Ca(2+) channel blocker benidipine promotes coronary angiogenesis and reduces both left-ventricular diastolic stiffness and mortality in hypertensive rats.

Abstract

The beneficial cardiac effects of some Ca(2+) channel blockers have been attributed to blood pressure reduction, but these pleiotropic effects require further investigation. We compared the effects of benidipine, which has beneficial cardiac effects, and nitrendipine, which does not, in an animal model of hypertensive diastolic heart failure (DHF).
Male Dahl salt-sensitive rats were fed a high-salt diet from age 7 weeks to induce hypertension and were either vehicle or orally administered benidipine (3 mg/kg daily) or nitrendipine (10 mg/kg daily) from age 10 to 18 weeks. Control rats were maintained on a low-salt diet. In vehicle-treated rats, left-ventricular (LV) fractional shortening was preserved but LV end-diastolic pressure was increased, indicative of DHF. Benidipine and nitrendipine had similar antihypertensive effects and reduced both LV weight and cardiomyocyte hypertrophy. Benidipine reduced LV diastolic stiffness and mortality to a greater extent than did nitrendipine. Benidipine, but not nitrendipine, also reduced lung weight. The extent of interstitial fibrosis and the abundance of mRNAs for prohypertrophic, profibrotic, or proinflammatory genes in the left ventricle were reduced by benidipine and nitrendipine. Benidipine, but not nitrendipine, increased capillary density and restored the expression of hypoxia-inducible factor 1alpha, vascular endothelial growth factor, and endothelial nitric oxide synthase in the left ventricle.
Benidipine reduced LV diastolic stiffness and increased survival, effects likely attributable predominantly to promotion of coronary angiogenesis rather than to attenuation of interstitial fibrosis. Benidipine may thus be more effective than purely L-type Ca(2+) channel blockers in preventing hypertensive DHF.

Full text

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Ca2þchannel blocker benidipine promotes coronary
angiogenesis and reduces both left-ventricular diastolic
stiffness and mortality in hypertensive rats
Takao Nishizawaa, Xian Wu Chenga,f,g, Zhehu Jinb,f, Koji Obatac,
Kohzo Nagatad, Akihiro Hirashikia, Takeshi Sasakib, Akiko Nodad,
Kyosuke Takeshitaa, Hideo Izawaa, Guo-Ping Shie, Masafumi Kuzuyab,
Kenji Okumuraaand Toyoaki Muroharaa
Background The beneficial cardiac effects of some Ca2þ
channel blockers have been attributed to blood pressure
reduction, but these pleiotropic effects require further
investigation. We compared the effects of benidipine, which
has beneficial cardiac effects, and nitrendipine, which does
not, in an animal model of hypertensive diastolic heart
failure (DHF).
Methodsandresults MaleDahl salt-sensitive rats werefed
a high-salt diet from age 7 weeks to induce hypertension
and were either vehicle or orally administered benidipine
(3mg/kg daily) or nitrendipine (10mg/kg daily) from age 10
to18weeks. Controlrats were maintained on alow-salt diet.
In vehicle-treated rats, left-ventricular (LV) fractional
shortening was preserved but LV end-diastolic pressure
was increased, indicative of DHF. Benidipine and
nitrendipine had similar antihypertensive effects and
reduced both LV weight and cardiomyocyte hypertrophy.
Benidipine reduced LV diastolic stiffness and mortality to a
greater extent than did nitrendipine. Benidipine, but not
nitrendipine, also reduced lung weight. The extent of
interstitial fibrosis and the abundance of mRNAs for
prohypertrophic, profibrotic, or proinflammatory genes in
the left ventricle were reduced by benidipine and
nitrendipine. Benidipine, but not nitrendipine, increased
capillary density and restored the expression of hypoxia-
inducible factor 1a, vascular endothelial growth factor, and
endothelial nitric oxide synthase in the left ventricle.
Conclusions Benidipine reduced LV diastolic stiffness and
increased survival, effects likely attributable predominantly
to promotion of coronary angiogenesis rather than to
attenuation of interstitial fibrosis. Benidipine may thus
be more effective than purely L-type Ca2þchannel
blockers in preventing hypertensive DHF. J Hypertens
28:1515–1526 Q 2010 Wolters Kluwer Health | Lippincott
Williams & Wilkins.
Journal of Hypertension 2010, 28:1515–1526
Keywords: angiogenesis, cardiac stiffness, diastolic heart failure, hypoxia-
induced factor-a, L-type Ca2þchannel
Abbreviations: b-MHC, b-myosin heavy chain; ACE, angiotensin-converting
enzyme; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin-
receptor blocker; DHF, diastolic heart failure; DHP, dihydropyridine; eNOS,
endothelial nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; HIF-1a, hypoxia-inducible factor-1a; IVST, interventricular
septum; LV dP/dtmax, maximum first derivative of left ventricular pressure;
LV dP/dtmin, minimal rate of left ventricular pressure; LVDd, left-ventricular
end-diastolic dimension; LVDs, left-ventricular end-systolic dimension; LVH,
left-ventricular hypertrophy; LV, left-ventricular; MR, mineralocorticoid
receptor; PCR, polymerase chain reaction; T1/2, the pressure half-time;
TGF-1b, transforming growth factor-1b; VEGF, vascular endothelial growth
factor
aDepartment of Cardiology,bDepartment of Geriatrics, Nagoya University
Graduate School of Medicine,cDepartment of Pharmacology, Aichi Gakuin
University School of Dentistry,dDepartment of Medical Technology, Nagoya
University School of Health Sciences, Nagoya, Japan,eDepartment of
Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical
School Boston, Massachusetts, USA,fDepartment of Cardiology, Yanbian
University Hospital, Yanji, Jilin Province, China andgDepartment of Internal
Medicine, Kyung Hee University Hospital, Seoul, Korea
Correspondence to Xian Wu Cheng, MD, PhD, Department of Cardiology, Nagoya
University School of Medicine, 65 Tsuruma-cho, Nagoya 466-8550, Japan
Tel: +0081 52 744 2427; fax: +0081 52 744 2371;
e-mail: chengxw0908@yahoo.com.cn or xianwu@med.nagoya-u.ac.jp
Received 23 August 2009 Revised 13 February 2010
Accepted 16 March 2010
Introduction
Chronic pressure overload due to hypertension results in
myocardial hypertrophy as an adaptive response to main-
taincardiacfunction.However,persistentleft-ventricular
hypertrophy (LVH) leads to diastolic heart failure (DHF)
[1,2], which accounts for 30–50% of all cases of heart
failure and has a poor prognosis [3,4]. DHF is character-
ized by abnormal LV relaxation, impaired LV filling, and
increasedLVdiastolic stiffness,with LVsystolic function
remaining largely unaffected.
Given that increased interstitial fibrosis has been associ-
ated with the onset of DHF [5], agents that block the
renin–angiotensin–aldosterone system are used for the
treatment of this condition [6]. However, given that
treatment with a single antihypertensive angiotensin-
converting enzyme inhibitor (ACEI) or angiotensin-
receptor blocker (ARB) is often insufficient to normalize
blood pressure, calcium channel blockers (CCBs) are
often used in combination with ARBs or ACEIs in the
clinical setting [7]. In addition to ACEI and ARB, recent
Original article 1515
0263-6352 ? 2010 Wolters Kluwer Health | Lippincott Williams & WilkinsDOI:10.1097/HJH.0b013e328339fd3a

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few studies in experimental animals have shown that
CCBs are also effective in preventing cardiac remodeling
and dysfunction [8]. In fact, it has been reported that
CCBs exert cardiovascular protective actions including
reduction of oxidative stress and proinflammatory
response [2,9]. However, the precise mechanisms under-
lying the cardioprotection afforded by a CCB in animals
or patients with hypertensive DHF remain largely
unknown.
The physiological role of T-type Ca2þchannel is diverse,
and clinical advantages of T-type channel blockade were
assessed in late 1990s using mibefradil, another class
Ca2þchannel antagonist. The drug had relatively selec-
tive blocking action on T-type Ca2þchannel, and
beneficial effects were shown in the treatment of cardiac
hypertrophy, angina pectoris and renal failure [10–12].
However, mibefradil, which is known to block all three
subtypes of T-type Ca2þchannel (a1G, a1Hand a1i) [13],
was abandoned for clinical usage because of frequent
drug–drug interaction. In addition, some studies show
that some dihydropyridines (DHPs) have blocking action
T-type Ca2þchannels in native tissues [14,15]. As sub-
type-specificblockingactionofDHPswasnotassessedin
these studies, different expression levels of each T-type
channel subtype would result in different blocking action
in case that DHP had subtype-specific blocking action.
Recent study has indicated that DHP Ca2þantagonists
have a different profile in blocking T-type Ca2þchannel
subtypes expressed in Xenopus oocytes [16,17]. Several
studies showed beneficial effect of efonidipine on renal
function, cardiac failure and aldosterone secretion as a
result of the T-type Ca2þchannel-blocking action of the
drug [18–20]. Accumulating evidence suggested that up-
regulation of T-type Ca2þchannels has been associated
with both LVH and hypertensive DHF [21,22]. To
elucidate the cardiac protective effects as well as the
mechanism of action of benedipine against the activated
T-type Ca2þchannel, we compared the effects of beni-
dipine, a blocker of T-type and L-type Ca2þchannels,
with those of nitrendipine, a blocker of L-type Ca2þ
channel, in a Dahl salt-sensitive rat model of hyper-
tensive DHF.
Methods
Animals
Male inbred Dahl salt-sensitive rats were obtained from
Japan SLC (Hamamatsu, Japan) and were handled in
accordance with the guidelines of Nagoya University
Graduate School of Medicine as well as with the Guide
for the Care and Use of Laboratory Animals (NIH pub-
lication no. 85–23, revised 1996). Weaning rats were fed
laboratory chow containing 0.3% NaCl until 7 weeks of
age. Animals on this latter diet served as models of
hypertensive LVH at 10 weeks of age and DHF at
18 weeks of age [1]. Both food and tap water were
provided ad libitum throughout the experiment. The rats
onthehigh-saltdietweredividedintothreegroups:those
orally given benidipine (3mg/kg body weight daily;
Kyowa Hakko Kirin CO., Ltd, Tokyo, Japan) from
10 to 18 weeks of age (n¼10); those orally given nitren-
dipine (10mg/kg daily; Sigma Aldrich) from 10 to 18
weeks of age (n¼10); and those orally given vehicle
(0.5% carboxymethylcellulose) from 10 to 18 weeks of
age (n¼10, untreated group). The benidipine, nitrendi-
pine, and vehicle were each given by oral gavage every
day. Rats maintained on the 0.3% NaCl diet until
18 weeks of age were studied as a control group
(n¼10). At 18 weeks of age, rats were anesthetized by
intraperitoneal injection of ketamine (50mg/kg) and
xylazine (10mg/kg) and were subjected to hemodynamic
and echocardiographic analyses. The heart and kidney
were subsequently excised, and LV and kidney tissues
werestoredat?808Cforeithermolecularanalyses,orfixed
with paraformaldehyde for pathological analysis.
Echocardiographic and hemodynamic analyses
Systolic blood pressure was measured weekly in con-
scious animals by tail-cuff plethysmography (BP-98A;
Softron, Tokyo, Japan) [23]. At 18 weeks of age, rats
were subjected to transthoracic echocardiography as pre-
viously described [23]. Echocardiography was performed
with a SONOS 7500 ultrasound system and an ultraband
transducer of 5–12MHz (Philips, Andover, Massachu-
setts, USA). LV end-diastolic (LVDd) and end-systolic
(LVDs) dimensions as well as the thickness of the inter-
ventricular septum (IVST) were measured. LV fractional
shortening was calculated as 100%?(LVDd?LVDs)/
LVDd. After echocardiography, a 2F micromanometer-
tipped catheter (SPR-407; Millar Instruments, Houston,
Texas, USA) that had been calibrated relative to atmos-
pheric pressure was inserted through the right carotid
artery into the left ventricle. We evaluated the maximum
first derivative of LV pressure (LV dP/dtmax) as an index
of contractility, minimal rate of LV pressure change (LV
dP/dtmin), and the pressure half-time (T1/2) as an index of
relaxation. Tracings of LV pressure and the electro-
cardiogram were digitized to determine the pressure
half-time and LV end-diastolic pressure as previous
described [2].
Histology
The left ventricle was fixed with ice-cold 4% parafor-
maldehyde for 16–24h, embedded in paraffin, sectioned
transversely (thickness 3mm), and stained either with
hematoxylin–eosin for evaluation of cardiomyocyte
hypertrophy or with Azan-Mallory solution for evaluation
of interstitial fibrosis. The cross-sectional areas of cardio-
myocytes and the areas of the fibrosis in the interstitial
region were calculated in 10 randomly chosen micro-
scopic fields from three different sections in each animal,
as previously described [24,25]. The sections of were
also immunostained with mouse monoclonal antibody
to rat CD31 (1:100 dilution; Pharmingen, San Diego,
1516
Journal of Hypertension
2010, Vol 28 No 7

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California, USA), and a Universal Immuno-Enzyme
Polymer kit (Nichirei Biosciences, Tokyo, Japan) was
used to visualize the coronary capillary endothelial cells.
The capillary endothelial cells were quantified by
measuring the number of CD31þcells per high-power
field (400?). The number of capillaries was measured in
15 randomly chosen microscopic fields from three differ-
ent sections in each animal. The evaluation of capillary
diameter was also analyzed in each high-power field
(400?).
Quantitation of gene expression
Total RNA was extracted from LV tissue, and the abun-
dance of specific mRNAs was determined by reverse
transcription and real-time polymerase chain reaction
(PCR) analysis with a Prism 7700 Sequence Detector
(Perkin-Elmer, Waltham, Massachusetts, USA). The
sequences of primers and TaqMan probes specific for
b-myosin heavy chain (b-MHC), angiotensin-converting
enzyme (ACE), transforming growth factor (TGF)-1b,
or collagen types I or III have been described previously
[25], as have been those for vascular endothelial growth
factor (VEGF), VEGF receptor-1 (Flt-1), hypoxia-
inducible factor (HIF)-1a, and endothelial nitric oxide
synthase (eNOS) [24,25]. PCR was also performed
with oligonucleotides specific for the a1G subunit of
T-typeCa2þchannels(50-CCTGCCTGTTGCCGA
GAG-30,50-CTGTCTGTGTTACTGGATTCCTTC
C-30, and 50-AGATTCCTGGTCGGCCTATATCTT
TCC-30astheforwardprimer,reverseprimer,andTaqMan
probe, respectively; GenBank accession no. AF027984).
TaqManrodentglyceraldehyde-3-phosphatedehydrogen-
ase (GAPDH) control reagents (Applied Biosystems,
FosterCity,California,USA)wereusedtodetectGAPDH
mRNA as an internal standard.
Immunoblot analysis
Tissue samples (80mg of protein) were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis on a 10% gel, and the separated proteins were trans-
ferred to a polyvinylidene difluoride membrane (Bio-Rad
Laboratories, Hercules, California, USA). The membrane
was incubated at room temperature, first for 1h with Tris-
buffered saline containing 5% nonfat milk and 0.1%
Tween-20 and then overnight with rabbit polyclonal anti-
bodies to VEGF-A, to VEGF-C (Santa Cruz Biotechno-
logy,Inc.,SantaCruz,California,USA),toHIF-1a(Novus
Biologicals,Littleton,Colorado,USA),toPhosepho-eNOs
(p-eNOs,Ser-1177),ortoeNOS(bothfromCellSignaling,
Danvers, Massachusetts, USA), all at a 1:1000 dilution in
the same solution. The membrane was washed and then
incubated at room temperature for 1h with a 1:1000
dilution of horseradish peroxidase-conjugated goat anti-
bodies to rabbit immunoglobulin G (MBL, Nagoya,
Japan), after which immune complexes were detected
and quantified as described previously [24]. The intensi-
ties of the VEGF, HIF-1a, and eNOS bands were quan-
tified by densitometry with ATTO CS Analyzer (version
1.01)software,andtheamountofeachproteinwasnormal-
ized against that of GAPDH determined with rabbit
antibodiestothisprotein(SantaCruzBiotechnology,Inc.).
Statistics
Data are presented as means?SEM. Differences in
various parameters between the four groups were eval-
uated by analysis of variance (ANOVA) followed by
Dunnett’s post-hoc test. Survival rate was analyzed by
the standard Kaplan–Meier method with a log-rank test.
A P value of less than 0.05 was considered statistically
significant.
Statement of responsibility
The authors had full access to the data and take respon-
sibility for data integrity. All authors have read and agree
to the manuscript as written.
Results
Systolic blood pressure
Dahl salt-sensitive rats fed a high-salt diet from 7 weeks
of age progressively developed hypertension [1,2]. Treat-
ment with benidipine or nitrendipine from 10 weeks of
age similarly lowered systolic blood pressure by approxi-
mately 15–20mmHg in the conscious state, with this
effect being apparent as early as 1 week after the
initiation of treatment (Fig. 1, Table 1).
Survival rate
Kaplan–Meier analysis revealed that the survival rate of
rats in the untreated group up to 18 weeks of age was half
that of animals in the control group (no deaths). The
survival rate was increased slightly in the nitrendipine
Benidipine prevents diastolic heart failure Nishizawa et al.
1517
Fig. 1
Benidipine or nitrendipine
Control
Untreated
Benidipine
Nitrendipine
270
Start of
8% NaCI
250
Systolic blood pressure (mmHg)
230
210
190
170
150
7891011 12
Age (weeks)
13 1415 16 1718
Time course of systolic blood pressure in Dahl salt-sensitive rats fed a
high-salt diet from 7 weeks of age and treated with vehicle (untreated
group), benidipine (3mg/kg daily), or nitrendipine (10mg/kg daily) from
10 weeks of age, as well as in age-matched controls fed a low-salt diet
(control group). Data are means?SEM (n¼10 per group).

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group (60%) compared with that in the untreated group
(50%), but no deaths were observed in the benidipine
group (Fig. 2).
Left-ventricular geometry and function
Heart rate was greater in the untreated group than in the
control group at 18 weeks of age, although this increase
was not significant. Heart rate was significantly reduced
by treatment with benidipine compared with that in the
untreated and nitrendipine groups (Table 1). Both the
thickness of the IVST and the ratio of LV weight to body
weight were significantly greater in the untreated group
than in the controls, indicative of LVH; both of these
effects were significantly reduced by nitrendipine or
benidipine.LVfractional shorteningdidnotdifferamong
thefourexperimentalgroups.TheLVdP/dtmaxwaslower
in the untreated group than in the control group; this
change was improved by benidipine and nitrendipine.
TheLVdP/dtwassmaller,whereastheT1/2wasgreaterin
the untreated group than in the controls. The changes in
these parameters were also improved by both treatments.
The LV end-diastolic pressure and the ratio of lung
weight to body weight were greater in the untreated
group than in the control group. Nitrendipine signifi-
cantly reduced the extent of both parameters, whereas
benidipine prevented it. In addition, the weight of the
right-ventricular free wall was slightly increased in
the untreated rats (0.21?0.04 versus 0.19?0.01g in
the controls; P>0.05); this change was not improved
by either treatment (0.22?0.04g with nitrendipine
and 0.20?0.03g with benidipine; P>0.05). Given that
the LVDd assessed by echocardiography did not differ
among the four experimental groups (Table 1), LV
diastolic stiffness (LV end-diastolic pressure/LVDd)
was significantly greater in the untreated group than in
the controls (Fig. 3). Nitrendipine reduced this increase
in stiffness to a level significantly lower than in the
untreated group but still significantly higher than in
the controls, whereas benedipine prevented it comple-
tely (Fig. 3).
Cardiomyocyte hypertrophy and interstitial fibrosis
Both the cardiomyocyte cross-sectional area (Fig. 4)
and the level of interstitial fibrosis (Fig. 5) in the left
ventricleweresignificantlygreaterinuntreatedratsthan
in control animals at 18 weeks of age. Benidipine or
nitrendipine reduced the extents of both cardiomyocyte
hypertrophy and interstitial fibrosis, but these para-
meters still remained significantly higher than the
control values.
Coronary capillary density and diameter
Immunostaning revealed that the ratio of the number of
coronary capillaries to that of cardiomyocytes was signifi-
cantly greater in the untreated than in the control group;
the ratio in thebenedipine group was significantlygreater
thanintheuntreatedandnitrendipinegroups(Fig.6a,b).
Capillary density, however, was significantly lower in the
untreatedgroupthaninthecontrolgroupasaresultofthe
cardiomyocyte hypertrophy evident in untreated animals
(Figs 4a and 6c). Benidipine restored capillary density
to the level in the control group, despite the residual
1518
Journal of Hypertension
2010, Vol 28 No 7
Table 1
weeks of age
Hemodynamic, echocardiographic, and pathological parameters in Dahl salt-sensitive rats of the four experimental groups at 18
Parameter ControlUntreated BenidipineNitrendipine
SBP (mmHg)
Heart rate (b.p.m.)
IVST (mm)
LVDd (mm)
LVFS (%)
LV dP/dtmax(mmHg/s)
LV dP/dtmin(mmHg/s)
T1/2(ms)
LVEDP (mmHg)
LV weight (mg)/BW (g)
Lung weight (mg)/BW (g)
172?3
466?18
1.91?0.05
8.07?0.16
44.7?1.5
8271?260
7654?324
3.8?0.2
3.9?0.2
2.11?0.02
2.76?0.07
253?5M
524?12
2.50?0.12M
7.81?0.11
42.5?3.6
7890?156M
7022?108M
6.2?0.3M
11.7?0.2M
4.20?0.12M
8.01?1.01M
234?9M,MM
450?13MM,MMM
2.10?0.06M,MM
7.97?0.33
44.0?2.8
10234?347M,MM
7634?434MM
4.9?0.3M,MM
3.8?0.4MM,MMM
2.94?0.07M,MM
3.51?0.12M,MM,MMM
235?7M,MM
511?7
2.20?0.08M,MM
8.00?0.40
44.2?3.8
10877?932M,MM
8023?663MM
5.1?0.3M,MM
8.1?0.3M,MM
3.05?0.16M,MM
6.11?1.06M,MM
SBP, systolic blood pressure; IVST, interventricular septum thickness; LVDd, left-ventricular end-diastolic dimension; LVFS, left-ventricular fractional shortening; LV dP/
dtmaxand LV dP/dtmin, first derivative of maximum or minimum left ventricular pressure, respectively, with respect to time; T1/2, time constant of LV pressure decay; LVEDP,
left-ventricular end-diastolic pressure; LV weight, weight of the left ventricle; BW, body weight. Data are means?SEM (n¼5 per group).
MMP<0.05 versus untreated group.MMMP<0.05 versus nitrendipine group.
MP<0.05 versus control group.
Fig. 2
1
0.8
0.6
Fraction surviving
0.4
Control
Untreated
Benidipine
Nitrendipine
0.2
0
10 111213 14 15
Age (weeks)
1617 18
P > 0.05
P > 0.05
Kaplan–Meier plots of survival rates of Dahl salt-sensitive rats in the
four experimental groups (n¼10 per group).

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cardiomyocytehypertrophy in benidipine-treated rats. As
indicated in the representative sections stained for CD31
(Fig. 6a), more coronary capillaries appeared to be dilated
inuntreatedor nitrendipine-treated ratsthan in control or
benidipine-treated animals. Indeed, quantitative analysis
revealed that the diameter of coronary capillaries was
significantly smaller in benidipine-treated rats than in
animals of the other three experimental groups (Fig. 6d).
Benidipine prevents diastolic heart failure Nishizawa et al.
1519
Fig. 3
Left-ventricular (LV) pressure recordings and LV diastolic stiffness values in Dahl salt-sensitive rats of the four experimental groups at 18 weeks of
age. (a) Representative waveforms obtained from a pressure manometer inserted into the left ventricle. (b) LV diastolic stiffness (LV end-diastolic
pressure/LV end-diastolic dimension). Data are means?SEM (n¼5 per group).?P<0.05 versus control group;yP<0.05 versus untreated group;
zP<0.05 versus nitrendipine group.
Fig. 4
Cardiomyocyte hypertrophy in Dahl salt-sensitive rats of the four experimental groups at 18 weeks of age. (a) Representative hematoxylin–eosin
staining of sections of the left ventricle. Scale bar, 100mm. (b) Cross-sectional cardiomyocyte area, as measured in 10 randomly chosen microscopic
fields from three different sections in each tissue block, similar to those shown in (a). Data are means?SEM (n¼5 per group).?P<0.05 versus
control group;yP<0.05 versus untreated group.

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Gene expression
Hemodynamic overload resulted in up-regulation of
the expression of the genes encoding b-MHC, ACE,
TGF-b1, and collagen types I and III in the left ven-
tricles of untreated rats at 18 weeks of age (Table 2). It
also increased the ratio of the amount of collagen type I
mRNA to that of collagen type III mRNA, an indicator of
LV stiffness [23,25]. These increases in gene expression,
with the exception of the increase in collagen type III
mRNA abundance, were partly inhibited by treatment
with benidipine or nitrendipine. Expression of the gene
encoding the a1G subunit of T-type Ca2þchannels was
also significantly higher in the untreated group than in
the controls; this increase was reduced to a greater extent
by treatment with benidipine than by that with nitren-
dipine (Table 2). The levels of VEGF, HIF-1a, eNOS,
and Flt-1 mRNAs were significantly lower in the left
ventricles of untreated-group rats than in the control-
group rats; these changes were improved by treatment
with benidipine but not with nitrendipine (Figs 7a–c and
8b). Similarly, benidipine, but not nitrendipine, signi-
ficantly increased the levels of VEGF, HIF-1a, and
p-eNOS proteins compared with those in the untreated
group (Fig. 7d–f). However, there were no differences in
the levels of eNOS protein among the four experimental
groups (Fig. 8a).
Renal fibrosis
Histological analysis showed that the level of interstitial
fibrosis was significantly greater in the kidneys of
untreated rats than in control animals at 18 weeks of
age; this change was improved by benidipine or nitren-
dipine, but these parameters remained significantly
higher than the control values (Fig. 8c and d). The level
of serum creatinine was higher in untreated rats than in
control rats (0.41?0.04 versus 0.28?0.3mg/dl, respec-
tively; P<0.05); this change was not improved by either
treatments(0.41?0.10mg/dl
0.45?0.09mg/dl with nitrendipine; P>0.05).
Discussion
We showed that benidipine, but not nitrendipine, pro-
moted coronary angiogenesis, likely accounting for the
reduction in both LV diastolic stiffness and death rate
induced by treatment with this drug in our animal model
of hypertensive DHF.
withbenidipineand
Effects of benidipine on diastolic dysfunction and
mortality
We inspected the general condition of Dahl salt-sensitive
rats in each experimental group every day, and we found
that high-salt diet rats developed rapid and labored
respiration. All animals that died were immediately sub-
jected to postmortem examination, including macro-
scopic inspection of the intracranial, thoracic, and
abdominal cavities. Cerebral hemorrhage or infarction,
aortic rupture, or colonic ischemia was not detected, but
all animals that died before 18 weeks of age manifested
marked pulmonary congestion (as evidenced by an
increase in the ratio of lung weight to body weight),
indicating that congestive heart failure – not stroke –
was the major cause of death.
1520
Journal of Hypertension
2010, Vol 28 No 7
Fig. 5
Interstitial fibrosis in the left ventricle of Dahl salt-sensitive rats in the four experimental groups at 18 weeks of age. (a) Representative Azan-Mallory
staining of sections of the left ventricle. Scale bar, 200mm. (b) Percentage area of interstitial fibrosis measured in 10 randomly chosen microscopic
fields from three different sections in each tissue block, similar to those shown in (a). Data are means?SEM (n¼5 per group).?P<0.05 versus
control group;yP<0.05 versus untreated group.

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The diastolic dysfunction that develops in this animal
model has been well characterized [5]. The time constant
of LV pressure decay increases in association with the
development of compensatory LVH. This time constant
does not increase further with the development of DHF,
which is accompanied instead by an increase in LV end-
diastolic pressure. The relaxation delay is thus not likely
to be a major contributor to the increase in end-diastolic
pressure [26]. Our untreated group exhibited significant
increases in both the time constant of LV pressure decay
and LV end-diastolic pressure. Although benidipine and
nitrendipine improved relaxation properties to similar
Benidipine prevents diastolic heart failure Nishizawa et al.
1521
Fig. 6
Coronary capillaries in the left ventricle of Dahl salt-sensitive rats in the four experimental groups at 18 weeks of age. (a) Representative CD31
immunostaining of sections of the left ventricle. Scale bar, 100mm. (b–d) Ratio of the number of coronary capillaries to that of cardiomyocytes (b);
capillary density (c); and capillary diameter (d) were measured in 15 randomly chosen microscopic fields from three different sections in each tissue
block, similar to those shown in (a). The number of capillaries was measured in 15 randomly chosen microscopic fields from three different sections
in each tissue block. Quantitative data are means?SEM (n¼5 per group).?P<0.05 versus control group;yP<0.05 versus untreated group;
zP<0.05 versus nitrendipine group.
Table 2
experimental groups at 18 weeks of age
Expression of prohypertrophic, profibrotic, or proinflammatory genes in the left ventricles of Dahl salt-sensitive rats in the four
GeneControlUntreated Benidipine Nitrendipine
b-MHC
TGF-b1
ACE
Collagen type I
Collagen type III
Collagen type I/III ratio
TCC a1G
1.0?0.2
1.0?0.1
1.0?0.2
1.0?0.2
1.0?0.2
1.0?0.1
1.0?0.1
3.7?0.7M
2.0?0.3M
1.7?0.1M
4.0?0.7M
3.0?0.5M
1.4?0.2M
3.2?0.6M
1.9?0.2M,MM
1.2?0.1MM
1.1?0.1MM
2.6?0.4M,MM
2.6?0.3M
1.1?0.1MM
1.2?0.2M,MM,MMM
1.9?0.2M,MM
1.2?0.2MM
1.0?0.1MM
2.5?0.3M,MM
2.5?0.2M
1.1?0.1MM
2.4?0.3M,MM
The amount of each mRNA in the left ventricle was determined by RT and real-time PCR analysis, normalized by that of GAPDH mRNA, and expressed relative to the
normalized value for the control group. TCC a1G, a1G subunit of T-type Ca2þchannels. Data are means?SEM (n¼5 per group).
MMP<0.05 versus untreated group.MMMP<0.05 versus nitrendipine group.
MP<0.05 versus control group.

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extents, benidipine induced a greater reduction in the
acute pressure increase from the end of relaxation to late
diastole (Fig. 3a), resulting in prevention of DHF and
improved survival.
Effect of benidipine on left-ventricular diastolic stiffness
Excess interstitial fibrosis or collagen deposition is associ-
ated with increased diastolic stiffness and pulmonary
edema or congestive heart failure [27,28]. Among various
mechanisms that contribute to LV diastolic stiffness [26],
abnormalitiesinthetranscriptional orpost-transcriptional
regulationofcollagen genesresultinthedisproportionate
accumulation of fibrous tissue during the development of
LVH. Increased LV diastolic stiffness during the devel-
opment of DHF has been associated with increased
interstitial fibrosis in hypertensive Dahl salt-sensitive
rats [2,5]. However, although we found that benidipine
reduced LV diastolic stiffness to a greater extent than did
nitrendipine, the inhibitory effects of the two drugs on
both interstitial fibrosis and the expression of profibrotic
genes were similar. These results thus suggested that the
superior effect of benidipine on LV diastolic stiffness was
attributable to an action other than inhibition of inter-
stitial fibrosis.
Effect of benidipine on coronary angiogenesis
In this study, we observed that the capillary-to-cardio-
myocyte ratio was increased by 14%, and the capillary
density was decreased by 18%, in the left ventricles of
Dahl salt-sensitive rats in the untreated group, compared
with the corresponding values in control animals. Beni-
dipine induced a further increase in the capillary-to-
cardiomyocyte ratio (40% increase compared with the
control group), resulting in complete restoration of capil-
lary density; nitrendipine had no such effects. Coronary
capillaries were significantly smaller in the benidipine
group than in the other three groups; most of the capil-
laries in the untreated and nitrendipine groups appeared
dilated. These observations suggest that the decreased
capillary ratio and density present in the untreated and
nitrendipine groups may dilate fully to maximize blood
flow, whereas the coronary reserve is sufficient in the
benidipine group. Alternatively, the large capillaries
observed in the untreated and nitrendipine groups may
1522
Journal of Hypertension
2010, Vol 28 No 7
Fig. 7
Expression of VEGF, HIF-1, and eNOS in the left ventricle of Dahl salt-sensitive rats in the four experimental groups at 18 weeks of age. (a–c)
Abundances of VEGF, HIF-1, and eNOS mRNAs, respectively, as determined by RT and real-time PCR analysis. Data were normalized by the
amount of GAPDH mRNA and then expressed relative to the normalized value for the control group; they are means?SEM (n¼5 per group). (d–f)
Abundances of VEGF, HIF-1, and eNOS proteins, respectively, as determined by immunoblot analysis. Representative blots are shown in the upper
panels, and quantitative data (means?SEM) are presented in the lower panels. The amount of each protein was normalized by that of GAPDH and
then expressed relative to the normalized value for the control group.?P<0.05 versus control group;yP<0.05 versus untreated group;zP<0.05
versus nitrendipine group (n¼5 per group). eNOS, endothelial nitric oxide synthase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1,
hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor.

Page 9

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be mature, whereas the small vessels in the benidipine
group may be newly generated. Given that angiogenesis
results in degradation of surrounding interstitial fibrosis,
benidipine might have been expected to reduce inter-
stitial fibrosis to a greater extent that did nitrendipine;
however, the two drugs had similar effects on cardiac
fibrosis.
Effects of benidipine on vascular endothelial growth
factor, hypoxia-inducible factor-1a, and endothelial
nitric oxide synthase expression
Cardiomyocyte hypertrophy is thought to increase diffu-
sion distance, contributing to a reduction in oxygen
supply to the myocardium. Neovascularization associated
with cardiac hypertrophy may be attributable to up-
regulation of the expression of angiogenic factors in
cardiomyocytes. HIF-1a is a transcription factor that
induces expression of the VEGF gene in response to
hypoxia–ischemia [29]. Angiogenesis in the vicinity of
hypertrophic cardiomyocytes played an important role in
preventing the transition from cardiac hypertrophy to LV
systolicdysfunctioninamousemodelofLVH[30].Inour
model, coronary angiogenesis associated with cardiomyo-
cyte hypertrophy was also implicated in preventing the
transition from cardiac hypertrophy to DHF. When car-
diac hypertrophy reaches a certain extent, even if ische-
mia persists, HIF-1a synthesis is down-regulated, with
the result that the production of VEGF and angiogenesis
also cease [30]. The VEGF-induced activation of protein
kinase AKT and consequent phosphorylation of eNOS
play a central role in angiogenesis [31,32]. Up-regulation
of eNOS has also been found to modify angiogenesis in
ischemic tissues [33]. It has previously been reported
that the drug had relatively selective blocking action
on T-type Ca2þchannel, and favorable actions were
shown in cardiovascular system [10,19]. Here, we have
Benidipine prevents diastolic heart failure Nishizawa et al.
1523
Fig. 8
Levels of total eNOS (t-eNOS) protein, Flt-1 mRNA, and renal interstitial fibrosis in the four experimental groups at 18 weeks of age. (a) Abundance
of t-eNOS protein, as determined by immunoblot analysis. (b) Abundance of Flt-1 mRNA, as determined by real-time PCR analysis. (c)
Representative Azan-Mallory stainings of sections of the left ventricle. Scale bar, 100mm. (d) Percentage area of interstitial fibrosis measured in
10 randomly chosen microscopic fields from three different sections in each tissue block, similar to those shown in (c). Data are means?SEM
(n¼5 per group).?P<0.05 versus control group;yP<0.05 versus untreated group;zP<0.05 versus nitrendipine group. eNOS, endothelial nitric
oxide synthase.

Page 10

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demonstrated that benidipine (but not nitrendipine)
increasedthelevelsofHIF-1a,VEGF,andeNOSmRNAs
and proteins in the LV tissues of DHF rats. Taken
together, these findings suggested that the blockade of
cardiac T-type Ca2þchannel promotes ischemia-induced
angiogenic response by enhancing HIF-1a-mediated
VEGF and eNOS expressions in the myocardium of
DHF rats. It should be noted that there was a discrepancy
incardiacT-typeCa2þchannelblockade-mediatedangio-
genicaction associatedwith HIF-1a/eNOS/VEGF signal-
ing pathway activation and morphological and functional
improvement with the two drug interventions. Dihydro-
pyridine nitrendipine has been shown to inhibit miner-
alocorticoid receptor activation in vitro and/or in vivo [34].
Recent studies have demonstrated that mineralocorticoid
receptor blockade by antagonists results in attenuation of
LV hypertrophy and heart failure in humans and in the
Dahl salt-sensitive rat model [35,36]. Taken together,
these findings suggest that the nitrendipine-mediated
improvements in LV hypertrophy, fibrosis, and function
are not attributable to the angiogenic action-linked HIF-
1a/eNOS/VEGF signaling pathway activation by T-type
Ca2þchannel blockade, but rather to the inhibition of
mineralocorticoid receptor activation.
Relationship between cardiomyocyte hypertrophy-
associated angiogenesis and left-ventricular diastolic
stiffness
Amismatchbetweenthenumberofcapillariesandthesize
of cardiomyocytes, resulting in myocardial hypoxia, is
thought to arise during the development of cardiac hyper-
trophy [37,38]. A relationship between cardiac angiogen-
esis, cardiac hypertrophy, and cardiac systolic function is
also thought to exist [39–41]. With regard to diastolic
function,thehypertrophicmyocardiumappearsespecially
susceptibletonitricoxidedonors;thisresultedinamarked
reductioninLVend-diastolicpressureinoneclinicalstudy
[42]. Our present results suggest that benidipine reduces
diastolic stiffness and prevents the transition from
compensatory LVH to DHF, not only by inhibiting the
development of interstitial fibrosis but also by promoting
coronary angiogenesis. Improved blood flow and function
areassociatedwithevidenceofangiogenesisinanischemic
regionofheart[43].Becausebenidipinepromotedcoronary
angiogenesis and improved LV diastolic stiffness, it is
plausible that benidipine increases myocardial blood flow
and myocardial function in a genomic and/or nongenomic
way. Hypoxia increases isovolumic resting tension in the
isolated guinea pig heart [44] and raises the diastolic PV
curve in humans during balloon coronary angioplasty [45].
Further studies to clarify the influence of ischemia on
cardiomyocyte resting tension or cardiomyocyte distensi-
bility in hypertensive DHF are warranted.
Study limitations
Up-regulation of T-type Ca2þchannels has been associ-
atedwithbothhypertensiveLVH[21]andDHF[12].We
compared the effects of benidipine, a blocker of T-type
and L-type Ca2þchannels, with those of nitrendipine, a
blocker of L-type Ca2þchannel [22], in a rat model of
hypertensive DHF. Inhibition of T-type Ca2þchannels
by benidipine may underlie the promotion of angiogen-
esis by this drug. Current through these channels and
expression of their a1G subunit are increased in associ-
ation with the development of LVH [12,37]. Here, we
could not measure T-type Ca2þchannel current in iso-
lated cardiomyocytes from rats in the four experimental
groups. Whereas benidipine and nitrendipine each inhib-
ited the progression of LVH to similar extents, benidi-
pine inhibited the increase in abundance of the a1G
subunit mRNA apparent in the left ventricle of untreated
Dahl salt-sensitive rats to a greater extent than did
nitrendipine, suggesting that current through these chan-
nels was reduced by benidipine treatment. Further
studies are warranted to clarify the relationship between
inhibition of T-type Ca2þchannel current and coronary
angiogenesis during the development of DHF. In
addition, it is better to calculate peak flow velocities at
the mitral level during rapid filling (E) and during atrial
contraction (A), as well as the E/A ratio, the deceleration
time, and the isovolumic relaxation time, from the pulsed
Doppler echocardiographic data for assessment of LV
diastolic function. Our study did not include an evalu-
ation of these cardiac diastolic function indices.
Clinical implications
We have demonstrated that benidipine reduced LV
diastolic stiffness and increased survival in hypertensive
Dahl salt-sensitive rats to a greater extent than did
nitrendipine. The prevention of DHF by benidipine
appeared to be due predominantly to the promotion
of angiogenesis rather than to inhibition of interstitial
fibrosis, and this effect on angiogenesis appeared to be
mediated by up-regulation of the production of HIF-1a,
VEGF, and eNOS. Benidipine may thus be more effec-
tive than purely L-type Ca2þchannel blockers in pre-
venting hypertensive DHF. Given that ARBs and ACEIs
are widely used and are more effective than CCBs for the
treatment of LVH, the latter drugs are considered to be
potentialsecond-lineagentsincombinationtherapy.The
effects of combinations of ARBs or ACEIs with different
types of CCBs, including a blocker of T-type and L-type
Ca2þchannels benidipine, thus warrant testing in clinical
trials.
Acknowledgements
We appreciate the technical assistance of A. Inoue, M.
Miyachi and M. Kato.
The work was supported in part by grants from the
Ministry of Education, Culture, Sports, Science, and
Technology of Japan (nos. 17590719 and 19590812 to
X.W.C.) and from the Japan Heart Foundation (no. 26-
7508 to X.W.C.); by a Japan Heart Foundation/Novartis
1524
Journal of Hypertension
2010, Vol 28 No 7

Page 11

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Research Award on Molecular and Cellular Cardiology
(no. 26-7523 to X.W.C); and by a grant from the Takeda
Science Foundation (no. 26-7527 to X.W.C).
The authors declare no conflict of interest with regard to
the present study. Benedipine was donated by Kyowa
Hakko Kirin Co., Ltd. (Tokyo, Japan).
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