Cardiovascular Research 49 (2001) 319–329
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Enhancement of Rho / Rho-kinase system in regulation of vascular smooth
muscle contraction in tachycardia-induced heart failure
Takayuki Hisaoka 1 , Masafumi Yano 1 , Tomoko Ohkusa, Masae Suetsugu, Kaoru Ono,
Masateru Kohno, Jyutaro Yamada, Shigeki Kobayashi, Michihiro Kohno, Masunori Matsuzaki*
The Department of Biomedical Regulation /Cardiovascular Medicine ( Second Department of Internal Medicine),
Yamaguchi University School of Medicine, 1 -1 -1 Minami-kogushi, Ube, Yamaguchi, 755 -8505, Japan
Received 18 August 2000; accepted 18 October 2000
Abstract
Objective: The Rho / Rho-kinase system regulates Ca 21 sensitivity in vascular smooth muscle. A new drug, Y-27632, specifically
inhibits Rho-kinase and hence decreases the phosphorylation of myosin light chain, thus reducing contraction. Here, we compare the
effects of Y-27632 and nifedipine on the vasoconstrictor response of the femoral artery in heart failure. Methods: Heart failure (HF) was
produced by chronic rapid RV pacing (250 bpm, 28 days, six dogs). Indo1-AM was loaded into endothelium-denuded femoral artery
segments for measuring intracellular [Ca 21 ]. Tension and changes in intracellular [Ca 21 ] [the change in the ratio (418 nm / 468 nm) of
Indo1 fluorescence (Fratio )] were simultaneously measured in Krebs–Ringer solution. Results: In HF: (i) norepinephrine (10 mM)
produced greater tension (784652 g / cm 2 ) than in control (502664 g / cm 2 ) despite a similar increase in Fratio , indicating increased Ca 21
sensitivity in vascular smooth muscle; (ii) nifedipine attenuated this enhanced response by only a maximum of 27% at 1 mmol / l with a
56% reduction in Fratio ; (iii) Y-27632 attenuated it by a maximum of 80% at 100 mmol / l without a significant change in Fratio ; (iv) RhoA
protein and mRNA expression levels in the femoral artery were up-regulated by 1110% and 156%, respectively, while those of
Rho-kinase were unchanged. Conclusions: The Ca 21 -sensitizing mechanism involving the Rho / Rho-kinase system may be deeply
involved in the enhanced arterial vasoconstriction seen in HF. Since Y-27632 attenuated this response in small arteries, it shows potential
as a novel, potent vasodilator for the treatment of HF.  2001 Elsevier Science B.V. All rights reserved.
Keywords: Adrenergic (ant)agonists; Calcium (cellular); Heart failure; Vasoconstriction / dilation
1. Introduction
In vascular smooth muscle, an increase in intracellular
[Ca 21 ] following stimulation to various receptors plays an
Abbreviations: HF, heart failure; CHF, congestive heart failure; MLC,
myosin light chain; GEFs, guanine–nucleotide exchange factors; GDI,
guanine–nucleotide dissociation inhibitor; GTPgS, guanosine 59-O-(gthiotriphosphate); GDPbS, guanosine 59-O-(g-thiodiphosphate; GEFs,
guanine–nucleotide exchange factors; GDI, guanine–nucleotide dissociation inhibitor; RAS, renin–angiotensin system; RT-PCR, reverse transcription
polymerase
chain
reaction
amplification;
PMSF,
phenylmethanesulfonyl fluoride; Tris, tris[hydroxymethyl]aminomethane;
ANP, atrial natriuretic-peptide
*Corresponding author. Tel.: 181-836-22-2248; fax: 181-836-222246.
E-mail address: [email protected] (M. Matsuzaki).
1
These authors contributed equally to this work.
important role in regulating vasomotor tone [1]. However,
because the cytosolic concentration of Ca 21 is not always
proportional to the extent of myosin light chain (MLC)
phosphorylation and contraction, an additional mechanism
— one that regulates the Ca 21 sensitivity of both processes
— has been proposed [2]. In this regard, an inhibition of
agonist-induced Ca 21 -sensitization by GDPbS [3] and a
Ca 21 -sensitizing effect of GTPgS in permeabilized smooth
muscle without a detectable change in [Ca 21 ] [4–6]
established that an upstream, membrane-coupled G-protein
was on a major pathway mediating Ca 21 -sensitization
[7,8].
Recently, RhoA — a small, monomeric G-protein, and a
member of the Rho subfamily of the Ras family of Gproteins — has been identified as the upstream component
Time for primary review 22 days.
0008-6363 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 00 )00279-0
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T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
of a major pathway for physiological Ca 21 -sensitization
[9]. Many of the same receptors that activate the phosphatidylinositol cascade also activate RhoA, and with the help
of GEFs, dissociate cytosolic RhoA?GDP from GDI. This
allows the exchange of GTP for GDP on RhoA. Active
RhoA-GTP activates Rho kinase, which then phosphorylates the regulatory subunit of myosin phosphatase
and inhibits its catalytic activity [10]. Uehata et al. [11]
demonstrated that Y-27632, an inhibitor of Rho kinase,
reduced both Ca 21 sensitization and vascular contraction
in response to a variety of G-protein-coupled receptor
agonists.
Congestive heart failure (CHF) is a clinical condition
associated with alterations in the normal balance of
neurohumoral agents and factors that act on the vascular
wall. In heart failure, the vasomotor tone increases in
response to an increased activity in the sympathetic
nervous system [12]. Several investigators have demonstrated that the sensitivity to the a1-adrenoceptor agonists,
phenylephrine and norepinephrine also increases during the
development of heart failure [13]. However, since such
changes were not seen with KCl, they may be unique to
a-adrenoceptor function in CHF, and not due to general
increases in contractile performance. It is also likely that
the enhanced vascular contractile activity seen in CHF is
due to events beyond receptor interaction, which hence
may involve the G-protein-mediated pathway.
The goal of this study was to examine the involvement
of the Rho / Rho-kinase system in the exaggerated vasoconstriction seen in heart failure.
2. Methods
The investigation conforms with the Guide for the Care
and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85–23,
revised 1996).
2.1. Production of pacing-induced heart failure
Heart failure was induced in beagle dogs of either sex
by 28 days of rapid right ventricular pacing at a rate of 250
bpm using an externally programmable miniature pacemaker (Medtronic inc., Minneapolis), as described previously [14–16]. In this model of heart failure, both LV
end–diastolic and end–systolic diameter are significantly
increased, in association with a decreased ejection fraction
(,35%) [14–16].
The study group consisted of 12 beagle dogs weighing
10–15 kg, of which six were designated CHF and six were
sham-operated controls. The care of the animals and the
protocols used were in accord with guidelines laid down
by the Animal Ethics Committee of Yamaguchi University
School of Medicine.
2.2. General tissue bath experiments
Lengths of femoral arteries were withdrawn under
anesthesia [2% isoflurane, 1.5 l / min, a mixture of nitrous
oxide and oxygen (2:1)] and placed in Krebs bicarbonate
solution containing (in mmol / l) 120 NaCl, 5.6 KCl, 2.5
CaCl 2 , 1.2 MgCl 2 , 1.17 NaH 2 PO 4 , 10 d–glucose, and 25
NaHCO 3 , which was continuously bubbled with 95% O 2 –
5% CO 2 . Each length of artery was cleaned of adhering
tissues, and a segment |2–3 mm long was cut, mounted on
wires, and suspended between two stainless-steel wires.
One wire was in a fixed position and the other was
connected to a force–displacement transducer (159901A
Isometric Force Transducer; Radnoti, Monrovia, CA). The
whole preparation was mounted in a two-hook 20-ml organ
chamber and bathed in Krebs bicarbonate solution aerated
with a mixture of 95% O 2 and 5% CO 2 at 378C via a
circulating water pump (RTE-100; NESLAB, NH). Segments were placed under a resting tension of 1.0 g and
allowed to equilibrate for 1 h, during which frequent
washing with Krebs bicarbonate solution was performed.
In 4–5 preparations from normal dogs and 4–6 from dogs
with heart failure, the endothelium was kept intact, while
in all other preparations the endothelium was denuded as
evidenced by a lack of relaxation to 1.0 mmol / l acetylcholine.
2.3. Experimental design
Concentration–effect curves for the response to norepinephrine (10 27 –10 4 mol / l) were constructed cumulatively.
Briefly, the lowest concentration of norepinephrine was
administered, and the resulting contraction was allowed to
develop until it reached a plateau, at which point another,
higher concentration of norepinephrine was introduced.
After control concentration–effect curves had been obtained, increasing concentrations of nifedipine (10 29 –10 26
mol / l) or Y-27632 (10 27 –10 24 mol / l) were added to the
organ bath 15 min before the reconstruction of the
concentration–effect curves. This equilibration period was
chosen on the basis of preliminary data showing that an
approximately 15-min exposure to nifedipine or Y-27632
was necessary to produce a peak inhibition of the norepinephrine-induced contraction. Consecutive concentration–effect curves were separated by a washout period of 1
h.
To assess whether the endothelium influences receptoroperated or membrane voltage-dependent contraction, the
contraction induced by norepinephrine (1 mmol / l) or KCl
(60 mmol / l) was assessed with an intact endothelium. The
vasorelaxant effects of nifedipine and Y-27632 were also
evaluated during the angiotensin II (0.1 mmol / l)-induced
contraction. Angiotensin II induces a peak contraction at
this dose [17]. Such an experiment could be performed
once only with angiotensin II because of tachyphylaxis, as
previously observed [17].
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
2.4. Calcium measurements
Arterial segments were loaded with Indo1-acetoxymethyl ester (Indo1-AM; Molecular Probes, Eugene, OR)
for 1–2 h at 208C by incubating with 2 ml Krebs
bicarbonate solution containing 10 mmol / l Indo1-AM
dissolved in 10% cremophore (Sigma Chemicals, St.
Louis, MO) [18,19]. The loaded segments were mounted
in the organ chamber, and the Indo1 fluorescence signal
during the vasoconstrictor response to norepinephrine was
measured using a spectrophotometer (RSP-601S; Unisoku,
Osaka [16]). Light from a 450-W xenon–mercury lamp,
filtered through a 360-nm interference filter, was used for
excitation. Emitted fluorescent light was monitored by two
photomultiplier tubes preceded by 418- and 468-nm interference filters with bandwidths of 65 nm. The ratio of the
418- and 468-nm signals (Fratio ) provides a measure of
intracellular [Ca 21 ] [19]. In an attempt to calibrate the
signals, in some experiments we obtained minimum (R min )
and maximum (R max ) values for the fluorescence ratio. For
this purpose, the tissues were incubated in the calcium
ionophore ionomysin (50 mmol / l) for 40–60 min to obtain
stable fluorescence signals [20]. The R min signal was
obtained using nominally a Ca 21 -free solution containing 2
mmol / l EGTA, while the R max signal was obtained using a
solution containing 10 mmol / l Ca 21 . However, ionomycin
may be less effective in tissues than in single cells, and
consistent R min and R max signals were obtained in less than
50% of all preparations, a value consistent with one in a
previous report (25%) [18]. For that reason, the results are
expressed as a fluorescence ratio rather than as absolute
[Ca 21 ].
2.5. Immunoblot analysis for determination of Rho A
and Rho kinase protein levels
Femoral arteries were immersed in iced Tris-buffer (5
mmol / l, pH 7.4) containing the protease inhibitors leupeptin (5 mg / l) and PMSF (0.1 mmol / l), then homogenized
for 30 s using a Brinkmann Polytron homogenizer. The
homogenates were centrifuged at 100 g for 10 min to
remove particulate matter and unbroken cells. The pellet
was resuspended in Tris-buffer, and this fraction was
rapidly frozen in liquid nitrogen and stored at 2808C. An
aliquot was retained for protein assay using the method of
Lowry et al. [21].
Immunoblot analysis was performed as previously described [22], with some modifications. The protein was
electrophoresed on SDS–polyacrylamide gels using a
Laemmli buffer system, and the proteins in the gel were
transferred to a protran nitrocellulose membrane
(Schleicher & Schuell, Dassel, Germany). The membranes
were then treated with 5% nonfat dry milk in phosphatebuffer, incubated with a solution containing commercially
available monoclonal antibodies (anti-Rho kinase from
321
Transduction Laboratories, Lexington, Sandiego, and antiRho A protein from Santa Cruz Biotechnology, CA), and
incubated further with peroxidase-conjugated secondary
antibody. The amount of protein recognized by the antibodies was quantified by means of an ECL immunoblotting detection system (Amersham, Bucks, UK), the
membrane being exposed to X-ray film. Quantitative
densitometry of immunoblots was performed using a
microcomputer imaging device (AE-6900M; ATTO,
Tokyo). The relative activity associated with Rho A or Rho
kinase in each sample was calculated by dividing the
activity associated with the Rho A or Rho kinase protein
products by the activity associated with the positive control
(rat cerebellum for Rho A [23] and RSV-3T3 cell lysate
(Rouse Sarcoma-infected 3T3 mouse fibroblast cell line)
for Rho kinase).
2.6. RT-PCR
Total cellular RNA was isolated from each frozen tissue
sample by the method of acid guanidinium thiocyanate /
phenol / chloroform extraction [24], then stored at 2808C.
cDNA was prepared using a Takara RNA PCR Kit
(Takara, Tokyo, Japan), as previously described [25], in a
buffer containing 10 mmol / l Tris–HCl, pH 8.3, 50 mmol / l
KCl, 5 mmol / l MgCl 2 , and 1 mmol / l each of dCTP,
dGTP, dTTP, and dATP, with 20 units of recombinant
ribonuclease inhibitor, 2.5 M random 9 mers, 1.3 mg of
total RNA, 5 units of avian myeloblastosis virus reverse
transcriptase, all in a volume of 20 ml. This reaction
mixture was incubated for 10 min at 308C followed by 30
min at 428C to initiate the synthesis of the cDNAs. Reverse
transcriptase was inactivated at 998C for 5 min, and this
mixture was then used for the amplification of specific
cDNAs by PCR. PCR was performed as follows: to 20 ml
of the RT reaction mixture was added 2 ml of 0.1 mol / l
forward primer, 2 ml of 0.1 mol / l reverse primer, 8 ml of
103 amplification buffer (100 mmol / l Tris–HCl, pH 8.3,
0.5 mol / l KCl), 12 ml of 25 mmol / l MgCl 2 , 55 ml of H 2 O,
0.5 ml of [a-32P]dCTP (Amersham), and 0.5 ml (2.5
U / 100 ml) of Taq polymerase. The primers for the
amplification of Rho A, Rho kinase and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were designed from
published sequences. For Rho A, they were based on
human sequences [26,27]: at positions 83–104 (sense
primer, 59-ACC AGT TCC CAG AGG TGT ATG T-39)
and 304–326 (antisense primer, 59-TTG GGA CAG AAG
TGC TTG ACT TC-39) (predicted length of PCR product,
244 bp). For Rho kinase, they were based on human
sequences [26,27]: at positions 495–521 (sense primer,
59-GAG CAA CTA TGA TGT GCC TGA AAA AT-39)
and 985–1006 (antisense primer, 59-GAT GTC GTT TGA
TTT CTT CTA C-39) (predicted length of PCR product,
512 bp). For GAPDH, they were based on human sequences [26,27]: at positions 102–125 (sense primer, 59CTT CAT TGA CCT CAA CTA CAT GGT-39) and
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T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
805–828 (antisense primer, 59-CTC AGT GTA GCC CAG
GAT GCC CTT-39) (predicted length of PCR product, 726
bp). Next, 100 ml of reaction mixture was overlaid with 20
ml mineral oil, and cycling was performed 25 times by
means of a thermal cycler (Perkin Elmer / Cetus, San
Diego, CA) using the following parameters: denaturation at
948C for 1 min, annealing at 608C for 1 min, extension at
728C for 2 min, followed by a final incubation at 728C for
7 min.
2.7. Quantitation of PCR products
The optimal number of amplification cycles needed to
allow quantitation of Rho A, Rho kinase, and GAPDH
gene PCR products was determined. The PCR products for
each cycle were subjected to 5% polyacrylamide gel
electrophoresis (PAGE) and autoradiography, and the
associated radioactivity was measured using an imaging
analyzer (model BAS-2000; Fuji Photo Film Co., Tokyo,
Japan). The optimal number of cycles was found to be 25
for Rho A, Rho kinase, and GAPDH.
2.8. Assessment of expression of Rho A and Rho kinase
mRNA
The relative radioactivity associated with Rho A and
Rho kinase PCR products in each sample was calculated
by dividing the radioactivity associated with the Rho A
and Rho kinase PCR products by the radioactivity associated with the GAPDH gene product (internal control;
amplified simultaneously). Each level of RT-PCR product
was obtained as the average of duplicate data. We used
GAPDH as an internal control because the densitometric
scores for the mRNAs did not differ between the groups of
dogs. Furthermore, this enzyme of the glycolytic pathway
is constitutively expressed in most tissues and is the most
widely accepted internal control in the molecular biology
literature [28].
2.9. Plasma neurohumoral measurements
Plasma norepinephrine, angiotensin II, and plasma atrial
neuro-peptide (ANP) were measured by means of highperformance liquid chromatography [29], angiotensin II
radioimmunoassay kit (SRL Co., Ltd., Tokyo, Japan) [30],
and Siono-RIA ANP kit (Shionogi Co., Ltd., Osaka, Japan)
[31], respectively.
two-way ANOVA and subsequent Scheffe’s post-hoc test.
Statistical significance was defined by P,0.05.
3. Results
3.1. Neurohormonal activity in congestive heart failure
As summarized in Table 1, plasma norepinephrine,
angiotensin II, and ANP were all higher with rapid chronic
pacing than in sham-operated dogs.
3.2. Contractile responses to norepinephrine and K 1
Fig. 1 shows norepinephrine- and K 1 -induced contractions of arterial segments in the presence or absence of
endothelium, and Table 2 summarizes the maximum
contractile responses to norepinephrine (1 mmol / l) and K 1
(60 mmol / l). In heart failure, the contractile response to
norepinephrine was greater than normal, whether or not the
endothelium was intact. However, in accord with previous
reports [13,32] there was no difference in the contractile
response to K 1 between normal and heart failure groups,
whether or not the endothelium was intact.
As shown in Fig. 2, the cumulative addition of norepinephrine in concentrations ranging from 10 27 to 10 24
mol / l produced concentration-dependent increases in tension in normal and in heart failure dogs. The maximum
response to norepinephrine was significantly greater in the
heart failure than in the normal group at all concentrations
of norepinephrine. However, there was no significant
difference in EC50 between normal (1.2160.10 mmol / l)
and heart failure (1.0460.2 mmol / l) groups.
3.3. Effects of nifedipine and Y-27632 on contractile
responses to norepinephrine
Fig. 3 shows concentration–effect curves for the effects
of increasing concentrations of nifedipine or Y-27632 on
the responses to two concentrations of norepinephrine.
Nifedipine and Y-27632 each produced a concentrationdependent decrease in the norepinephrine-induced tension
at each concentration of norepinephrine. However, the
inhibition was much larger with Y-27632 than with
nifedipine. In fact, in heart failure the enhanced contractile
response to norepinephrine was almost completely inTable 1
Hormonal data a
2.10. Statistics
An unpaired t-test was used to compare data between
normal and heart failure. Changes within the same group
were analyzed by one-way analysis of variance (ANOVA)
for repeated measures and subsequent Scheffe’s post-hoc
test. Differences between two groups were analyzed by
Normal (n55)
Heart failure (n55)
a
Norepinephrine
(pg / ml)
Angiotensin II
(pg / ml)
ANP
(pg / ml)
1866104
6126165*
25610
145627*
3167
205659*
Data represent mean6S.D. The number of experiments is indicated in
parenthesis.
*P,0.01 vs. normal.
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
323
Fig. 1. Time course of vasoconstriction in response to 1 mmol / l norepinephrine (A, B) or 60 mmol / l K 1 (C, D) in the presence or absence of endothelium
(ET). Normal (—), Heart failure (---). Note that the contractile response to norepinephrine was greater in heart failure than in the normal condition whether
or not the endothelium was intact.
hibited by Y-27632, whereas nifedipine only partially
inhibited it.
3.4. Effects of nifedipine and Y-27632 on tension–
[ Ca 21 ] relationship for norepinephrine-induced
contraction
Fig. 4 shows simultaneous time courses for tension and
the Indo-1 fluorescence ratio. In normal and heart failure
groups, both tension and Fratio increased after the addition
of norepinephrine. In the presence of nifedipine (1 mmol /
l), tension and Fratio both showed a smaller increase in
response to norepinephrine, the Fratio response being more
strongly affected than the tension response. In the presence
of Y-27632, however, the tension response was decreased
with no change in the Fratio response. Fig. 5 summarizes
the relation between tension and Fratio after the addition of
norepinephrine in the absence or presence of nifedipine
and Y-27632. In heart failure group, norepinephrine (1
mmol / l) produced a higher tension than in the normal
Table 2
Maximum contractile responses to norepinephrine or K 1 in the presence or absence of endothelium a
Tension (g?cm 22 )
Norepinephrine (1 mmol / l)
Normal
Heart failure
KCl (60 mmol / l)
ET (2)
ET (1)
ET (2)
ET (1)
297641 (5)
486638* (5)
289638 (4)
481648* (4)
308617 (5)
311624 (5)
300610 (6)
289622 (5)
a
Data represent mean6S.D. The number of experiments is indicated in parenthesis.
*P,0.01 vs. normal.
324
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
group despite a similar increase in Fratio , indicating an
increase in the Ca 21 sensitivity of the vascular smooth
muscle in heart failure. In the heart failure group (i)
nifedipine inhibited this norepinephrine-induced enhanced
vasoconstriction by only a maximum of 27% at 1 mmol / l,
although this was associated with a 56% reduction in Fratio ;
(ii) in contrast, Y-27632 inhibited it by a maximum of 80%
at 100 mmol / l without a significant change in Fratio.
3.5. Effects of nifedipine and Y-27632 on angiotensin
II-induced contraction
Fig. 2. Concentration–effect curves for response to norepinephrine
(10 28 –10 4 mol / l). Normal (—s—; n56), Heart failure (—d—; n56).
Data represent mean6S.D. *P,0.01 vs. baseline. [P,0.01 vs. normal.
Fig. 6 shows the time course of the tension response to
angiotensin II (0.1 mmol / l). In both normal and heart
failure groups, the tension curve showed a spontaneous
decline after reaching a peak in response to angiotensin II.
As shown in Fig. 7, angiotensin II induced a higher peak
tension in heart failure than in the normal group, as in the
case of norepinephrine. Once again, Y-27632 caused a
larger inhibition of the contraction (this time, induced by
angiotensin II) than nifedipine.
3.6. mRNA and protein expression levels for RhoA and
Rho-kinase
Fig. 8 shows the expression levels of Rho A protein (A)
and Rho A mRNA (B) in femoral arteries isolated from
heart failure dogs and from normal dogs. The level of Rho
A protein was significantly higher in heart failure
(0.8260.09) than in the normal animals (0.4060.20), as
was the expression level of the mRNA encoding Rho A
(0.2860.08 vs. 0.1860.04). However, there was no significant difference in the Rho-kinase protein or mRNA
expression levels between the heart failure and normal
groups.
4. Discussion
Fig. 3. (A) Effect of nifedipine on norepinephrine-induced vasoconstriction. Normal (n54) (—s—), 1 mmol / l; (– –h– –), 10 mmol / l
norepinephrine. Heart failure (n54) (—d—), 1 mmol / l; (– –j– –), 10
mmol / l norepinephrine. Data represent mean6S.D. (B) Effect of Y27632 on norepinephrine-induced vasoconstriction. Normal (n54)
(—s—), 1 mmol / l; (– –h– –), 10 mmol / l norepinephrine. Heart failure
(n55) (—d—), 1 mmol / l; (– –j– –), 10 mmol / l norepinephrine. Data
represent mean6S.D.
It is commonly accepted that contraction of the smooth
muscle cell mainly depends upon the concentration of
Ca 21 within the cytoplasm. Two major pathways exist at
the level of the sarcolemma for the entry of Ca 21 in
response to appropriate stimuli: voltage-operated Ca 21
channels and receptor-operated Ca 21 channels [33]. Membrane depolarization and ligand binding to the receptor,
respectively, result in the opening of these channels,
allowing Ca 21 to diffuse into the cell. Studies in which
force and Ca 21 have been simultaneously measured in
intact smooth muscle tissue have suggested that the force /
Ca 21 ratio is greater in agonist-stimulated contractions
than in [K 1 ]-stimulated contractions [34]. Indeed, there is
evidence showing that stimulation of receptors significantly increases the myofilament Ca 21 sensitivity of
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
325
Fig. 4. Effects of nifedipine (1 mmol / l) and Y-27632 (100 mmol / l) on simultaneous time courses of changes in tension and Indo-1 fluorescence ratio in
response to norepinephrine (1 mmol / l). Segments loaded with Indo1-AM were mounted in an organ chamber. Then, the Indo1 fluorescence signal during
the vasoconstrictor response to norepinephrine was measured using a spectrophotometer. The ratio of the 418- and 468-nm signals (Fratio ) provides a
measure of intracellular [Ca 21 ].
smooth muscle [34]. Recently, the key element in the
modulation of Ca 21 sensitivity was identified as a small,
monomeric G-protein, RhoA [10].
Changes in peripheral vascular sensitivity to a-adrenoceptor agonists have been demonstrated in certain models
of hypertension and diabetes [35,36]. Further, in pacinginduced canine heart failure an enhanced reactivity of
blood vessels to a-adrenoceptor agonists has been noted
[37], and this enhanced response occurred early in the
development of the heart failure [38]. We confirmed here
that the vascular reactivity to norepinephrine was significantly enhanced in heart failure, by comparison with
the normal condition. Moreover, the present study — by
assessing the Ca 21 -desensitizing effect of Y-27632 upon
norepinephrine-induced vasoconstriction in parallel with
the effect of a Ca 21 -antagonist, nifedipine — permitted a
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T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
Fig. 5. Effects of nifedipine (1 mmol / l) and Y-27632 (100 mmol / l) on
norepinephrine (1 mmol / l)-induced change in tension–[Ca 21 ] relationship. Normal (n55) (—s—), heart failure (n55) (—d—). Concentration of Ca 21 is expressed as Indo1 fluorescence ratio (418 nm / 468 nm)
as described in ‘Methods’. Abbreviations: nor, norepinephrine; nif,
nifedipine; Y, Y-27632. Data represent mean6S.D. Tension: *P,0.01 vs.
norepinephrine alone; Indo1 fluorescence ratio: [P,0.01 vs. norepinephrine alone.
greater insight into the mechanism underlying this enhanced vasoconstriction.
The major findings of this study are as follows. First, the
enhancement of the norepinephrine-induced vasoconstriction in heart failure was largely ascribable to an increase in
Ca 21 sensitivity rather than to an increase in cytoplasmic
free Ca 21 . Second, nifedipine inhibited this enhanced
contraction by only a maximum of 27% in association with
Fig. 7. Effects of nifedipine (A) and Y-27632 (B) on maximum tension
induced by angiotensin II (0.1 mmol / l). Normal (n54), Heart failure
(n54). Data represent mean6S.D.
Fig. 6. Time course of tension response to angiotensin II (0.1 mmol / l).
Normal (—), Heart failure (---).
a decrease of 56% in free [Ca 21 ]. In contrast, Y-27632
inhibited it by a maximum of 80% with no change in free
[Ca 21 ], indicating that the enhancement of the vasoconstriction was indeed induced by a marked increase in Ca 21
sensitivity mediated through activation of the Rho / Rhokinase system. Third, immunoblot and RT-PCR analyses
showed that the expressions of RhoA protein and its
mRNA were increased in heart failure, whereas the
expressions of Rho kinase protein and mRNA were
unchanged (Fig. 9). Possibly, the mode of expression of
these two proteins may be differently regulated in heart
failure.
Another subject of this study was the role of the renin–
angiotensin system (RAS) in the evolution of heart failure.
This system has been considered a compensatory mechanism that initially acts to increase blood pressure and renal
perfusion, although it appears to have an important role in
elevating systemic vascular resistance in advanced heart
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
Fig. 8. Expression level of Rho A protein (A) and mRNA (B). Normal
(n56), Heart failure (n56). Data represent mean6S.D.
failure [39]. Recently, a local type of RAS has been
identified in a variety of tissues, including vascular smooth
muscle, and several lines of evidence suggest that the
angiotensin II produced by local RAS is heavily involved
in the increase in systemic vascular resistance caused by
the enhanced vasoconstriction seen in heart failure [40].
Interestingly, Y-27632 exhibited a counteracting (vasodilating) action against both agonists (norepinephrine and
angiotensin II), suggesting that an exaggerated activation
of the Rho / Rho-kinase system is common to the mechanisms underlying the enhanced vasoconstrictor response to
these two agonists.
Since endothelial dysfunction has been said to contribute
to the increased vascular resistance seen in heart failure
[41,42], we examined the vasoconstrictor responses to
norepinephrine and K 1 under both endothelium-intact and
327
Fig. 9. Expression level of the Rho kinase protein (A) and mRNA (B).
Normal (n56), Heart failure (n56). Data represent mean6S.D.
endothelium-denuded conditions. In fact, the contractile
response of vascular segments to norepinephrine was
augmented in heart failure to a degree that did not depend
on the existence of the endothelium (Table 2). This finding
is consistent with the observation that the acetylcolineinduced relaxation response in the dorsal pedal artery
remains unaltered in experimental heart failure [13].
Before drawing firm conclusions, several issues remain
to be resolved. First, a Ca 21 channel blocker, nifedipine,
also significantly inhibited the enhanced vasoconstriction
induced by norepinephrine in heart failure, although the
inhibition was smaller than that produced by the Rhokinase inhibitor, Y-27632. In accord with this, Forster et al.
[43] showed a significant inhibition by nifedipine of the
enhanced vasoconstriction induced by norepinephrine in
the same model of heart failure. A relevant finding may be
that of Han et al. [44], who demonstrated that two distinct
328
T. Hisaoka et al. / Cardiovascular Research 49 (2001) 319 – 329
subtypes of a1-adrenoceptor cause contractile responses,
one of which causes contractions that require an influx of
extracellular Ca 21 through dihydropyridine-sensitive channels.
Second, a caused link needs to be established between
the observed up-regulation of RhoA in heart failure and the
increased Ca 21 sensitivity of vascular smooth muscle (and
the resulting enhanced vasoconstriction). With regard to
this point, Kimura et al. [10] demonstrated a few years ago
that an over-expression of Rho A increased the phosphorylation of the myosin-binding subunit of myosin
phosphatase, an event that leads to the inactivation of
myosin phosphatase.
Third, in heart failure structural and functional alterations in the components of vascular beds (such as smooth
muscle, endothelium, and nerve endings) occur under the
control of various neurohumoral factors [1]. Therefore,
more work is clearly needed to elucidate whether, and to
what extent, the Rho / Rho-kinase system contributes to
enhanced vasoconstriction in the intact circulation in heart
failure.
Fourth, enhanced vascular contractile activity seen in
heart failure may be due to the quantitative or qualitative
alteration at receptor level in tachycardia-induced heart
failure. Luchner et al. [45] demonstrated that in tachycardia-induced heart failure the tissue angiotensin II was
increased in heart and aorta, suggesting that local renin–
angiotensin-system is activated in this model. Also, Foster
et al. [13] showed that vaso-contractile response to selective a1-agonist, phenylephrine was enhanced in tachycardia-induced heart failure, suggesting that a1-adrenoceptor
activity in arterial smooth muscle increases in congestive
heart failure. To our knowledge, there is no direct evidence
that the density of angiotensin II receptor or a receptor in
vascular wall changes in a model of tachycardia-induced
heart failure. However, since the receptor density has been
shown to change dramatically in other cardiac hypertrophy
and / or failure (up-regulation of AT1 receptor in experimental infarct myocardium [46], down-regulation of
AT1 receptor in human failing myocardium [47,48],
unchanged a1 receptor density in human failing heart
[49]), it is likely that these changes in the receptor density
also occur in vascular wall, contributing to the hypervasoconstriction seen in this model of heart failure.
Fifth, although this model of tachycardia-induced heart
failure causes well-defined, predictable, and progressive
LV dilatation, contractile dysfunction, and neurohumoral
activation [50], it does not resemble chronic heart failure
inasmuch as the pacing-induced failure lacks a hypertrophic compensatory phase [51], and disappears if pacing
is discontinued after 3 weeks [52]. Clearly, research using
other models of heart failure is needed if we are fully to
elucidate the alterations in vascular contractile function
seen in chronic heart failure.
In conclusion, a G-protein-coupled increase in myofilament Ca 21 sensitivity mediated through the Rho / Rho-
kinase system is heavily involved in the mechanism
underlying the enhanced vasoconstriction observed in heart
failure. This finding may point the way to a new vasodilator therapy, a form of therapy now recognized as the most
important in severe congestive heart failure.
Acknowledgements
Y-27632 was kindly provided by Welfide Co. Ltd.
(Tokyo, Japan). We thank Drs. Takeshi Yamamoto and
Yasuhiro Ikeda for superior technical advice. Support:
Grant-in-Aid for Scientific Research from The Ministry of
Education in Japan (11670684) and Health Sciences
Research Grant for Comprehensive Research on Aging and
Health from the Ministry of Health and Welfare, Japan.
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