases in
contractility is Ca21 independent. The novel finding of
the potentiation and restoration effects of Akt inhibition on
CS-induced increases in heart contractility in WT and acc
mutants, respectively, may result from changes in CS-induced
Ca21 transients or other Ca21 independent mechanisms. To
ERK and Akt Signaling in Cardiac Steroid Inotropic Effect
353
zebrafish larvae to address the hypothesis that MAPK and
Akt signaling pathways play a role in CS-induced increases in
heart contractility. To address this issue, an in vivo cardiac
function system for zebrafish larvae was established. The
effects of CS and kinase inhibitors were studied using live
imaging of the fully developed cardiovascular zebrafish larvae. The method, originally developed by Shin et al. (2010),
was based on the assumption that the heart ventricle has an
elliptic shape and construction of the changes in the area
between systole and diastole (Shin et al., 2010). In the present
study, the measurements were improved by determinations of
the ventricular area using continuous drawings of the polygon
TABLE 1
Contractility parameters of ouabain-induced increase in primary
cardiomyocyte contractility in the presence and absence of ERK or Akt
inhibitors
The experiments were performed as described in the legend to Fig. 9. Average twitch
amplitude, contraction, and relaxation rates were measured in 9 cells in each
group.
Amplitude
Control
0.1 nM Oua
10 nM PD98059
Oua & PD98059
Control
0.1 nM Oua
Oua & MK
1 nM MK2066
Acc mutant
Control
0.1 nM Oua
Oua & MK
1 nM MK2066
Contraction
Rate (2dv/dt)
Relaxation
Rate (+dv/dt)
0.14
0.53
0.13
0.15
0.13
0.38
0.43
0.19
6
6
6
6
6
6
6
6
0.04
0.1#
0.01
0.02
0.01
0.05*
0.04*#
0.03
0.63
3.65
0.87
1.06
0.77
1.84
3.37
0.98
6
6
6
6
6
6
6
6
0.16
0.38*
0.09*
0.09
0.14
0.28*
0.27*#
0.19
0.41
2.92
0.78
0.79
0.50
1.64
2.78
0.87
6
6
6
6
6
6
6
6
0.09
0.37*
0.14*
0.08
0.11
0.23*
0.32*#
0.20
0.13
0.14
0.22
0.15
6
6
6
6
0.01
0.03
0.02*
0.02
1.58
2.43
3.65
2.61
6
6
6
6
0.29
0.84
0.71*
0.74
0.56
0.07
0.52
0.98
6
6
6
6
0.19
0.05*
0.26
0.34
*Significantly higher than control P , 0.05, #significantly higher than ouabaintreated cells (post hoc Bonferroni-adjusted between subjects ANOVA).
border of the ventricle, yielding accurate FAC and EF of the
heart in vivo (Fig. 1, B–E). The system was validated by testing
the effects of adrenergic and cholinergic agonists added to the
swimming media on zebrafish heart contractility. As expected,
adrenalin (1 mM) induced a significant increase in heart
contractility and rate (Fig. 1, F–I). This classic response
resembles that seen in other species. Similarly, cholinergic
agonists (i.e., acetylcholine, 1 mM) induced a decrease in heart
contractility and rate (Fig. 1) comparable to its effects in other
animal models. Hence, this method of cardiac measurement
enables efficient determination of key aspects of cardiac function, such as FAC, EF, heart rate, and calculation of CO of
zebrafish larvae, and can be used for in vivo physiologic and
pharmacological investigations.
The addition of ouabain, digoxin, or bufalin to zebrafish
larvae swimming medium increased dose dependently the
force of contraction of heart muscle. Whereas 0.1 nM bufalin or
ouabain produced a significant increase in heart contractility,
an about tenfold higher concentration of digoxin was required
to yield a similar effect (Figs. 2, 3, and 4). This effect and the
differential potencies are similar to the one seen in mammals,
demonstrating the suitability of the zebrafish model for
the study of CS action. CS-induced toxicity, manifested by
reduced FAC, EF, or mortality, was observed at concentrations 1000 times higher than those required for a significant
positive inotropic effect, indicating a wider dose response for
CS in this organism.
Inhibition of Na1, K1-ATPase by CS is the recognized
mechanism of action for their ability to increase the force of
contraction of heart muscle. The exclusiveness of this mechanism was challenged by the demonstration that the interaction of CS with Na1, K1-ATPase elicits the activation
of several major signaling cascades, including ERK, Akt, and
iNOS (Tian et al., 2001; Gan et al., 2012; Wu et al., 2013).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 9. Effect of ERK and Akt inhibitors on CS-induced increase in primary cardiomyocyte contractility. Primary adult zebrafish cardiomyocytes were
prepared as described in Materials and Methods and exposed to ouabain, ERK, or Akt inhibitor or a combination thereof for 20 minutes at room
temperature. The cardiomyocytes were stimulated at 0.5 Hz, and cell shortening was recorded. Nine cells per each group were measured. A
representative twitch of % shortening of cells exposed to ouabain with or without ERK inhibitor (A) or to ouabain with or without Akt inhibitor (C) is
shown. Quantification of the data as % of control is shown in (B) and (D). A representative twitch of % shortening of cells from acc mutant exposed to
ouabain with or without Akt inhibitor is shown in (E), and quantification of the data as % of control of responses is shown in (F). *Significantly higher
than the control, P , 0.05, #significantly higher than ouabain-treated group P , 0.05 (post hoc Bonferroni-adjusted between subjects ANOVA).
354
Buzaglo et al.
These signaling pathways, once activated by CS, participate in
numerous physiologic functions including cell viability, kidney and muscle function, and cardiac hypertrophy (Liu et al.,
2007; Xie et al., 2013; Wang et al., 2014). Indeed, using an ex
vivo experimental system, it was shown that inhibition of Src
and ERK attenuated the CS-induced increase in heart
contractility 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 proximal
tubular cells (Khundmiri et al., 2007). Similarly, the exposure of
adult zebrafish to CS caused an about twofold increase in ERK
and Akt phosphorylation in heart tissue in the WT fish and in
the acc mutants (Fig. 6). The protein phosphorylation may
result in conformational changes after the interaction of CS
with the Na1, K1-ATPase. Alternatively, the phosphorylation
may be the consequence of indirect mechanisms, such as
changes in intracellular Ca12 (Agell et al., 2002) or in muscle
tension (Xu et al., 1996). The observation that CS did not
increase the heart force of contraction nor Ca21 transients but
did augment ERK and Akt phosphorylation in the acc mutants
favors the first mechanism. In addition, the observation that CS
did not induce an increase in heart contractility in the acc
mutant but did stimulate ERK phosphorylation indicates that
the CS-induced MAPK activation per se is not sufficient to elicit
an increase in contractility.
The inhibition of MAPK pathway by the addition of Src
kinase or ERK inhibitors did not affect the force of contraction of
the zebrafish larvae hearts or cardiomyocytes. These treatments, however, completely abolished the increased contraction
caused by CS in vivo and ex vivo (Fig. 7 and Fig. 9, A and B,
respectively). These findings are in accordance with the notion
that MAPK activation is required for the CS-induced increase
in heart contractility. A similar conclusion was drawn in a
previous study demonstrating that ERK inhibitors attenuate a
CS-induced increase in contractility in rat cardiomyocytes (Tian
et al., 2001). The possible mechanism involved in this effect
may be mediated by the established link between ERK
TABLE 2
Effect of ouabain and Akt inhibitor on Ca2+ transients in adult zebrafish heart
The 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
Rate (wave/min)
Amplitude (Maximal deflection from baseline)
Area (Area under the transient relative to baseline)
Rise slope (+D Fluorescence in the linear phase/sec)
Decay slope (-D Fluorescence in the linear phase/sec)
Acc mutant
Rate (wave/min)
Amplitude (Maximal deflection from baseline)
Area (Area under the transient relative to baseline)
Rise slope (+D Fluorescence in the linear phase/sec)
Decay slope (-D Fluorescence in the linear phase/sec)
Ouabain 200 mM
MK-2206 (1 nM)
Ouabain & MK-2206
58
5.28
2.52
4.85
10.73
6
6
6
6
6
7.4
0.32
0.28
1.29
7.54
52
6.59
2.94
7.84
20
6
6
6
6
6
6.8
0.71*
0.44*
2.02*
3.51*
48
4.8
2.81
1.65
4.65
6
6
6
6
6
4.8
0.13
0.19
0.28*
0.54
48
5.91
2.08
2.68
7.87
6
6
6
6
6
5
0.28*#
0.23
0.34*#
1.11
31
1.79
2.64
0.48
1.88
6
6
6
6
6
5
0.11
0.65
0.07
0.33
35
1.67
2.54
0.47
1.92
6
6
6
6
6
8.4
0.15*
0.56
0.1
0.46
25
1.85
2.79
0.51
1.72
6
6
6
6
6
3
0.05
0.23
0.04
0.22
31
1.76
2.92
0.56
1.61
6
6
6
6
6
4.4
0.05
0.4
0.05
0.19
*Significantly higher than control P , 0.05; #significantly higher than MK-2206 P , 0.05 (post hoc Bonferroni-adjusted between subjects ANOVA).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
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 control
and 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 acc
mutant is shown in (C) and (D), respectively. The quantitative analyses of these Ca2+ oscillations are depicted in Table 2.
ERK and Akt Signaling in Cardiac Steroid Inotropic Effect
Acknowledgments
We thank Dr. Michal Horowitz for advice and support in cell
motility measurements and Dr. Yuval Kalish for assistance in the
statistical 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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Andrikopoulos et al., 2011). The identical dependence of the CSinduced increase in contractility in vivo and in cardiomyocytes
points to the direct effect of the steroid on the zebrafish heart.
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gene expression or Ca21 sensitivity.
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Address correspondence to: Dr. David Lichtstein, Department of Medical
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Supplemental Data
Title: Essential Opposite Roles of ERK and Akt Signaling
in Cardiac Steroid-Induced Increase in Heart
Contractility
Nahum Buzaglo, Haim Rosen, Hagit Cohen-Ben Ami,
Adi Inbal and David Lichtstein
Journal of Pharmacology and Experimental
Therapeutics #230763
Supplement Figure 1
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190
HR (beats/minuts)
FAC ((EDA-ESA)/EDA*100)
45
Control
1μM
Acth
10μM
Acth
1μM
NorAd
10μM
NorAd
Effect of Acetylcholine and Noradrenalin on zebrafish heart contractility.
The effect of acetylcholine and noradrenalin were determined by placing zebrafish larvae in standard
swimming medium (E3) containing 1 and 10 µM acetylcholine, for 60 min, or 1 and 10 µM of
noradrenalin for 90 min. The larvae were then anaesthetized and images of the heart were obtained
and analyzed as described above. A minimum five pairs of systolic and diastolic cycles were
measured and analyzed in 10 larvae for each treatment. FAC (A) and EF (B), Heart rate (C) and
calculated cardiac output (D) were measured as described in the Methods. *Significantly different
from control, P<0.05
Supplement Figure 2
200
180
HR (beats/minuts)
160
140
120
100
80
60
40
20
0
Ctrl
50nM PP2
1mM
m u0126
10nM MK-2206
Effect of Src, ERK and Akt Inhibitors on Zebrafish Larvae Heart Rate.
Zebrafish larvae aged 72 hpf were placed in standard swimming medium containing pp2 (Src
inhibitor) or U0126 (ERK inhibitor) or MK-2206, for 30 min at 28oC. The larvae were then
anaesthetized and images of the heart were obtained and analyzed as described in the
legend to Fig. 1. Heart rate was measured for 1 min.