Phenylephrine-Induced Cardiomyocyte Injury Is Triggered
by Superoxide Generation through Uncoupled Endothelial
Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-
(DY-9836), a Novel Calmodulin Antagonist□
Ying-Mei Lu, Feng Han, Norifumi Shioda, Shigeki Moriguchi, Yasufumi Shirasaki,
Zheng-Hong Qin, and Kohji Fukunaga
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (Y.-M.L., N.S.,
S.M., K.F.); Daiichi-Sankyo Pharmaceutical Co., Ltd. Tokyo, Japan (Y.S.); Institute of Pharmacology & Toxicology and
Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (F.H.);
Department of Pharmacology, Soochow University School of Medicine, Suzhou, China (Z.-H.Q.); and Tohoku University 21st
Century COE Program “CRESCENDO”, Sendai, Japan (K.F.)
Received July 17, 2008; accepted October 24, 2008
The pathophysiological relevance of endothelial nitric-oxide
synthase (eNOS)-induced superoxide production in cardiomyo-
cyte injury after prolonged phenylephrine (PE) exposure re-
mains unclear. The aims of this study were to define the mech-
anism of O2.production by uncoupled eNOS and evaluate the
therapeutic potential of a novel calmodulin antagonist 3-[2-[4-
indazole (DY-9836) to rescue hypertrophied cardiomyocytes
from PE-induced injury. In cultured rat cardiomyocytes, pro-
longed exposure for 96 h to PE led to translocation from mem-
brane to cytosol of eNOS and breakdown of caveolin-3 and
dystrophin. When NO and O2.production were monitored in
PE-treated cells by 4-amino-5-methylamino-2?,7?-difluorofluo-
rescein and dihydroethidium, respectively, Ca2?-induced NO
production elevated by 5.7-fold (p ? 0.01) after 48-h PE treat-
ment, and the basal NO concentration markedly elevated (16-
fold; p ? 0.01) after 96-h PE treatment. On the other hand, the
O2.generation at 96 h was closely associated with an in-
creased uncoupled eNOS level. Coincubation with DY-9836
(3 ?M) during the last 48 h inhibited the aberrant O2.generation
nearly completely and NO production by 72% (p ? 0.01) after
96 h of PE treatment and inhibited the breakdown of caveolin-
3/dystrophin in cardiomyocytes. PE-induced apoptosis as-
sessed by TdT-mediated dUTP nick-end labeling staining was
also attenuated by DY-9836 treatment. These results suggest
that O2.generation by uncoupled eNOS probably triggers PE-
induced cardiomyocyte injury. Inhibition of abnormal O2.and
NO generation by DY-9836 treatment represents an attractive
therapeutic strategy for PE/hypertrophy-induced cardiomyo-
NO toxicity is in part mediated by the generation of per-
oxynitrite (ONOO?) with concomitant production of O2.un-
der pathological conditions common to heart disease, such as
ischemia (Bauersachs et al., 1999) and heart failure (Mu ¨nzel
and Harrison, 1999; Dixon et al., 2003). ONOO?can trigger
lipid peroxidation, protein tyrosine nitration, and DNA
strand breakdown (Schulz et al., 1997; Yasmin et al., 1997).
The increase in reactive oxygen species, in particular O2., is
mainly due to activation of plasma membrane NADPH oxi-
dase, mitochondrial respiratory chain enzymes, cyclooxygen-
This work was supported by the Ministry of Education, Science, Sports and
Culture of Japan [Grant 19390150] and the Smoking Research Foundation.
Article, publication date, and citation information can be found at
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; iNOS, inducible
nitric-oxide synthase; BH4, tetrahydrobiopterin; PE, phenylephrine; DMEM, Dulbecco’s modified Eagle’s medium; DAF-FM, 4-amino-5-methyl-
amino-2?,7?-difluorofluorescein; DHE, dihydroethidium; TUNEL, TdT-mediated dUTP nick-end labeling; FBS, fetal bovine serum; PBS, phosphate-
buffered saline; PAGE, polyacrylamide gel electrophoresis; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy; MES, 2-(N-morpholino)eth-
anesulfonic acid; L-NAME, N?-nitro-L-arginine methyl ester; A23187, calcimycin; DY-9836, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-
5,6-dimethoxyindazole; DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-
indazole dihydrochloride 3.5 hydrate.
Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 75:101–112, 2009
Vol. 75, No. 1
Printed in U.S.A.
Supplemental material to this article can be found at:
at ASPET Journals on November 7, 2015
ase, and xanthine oxidase, and it causes oxidative stress
during heart failure. In addition, uncoupled nitric-oxide syn-
thase (NOS) also mediates O2.generation in pathological
conditions (Vasquez-Vivar et al., 1998; Mollnau et al., 2002).
However, the precise mechanism of superoxide production
during heart failure remains unclear.
Among three NOS isoforms (neuronal, inducible, and en-
dothelial), eNOS is easily uncoupled under conditions of
cofactor tetrahydrobiopterin (BH4) depletion, in which dis-
sociation of eNOS dimer also promotes O2.generation (Fo ¨r-
stermann and Mu ¨nzel, 2006). In the absence of BH4, eNOS
generates O2.from breakdown of the heme-dioxygen complex
at the oxygenase domain of the enzyme (Fig. 8), whereas
electron flow from the reductase to the oxygenase domain is
diverted to molecular oxygen rather than to L-arginine (Ver-
haar et al., 2004). O2.generation by uncoupled eNOS cannot
be prevented by supplementary L-arginine (Xia et al., 1998).
By contrast, nNOS and iNOS isoforms are relatively difficult
to uncouple, even in the absence of cofactors. O2.generation
by nNOS and iNOS is minimally induced by L-arginine de-
pletion (Pou et al., 1992; Xia et al., 1996). Thus, eNOS un-
coupling accompanied by BH4depletion is believed to be
crucial for heart damage after cardiovascular disorders, in-
cluding hypertension (Fo ¨rstermann and Mu ¨nzel, 2006), ath-
erosclerosis (Lahera et al., 2007), and diabetes (Thum et al.,
2007). Furthermore, eNOS uncoupling has been documented
in mice subjected to sustained pressure overload, thereby
resulting in increased O2.production with concomitant dis-
ruption of the eNOS dimer (Takimoto et al., 2005). Likewise,
oxidative stress in an experimental model of idiopathic di-
lated cardiomyopathy is mediated by uncoupled eNOS
through activation of the renin-angiotensin systems (Moll-
nau et al., 2005).
In cardiac myocytes, the eNOS isoform mostly localizes to
caveolae, where it associates with caveolin-3 (Feron et al.,
1996; Garcia-Cardena et al., 1996). Unlike eNOS, nNOS lo-
calizes primarily to sarcoplasmic reticulum (Xu et al., 1999).
In resting conditions, binding to caveolin-3 inhibits eNOS
activity, thereby limiting NO production, whereas calmodu-
lin binding to eNOS disrupts caveolin-3 binding, leading to
eNOS activation (Michel and Feron, 1997). Moreover, in car-
diomyocytes, G-protein-coupled ?1-adrenergic receptors are
enriched in caveolae and colocalize with caveolin-3 (Fujita et
al., 2001). Stimulation of ?1-adrenergic receptors with PE
up-regulates caveolin-3 in cardiomyocytes (Kikuchi et al.,
2005). Indeed, after PE stimulation, eNOS dissociates from
caveolin-3 and translocates to the cytosol (Michel and Feron,
1997). Molnar et al. (2005) reported that pathological inter-
ference with eNOS dimerization impairs NO-dependent en-
dothelial function without changing eNOS phosphorylation
status. However, the physiological relevance of caveolin-3
and calmodulin to eNOS uncoupling upon PE stimulation
Our previous studies demonstrated that exposure of cardi-
omyocytes to angiotensin II, endothelin-1, or PE markedly
increased cell size after 48 h of treatment with these re-
agents, most likely representing compensatory hypertrophy.
More prolonged exposure to these factors disrupts Ca2?reg-
ulation by the sarcoplasmic reticulum, causing cardiomyo-
cyte dysfunction, including caveolin-3/dystrophin breakdown
(Lu et al., 2007). These in vitro models allowed us to test the
effects of novel cardioprotective drugs on caveolin-3/dystro-
phin breakdown in humoral factor-induced cardiac injury.
We showed previously that DY-9760e, a novel calmodulin
antagonist, inhibited nNOS and eNOS and reduced NO pro-
duction in N1E-115 cells (Fukunaga et al., 2000). However,
DY-9760e also inhibits cytochrome P450 enzymes in the
liver. Thus clinically achievable efficacy is limited by pre-
dicted drug interaction by DY-9760e in drug metabolism in
humans (Tachibana et al., 2005). It is noteworthy that we
showed that DY-9836, as an N-dealkylated DY-9760e and
active metabolite, produced cardioprotective effects against
cardiac hypertrophy induced by endothelin-1 and angioten-
sin II (Lu et al., 2007). Unlike DY-9760e, DY-9836 does not
interfere with the metabolism of other drugs in the liver
(Tachibana et al., 2005), suggesting that it is a more attrac-
tive candidate than DY-9760e for clinical therapy.
To understand the precise mechanisms of oxidative car-
diomyocyte injury induced by humoral factors causing hyper-
trophy, we first focused on spatiotemporal changes in eNOS
and caveolin-3 expression during prolonged exposure to PE
and determined whether eNOS accounts for both NO and
O2.production in the pathological conditions. We also ad-
dress whether inhibiting uncoupling of eNOS by a novel CaM
inhibitor, DY-9836, ameliorates PE-induced cardiomyocyte
Materials and Methods
Cell Culture. Neonatal ventricular myocytes were isolated from
hearts of 1- to 3-day-old Wistar rats by collagenase digestion and
cultured according to the method of Waspe et al. (1990). In brief,
myocytes were dissociated from ventricles by serial digestion with
0.1% trypsin and 0.05% DNase I in Hanks’ balanced salt solution.
After each digestion, digested cardiomyocytes were collected and
suspended in DMEM with 10% fetal bovine serum (FBS) and 0.02%
trypsin inhibitor to block further trypsin digestion. Cells were col-
lected by centrifugation (4°C, 1000g for 10 min). After the superna-
tant was removed, DMEM containing 10% FBS was added. Cells
were gently agitated and plated on uncoated 90-mm culture dishes.
Plates were allowed to stand for 30 min in a CO2incubator at 37°C
to remove nonmyocytes attached to the culture plates. Unattached
myocytes were collected and plated at 1 to 2 ? 106cells/35-mm dish
and incubated with DMEM and 10% FBS in a humidified incubator
with 5% CO2at 37°C for 24 h. Cells were cultured in serum-free
DMEM for 24 h before treatment with PE (10 ?M).
Membrane Fractionation. Membrane fractionation from cul-
tured cardiomyocytes was performed as described previously (Os-
trom et al., 2001). In brief, cells were homogenized in buffer contain-
ing 500 mM sodium carbonate plus 50 ?g/ml leupeptin, 25 g/ml
pepstatin A, 100 nM calyculin A, 50 g/ml trypsin inhibitor, and 1 mM
dithiothreitol. The homogenate was adjusted to 45% sucrose by add-
ing 90% sucrose in 25 mM MES and 150 mM NaCl, pH 6.5, and
loaded in an ultracentrifuge tube. A discontinuous sucrose gradient
was layered on top of the sample by placing 4 ml of 35% sucrose and
then 4 ml of 5% sucrose. The gradient was centrifuged at 39,000 rpm
on a SW41Ti rotor (Beckman Coulter, Fullerton, CA) for 16 h at 4°C.
Fractions were collected in 1-ml aliquots from the top of the gradient.
Western Blot Analysis. Cultured cells were washed with PBS at
4°C and stored at ?80°C until immunoblotting analyses were per-
formed as described previously (Lu et al., 2007). In brief, equal
amounts of proteins were separated on 7.5 to 10% SDS-PAGE gels
and transferred to a polyvinylidene difluoride membrane (Millipore
Corporation, Billerica, MA). Blots were stained with 0.1% Ponceau S
solution to visualize protein bands and confirm equal protein load-
ing. After blocking in 5% nonfat milk, blots were incubated overnight
at 4°C with antibodies against anti-eNOS and iNOS (Sigma, St.
Lu et al.
at ASPET Journals on November 7, 2015
vated by Ca2?/CaM in nNOS (Craig et al., 2002). Likewise,
electron transfer in the reductase domain is also CaM-sensi-
tive in human endothelial NOS (Nishino et al., 2007). There-
fore, we hypothesized that the CaM antagonist, DY-9836,
would prevent superoxide generation and NO generation
during prolonged PE treatment. Consistent with our hypoth-
esis, aberrant NO elevation at 96 h was blocked by prolonged
incubation with DY-9836. Likewise, aberrant superoxide pro-
duction was also largely attenuated by DY-9836 treatment.
The inhibitory dose of DY-9836 for both NO and superoxide
generation was similar, suggesting that CaM sensitivity is
almost equivalent in both coupled and uncoupled eNOS.
These observations are potentially important because uncou-
pled eNOS generates superoxide.
L-NAME and Tempol totally inhibited the NO and super-
oxide generation, respectively, at 96 h after PE exposure (Fig.
4). We addressed the question of whether NO and superoxide
mediate cardiomyocyte apoptosis and hypertrophy. It is in-
teresting that the treatment with L-NAME or Tempol totally
inhibited the apoptosis after prolonged PE exposure (Fig. 6),
whereas it failed to inhibit cardiomyocyte hypertrophy (Fig.
7). Thus NO and superoxide probably trigger the PE-induced
apoptosis but not mediate PE-induced cardiomyocyte hyper-
trophy. The inhibition of both apoptosis and hypertrophy by
DY-9836 suggests its diverse actions to protect cardiomyo-
cytes from oxidative and hypertrophic injuries.
The eNOS-derived superoxide has been shown to be asso-
ciated with the development and progression of atheroscle-
rosis and hypertension (Cosentino et al., 1998; Ozaki et al.,
2002). Under physiological conditions, eNOS is localized to
caveolae through caveolin-3 in cardiomyocytes. Binding of
CaM to eNOS promotes the dissociation of eNOS from caveo-
lin-3 concomitant with activation. Because the cardiac mus-
cle fibers of caveolin-3 knockout mice showed a loss of caveo-
lae and impaired excitation-contraction coupling, thereby
leading to cardiac hypertrophy (Woodman et al., 2002), trans-
location of caveolin-3 from the membrane to the cytosol seen
at 96 h probably accelerates the disruption of caveolae. In
addition, caveolae structure is essential for coupling between
voltage-dependent Ca2?channels and intracellular Ca2?
channels such as the ryanodine receptor in sarcoplasmic
reticulum (Scriven et al., 2005). Thus caveolin-3 breakdown
may impair intracellular Ca2?mobilization by excitation-
contraction coupling in cardiomyocytes after prolonged PE
treatment. Indeed we observed previously impaired caffeine-
induced Ca2?mobilization at 96 h under the same conditions
(Lu et al., 2007). Although precise mechanisms of eNOS
translocation and uncoupling remain unclear, perturbed
caveolae structure after caveolin-3 breakdown also promotes
eNOS translocation. The eNOS phosphorylation at Thr496
by protein kinase C and at Ser1177 by protein kinase B and
Ca2?/CaM-dependent protein kinase II also regulates eNOS
activity (Mount et al., 2007). Therefore, further extensive
studies are required to understand the pathological rele-
vance of these phosphorylation steps in aberrant NO and
superoxide production in PE-induced injury.
We also found that dystrophin breakdown precedes caveo-
lin-3 dissociation from the plasma membrane (Supplemen-
tary Fig. 1B). Because caveolin-3 binds directly to the dys-
trophin-glycoprotein complex through the PPXY motif in the
C-terminal tail of ?-dystroglycan (Sotgia et al., 2000), dystro-
phin breakdown may trigger the translocation of caveolin-3
from caveolae. In addition, we documented previously that
DY-9836 inhibits dystrophin breakdown in cultured cardio-
myocytes (Lu et al., 2007). Therefore, we propose that the
cardioprotective effects of DY-9836 partly due to the inhibi-
tion of dystrophin break down, as proposed previously (Lu et
In conclusion, we demonstrate that superoxide generation
resulting from uncoupling of eNOS accounts for PE-induced
cardiomyocyte injury. Dissociation of caveolin-3 and dystro-
phin from the plasma membrane leads to eNOS uncoupling.
Breakdown of caveolin-3 and dystrophin was also elicited by
calpain activation as described previously (Lu et al., 2007).
Both inhibition of superoxide generation and breakdown of
caveolin-3 and dystrophin by DY-9836 probably mediate its
cardiomyocyte protective effects in hypertrophied cardio-
myocytes (Fig. 9). These mechanisms provide a novel thera-
peutic intervention for cardiac hypertrophy and heart fail-
ure. In this context, we strongly suggest the effectiveness of
DY-9836 treatment on the progression of cardiac pathology
in vivo, including cardiac dysfunction, injury, and mortality
associated with heart failure.
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Address correspondence to: Dr. Kohji Fukunaga, Department of Pharma-
cology, Graduate School of Pharmaceutical Sciences, Tohoku University, Ara-
maki-Aoba Aoba-ku, Sendai 980-8578, Japan. E-mail: fukunaga@mail.
Lu et al.
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