J Mol Cell Cardiol 33, 131–139 (2001)

doi:10.1006/jmcc.2000.1285, available online at http://www.idealibrary.com on

Reactive Oxygen Species Mediate Alpha-

adrenergic Receptor-stimulated
Hypertrophy in Adult Rat Ventricular
Myocytes
Jay K. Amin, Lei Xiao, David R. Pimental, Patrick J. Pagano,
Krishna Singh, Douglas B. Sawyer and Wilson S. Colucci
The Cardiovascular Section, Department of Medicine, Boston Medical Center and The Myocardial
Biology Unit, Boston University School of Medicine, Boston, MA, USA
(Received 11 October 2000, accepted in revised form 16 October 2000, published electronically 4 December 2000)

J. K. A, L. X, D. R. P, P. J. P, K. S, D. B. S  W. S. C. Reactive Oxygen
Species Mediate Alpha-adrenergic Receptor-stimulated Hypertrophy in Adult Rat Ventricular Myocytes. Journal
of Molecular and Cellular Cardiology (2001) 33, 131–139. Norepinephrine (NE) causes hypertrophic growth of
cardiac myocytes via stimulation of alpha1-adrenergic receptors (
1-AR). Reactive oxygen species (ROS) can act
as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate
1-AR-stimulated
hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as
assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the
superoxide dismutase (SOD)-mimetic MnTMPyP. NE also caused the induction of MnSOD mRNA.
1-AR stimulation
with NE (1 ) in the presence of propranolol (2 ) for 48–96 h caused a hypertrophic growth phenotype
characterized by a 36±3% increase in 3H-leucine incorporation, a 49±14% increase in protein accumulation,
a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of MnSOD
mRNA. These responses were all prevented by pretreatment with the
1-AR-selective antagonist prazosin (100 n)
or the SOD-mimetics MnTMPyP (50 ) and Euk-8 (100 ). MnTMPyP had no effect on
1-AR-stimulated 3H-
inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-
myristate-13-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to
1-AR-
stimulation in ARVM.  2000 Academic Press
K W:
1-adrenergic receptor; Myocyte; Reactive oxygen species; Superoxide dismutase; Hypertrophy.

Introduction and members of the mitogen-activated protein kin-

The adrenergic agonist norepinephrine (NE) stimu-
lates a variety of biological responses in cardiac
myocytes including hypertrophic growth and the
re-expression of fetal genes.1–3 These effects are
mediated by the activation of
1-adrenergic re-
ceptors (
1-AR) that are coupled via G proteins to
a variety of signaling molecules including phos-
pholipase C, protein kinase C, calcium, PI-3-kinase
ase family.4–6
It is now apparent that reactive oxygen species
(ROS) such as superoxide and hydrogen peroxide
may act as intracellular signaling molecules for
biological events including cell growth.7 For ex-
ample, ROS have been shown to mediate the hy-
pertrophic effect of angiotensin in both vascular
smooth muscle cells8 and cardiac myocytes.9 Like-
wise, we found that a modest increase in intra-

Please address all correspondence to: Wilson S. Colucci, M.D., Cardiovascular Section, Boston University Medical Center, 88 East
Newton Street, Boston, MA 02118, USA. Fax: 617-638-8712. E-mail: wilson.colucci@bmc.org

0022–2828/01/010131+09 $35.00/0  2000 Academic Press

132 J. K. Amin et al.
cellular ROS caused by inhibition of superoxide
dismutase (SOD) can stimulate growth, fetal pro-
gram expression and apoptosis in neonatal rat car-
diac myocytes.10 It is not known whether ROS are
involved in mediating the effects of
1-AR in cardiac
myocytes or other cell types. Therefore, the purpose
of this study was to test the hypothesis that ROS
mediate the hypertrophic effect of
1-AR stimulation
in adult rat ventricular myocytes (ARVM).
Methods
Myocyte isolation and culture
ARVM were prepared from male Sprague–Dawley
rats as previously described.11 Rod-shaped myocytes
were plated in serum-free, defined medium (DMEM
supplemented with 2 mg/ml BSA, 2 mmol/l -car-
nitine, 5 mmol/l creatine, 5 mmol/l taurine, 0.1 
insulin, 100 IU/ml penicillin, and 100 g/ml strep-
tomycin) on 100 mm laminin-coated plates at a
density 100–150 cells mm2 or on 24 well plates at
a density of 50 000 cells/well. The media was
changed 1 h after plating to remove unattached
cells, and the attached cells were incubated (37°C)
for 18 h prior to addition of NE or other reagents.
Ascorbic acid (10 mol/l) was added to all plates
to prevent the auto-oxidation of NE.
Lucigenin-enhanced chemiluminescence
ROS production was measured by lucigenin-en-
hanced chemiluminescence.12 After treatment with
NE, ARVM in serum-free DMEM were lysed in
physiological buffer (in mmol/l: NaCl 119, HEPES
20, KCl 4.6, MgSO4 1, Na2HPO4 0.15, KH2PO4 0.4,
NaHCO3 5, CaCl2 1.2, glucose 11.1, pH 7.4) and
sonicated with three 10-s bursts on ice. Protein
concentration was determined using the Bradford
assay, and aliquots containing 50 g of protein
were brought to a volume of 170 l with lysis
buffer. Lucigenin (50 mol/l; bis-N-methyl-
acridinium nitrate; Sigma, St. Louis, MO, USA) was
added to the aliquots and allowed to equilibrate for
several minutes at 37°C. Luminescence, measured
with a Turner 20/20 Luminometer (Turner, Sunny-
vale, CA), was integrated over 30 s intervals for
a total of 2.5 min at room temperature. NADH
(100 ) was added to the reaction and the meas-
urements were continued at 30-s intervals for an
additional 5–10 min until a maximal lumines-
cence was observed. In some experiments the SOD-
mimetic Mn(II/III)tetrakis(1-methyl-4-peridyl)por-
phyrin (MnMnTMPyP)13 was added at a final con-
centration of 50 .
Cytochrome c reduction
Cytochrome c reduction was measured as de-
scribed.14 The media was changed to Tyrode’s with
glucose. Cytochrome c was added at a final con-
centration of 80 , with or without SOD at a final
concentration of 300 U/ml. NE was added for 1 h
and the media was removed, centrifuged to remove
any debris, and analyzed for the proportion of
reduced cytochrome c by measuring the absorbence
at 550 and 600 n. The difference between paired
plates with and without SOD was taken as SOD-
inhibitable cytochrome c reduction. As NE can
redox cycle and react with O2− itself, cytochrome
c reduction was also measured in cell-free plates
that contained media and NE.
3
H-leucine uptake and protein accumulation
3
H-leucine uptake and protein accumulation were
measured as previously described.15 3H-leucine (1
Ci/ml), NE and other reagents were added to
ARVM plated in 24-well plates. After 48 h, cells
were washed twice with PBS, precipitated with 5%
trichloroacetic acid at 4°C for 1 h, and tri-
chloroacetic acid-precipitable radioactivity was
measured by scintillation counting. To measure
total cellular protein content, ARVM were washed
with cold water, solubilized with NaOH (0.4 N) at
room temperature for 1 h, and an aliquot was
analyzed for total protein using the Bradford method
(Biorad).
Northern blot analysis
Total cellular RNA was isolated as previously de-
scribed.15 Total RNA (20 g) was size-fractionated
by gel electrophoresis, blotted onto a nylon mem-
brane, and hybridized for 24 h with cDNAs for rat
prepro-atrial natriuretic factor (0.6 kilobase pair of
coding region) or human MnSOD (courtesy of J.
Wispe, Cincinnati, Ohio, USA)16 labeled with alpha
32
P-dCTP by the random priming method. Signal
intensity was determined by densitometry, and blots
were reprobed with a cDNA for 18 S ribosomal
RNA to normalize for RNA loading.17
 ROS-dependent
-adrenergic Signaling in Cardiac Myocytes 133

Histochemistry

ARVM plated on laminin-coated glass coverslips

were fixed in 3.7% (w/v) paraformaldehyde for
30 min at room temperature, rinsed with PBS,
and permeabilized with 0.1% Triton X-100 for an
additional 15 min. Following an additional rinse
with PBS, cells were incubated with fluorescent
phallotoxin (Molecular Probes, Eugene, OR, USA)
to stain for filamentous-actin. Coverslips were
mounted on slides with Slow-Fade (Molecular
Probes) and viewed using fluorescence microscopy
(Nikon Diaphot microscope).

Inositol phosphates

Total inositol phosphate formation was measured

as described.18 Briefly, ARVM were labeled with 3H-
inositol (3 uCi/ml for 24 h) in inositol-free media.
The cells were then washed twice with HEPES-
buffered Krebs buffer (20 m HEPES, 4 m
NaHCO3, pH 7.4, 37°C) and preincubated at 37°C
for 15 min in the same buffer containing 20 m LiCl
substituted for NaCl. Prazosin (100 n), MnTMPyP Figure 1 Hypertrophic effect of
1-AR stimulation in
(50 ), propranolol (2 ), MnTMPyP (50 ) ARVM. Treatment with NE (1 ) for 48h stimulated
protein synthesis as reflected by 3H-leucine uptake (A) and
and combinations thereof were added 15 min prior cellular protein accumulation (B). The
1-AR-selective
to the addition of NE (1 ). The reaction was antagonist prazosin (Pz; 100 n) prevented NE-stimu-
stopped 15 min after addition of NE by adding 1 ml lated protein synthesis, whereas the -AR antagonist
of ice-cold choloroform:methanol solution (1:2, v/ propranolol (Pro; 2 ) had no significant effect. Data
v). After incubation at −20°C for 2 h, cells were presented are the means of 5–7 experiments. (∗=P<0.01
v controls; #=P<0.05 v NE).
collected and lysed by sonication. The samples were
mixed with 0.5 ml of water, and 1 ml of the aqueous
phase was applied to a column (3 ml Dowex AGI-
X8 anion exchange resin, 200–400 mesh, formate or a one-way analysis of variance followed by the
form). The inositol phosphates were eluted and Student–Newman–Keuls test for multiple com-
radioactivity was quantified in a 0.5 ml aliquot of parisons, as appropriate. A P-value <0.05 was
the final elution by scintillation counting. considered significant.

Materials
-norepinephrine, dl-propranolol, ascorbate, and
phorbol 12-myristate 13-acetate (PMA) were from
Sigma (St. Louis, MO, USA). MnTMPyP13 was from
Calbiochem, Inc (San Diego, CA, USA). Euk-819 was
a gift from Eukarion, Inc. (Beverly, MA, USA). 32P-
CTP was from New England Nuclear, Inc (Boston,
MA, USA).
Statistical methods
All data are presented as mean±... Statistical
analysis was performed using the Student’s t-test
Results
Alpha1-AR stimulation causes hypertrophy in ARVM
Exposure to NE (1 ) for 48 h increased 3H-leucine
uptake by 42±6% (P<0.01 v control; n=8) and
increased total cellular protein content by 30±8%
(P<0.01 v control; n=6) (Fig. 1). As previously
described,6 this hypertrophic effect was abolished
by the
1-AR-selective antagonist prazosin (100 n)
and was not significantly affected by the 1-AR
antagonist propranolol (2 ) (Fig. 1), confirming
that NE-stimulated protein synthesis is mediated
predominantly by
1-AR in ARVM.
134 J. K. Amin et al.

Figure 2 Effect of NE on ROS in ARVM. (A) ARVM were

exposed to NE (1 , 1 h), washed and lysed. Baseline
lucigenin chemiluminescence was recorded and NADH
(100 ) was added at 2.5 min (solid arrow). The ex-
ample shown is representative of three similar ex-
periments in which NE increased chemiluminescence
55±11% (P<0.05 v untreated cells). Addition of the SOD-
mimetic MnTMPyP (50 ) at 7.5 min (dashed arrow)
returned chemiluminescence to baseline. (B) Exposure to
NE (1 ; 24 h) caused a three- to four-fold induction of
MnSOD mRNA in ARVM. Shown is the most abundant
1.0 kb message from one of two similar experiments.

NE stimulates ROS production in ARVM

In myocytes exposed to NE (1 ; 1 h) NADH-

dependent O2− production was increased by
55±19% (P=0.03; n=3) as assessed by lucigenin-
enhanced chemiluminescence (Fig. 2A). Addition
of NADPH as a substrate resulted in minimal lu-
cigenin-enhanced chemiluminescence under either
control or NE-treated conditions (data not shown).
The subsequent addition of the SOD-mimetic
MnTMPyP (50 ) caused a rapid decrease in
chemiluminescence to the baseline value (Fig. 2A).
ROS were also assessed by cytochrome c re-
duction14. Exposure to NE (1 ; 1 h) increased
the magnitude of SOD-inhibitable cytochrome c
reduction by 320±41% (P<0.05; n=3) (Fig. 2B).
Ascorbate (10 ), which was added to the reaction
to prevent the oxidation of NE, had no effect on
basal SOD-inhibitable cytochrome c reduction (data
not shown). Likewise, NE alone, in the absence of
cells, had no effect on SOD-inhibitable cytochrome
c reduction (data not shown).
The expression of MnSOD, an important anti-
oxidant enzyme in cardiac myocytes, is increased
by oxidant stress in several cell types, including
Figure 3 Effect of the SOD-mimetic MnTMPyP (MnT;
50 ) on
-AR-stimulated 3H-leucine uptake (A) and
cellular protein accumulation (B). (C) depicts the effect
of the SOD-mimetic Euk-8 (Euk) on
-AR-stimulated 3H-
leucine uptake. Data presented are the means of 4–6
experiments, each performed in triplicate (P<0.05 v NE/
Pro). (∗=P<0.05 v controls; #=P<0.05 v NE/Pro).
cardiac myocytes.20 In ARVM, treatment with NE
(1 ; 24 h) increased the level of MnSOD mRNA
by three- to four-fold (v control; n=2) (Fig. 2C).
Effect of SOD-mimetics on
1-AR-stimulated protein
synthesis
Pretreatment with the SOD-mimetic MnTMPyP
(50 ; 30 min)13 completely inhibited the
1-AR-
stimulated increases in 3H-leucine uptake and total
protein accumulation (Fig. 3). MnTMPyP alone had
 ROS-dependent
-adrenergic Signaling in Cardiac Myocytes
Figure 4 Effect of the SOD-mimetic MnTMPyP (MnT; 50 ) on the
-AR-stimulated induction of ANP mRNA in
ARVM. (A) shows a representative northern blot using cDNAs for ANP and 18 S RNA. (B) shows the mean data
normalized to 18 S RNA (∗P<0.03 v NE/Pro; n=3).
no effect on
1-AR-stimulated 3H-leucine in-
corporation or protein accumulation.
Pretreatment with Euk-8 (100 ; 30 min), an
SOD-mimetic that is chemically-distinct from
MnTMPyP,19–21 likewise abolished the
1-AR-stimu-
lated increases in 3H-leucine uptake (Fig. 3C). Euk-
8 alone had no effect on
1-AR-stimulated 3H-
leucine uptake.
SOD-mimetics inhibit the effects of
1-AR stimulation on
molecular and structural phenotype
Exposure to NE (1 ) with propranolol (2 )
for 48 h induced the expression of prepro-atrial
natriuretic peptide (ANP) mRNA by approximately
six-fold (Fig. 4). Pretreatment with MnTMPyP
(50 ; 30 min) alone had no effect, but completely
prevented the
1-AR-stimulated increase in ANP
mRNA level (P=0.03 v NE/propranolol, n=3).
In ARVM, hypertrophic stimuli induce a typical
phenotype characterized by cytoskeletal rearrange-
ment and cell spreading.27
1-AR-stimulation for
72 h caused actin reorganization associated with
cell spreading, whereas control myocytes remained
rod-shaped with retention of an organized sar-
comeric pattern (Fig. 5A–C). Pretreatment with
MnTMPyP (50 ; 30 min) completely prevented

1-AR-stimulated cytoskeletal rearrangement.
MnTMPyP alone had no effect on cytoskeletal or-
ganization. Euk-8 (100 ) likewise prevented
1-
AR-stimulated cytoskeletal rearrangement (data
not shown).
Inositol phosphate turnover
Stimulation of
1-AR activates phospholipase C via
Gq, leading to the turnover of inositol phosphates.21
135
NE (1 ; 15 min) caused an 82±34% (P<0.01)
increase in 3H-inositol incorporation which was
abolished by prazosin (100 n), confirming that
this effect is mediated by
1-AR (Fig. 6A). MnTMPyP
(50 ) had no effect on NE stimulated 3H-inositol
incorporation.
Phorbol ester-stimulated hypertrophy
Exposure to phorbol-12-myristate-13-acetate
(PMA); 25 ng/ml) for 48 h stimulated a 30±7%
increase in 3H-leucine incorporation (Fig. 6B) and
caused actin reorganization, though not as striking
as that seen with
1-AR stimulation (Fig. 5E). Nei-
ther PMA-stimulated 3H-leucine uptake (Fig. 6B)
nor cytoskeletal reorganization (Fig. 5F) was in-
hibited by pretreatment with MnTMPyP.
Neonatal rat ventricular myocytes
In neonatal rat cardiac myocytes,
1-AR stimulation
with NE 1 ) with propranolol (2 ) caused hy-
pertrophic growth as reflected by an 48±4% in-
crease in 3H-leucine uptake (P<0.05; n=3).
1-
AR-stimulated 3H-leucine uptake was abolished by
pretreatment with MnTMPyP (−4±7%; P<0.05 v
NE/propranolol; n=4).
Discussion
The major new findings of this study are (1) that

1-AR stimulation increases the level of ROS in
ARVM, and (2) that SOD-mimetics prevent both
the increase in ROS and the hypertrophic response
caused by
1-AR stimulation. Taken together, these
136 J. K. Amin et al.

Figure 5 Effect of an SOD-mimetic on
1-AR and PMA stimulated actin filament reorganization in ARVMs. ARVMs
were cultured on laminin-coated glass coverslips for 96 h. Filamentous actin was stained with rhodamine- or bodipy-
phalloidin. Panels A–C and D–F represent two separate experiments. (A) untreated; (B) treatment with NE (1 ) and
propranolol (2 ); (C) NE, propranolol, after pretreatment with the SOD-mimetic MnTMPyP (50 ). (D) untreated;
(E) treatment with PMA (25 ng/ml); (F) PMA after pretreatment with MnTMPyP (50 ). Photomicrographs are
representative of at least 4 similar experiments. Scale bar=50 m.

observations suggest that ROS play a central role
in mediating the hypertrophic phenotype caused
by
1-AR stimulation in adult cardiac myocytes.

1-AR-stimulated growth in ARVM
NE is a potent stimulus for the hypertrophic growth
of ventricular myocytes.1,2,6 This effect is associated
with the induction of protooncogenes such as c-
myc,22 the re-expression of fetal genes such as skel-
etal
-actin3 or atrial natriuretic peptide,23 and the
reorganization of actin filaments.24 In short-term
(i.e. 1–3 days) cultures of ARVM25 and neonatal
rat ventricular myocytes,26 it has been shown that
NE causes hypertrophic growth entirely due to
stimulation of
1-AR. With prolonged time in cul-
ture (e.g. [6 days), ARVM exhibit a change in
phenotype that is associated with the development
of a growth response to 2-adrenergic stimulation.27
The ability of the highly
1-AR-selective antagonist
prazosin to prevent NE-stimulated protein synthesis
confirmed that, under the conditions used in these
experiments (i.e. short-term culture), the hy-
pertrophic effect of NE is mediated predominantly
by
1-AR.

1-AR stimulation increases ROS in ARVM
By lucigenin chemiluminescence there was in-
creased NADH-dependent ROS generation in lysates
of cells treated with NE for 60 min. Addition of the
SOD-mimetic rapidly quenched the lucigenin signal.
Likewise, in intact cells NE caused an increase in
SOD-inhibitable cytochrome c reduction, another
measure of cellular generation of ROS. Because NE
can redox cycle, it was important to demonstrate
that NE alone, in the absence of cells, had no effect
on SOD-inhibitable cytochrome c reduction. While
ascorbate, which was used to prevent the oxidation
of NE, can reduce cytochrome c directly, we found
no effect of ascorbate on basal SOD-inhibitable cyto-
chrome c reduction. Moreover, ascorbate was added
to both the control and NE-treated cells to prevent
any direct effect of ascorbate on cytochrome c that
might confound these results.
We further found that
1-AR stimulation for 24 h
increased the expression of MnSOD mRNA. MnSOD,
the predominant isoform of SOD in cardiac myo-
cytes, is upregulated by oxidant stress in a variety
of cell types including cardiac myocytes.20 This
observation provides further support for the con-
clusion that
1-AR stimulation increased in-
tracellular oxidative stress.
 ROS-dependent
-adrenergic Signaling in Cardiac Myocytes
Figure 6 Effect of MnTMPyP (MnT) on
-AR stimulated
3
H-inositol incorporation and PMA stimulated 3H-leucine
incorporation in ARVM. (A) 3H-inositol incorporation.
Data are shown as the mean of three experiments nor-
malized with NE treatment as 100% response v untreated
as 0% response. (∗P<0.01 v NE; #P=ns v NE, n=3).
(B) PMA stimulated 3H-leucine incorporation. Data are
shown as the mean of three experiments and presented
as the percent change from unstimulated cells. (∗P<0.05
v untreated; #P=ns v PMA alone; n=3).
These observations do not delineate the source of

1-AR-stimulated ROS. The lucigenin data suggest
that a NADH/NAD(P)H oxidase system may be
involved. This system has been shown to play an
important role in mediating the growth response
to angiotensin in vascular smooth muscle cells.8
However in ARVM, there was little lucigenin signal
with the addition of NAD(P)H (relative to that
generated by the addition of NADH). An important
source of NADH-dependent enzymatic activity is the
NADH-ubiquinone-reductase complex, or complex
I of the mitochondrial electron transport system.
Interestingly, mitochondrial respiration is a po-
tential source of ROS that was recently implicated
in failing myocardium,28,29 apparently due to dys-
regulation of complex I activity.
ROS as an intracellular signaling molecule
ROS are well-recognized mediators of cell damage.30
There is growing evidence that ROS may also serve
137
as intracellular signaling molecules.7–9,31–33 For ex-
ample, in fibroblasts ROS have been implicated in
mediating the growth effects of stimuli that act
through ras.7 In vascular smooth muscle cells ROS
generation by NAD(P)H has been shown to be a
critical step in angiotensin-stimulated cell pro-
liferation.8,31 Likewise, the hypertrophic growth re-
sponses to angiotensin and tumor necrosis factor-

appear to be ROS-dependent in neonatal rat
cardiac myocytes.9 We recently showed in cardiac
myocytes that a modest increase in ROS due to
partial inhibition of SOD caused a hypertrophic
phenotype, whereas a larger increase in ROS res-
ulted in apoptosis,10 thus supporting the thesis that
ROS can act as signaling molecules in cardiac
myocytes.
Role of ROS in hypertrophic growth
In ARVM
1-AR-stimulated hypertrophy, ANF
mRNA expression and actin re-organization were
all inhibited completely by the concurrent addition
of either of two chemically-distinct cell-permeable
SOD-mimetics, MnTMyP13 and Euk-8.19 Taken to-
gether with the demonstrated ability of
1-AR-
stimulation to increase ROS in ARVM, these ob-
servations strongly implicate ROS as mediators of
the hypertrophic response to
1-AR stimulation.
Using a similar approach, it was demonstrated
that ROS mediate the hypertrophic response to
angiotensin and tumor necrosis factor-
in neonatal
rat cardiac myocytes.9

1-AR-stimulated hypertrophy has been studied
extensively in cardiac myocytes from neonatal rat
ventricle.1,2 We found that SOD-mimetics inhibited
fully the ability of
1-AR stimulation to induce the
hypertrophic phenotype in both adult and neonatal
rat ventricular myocytes, indicating that the role
of ROS is not developmentally-determined.
ROS-dependent
1-AR signaling
The redox sensitive step(s) in the signaling pathway
for
1-AR-stimulated hypertrophy remains to be
determined. An SOD-mimetic had no effect on the

1-AR-stimulated turnover of inositol phosphates,
suggesting that the ROS-sensitive step is down-
stream from the receptor and does not involve
coupling to inositol phosphates. In cardiac myo-
cytes,
1-AR also couple to mitogen-activated pro-
tein kinases (MAPK)5,34,35 and one or more isoforms
of protein kinase C.36 Since some MAPKs37–39 and
protein kinase C isoforms40 can be activated by
138 J. K. Amin et al.
oxidative stress, one or more of these signaling
molecules may be downstream of ROS.
We found that a phorbol ester stimulated protein
synthesis and actin re-organization, but that neither
effect was sensitive to an SOD-mimetic. This finding
does not allow us to exclude protein kinase C as
an ROS-dependent step in
1-AR-stimulated hyper-
trophy. However, it suggests that molecules down-
stream of protein kinase C that are involved in
protein synthesis and actin re-organization are not
ROS-dependent.
Implications
These data support the hypothesis that
1-AR-
stimulation in cardiac myocytes causes hyper-
trophic growth and an associated phenotype via a
ROS-dependent step. This observation adds to the
list of signaling pathways that are ROS-dependent.
Given the ability of ROS to mediate important
biologic effects in cardiac myocytes,10,41 these ob-
servations raise the possibility that pathophysiologic
states associated with increased sympathetic ac-
tivity in the myocardium (e.g. myocardial failure)
may be associated with increased oxidative stress,
and conversely, that the resulting phenotype may
be responsive to interventions that reduce the level
of ROS.
Acknowledgements
Supported in part by grants HL-42539 and HL-
61639 (WSC), HL057947 (KS), HL03878 (DBS),
HL55425 (PJP) and HL07724 (JA, DRP) from the
National Institutes of Health; a Grant-in-Aid from
the AHA, Massachusetts Affiliate (DBS); and a Merit
grant from the Department of Veterans Affairs (KS).
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