0022-3565/10/3322-421–428$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET 332:421–428, 2010

Vol. 332, No. 2
161646/3550644
Printed in U.S.A.

Inhibition of Dystrophin Breakdown and Endothelial NitricOxide Synthase Uncoupling Accounts for Cytoprotection by
3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6dimethoxy-1-(4-imidazolylmethyl)-1H-indazole Dihydrochloride
3.5 Hydrate (DY-9760e) in Left Ventricular Hypertrophied Mice
Feng Han, Ying-Mei Lu, Hideyuki Hasegawa, Hiroshi Kanai, Erika Hachimura,
Yasufumi Shirasaki, and Kohji Fukunaga
Institute of Pharmacology, Toxicology, and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, China (F.H.);
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (Y.-M.L., E.H.,
K.F.); Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan (H.H.); Graduate School of Engineering,Tohoku University, Sendai, Japan (H.K.); and Daiichi-Sankyo Co., Ltd., Tokyo, Japan (Y.S.)
Received September 15, 2009; accepted November 2, 2009

ABSTRACT
Using a heart ischemia/reperfusion model in rats, we recently demonstrated that 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride
3.5 hydrate (DY-9760e), a calmodulin inhibitor, is a cardioprotective
drug. Here, we examined cardioprotective mechanisms of DY-9760e
in hypertrophy and heart failure using a mouse transverse aortic
constriction (TAC) model. Mice were subjected to TAC and 2 weeks
later they were administered DY-9760e for another 6 weeks (at 10 or
20 mg/kg/day p.o.). Chronic administration inhibited TAC-induced
increased heart-to-body weight ratio dose-dependently. Consistent
with inhibition of hypertrophy, fraction shortening, an indicator ofheart contractile function, assessed by echocardiography was completely restored by DY-9760e (20 mg/kg/day) administration. Inhibition of TAC-induced atrial natriuretic peptide (ANP) up-regulation
further confirmed an antihypertrophic effect of DY-9760e. It is note-

Cardiac hypertrophy is an adaptive response to prolonged
increases in hemodynamic pressure overload and humoral
stresses. Although this compensatory process is initially aphysiological event to reduce wall stress and oxygen conThis work was supported in part by the Ministry of Education, Science, Sports
and Culture of Japan [Grant 19390150] (to K.F.); and by the Uehara Memorial
Foundation (to Y.-M.L.) and the Smoking Research Foundation (to K.F.).
F.H. and Y.-M.L. contributed equally to this work.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.161646.

worthy that we found that breakdown of dystrophin and spectrin bycalpain was associated with heart failure in TAC mice. Caveolin-3
breakdown was closely associated with endothelial nitric-oxide synthase (eNOS) dissociation from the plasma membrane and its subsequent uncoupling. Uncoupled monomeric eNOS formation was
associated with increased protein tyrosine nitration, suggesting peroxynitrite production and NO and superoxide formation. It is important to note that 6 weeks of DY-9760e treatment significantly blocked
hypertrophic responses, such as increased heart weight and ANP
induction. Overall, we show that inhibition of both dystrophin/spectrin
breakdown and uncoupling of eNOS probably underlies the cardioprotective mechanisms of DY-9760e. The observed protection of
sarcolemmal proteins and eNOS by DY-9760e during pressure overload suggests a novel therapeutic strategy to rescue the heart fromhypertrophy-induced failure.

sumption, progressive left ventricular (LV) hypertrophy significantly increases the risk of developing heart failure andsudden death in humans (Levy et al., 1996; Haider et al.,
1998; Brown et al., 2000; Meijs et al., 2007). During continuous pressure overload, degradation of sarcolemma andcaveolae components, including dystrophin, spectrin, andcaveolin-3, in cardiomyocytes triggers cardiomyocyte injury(Weber, 1989; Jugdutt, 2003; Spinale, 2007).
Dystrophin, a 430-kDa rod-shaped protein, is a componentof the sarcolemma cytoskeleton in both cardiac and skeletal

ABBREVIATIONS: LV, left ventricle; eNOS, endothelial nitric-oxide synthase; CaM, calmodulin; ET, endothelin; DY-9836, 3-(2-(4-(3-chloro-2methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxyindazole; DY-9760e, 3-[2-[4-(3-chloro-2-methlphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate; TAC, transverse aortic constriction; PO, pressure overload; HW, heart weight; BW,body weight; LVEDD, left ventricle end-diastolic diameter; LVESD, left ventricle end-systolic diameter; FS, fraction shortening; PCR, polymerase
chain reaction; ANP, atrial natriuretic peptide; PBS, phosphate-buffered saline; BH4, tetrahydrobiopterin.
421

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Han et al.

muscle. Dystrophin is attached to the ␤-subunit of dystroglycan, a member of a complex of dystrophin-associated glycoproteins. Dystrophins are also associated with other cytoskeletal elements, such as spectrin, through filamentous actin,
thereby supporting the submembranous cytoskeletal structure to maintain heart function. Thus, disruption of myocardial dystrophin is a critical event in progression to advanced
heart failure (Toyo-oka et al., 2004). Both calpains 1 and 2
account for degradation of dystrophin (Yoshida et al., 2003)
and spectrin (Saido et al., 1994).
Caveolin-3 is a component of caveolae in cardiac and skeletal muscles. Caveolin-3 is localized to the sarcolemma andfunctions in formation of caveolae membranes, serving as a
scaffolding protein to interact with and organize lipid andprotein constituents, including endothelial nitric-oxide synthase (eNOS), in caveolae. As in endothelial cells, eNOS istargeted to sarcolemmal caveolae through both post-translational myristoylation and later palmitoylation in cardiomyocytes. Caveolin-3 binds to eNOS in caveolae, thereby inhibiting its NO-generating activity. When Ca2⫹ is mobilized into
cardiomyocytes through stimulation of calcium channels or
GTP-binding protein-coupled receptors localized in caveolae,eNOS is efficiently activated by Ca2⫹/calmodulin (CaM)(Feron et al., 1996).
We have documented that prolonged exposure of cultured
cardiomyocytes to endothelin (ET)-1 causes biphasic changes
in dystrophin levels (Lu et al., 2007). We subsequently foundthat dystrophin levels were markedly elevated during thefirst 48 h of ET-1 exposure, whereas dystrophin was significantly degraded after 96 h of ET-1 treatment, resulting ininstability of sarcolemmal structures, dissociation of caveolin-3 and eNOS from caveolae and eNOS uncoupling, andultimately apoptosis (Lu et al., 2009a). These in vitro studies
prompted us to seek novel cardioprotective drugs to prevent
dystrophin breakdown and eNOS dysregulation.
We have found that the novel calmodulin antagonist DY9760e can protect the heart from ischemic/reperfusion injury(Hashimoto et al., 2005) and that its active metabolite DY9836 prevents phenylephrine-induced injury to cultured cardiomyocytes (Lu et al., 2009a). It is more important that
DY-9836 treatment totally inhibited aberrant NO production
and superoxide generation by uncoupled eNOS after phenylephrine treatment of these cells (Lu et al., 2009a). We also
found that DY-9760e treatment of cultured cardiomyocytes
prevents ET-1-induced hypertrophy (Lu et al., 2009b). However, potential antihypertrophic and cardioprotective effects
of DY-9760e had not been tested in vivo on pressure overloadinduced hypertrophy.
Here, we used a mouse heart failure model induced bytransverse aortic constriction (TAC)-induced hypertrophy toanalyze potential antihypertrophy and cardioprotective effects of DY-9760e in vivo. DY-9760e markedly inhibited TACinduced cardiac hypertrophy with concomitant restoration ofcontractile function. Our findings strongly suggest that protection of dystrophin and spectrin from calpain-induced degradation as well as inhibition of eNOS uncoupling probably
mediates these cardioprotective effects in vivo.

Materials and Methods
Animals. Adult male DDY mice weighing 35 to 40 g were obtained
from Nippon SLC (Hamamatsu, Japan). Ten-week-old males wereacclimated to the local environment for 1 week, which included
housing in polypropylene cages at 23 ⫾ 1°C in a humidity-controlledenvironment maintained on a 12-h light/dark schedule (lights on
8:00 AM– 8:00 PM). Mice were provided food and water ad libitum.
Animal experiments were in accordance with the Guide for the Careand Use of Laboratory Animals at Tohoku University. The Medical
Experimental Animal Administrative Committee of Tohoku University approved all experiments. All efforts were made to minimize
animal suffering and reduce the number of animals used.
Surgical Procedures and Drug Treatment. Transverse aortic
constriction (TAC) was performed on males as described previously(Hu et al., 2003). After acclimatization for 7 days, animals wereanesthetized with tribromoethanol (0.25– 0.3 g/kg i.p.). The chest
cavity was opened with scissors by a small incision at the level of thesecond intercostal space. After isolating the aortic arch, a 6-0 silk
suture was placed around the aorta with a 27-gauge needle. The
needle was immediately removed to produce an aorta with a stenotic
lumen. The chest cavity was then closed with one 6-0 nylon suture,
during which negative pressure in the thorax was re-established byremoving air with a polyethylene-50 chest tube attached to a syringe.
Sham-operated animals, which underwent surgery without the final
tightening of the constrictive suture, served as controls.
After 2 weeks of TAC or sham surgery, mice were randomly
divided into five groups: 1) sham receiving vehicle (0.5% methyl
cellulose; n ⫽ 6), 2) sham receiving DY-9760e at 20 mg/kg (n ⫽ 6),3) a pressure overload (PO) group receiving vehicle (n ⫽ 6), 4) a PO
group receiving DY-9760e at 10 mg/kg (n ⫽ 6), and 5) a PO group
receiving DY-9760e at 20 mg/kg (n ⫽ 6). Mice were orally administered vehicle or DY-9760e daily for another 6 weeks, as indicated.
Measurement of Cardiac Hypertrophy. After 6 weeks of DY9760e or vehicle administration, animals were subjected to terminal
surgery. Mice were weighed and anesthetized with tribromoethanol(0.25– 0.3 g/kg i.p.). The thoracic cavity was opened, and hearts wereimmediately harvested and weighed. Cardiac indices, expressed as
the ratio of heart (in milligrams) to body (in grams) weight (HW/BW),
were used to estimate the degree of cardiac hypertrophy.
Transthoratic Echocardiography. Noninvasive echocardiographic measurements were performed using ultrasonic diagnostic
equipment (SSD-6500; Aloka, Tokyo, Japan). In motion-mode images
obtained using a 10-Hz linear type ultrasonic probe (UST-5545;
Aloka), the following parameters, including LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD), were used to
calculate fraction shortening (FS), as follows: %FS ⫽ ([LVEDD ⫺LVESD]/LVEDD) ⫻ 100. Animals (n ⫽ 4 in each group) were subjected to echocardiographic analyses after sham operation or TAC at
8 weeks with or without DY-9760e administration.
Reverse Transcription-Polymerase Chain Reaction. Cardiac
gene expression was analyzed by reverse transcription-PCR. Total
RNA was extracted from heart after 8 weeks of TAC using TRIzol
Reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA
was reverse-transcribed using the Reverse Transcription System(Promega, Madison, WI) according to the manufacturer’s protocol.
Primer sequences for ANP were as follows: 5⬘-GTCCAACACAGATCTGATGG-3⬘ and 5⬘-GATTTGGCTGTTATCTTCGG-3⬘, generating a 377-base pair product. Primer sequences for ␤-actin were as
follows: 5⬘-CGTCCACCCGCGAGTACAAC-3⬘ and 5⬘-TCCTTCTGACCCATACCCAC-3⬘, generating a 220-base pair product. PCR amplification was performed using the GeneAmp PCR system 9700 (Applied Biosystems Japan, Chiba, Japan). PCR conditions (ANP: 26
cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s; brain
natriuretic peptide ␤-actin: 30 cycles of 94°C for 30 s, 55°C for 30 s,
and 72°C for 30 s) were determined within the linear amplification
range. After separation of PCR products on a 2% agarose gel containing ethidium bromide, products were quantified using an imaging analyzer (ChemiDoc XRS; Bio-Rad Laboratories, Hercules, CA),and expression levels were normalized to those of ␤-actin.
Western Blot Analysis. All animals were sacrificed after 6 weeks
of drug administration. After weighing the heart, the left ventricle

DY-9760e Inhibits Dystrophin Breakdown in Cardiac Hypertrophy
was isolated from each heart (n ⫽ 6) and stored at ⫺80°C until
immunoblotting analyses were performed as described previously(Lu et al., 2007). In brief, equal amounts of protein were separated on
7.5 to 10% SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica,
MA). Blots were then stained with 0.1% Ponceau S solution to visualize protein bands and confirm equal protein loading among groups.
After blocking in 5% nonfat milk, blots were incubated overnight at
4°C with antibodies against dystrophin (mouse monoclonal antibody,
1:1000; Millipore Bioscience Research Reagents, Temecula, CA),
spectrin, caveolin-3, eNOS, and nitrotyrosine (rabbit polyclonal antibody, 1:500; Hashimoto et al., 2005; Lu et al., 2009a). Immunoreactive proteins on membranes were visualized with an enhanced
chemiluminescence (ECL) detection system (GE Healthcare, Little
Chalfont, Buckinghamshire, UK). Images were scanned and analyzed semiquantitatively using Image (National Institutes of Health,Bethesda, MD) and Image Gauge software (Fujifilm, Tokyo, Japan).
Immunohistochemistry. For immunohistochemical studies, anadditional three mice in each experimental condition were anesthetized with pentobarbital and perfused via the ascending aorta with
0.1 M phosphate-buffered saline, pH 7.4, until the outflow became
clear followed by 0.1 M phosphate buffer, pH 7.4, containing 4%paraformaldehyde for 15 min. The left ventricle was removed andpostfixed in the same solution for 24 h at 4°C and then sliced at 35
␮m using a Vibratome sectioning system (Dosaka EM Co. Ltd.,
Kyoto, Japan). Sections were incubated at room temperature with
0.01% Triton X-100 in phosphate-buffered saline for 30 min and foranother hour in 3% bovine serum albumin in phosphate-bufferedsaline. For immunolabeling, slices were probed with anti-dystrophin
antibody (1:500) overnight at 4°C. After washing, sections wereincubated with biotinylated anti-rabbit IgG (1:5000) in TNB buffer(ECL detection system) for 1 h, followed by streptavidin-horseradish
peroxidase (1:5000) labeling for 2 h. Sections were then stained withtetramethylrhodamine tyramide for 10 min using the TSA-Direct kit(PerkinElmer Life and Analytical Sciences, Boston, MA). Immunofluorescent images were acquired using a confocal laser scanningmicroscope (TCS SP; Leica Microsystems, Inc., Deerfield, IL).
Statistical Analyses. All values are expressed as means ⫾S.E.M. Multiple comparisons between experimental groups weremade by analysis of variance followed by Dunnett’s test. P ⬍ 0.05
was considered significant.

Results
Inhibitory Effect of DY-9760e on Pressure OverloadInduced Myocardial Hypertrophy in Vivo and Echocardiography Analysis. After 2 weeks of TAC, the heart
was slightly enlarged and the wall of ventricle was thickened(Fig. 1A). After 8 weeks of TAC, the ventricular cavity waslarger than that seen at 2 weeks. DY-9760e administration(10 or 20 mg/kg orally) for 6 weeks beginning after the first 2
weeks of TAC dose-dependently inhibited heart weight increases, as indicated by the HW/BW ratio, and decreased thesize of the left ventricular cavity (Fig. 1B), suggesting that
DY-9760e inhibits TAC-induced hypertrophy in vivo.
Next, we used echocardiography to determine whether inhibition of hypertrophy is accompanied by functional recovery ofheart contraction. After 8 weeks of TAC, two-dimensional imaging using LV echocardiography showed a significant increase
both in LVEDD and LVESD in animals not treated with DY9760e. By contrast, animals administered DY-9760e (20 mg/kg)
showed values comparable with the sham-operated group (Fig.
1C). TAC also resulted in a significant decrease in FS% compared with the sham group in untreated animals. However, this
impairment was rescued by 6 weeks of DY-9760e treatment (20mg/kg) beginning after 2 weeks of TAC (Fig. 1C).

423

Inhibition of TAC-Induced ANP Expression by DY9760e Administration. We next confirmed antihypertrophic action of DY-9760e in vivo by measuring ANP mRNAlevels in the LV. Although DY-9760e treatment had no effecton baseline ANP expression in sham-operated animals, TACinduced ANP up-regulation was totally blocked by DY-9760e(20 mg/kg) treatment (Fig. 2).
Inhibitory Effect of DY-9760e on TAC-Induced Degeneration of Dystrophin. We showed previously that DY-9760e
treatment inhibits dystrophin breakdown seen in cultured cardiomyocytes after prolonged ET-1 treatment (Lu et al., 2007).
Therefore, we evaluated dystrophin levels in LV after TAC
in vivo using immunohistochemical analyses of LV cardiomyocytes at 2, 4, and 8 weeks after TAC surgery (Fig. 3A). Compared with strong dystrophin immunoreactivity seen in theplasma membrane of sham-operated animals, membrane dystrophin immunoreactivity in experimental animals was weak
and accompanied by increased cardiomyocyte size during theentire period after TAC surgery. Notably, cytoplasmic dystrophin immunoreactivity was weak in cardiomyocytes at 2 and 4
weeks but strong in some cells by 8 weeks (Fig. 3A). We thentested the effect of DY-9760e treatment on dystrophin levels byWestern blotting using LV extracts prepared after 8 weeks of
TAC. As shown in Fig. 3B, TAC alone markedly reduced dystrophin levels (50 ⫾ 3% of the sham group), whereas DY-9760e
treatment (20 mg/kg) blocked TAC-induced dystrophin breakdown (75 ⫾ 3% of sham group; p ⬍ 0.01 versus the PO group)(Fig. 3B). These findings indicate that inhibition of dystrophin
breakdown by DY-9760e treatment could protect cardiomyocytes in conditions of heart failure.
Effects of DY-9760e on TAC-Induced Spectrin andCaveolin-3 Breakdown. To confirm that TAC-induced dystrophin breakdown is mediated by calpain activity, we investigated calpain activity by assessing a specific, calpain-dependent 150-kDa spectrin breakdown product. As expected,
after 8 weeks of TAC, levels of full-length, 240-kDa spectrin
were significantly reduced in the absence of DY-9760e (Fig. 4,
A and B). These levels recovered dose-dependently after DY9760e treatment. Inversely, levels of the TAC-induced 150kDa breakdown product were markedly increased by 270%(p ⬍ 0.05) and reduced dose-dependently by DY-9760e treatment (Fig. 4, A and C).
Caveolin-3 breakdown by phenylephrine-induced hypertrophy is associated with injury in cultured cardiomyocytes(Lu et al., 2009a). As expected, we observed significant TACinduced caveolin-3 breakdown in the LV compared withsham-operated animals. Significantly, DY-9760e (20 mg/kg)
treatment strongly inhibited this effect (Fig. 4, D and E),
suggesting that dystrophin breakdown is accompanied withbreakdown of spectrin and caveolin-3.
TAC-Induced eNOS Redistribution after Transverse
Aortic Constriction. Because eNOS is localized to caveolae
through caveolin-3 in cardiomyocytes (Michel and Feron,1997), we asked whether dystrophin and caveolin-3 breakdown triggers eNOS dissociation from caveolae. Although
total eNOS protein levels in LV extracts remained unchanged in TAC versus sham-operated animals (Fig. 5A),
membrane-associated eNOS levels were markedly reducedby TAC, whereas DY-9760e treatment dose-dependently restored plasma membrane eNOS levels (Fig. 5B). We hypothesized that caveolin-3 breakdown in caveolae and concomitant eNOS dissociation from the plasma membrane might

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Han et al.

ShamTAC(2W) TAC(8W) TAC(8W)++
DY(20)
vehicleB 16**
LW/BW(mg/g)

A

Sham

TAC(2W) TAC(8W) TAC(8W)++
vehicleDY(20)

12
#

8

##

4
0
Sham

5
*

#

4

3

2

1

Sham

TAC
+
vehicle)

TAC
TAC
TAC
+
+
+
vehicle) DY(10) DY(20)TAC+DY20

LVEDD

TAC
+
DY(20)

LV end-systolic diameter (mm)LVESD

LV end-diastolic diameter (mm)TAC+vehicle

Sham

5

4
*
#

3

2

1

Sham

TAC
+
vehicle)TAC+
DY(20)

LV fraction shortening (%)

C

DY
(20)

#

50

40
**

30
20

10

Sham

TAC
+
vehicle)TAC+
DY(20)

Fig. 1. DY-9760e treatment effectively inhibits pressure overload-induced cardiomyocyte hypertrophy. After 2 weeks (W) of TAC, DY-9760e [10 or 20
mg/kg (DY10 or DY20)] was orally administered daily for the next 6 weeks of TAC. A, after isolation of whole heart, the sections are fixed inparaformaldehyde solution. B, HW/BW ratio was used as an index of cardiac hypertrophy. Data are expressed as percentage of values of shamoperated animals (mean ⫾ S.E.M.; n ⫽ 6). ⴱⴱ, P ⬍ 0.01 versus sham; #, P ⬍ 0.05; ##, P ⬍ 0.01 versus vehicle. C, top, representative motion-mode
echocardiographic images were obtained in conscious mice 8 weeks after aortic banding with or without DY-9760e (20 mg/kg) treatment. Bottom, data
are expressed as percentage of values of sham-operated animals (mean ⫾ S.E.M.; n ⫽ 4). ⴱ, P ⬍ 0.05 versus sham; #, P ⬍ 0.05 versus vehicle.

induce eNOS uncoupling and generate superoxide rather
than nitric oxide. As expected, reduced levels of eNOS dimers
and reciprocally increased levels of eNOS monomers wereobserved in LV extracts 8 weeks after TAC (Fig. 5C). DY9760e treatment dose-dependently restored the dimeric formof eNOS, suggesting formation of physiological coupled eNOS
(Fig. 5C). Likewise, peroxynitrite formation, as assessed byprotein tyrosine nitration in LV extracts, was markedly increased by TAC and blocked by DY-9760e dose-dependently(Fig. 5D). Increased peroxynitrite formation suggests generation of superoxide by uncoupled eNOS, as we have shown in
cultured cardiomyocytes (Lu et al., 2009a).

Discussion
Calpain activation triggers cleavage of several membrane-associated cytoskeletal proteins such as dystrophin

and spectrin. Disruption of the submembranous structureof cardiomyocytes triggered by dystrophin breakdown
causes contractile dysfunction in the muscle of dystrophic
patients (Toyo-oka et al., 2004). Here, we demonstrated
that calpain activation upon pressure overload leads todystrophin breakdown. In addition, disruption of eNOS
localization by caveolin-3 breakdown in caveolae structures probably induces aberrant eNOS uncoupling andsuperoxide generation, contributing to cardiomyocyte injury and contractile dysfunction. Thus, inhibiting the calpain/dystrophin cascade and eNOS uncoupling could represent a novel therapeutic approach to inhibit heart
dysfunction and heart failure. A critical finding of thepresent study is that DY-9760e, a novel calmodulin antagonist, effectively blocked both dystrophin breakdown andeNOS uncoupling in vivo in a mouse TAC model. DY-9760e

DY-9760e Inhibits Dystrophin Breakdown in Cardiac Hypertrophy

ANP

377 bpβ β-actin

220 bpA

Sham DY(20) TAC TAC+
DY(20)ANP mRNA (% of Sham)

250
200

425

Sham

2W

4W

8W

**

150

#

100
50
0

Sham DY(20) TAC

TAC+
DY(20)

Fig. 2. DY-9760e treatment inhibits TAC-induced ANP expression. After
2 weeks of sham or TAC treatment, DY-9760e (20 mg/kg) was orally
administered for 6 weeks to sham or experimental groups. Top, reverse
transcription-PCR products of ANP and ␤-actin are shown. Bottom, quantitation of ANP and ␤-actin mRNA levels shown as a percentage of valuesof sham-operated animals (mean ⫾ S.E.M.; n ⫽ 6). ⴱⴱ, P ⬍ 0.01 versus
sham-operated mice; #, P ⬍ 0.05 versus vehicle-treated TAC mice.

B
430 kDa

Dystrophin-tubulin

50 kDa(-)

DY(20) ( - ) DY(10) DY(20)Dystrophin (% of Sham)

Shamprotective activity was further evidenced by inhibition ofspectrin breakdown and protein tyrosine nitration in the
LV after TAC.
Our previous in vitro study demonstrated that DY-9760e
inhibits ET-1-induced cardiomyocyte hypertrophy and dystrophin breakdown in cultured cardiomyocytes (Lu et al.,2007). In addition, DY-9760e significantly inhibits activation of Ca2⫹/CaM-dependent nitric-oxide synthase after
brain and heart ischemia (Hashiguchi et al., 2004; Hashimoto et al., 2005; Han et al., 2006). The antihypertrophic
effect of DY9760e is mediated primarily by inhibition ofCa2⫹/CaM-dependent activation of CaMKII␦ in cardiomyocytes (Lu et al., 2009b). This pathway, together withdystrophin/spectrin breakdown and eNOS uncoupling, underlies cardiomyocyte injury after cardiac hypertrophy.
Dystrophin is essential to maintain membrane integrityin cardiac and skeletal myocytes. Its disruption in thesubmembranous structure triggers heart failure after cardiac hypertrophy (Toyo-Oka et al., 2004; Kawada et al.,2005). The idea that membrane dystrophin is lost to thecytosol during the early weeks after TAC was supported byimmunohistochemistry after prolonged TAC. Calpains arecalcium- and thiol-dependent proteases whose overactivation is implicated in several diseases, including cardiovascular disease (Matsumura et al., 1996) and ischemic stroke(Yoshida et al., 1995). Calpain activation followed by dystrophin breakdown leads to myocardial injury. Indeed,
breakdown of dystrophin and spectrin correlates with myocardial injury after cardiac ischemia (Armstrong et al.,
2001; Hashimoto et al., 2005). In cardiac ischemia, calpain
activity mediates impaired Na⫹/K⫹-ATPase activity withconcomitant spectrin breakdown (Inserte et al., 2005). Because Na⫹/K⫹-ATPase interacts with the cytoskeletal protein
spectrin through ankyrin, spectrin breakdown probably mediates impairment of Na⫹/K⫹-ATPase after cardiac ischemia. It is

TAC

150

100

##
**

**

50

0
Sham

DY
(20)

TAC

TAC TAC++
DY(10)
DY(20) degradation.
Fig. 3. DY-9760e treatment blocks TAC-induced
dystrophin
A, immunohistochemical localization of dystrophin in sham- and TACoperated animals. Localization of dystrophin in the plasma membrane ofcardiomyocytes indicates enlargement of cardiomyocytes 2 weeks (2W)
after TAC and redistribution of dystrophin at 4 and 8 weeks after TAC.
Scale bar, 40 ␮m. B, immunoblotting analyses of LV extracts (top) show
markedly reduced levels of dystrophin 8 weeks after TAC and indicatethat 6 weeks of DY-9760e treatment (20 mg/kg) began after 2 weeks of
TAC significantly inhibit dystrophin breakdown. Bottom, quantitative
analyses are shown in the bar graph as percentage of values of shamoperated animals (mean ⫾ S.E.M.; n ⫽ 6). ⴱⴱ, P ⬍ 0.01 versus shamoperated mice; #, P ⬍ 0.01 versus vehicle-treated TAC mice.

interesting to note that overexpression of calpastatin, an endogenous calpain inhibitor, blunts angiotensin II-induced cardiac
hypertrophy and perivascular fibrosis in heart (Letavernier
et al., 2008). However, the precise mechanism underlying
DY-9760e-induced inhibition of spectrin breakdown remains
unclear.
Spectrin contains a calmodulin binding site, and calmodulin/␣II-spectrin interaction regulates cleavage efficacy by
calpains and caspases (Rotter et al., 2004). Accumulating
evidence also demonstrates that Ca2⫹/calmodulin stimulates degradation of brain spectrin by calpain (Seubert etal., 1987; Harris et al., 1989). Thus, as a CaM antagonist,
DY-9760e probably indirectly inhibits calpain-mediated426

Han et al.

A

DSpectrin

Caveolin-3

240 kDa

18 kDa150 kDa
(-)
Sham

(-)

DY(10) DY(20)

DY(20) ( - ) DY(10) DY(20)
Sham

TAC

TAC

E
150

150
##

100
**

50

*

0

Sham

TAC

TAC
TAC
+
+
DY(10) DY(20)

Cav-3 (% of Sham)

Spectrin 240 kDa (% of Sham)

B
200

##

100
**
50

0

Sham

Spectrin 150kDa (% of Sham)

C
400
**

300

**

##

**

200
##

DY
(20)

TAC

TAC TAC++
DY(10) DY(20)Fig. 4. DY-9760e treatment antagonizes
TAC-induced spectrin and caveolin-3 breakdown. A, after 8 weeks of TAC, immunoblotting of LV extracts with anti-spectrin antibody showed that levels of 240-kDa spectrin
were markedly reduced in untreated (⫺) animals compared with sham-operated animals. Six weeks of DY-9760e [10 and 20
mg/kg (DY10 or DY20)] treatment after 2
weeks of TAC significantly and dose-dependently inhibited TAC-induced spectrin
breakdown. Inversely, the level of calpaincleaved 150-kDa spectrin was increased by
TAC and reduced by DY-9760e treatment
dose-dependently. B and C, quantitative
analyses of levels of 240- and 150-kDa spectrin are summarized, respectively, as a percentage of values seen in sham-operated
animals (mean ⫾ S.E.M.; n ⫽ 6). D, Immunoblotting of LV extracts with an anticaveolin-3 antibody indicates that levels of
18-kDa caveolin-3 were significantly reduced 8 weeks after TAC (⫺) compared withsham-operated animals. Six weeks of DY9760e treatment (20 mg/kg) began after 2
weeks of TAC significantly inhibited TACinduced caveolin-3 breakdown. E, quantitative analyses are presented as the percentage of values seen in sham-operated animals(mean ⫾ S.E.M.; n ⫽ 6). ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍0.05 versus sham-operated mice; ##, P ⬍0.01 versus vehicle-treated TAC mice.

100
0

Sham

TAC

TAC
TAC
+
+
DY(10) DY(20)spectrin cleavage by inhibiting calmodulin-binding tospectrin.
DY-9760e cardioprotection also includes protection of
eNOS/caveolin-3 signaling in caveolae. In cardiac myocytes, the eNOS isoform mostly localizes in caveolae where
it associates with caveolin-3 (García-Cardeña et al., 1996;
Feron et al., 1996). Caveolin-3 inhibits eNOS activity,
whereas calmodulin binding to eNOS after Ca2⫹ mobilization disrupts caveolin-3 binding, leading to eNOS activation and nitric oxide production (Michel and Feron, 1997).
Our previous study documented that dystrophin breakdown was closely associated with translocation of caveolin-3 from caveolae and decreased Ca2⫹/CaM-dependent
nitric oxide generation by eNOS in cultured cardiomyocytes (Lu et al., 2009a). Likewise, dystrophin knockout miceshow impaired nitric oxide-dependent and endotheliumdependent vasodilation in arteries (Loufrani et al., 2004). In thepresent study, we demonstrated in vivo that disruption of eNOS
association with caveolin-3 is associated with eNOS uncoupling,
thereby increasing peroxynitrite formation, as assessed by protein tyrosine nitration. Indeed, superoxide generation isclosely associated with eNOS uncoupling in cultured cardiomyocytes exposed to phenylephrine (Lu et al., 2009a). Furthermore, treatment with DY-9836, an active metabolite of

DY-9760e, largely inhibited elevation both of superoxide andnitric oxide generation after prolonged exposure to phenylephrine in cultured cardiomyocytes. Under the same conditions, generation of monomeric eNOS was also inhibited by
DY-9836 treatment (Lu et al., 2009a). It is noteworthy that
Tempol (a radical scavenger), DY-9836, and N␻-nitro-Larginine methyl ester (a NO synthase inhibitor) all completely inhibited apoptosis of cardiomyocytes exposed tophenylephrine (Lu et al., 2009a).
Accumulating evidence confirms the pathophysiologicalrelevance of eNOS uncoupling and peroxynitrite generation to TAC-induced heart failure. Tetrahydrobiopterin(BH4) treatment in TAC-induced hypertrophy inhibitedpressure overload-induced oxidative stress through reversal of eNOS uncoupling, thereby ameliorating cardiac hypertrophy/dysfunction in mice (Takimoto et al., 2005;
Moens et al., 2008). The reversal of eNOS uncoupling byexogenous BH4 was more effective than Tempol in preventing heart failure (Moens et al., 2008). Likewise, infusion of peroxynitrite or a peroxynitrite donor such as
3-morpholino sydnonimine impaired contractile function by altering Ca2⫹ handling and/or ␤-adrenergic responsiveness in
isolated myocytes (Katori et al., 2006; Kohr et al., 2008). Taken
together, in addition to dystrophin/spectrin breakdown, de-DY-9760e Inhibits Dystrophin Breakdown in Cardiac Hypertrophy

A

Total extracts

B
135 kDa(-)

DY(20) ( - ) DY(10) DY(20)150eNOS (% of Sham)

Sham

TAC

100

50

0

Sham

150

Sham

DY(10)

DY(20)

##

*

100

##

**

50

0

Sham

D

C

(-)

TAC

TAC
+
DY(20)TAC+
TAC
DY(10)

DY(20)

Membrane fractions

eNOS

membrane eNOS (% of Sham)

eNOS

TAC
+
DY(10)TAC

TAC+
DY(20)

Nitrotyrosine
45 kDadimer
monomer

eNOS
Sham

(-)

DY(10)

427

DY(20)
Sham

TAC

(-)

DY(10) DY(20)

150

Dimer
Monomer

##

100

$$

#

**

$$

**

50

§§

0

Nitrotyrosine (% of Sham)eNOS (% of Sham dimer)

TAC

400
**

300

##

**

200

##

100
0

Sham

TAC

TAC
+
DY(10)TAC+
DY(20)

Sham

TAC

TAC
+
DY(10)TAC+
DY(20)

Fig. 5. Effects of DY-9760e on TAC-induced membrane eNOS levels, eNOS uncoupling, and nitrotyrosine formation. A, representative image (top) andbar graph (bottom) showing results of immunoblotting of total heart extracts with anti-eNOS antibody. Analysis indicates no apparent changes in totaleNOS levels among conditions. B, representative image (top) and bar graph (bottom) showing results of immunoblotting with anti-eNOS antibody.
Analysis of cell membrane extracts indicates marked reduction in eNOS levels in TAC animals and rescue of eNOS levels after DY-9760e treatment
dose-dependently. Data are expressed as a percentage of values seen in sham-operated animals (mean ⫾ S.E.M.; n ⫽ 6). ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01 versus
sham-operated mice; ##, P ⬍ 0.01 versus vehicle-treated mice. C, levels of uncoupled (monomer) eNOS are shown as analyzed by low-temperature
SDS-polyacrylamide gel electrophoresis (top). Quantitation of dimeric and monomeric eNOS (lower) was performed by densitometric analysis. DimericeNOS from sham-operated animals was arbitrarily set at 100%. Data are expressed as percentage of values seen in sham-operated animals (mean ⫾S.E.M.; n ⫽ 6). ⴱⴱ, P ⬍ 0.01 versus sham-operated mice (dimer); #, P ⬍ 0.05 and ##, P ⬍ 0.01 versus vehicle-treated mice (dimer); $$, P ⬍ 0.01 versus
sham-operated mice (monomer); and §§, P ⬍ 0.01 versus vehicle-treated mice (monomer). D, representative image (top) and bar graph (bottom) of
immunoblots of total heart extracts with anti-nitrotyrosine antibody. Analysis indicates that protein tyrosine nitration was markedly increased after
TAC compared with sham-operated animals and decreased by DY-9760e treatment dose-dependently. ⴱⴱ, P ⬍ 0.01 versus sham-operated mice; ##, P ⬍0.01 versus vehicle-treated TAC mice.

creased BH4 levels in cardiomyocytes probably underlies eNOS
uncoupling, leading to heart failure by oxidant stress, including
peroxynitrite generation.
In summary, we report that a CaM antagonist, DY-9760e,
inhibits in vivo the progression of cardiac hypertrophy-induced
cardiac injury by inhibiting calpain-mediated dystrophin/spectrin degeneration and loss of caveolae integrity in cardiomyo-cyte plasma membranes (Fig. 6). Protein tyrosine nitration
through superoxide generation by uncoupled eNOS was alsoameliorated by DY-9760e treatment. Loss of caveolae integrity
probably triggers eNOS uncoupling by dissociation from theplasma membranes. Taken together, evidence gathered using
the TAC mouse model is consistent with our previous results
obtained in cultured cardiomyocytes, supporting the idea that428

Han et al.

Pressure overload
Shear stress

Ca2+
Calpain activationeNOS dysregulation

DY-9760e

DY-9760e

Dystrophin/spectrin breakdown
Loss of caveolae integrity

Protein tyrosine nitration
Superoxide generation

Fig. 6. Potential cardioprotective mechanism of DY-9760 on
TAC-induced heart injury. DY-9760e in vivo inhibits progression of cardiac hypertrophy-induced cardiac injury by inhibiting calpain-mediated dystrophin/spectrin degeneration andloss of caveolae integrity in cardiomyocyte plasma membranes.
Protein tyrosine nitration through superoxide generation byuncoupled eNOS is also ameliorated by DY-9760e treatment.
Loss of caveolae integrity probably triggers eNOS uncouplingby dissociation from plasma membranes.

Progression to heart failure

DY-9760e treatment could provide a novel therapeutic strategy
to treat hypertrophy and heart failure.
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Address correspondence to: Dr. Kohji Fukunaga, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University,
Aramaki-Aoba Aoba-ku, Sendai 980-8578, Japan. E-mail: fukunaga@mail.
pharm.tohoku.ac.jp