Download Decreased Nox4 levels in the myocardium of patients with aortic

Clinical Science (2013) 125, 291–300 (Printed in Great Britain) doi: 10.1042/CS20120612
Decreased Nox4 levels in the myocardium
of patients with aortic valve stenosis
Marı́a U. MORENO∗ , Idoia GALLEGO∗ , Begoña LÓPEZ∗ , Arantxa GONZÁLEZ∗ , Ana FORTUÑO∗ ,
Gorka SAN JOSÉ∗ , Félix VALENCIA†, Juan José GÓMEZ-DOBLAS†, Eduardo DE TERESA†,
Ajay M. SHAH‡, Javier DÍEZ∗ § and Guillermo ZALBA∗
∗
Division of Cardiovascular Sciences, Centre for Applied Medical Research, University of Navarra, Avda. Pı́o XII 55, 31008 Pamplona, Spain
†Division of Cardiology, Vı́rgen de la Victoria University Hospital, Campus Universitario de Teatinos, s/n. 29010 Málaga, Spain
‡Cardiovascular Division, King’s College London British Heart Foundation Centre of Excellence, Denmark Hill Campus, 125 Coldharbour Lane,
London SE5 9NU, U.K.
§Department of Cardiology and Cardiac Surgery, University Clinic, University of Navarra, Avda. Pı́o XII, 36, 31008 Pamplona, Spain
Abstract
The NADPH oxidases are a key family of ROS (reactive oxygen species)-producing enzymes which may differentially
contribute to cardiac pathophysiology. Animal studies show uncertain results regarding the regulation of cardiac
Nox4 by pressure overload and no data are available on human myocardial Nox4. In the present study, we
evaluated Nox4 expression and its relationship with myocardial remodelling and LV (left ventricular) function in
patients with severe AS (aortic valve stenosis). Endomyocardial biopsies from 34 patients with AS were obtained
during aortic valve replacement surgery. LV morphology and function were assessed by echocardiography.
Myocardial samples from subjects deceased of non-CVDs (cardiovascular diseases) were analysed as controls.
Nox4 localization was evaluated by immunohistochemistry and quantified by Western blot. Myocardial capillary
density, fibrosis and cardiomyocyte dimensions and apoptosis were assessed histologically to evaluate myocardial
remodelling. Nox4 was present in samples from all subjects and expressed in cardiomyocytes, VSMCs (vascular
smooth muscle cells), endothelium and fibroblasts. Nox4 levels were reduced 5-fold in AS patients compared with
controls (P < 0.01). Nox4 levels directly correlated with cardiomyocyte cross-sectional area (r = 0.299, P<0.05) and
diameter (r = 0.406, P < 0.05) and capillary density (r = 0.389, P < 0.05), and inversely with cardiomyocyte
apoptosis (r = − 0.316, P < 0.05) in AS patients. In addition, Nox4 levels correlated with echocardiographic
parameters (LV ejection fraction: r = 0.353, P < 0.05; midwall fractional shortening: r = 0.355, P < 0.05;
deceleration time: r = − 0.345, P < 0.05) in AS patients. Nox4 is expressed in human myocardium and reduced in
AS patients. The observed associations of Nox4 with cardiomyocyte parameters and capillary density in AS patients
suggest a potential role of Nox4 deficiency in the myocardial remodelling present in the human pressure-overloaded
heart.
Key words: aortic valve stenosis, myocardial remodelling, NADPH oxidase, Nox4, pressure overload
INTRODUCTION
AS (aortic valve stenosis) is the most common valve disease
in developed countries, affecting 2–3 % of the population over
65 years, and its prevalence is likely to rise in the future because
it increases with advancing age [1]. Chronic pressure overload
in AS patients induces a structural remodelling of the LV (left
ventricular) myocardium characterized by alterations of cardiomyocytes, the extracellular matrix and the coronary microcirculation, which contributes to LV functional impairment [2,3].
Oxidative stress is implicated in the development of myocardial remodelling [4]. ROS (reactive oxygen species) contribute at
the molecular level to alter cardiac structure and function, damaging macromolecules and altering redox-sensitive signalling
pathways, thus contributing to myocardial remodelling [4,5]. Although there are multiple cardiac sources of ROS, the NADPH
oxidase family of enzymes is the only one whose primary role
is ROS generation. In general terms, they consist of a distinct
catalytic subunit (Nox1–5, Duox1–2), usually bound to the smaller p22phox subunit, and have varying regulation depending upon
Abbreviations: AngII, angiotensin II; AS, aortic valve stenosis; AU, arbitrary units; CVF, collagen volume fraction; eNOS, endothelial nitric oxide synthase; LV, left ventricular; LVEF, LV
ejection fraction; LVMI, LV mass index; Nrf2, nuclear factor-erythroid 2-related factor 2; NT-pro-BNP, amino-terminal propeptide of brain natriuretic peptide; Poldip2, polymerase
(DNA-directed) delta-interacting protein 2; ROS, reactive oxygen species; TBST, Tris-buffered saline with 1 % (v/v) Tween 20; VSMC, vascular smooth muscle cell; vWF, von Willebrand
factor.
Correspondence: Dr Maria U. Moreno (email [email protected]) or email Dr Guillermo Zalba (email [email protected]).
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291
M. U. Moreno and others
the isoform [6]. Nox4 requires the association of p22phox and is
constitutively active [6–8], although its activity is increased by
Poldip2 [polymerase (DNA-directed) delta-interacting protein 2]
in VSMCs (vascular smooth muscle cells) [9]. Several reports indicate that Nox4 predominantly generates H2 O2 [7,8]. In human
cells, there is in vitro evidence of Nox4 expression in microvascular endothelial cells [10], cardiac fibroblasts [11] and coronary
artery smooth muscle cells [12].
Regarding the modulation of Nox4 expression in experimental
cardiac pressure overload, there is no undisputable evidence.
Studies in mice subjected to suprarenal constriction show no
changes in Nox4 protein levels [13], an increase in Nox4 mRNA
levels [14] or an increase in Nox4 protein levels [15]. Models
of pressure overload by thoracic aortic constriction report an increase in cardiac Nox4 mRNA [16] or Nox4 protein levels [17].
Currently, there is no information regarding the myocardial
localization of Nox4 in humans or of changes of its levels in
pressure overload. Therefore the aim of the present study was to
evaluate the presence of Nox4 in the myocardium of patients with
pressure overload due to AS and assess the associations of Nox4
with myocardial remodelling and LV morphology and function.
MATERIALS AND METHODS
Study population
All patients gave written, informed consent to participate in the
present study. The study was carried out in accordance with the
Helsinki Declaration and the Ethics Committee of the Vı́rgen
de la Victoria University Hospital (Málaga, Spain) approved the
protocol.
The study population consisted of 34 patients with clinically
diagnosed severe isolated AS, defined in accordance with the following criteria [18]: mean transvalvular pressure gradient 40
mmHg, aortic valve area <1 cm2 or aortic valve area index <0.6
cm2 /m2 . The patients were referred for valve replacement due to
the presence of characteristic clinical symptoms (i.e. angina, syncope or heart failure manifestations) and/or LVEF (LV ejection
fraction) <50 %. Patients with cardiac valve disease other than
AS and/or history of acute myocardial infarction were excluded
after complete medical examination. Heart failure was clinically
diagnosed on the basis of the presence of at least one major and
two minor Framingham criteria [19] and was reinforced by either
echocardiographic signs of systolic dysfunction (LVEF <50 %)
or by the presence of elevated levels of the NT-pro-BNP (Nterminal propeptide of brain natriuretic peptide) and alterations
in cardiac morphology and function indicative of heart failure
with normal ejection fraction according to the European Society
of Cardiology [20].
Cardiac samples were collected from 18 subjects who had died
from non-cardiovascular-related diseases, nine of which were
processed for protein studies [six men and three women, average
age 54 (range 46–62) years] and nine processed for histomorphological studies [six men and three women, average age 59 (range
40–68) years], to be used as controls.
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Echocardiographic assessment
Two-dimensional echocardiographic imaging, targeted M-mode
recordings, and Doppler ultrasound measurements were obtained
in each AS patient. LV mass was measured from M-mode recordings according to the American Society of Echocardiography
criteria [21], and LVMI (LV mass index) was calculated by dividing LV mass by the body surface area. The presence of LV
hypertrophy was established when the LVMI was >149 g/m2 for
men and >122 g/m2 for women [22]. The presence of concentric LV hypertrophy was established when relative wall thickness
was >0.42 [22]. LV midwall fractional shortening and ejection
fraction were calculated according to the method of Quinones
et al. [23]. The following pulsed Doppler measurements were obtained: maximal early transmitral diastolic velocity, maximal late
transmitral diastolic velocity, the deceleration time of the early
mitral filling wave and the isovolumic relaxation time. Mean and
maximal transvalvular pressure gradient and aortic valve area
were assessed in all patients.
Myocardial sampling
During surgical aortic valve replacement two endomyocardial
biopsies were taken from the interventricular septum. The biopsy
procedure was well tolerated in all cases and no complications
were recorded. The first sample, for molecular studies, was divided into two pieces, immediately frozen in liquid nitrogen and
processed for mRNA and for protein extraction. The second
sample was processed for histomorphological and immunohistochemical studies. The biopsies were taken from adjacent areas,
so that they were equivalent. The average biopsy size was 21 +
−2
mm3 .
Histomorphological and immunohistochemical
studies
After formalin fixation, the biopsies were embedded in paraffin
and serially sectioned in 4-μm-thick sections. In all the cases, the
endogenous peroxidase was inactivated by 30 min incubation in
0.01 % H2 O2 in methanol. Slides were blocked in 20 % (v/v) normal goat serum (Dako) for 30 min. Primary antibodies used were
Nox4 (sc-30141 diluted 1:100; SantaCruz Biotechnology), noncommercial Nox4 [14] (diluted 1:100), Poldip2 (ab85364 diluted
1:200; Abcam) and vWF (von Willebrand factor) (A0082 diluted
1:250; Dako). Immunohistochemistry to confirm cell type was
performed in serial sections with antibodies against vWF (to
detect endothelium), α and β myosin heavy chain (to detect
cardiomyocytes) (Ab15 diluted 1:400; Abcam), smooth muscle
α-actin (to detect smooth muscle cells) (A5228 diluted 1:400
Sigma) or vimentin (to detect fibroblasts) (sc-6260 diluted 1:500;
Santa Cruz Biotechnology). Secondary antibodies were Envision
anti-rabbit or anti-mouse, as appropriate, incubated for 30 min.
Signal was detected by 3,3 -DAB (diaminobenzidine) (Dako).
Slides were counterstained with Harris haematoxylin (Sigma).
Negative controls were carried out with primary antibody
omission.
Cardiac capillarization was quantified in sections by immunohistochemistry against vWF. Images were captured at ×20 magnification with an Eclipse 80i microscope (Nikon) and the
average capillary number was calculated from six fields. Cardiac
Decreased Nox4 in human aortic stenosis
capillarization was calculated as the number of capillaries per
area (capillaries/mm2 ) and the capillary-to-cardiomyocyte ratio
(by dividing the number of capillaries present in each field by the
number of cardiomyocyte nuclei present in each field).
Collagen was identified by Picrosirius Red staining and quantified as described previously [24], which allowed us to calculate
the area occupied by collagen [CVF (collagen volume fraction)].
Cell apoptosis was assessed by TUNEL (terminal
deoxynucleotidyltransferase-mediated dUTP nick-end labelling)
as described previously [25].
Cardiomyocyte mean cross-sectional diameter and area were
assessed in Masson’s trichrome-stained sections, captured at ×40
magnification, with the AnalySIS 3.1 software (Soft Imaging
System).
Quantification of Nox4 immunostaining was carried out by
quantitative morphometry with an automated image analysis system (AnalySYS; Soft Imaging System).
Biochemical determinations
Venous blood samples were obtained in each patient from the
left antecubital vein. The NT-pro-BNP was measured in plasma
samples by an ELISA method (Biomedica Gruppe) [26].
Protein studies
Protein was extracted in lysis buffer [7 mol/l urea, 2 mol/l
thiourea, 4% (w/v) CHAPS and 1% (w/v) DTT (dithiothreitol)]
and quantified (Pierce BCA Protein Assay Kit; Thermo Scientific). Then, 10 μg of the protein homogenate were mixed with
adequate volumes of Laemmli buffer and subjected to SDS/PAGE
(10 % gel). Gels were blotted on to nitrocellulose membranes
(Hybond ECL; GE Healthcare). Membranes were then washed
in TBST [Tris-buffered saline with 1% (v/v) Tween 20] for 5 min
at room temperature (22 ◦ C) and incubated in blocking solution
[5% (w/v) non-fat dried skimmed milk powder in TBST] for 2 h
at room temperature. Afterwards, the blots were incubated with
the primary antibody in blocking solution [Nox4: sc-30141 diluted 1:1000 (Santa Cruz Biotechnology); and Poldip2: ab85364
diluted 1:1000 (Abcam)] overnight at 4 ◦ C with slow rocking.
The blots were then washed five times for 5 min in TBST and
incubated with an HRP (horseradish peroxidase)-conjugated secondary antibody in blocking solution. After washing five times for
5 min in TBST, bands were identified by chemiluminescence with
the ECL Advanced kit (GE Healthcare). The membranes were
stripped using Restore Western Blot Stripping Buffer (Pierce)
according to instructions and reblotted for α-tubulin (sc-5286 diluted 1:10000). Band intensities were quantified with the MultiAnalyst software (Bio-Rad Laboratories). Data are expressed as
AU (arbitrary units) relative to α-tubulin levels.
The specificity of the Nox4 band was confirmed with a noncommercial antibody against Nox4 (diluted 1:2000) that had been
validated using Nox4-transfected cells and Nox4-knockout mice
as positive and negative controls respectively [14,27].
Table 1 Clinical characteristics of the patients
Results are expressed as means +
− S.E.M or percentage of subjects.
V E /V A , maximal early transmitral diastolic velocity/maximal late transmitral diastolic velocity ratio.
Parameter
Patients (n = 34)
71 +
−1
Age (year)
Sex (male/female) (n)
10/24
28.2 +
− 0.9
120 +
−3
69 +
−1
Body mass index (kg/m2 )
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Hypertension (%)
71
Diabetes mellitus (%)
35
Coronary artery disease (%)
35
Heart failure (%)
56
Atrial fibrillation (%)
32
Treatment (%)
Warfarin
21
Diuretics
70
Digitalis
9
Angiotensin-converting enzyme inhibitors
18
AngII type1 receptor antagonists
15
β-Blockers
36
Aldosterone antagonists
6
Ca2 + antagonists
18
Aortic valve area (cm2 )
Aortic valve area index (cm2 /m2 )
Mean transvalvular pressure gradient (mmHg)
Maximal transvalvular pressure gradient (mmHg)
LVMI (g/m2 )
Relative wall thickness
Concentric LV hypertrophy (%)
LVEF (%)
LV midwall fractional shortening (%)
End-systolic wall stress (kdyn/cm2 )
V E /V A
Isovolumic relaxation time (ms)
Deceleration time (ms)
NT-pro-BNP (fmol/ml)
0.63 +
− 0.04
0.38 +
− 0.02
53.45 +
− 2.89
82.24 +
− 4.37
147.7 +
− 7.5
0.612 +
− 0.025
58
66.15 +
− 2.84
38.96 +
− 1.91
59.66 +
− 4.28
1.05 +
− 0.18
97.73 +
− 7.37
291.24 +
− 20.50
829 +
− 72
spectrophotometer). Retrotranscription of 1 μg of total RNA
was carried out with random hexamers and Superscript III
(Invitrogen). Quantification of the cDNA was performed by
Real time PCR with TaqMan probes (4333760F for 18S,
Applied Biosystems) and SYBR green technology (Promega)
with primers 5 -GTGGCTGTCTGCATGGACCT-3 and 5 CCACGATGGTGACTTTGGCT-3 for eNOS (endothelial nitric
oxide synthase) in an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Data are expressed as AU relative to
the 18S ribosomal RNA.
Statistical analysis
RNA studies
Extraction of total RNA was performed with TRIzol® (Invitrogen) according to the manufacturer’s instructions. RNA
quantity and quality were assessed by absorbance (Ultraspec
Results are expressed as means +
− S.E.M. Differences between
the groups were evaluated using a Student’s t test for unpaired
data or Mann–Whitney U test, as appropriate after evaluating normality (Shapiro–Wilks test). Correlations between continuously
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Figure 1
Myocardial Nox4 expression
Nox4 was detected in myocardial biopsies from controls and AS patients, with a lower intensity in the latter. (A) Myocardium from a control subject, Nox4 staining (left) and immunohistochemistry negative control (right) (×10 magnification).
(B) Myocardium from a patient with AS, Nox4 staining (left) and immunohistochemistry negative control (right) (×10
magnification).
distributed variables were tested by bivariate association and univariate and multivariate regression analysis. A P value <0.05
was considered statistically significant. Analyses were performed
with the SPSS 15.0 statistical package.
capillary-to-cardiomyocyte ratio (1.18 +
− 0.08 compared with
+ 0.15; P < 0.001). Patients showed greater CVF than con2.63 −
trols (17.33 +
− 1.90 % compared with 1.95 +
− 0.07 %; P < 0.001).
Nox4 and Poldip2 expression
RESULTS
Characteristics of patients
The clinical characteristics of patients are summarized in Table 1.
Around half of them had clinical heart failure and almost onethird exhibited atrial fibrillation.
Myocardial remodelling
Compared with controls, patients had increased cardiomyocyte cross-sectional area (498.48 +
− 95.91 compared with
2
203.09 +
− 0.44
− 17.36 μm ; P < 0.001) and diameter (26.13 +
compared with 16.61 +
− 0.69 μm; P < 0.001). The cardiomyocyte
apoptotic index was increased in patients compared with controls
(0.101 +
− 0.016 compared with 0.0045 +
− 0.0 016 %; P < 0.001).
The cardiac capillary density was reduced in patients compared with controls, whether calculated as the capillaries/mm2
(522 +
− 48 compared with 1531 +
− 78; P < 0.001) or the
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Detection of Nox4 by immunohistochemistry showed that it is
expressed in the human heart, both in controls and AS patients, although with greater stained area in controls (23.3 +
− 2.2 compared
with 9.5 +
2.5
%;
P
<
0.01)
(Figure
1).
Serial
immunohistoche−
mistry with cell-type markers confirmed the expression of Nox4
in cardiomyocytes, VSMCs, endothelial cells and fibroblasts,
both in controls and in AS patients (Figure 2). In cardiomyocytes, Nox4 staining was present in the cytoplasm and some
nuclei (Figure 3A).
Quantification in cardiac homogenates showed that Nox4 protein levels were 5-fold lower in AS patients than in controls
(0.48 +
− 0.03 compared with 2.45 +
− 0.50 AU; P < 0.01) (Figure 3B). We observed in patients that Nox4 levels were higher
in women than in men (0.50 +
− 0.04 compared with 0.34 +
− 0.02
AU; P < 0.05). However, there were no differences in Nox4
levels between patients without (n = 14, 0.437 +
− 0.057 AU)
and with (n = 20, 0.502 +
0.036
AU)
LV
hypertrophy,
between
−
patients without (n = 20, 0.508 +
0.046
AU)
and
with
heart
−
failure (n = 14, 0.428 +
0.037
AU),
between
patients
without
−
Decreased Nox4 in human aortic stenosis
Figure 2
Co-expression of Nox4 and cell-type markers
Serial immunohistochemical staining of Nox4 and cell-type markers α- and β-myosin heavy chain for cardiomyocyte (×20
magnification), vWF for endothelium (×40 magnification), α-actin for vascular smooth muscle (×40 magnification) and
vimentin for fibroblasts (×100 magnification) showed that Nox4 was expressed in these cell types in myocardial samples
from (A) controls and (B) patients with AS
(n = 23, 0.482 +
− 0.045 AU) and with (n = 11, 0.461 +
− 0.030 AU)
arrhythmias, between patients without (n = 22, 0.459 +
− 0.037
AU) and with (n = 12, 0.505 +
− 0.061 AU) coronary artery disease, between patients without (n = 10, 0.457 +
− 0.055 AU) and
with (n = 24, 0.483 +
0.039
AU)
hypertension,
or between pa−
tients without (n = 22, 0.471 +
0.039
AU)
and
with (n = 12,
−
0.482 +
0.057
AU)
diabetes.
There
were
no
differences
in Nox4
−
levels between patients taking medications interfering with the
renin–angiotensin–aldosterone system (n = 13, 0.498 +
− 0.041
AU) and those who were not taking such medication (n = 20,
0.462 +
− 0.047 AU), or between patients under statin treatment
(n = 21, 0.451 +
− 0.031 AU) and those who were not (n = 12,
0.521 +
0.072
AU).
−
In addition, we assessed Poldip2, a regulator of Nox4 activity
[9]. Immunohistochemical studies showed that it was expressed
in the human heart, in the cytoplasm and nucleus of cardiomyocytes (Figure 3C). Quantification in homogenates showed that
Poldip2 expression was 8-fold lower in AS patients than in
controls (0.29 +
− 0.02 compared with 2.33 +
− 0.51 AU; P < 0.01)
(Figure 3D).
Association studies
Nox4 levels directly correlated with the cardiomyocyte crosssectional area (Figure 4A) and diameter (Figure 4B) in AS patients. Furthermore, Nox4 levels correlated inversely with the
apoptotic index in AS patients (Figure 4C). There was a direct
correlation between Nox4 levels and the degree of capillary density expressed as the capillary-to-cardiomyocyte ratio (Figure 4D)
in AS patients. These associations were independent of age, sex
and the severity of AS (transvalvular gradient and valve area).
Nox4 levels did not correlate with the CVF (r = − 0.181, P =
not significant).
It has been proposed that Nox4 may regulate eNOS expression
and contribute to vessel generation [28,29]. We observed in AS
patients that Nox4 levels directly correlated with the mRNA levels
of eNOS (r = 0.402, P < 0.05).
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M. U. Moreno and others
Figure 3
Localization of Nox4 and Poldip2 within the myocardium
(A) Immunohistochemistry of Nox4 in patients with AS. Nox4 was detected mainly in cardiomyocyte cytoplasm and
some nuclei (arrows) (left, ×10 magnification; right, ×40 magnification). (B) Representative Western blot images and
histograms quantifying Nox4 in homogenates. Nox4 expression was lower in patients with AS than in controls. (C)
Immunohistochemistry of Poldip2 in patients with AS. Poldip2 was detected in cardiomyocyte cytoplasm, by the plasma
membrane (dotted arrows) and some nuclei (closed arrows) (left, ×10 magnification; right, ×40 magnification). (D)
Representative Western blot images and histograms quantifying Poldip2 in homogenates. Poldip2 expression was lower
in patients with AS than in controls. ∗ P<0.01.
Nox4 levels correlated with echocardiographic parameters
in AS patients. Nox4 levels directly correlated with the LVEF
(Figure 5A) and the LV midwall fractional shortening (Figure 5B)
and inversely with the deceleration time (Figure 5C). These
associations were independent of age, sex and the severity of
AS (transvalvular gradient and valve area).
Poldip2 levels did not correlate with Nox4 levels, parameters
of myocardial remodelling or parameters of cardiac function.
DISCUSSION
The main novel findings of our study are the following: (i) Nox4
is expressed in human cardiac cells; (ii) Nox4 levels are abnormally decreased in patients with severe AS; (iii) Nox4 levels are
associated with alterations in parameters assessing myocardial
structure in AS patients; and (iv) Nox4 levels are associated with
parameters of LV systolic and diastolic function in these patients.
Taken together, these results suggest that Nox4 deficiency may
contribute to the remodelling of the myocardium present in human cardiac pressure overload.
In the present study, we provide the first data on the
expression of Nox4 in the human myocardium. Regarding
its localization, we detected Nox4 expression in cardiomyocytes, as well as in VSMCs, endothelial cells and fibroblasts,
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in both controls and AS patients. These data expand previous observations in animal studies showing Nox4 expression
in cardiac cells [30]. Moreover, we observed Nox4 in both
the cytoplasm and the nucleus of cardiomyocytes, supporting data from other studies identifying Nox4 in the nuclei
[31] and cellular organelles {i.e. ER (endoplasmic reticulum
[30,32] and mitochondria [17,30]}.
Currently, there is conflicting evidence on the myocardial
changes of Nox4 in experimental pressure overload, with studies showing no changes in Nox4 protein levels [13], an increase
in Nox4 mRNA levels [14,16], or an increase in Nox4 protein
levels [15,17]. Surprisingly, our data indicate that patients with
pressure overload secondary to AS present a marked reduction of
myocardial Nox4 levels compared with controls. Nox4 seems to
be modulated by a number of factors that may also be of relevance
for myocardial remodelling in the setting of pressure overload, for
instance, AngII (angiotensin II) [30,33]. Although some authors
have shown that AngII administration decreases Nox4 expression in VSMCs [34], others failed to show any effect [35], and
yet others reported increased Nox4 expression in VSMCs [36]
and endothelial cells [37]. In this regard, no relationship was
found between Nox4 expression and the treatment with drugs
interfering with the renin–angiotensin–aldosterone system in the
patients from the present study. In addition, down-regulation of
Nox4 expression in the vasculature has been reported following
Decreased Nox4 in human aortic stenosis
Figure 4
Correlations of Nox4 with parameters assessing myocardial remodelling
Myocardial Nox4 expression correlated with (A) the cardiomyocyte cross sectional area (y = 103.6x + 434.59),
(B) the cardiomyocyte cross-sectional diameter (y = 6.6682x + 23.335), (C) the cardiomyocyte apoptotic index
(y = − 0.0988x + 0.1252) and (D) the capillary-to-cardiomyocyte ratio (y = 0.9479x + 0.7299) in patients with AS.
pathophysiological shear stress [38,39], although the potential
contribution of this factor to reduced Nox4 expression in the
myocardium of AS patients remains to be investigated.
Nox4 seems to be constitutively active [7] and thus Nox4
levels may be a good indicator of the Nox4-dependent NADPH
oxidase activity. In addition, we also evaluated the levels of
Poldip2, an enhancer of Nox4 activity [9]. We identified for the
first time that Poldip2 is expressed in human myocardium and that
Poldip2 levels were lower in AS patients than in controls, thus
suggesting that besides reduced Nox4 availability, the activity of
the enzyme dependent on this factor may also be reduced in the
myocardium of AS patients.
Several findings reported in the present study deal with the
potential consequences of Nox4 deficiency. On the one hand, we
find an association between reduced Nox4 levels and decreased
capillarization in patients with AS. This mirrors the data from
Zhang et al. [14] showing that cardiac Nox4 overexpression is
associated with increased capillary density in mice subjected to
pressure overload. Craige et al. [28] demonstrated that an augmented endothelial Nox4 expression promotes angiogenesis in
an eNOS-dependent manner, which was recently corroborated
by Schröder et al. [29] in a study on the role of endogenous Nox4
during ischaemic or AngII-induced stress. In this regard, we have
detected in AS patients an association between reduced Nox4
levels and decreased eNOS mRNA expression. Thus, it can be
hypothesized that Nox4 deficiency contributes to diminished capillarization via reduced eNOS availability among other mechanisms in the myocardium of AS patients. On the other hand,
we detect an association between reduced Nox4 levels and reduced cardiomyocyte hypertrophy and increased cardiomyocyte
apoptosis. It is known that H2 O2 may determine cardiomyocyte
fate, either death (apoptosis) or survival (hypertrophy), in a dosedependent manner [40–42], with the involvement of Nrf2 (nuclear factor-erythroid 2-related factor 2) and antioxidants [41,43].
Interestingly, Brewer et al. [44] in cardiomyocytes and Schröder
et al. [29] in blood vessels, showed that Nox4 contributes to the
antioxidant defences via the Nrf2 transcription factor, supporting
a beneficial role of Nox4. In this conceptual framework, it can be
suggested that reduced Nox4 may contribute to the cardiomyocyte alterations seen in AS patients.
Some considerations suggest that the association of Nox4 with
myocardial remodelling reported in the present study may be of
potential pathophysiological relevance. First, there is evidence of
reduced capillarization in AS [45] and of its negative impact on
cardiac function [46,47]. Similarly, there is evidence of increased
apoptosis in AS, which is associated with the preoperative functional class and valve-replacement postoperative outcome [48].
These findings fit well with our results showing that Nox4 levels
are associated with parameters of LV systolic and diastolic function, suggesting that reduced Nox4 levels may contribute to the
functional alterations of the left ventricle in AS patients.
Several limitations of our study must be acknowledged. First,
this is a clinical study involving relatively small amounts of tissue obtained from patients, making it difficult to undertake an
extensive range of analyses as would be possible in an animalbased study. Secondly, we studied AS patients at the time of
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M. U. Moreno and others
r
r
In the present study, we provide novel information on cardiac
Nox4 in humans. In particular, our findings suggest that a
decrease in Nox4 levels is associated with myocardial remodelling in patients with severe AS.
Although descriptive in nature, these findings set the stage for
future investigations to assess the role of Nox4 in the development and progression of myocardial remodelling in human
cardiac pressure overload, as well as to explore its modulation as a potential therapeutic target to prevent myocardial
remodelling.
AUTHOR CONTRIBUTION
Marı́a Moreno, Javier Dı́ez, Guillermo Zalba conceived, designed,
analysed and interpreted the data, drafted and revised the paper
critically for important intellectual content, and gave final approval.
Idoia Gallego, Begoña López and Arantxa González conceived, designed, analysed and interpreted the data, and revised the paper
critically for important intellectual content and gave final approval.
Ana Fortuño, Gorka San José, Félix Valencia, Juan Gómez-Doblas,
Eduardo de Teresa and Ajay Shah revised the paper critically for
important intellectual content and gave final approval.
ACKNOWLEDGEMENTS
We gratefully acknowledge the technical assistance of Idoia
Rodrı́guez, Ana Montoya, Sonia Martı́nez and Marı́a González.
FUNDING
Figure 5 Correlation of Nox4 with cardiac function
Myocardial Nox4 expression correlated with (A) ejection fraction (y =
31.769x + 51.056), (B) midwall fractional shortening (y = 20.499x
+ 29.079) and (C) deceleration time (y = − 1.5546x + 3.5834) in patients with AS.
valve replacement, hence we lack information regarding the progression of the disease. Thus, although novel and valuable, the
information provided in the present study regarding Nox4 expression is rather a snapshot of the late state of the valve disease. In
addition, the study involved a relatively small number of patients,
but because of the nature of the aims of the study, the design was
appropriate. Moreover, we are aware that patients were under
treatment, which may influence the results, although Nox4 levels
did not seem to be influenced by therapy. Finally, as there was
no material available for mRNA studies in controls, we could not
evaluate if the decrease in Nox4 levels in AS patients was due to
decreased Nox4 transcription.
This study was supported through the agreement between the
Foundation for Applied Medical Research and ‘UTE project CIMA’,
the Ministry of Economy and Competitiveness [grant numbers RECAVA RD06/0014/0008, SAF-2010-20367, RYC-2010-05797 (to
A.G.)], the European Union [MEDIA project grant HEALTH-F2-2010261409], the British Heart Foundation and a Foundation Leducq
Transatlantic Network of Excellence award (to A.M.S.).
REFERENCES
1
2
3
CLINICAL PERSPECTIVES
r
298
NADPH oxidases are a key family of ROS-producing enzymes, but there is currently no information regarding the
presence of Nox4 in the myocardium of patients with pressure
overload due to aortic valve stenosis (AS) or changes in its
levels during pressure overload.
C The Authors Journal compilation C 2013 Biochemical Society
4
5
Lindroos, M., Kupari, M., Heikkila, J. and Tilvis, R. (1993)
Prevalence of aortic valve abnormalities in the elderly: an
echocardiographic study of a random population sample. J. Am.
Coll. Cardiol. 21, 1220–1225
Hein, S., Arnon, E., Kostin, S., Schonburg, M., Elsasser, A.,
Polyakova, V., Bauer, E. P., Klovekorn, W. P. and Schaper, J. (2003)
Progression from compensated hypertrophy to failure in the
pressure-overloaded human heart: structural deterioration and
compensatory mechanisms. Circulation 107, 984–991
Schwartzkopff, B., Frenzel, H., Dieckerhoff, J., Betz, P., Flasshove,
M., Schulte, H. D., Mundhenke, M., Motz, W. and Strauer, B. E.
(1992) Morphometric investigation of human myocardium in
arterial hypertension and valvular aortic stenosis. Eur. Heart J.
13, 17–23
Seddon, M., Looi, Y. H. and Shah, A. M. (2007) Oxidative stress
and redox signalling in cardiac hypertrophy and heart failure.
Heart 93, 903–907
Takimoto, E. and Kass, D. A. (2007) Role of oxidative stress in
cardiac hypertrophy and remodeling. Hypertension 49, 241–248
Decreased Nox4 in human aortic stenosis
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Bedard, K. and Krause, K. H. (2007) The NOX family of
ROS-generating NADPH oxidases: physiology and
pathophysiology. Physiol. Rev. 87, 245–313
Nisimoto, Y., Jackson, H. M., Ogawa, H., Kawahara, T. and
Lambeth, J. D. (2010) Constitutive NADPH-dependent electron
transferase activity of the Nox4 dehydrogenase domain.
Biochemistry 49, 2433–2442
Takac, I., Schroder, K., Zhang, L., Lardy, B., Anilkumar, N.,
Lambeth, J. D., Shah, A. M., Morel, F. and Brandes, R. P. (2011)
The E-loop is involved in hydrogen peroxide formation by the
NADPH oxidase Nox4. J. Biol. Chem. 286, 13304–13313
Lyle, A. N., Deshpande, N. N., Taniyama, Y., Seidel-Rogol, B.,
Pounkova, L., Du, P., Papaharalambus, C., Lassegue, B. and
Griendling, K. K. (2009) Poldip2, a novel regulator of Nox4 and
cytoskeletal integrity in vascular smooth muscle cells. Circ. Res.
105, 249–259
Montezano, A. C., Burger, D., Paravicini, T. M., Chignalia, A. Z.,
Yusuf, H., Almasri, M., He, Y., Callera, G. E., He, G., Krause,
K. H. et al. (2010) Nicotinamide adenine dinucleotide phosphate
reduced oxidase 5 (Nox5) regulation by angiotensin II and
endothelin-1 is mediated via calcium/calmodulin-dependent,
rac-1-independent pathways in human endothelial cells. Circ.
Res. 106, 1363–1373
Cucoranu, I., Clempus, R., Dikalova, A., Phelan, P. J., Ariyan, S.,
Dikalov, S. and Sorescu, D. (2005) NAD(P)H oxidase 4 mediates
transforming growth factor-beta1-induced differentiation of
cardiac fibroblasts into myofibroblasts. Circ. Res. 97, 900–907
Sorescu, D., Weiss, D., Lassegue, B., Clempus, R. E., Szocs, K.,
Sorescu, G. P., Valppu, L., Quinn, M. T., Lambeth, J. D., Vega, J. D.
et al. (2002) Superoxide production and expression of nox family
proteins in human atherosclerosis. Circulation 105, 1429–1435
Byrne, J. A., Grieve, D. J., Bendall, J. K., Li, J. M., Gove, C.,
Lambeth, J. D., Cave, A. C. and Shah, A. M. (2003) Contrasting
roles of NADPH oxidase isoforms in pressure-overload versus
angiotensin II-induced cardiac hypertrophy. Circ. Res. 93,
802–805
Zhang, M., Brewer, A. C., Schroder, K., Santos, C. X., Grieve,
D. J., Wang, M., Anilkumar, N., Yu, B., Dong, X., Walker, S. J.
et al. (2010) NADPH oxidase-4 mediates protection against
chronic load-induced stress in mouse hearts by enhancing
angiogenesis. Proc. Natl. Acad. Sci. U.S.A. 107, 18121–18126
Henderson, B. C., Sen, U., Reynolds, C., Moshal, K. S.,
Ovechkin, A., Tyagi, N., Kartha, G. K., Rodriguez, W. E. and Tyagi,
S. C. (2007) Reversal of systemic hypertension-associated
cardiac remodeling in chronic pressure overload myocardium by
ciglitazone. Int. J. Biol. Sci. 3, 385–392
Mohammed, S. F., Ohtani, T., Korinek, J., Lam, C. S., Larsen, K.,
Simari, R. D., Valencik, M. L., Burnett, Jr, J. C. and Redfield, M.
M. (2010) Mineralocorticoid accelerates transition to heart
failure with preserved ejection fraction via ‘nongenomic effects’.
Circulation 122, 370–378
Ago, T., Kuroda, J., Pain, J., Fu, C., Li, H. and Sadoshima, J.
(2010) Upregulation of Nox4 by hypertrophic stimuli promotes
apoptosis and mitochondrial dysfunction in cardiac myocytes.
Circ. Res. 106, 1253–1264
Bonow, R. O., Carabello, B. A., Chatterjee, K., de Leon, Jr, A. C.,
Faxon, D. P., Freed, M. D., Gaasch, W. H., Lytle, B. W., Nishimura,
R. A., O’Gara, P. T. et al. (2008) Focused update incorporated into
the ACC/AHA 2006 guidelines for the management of patients
with valvular heart disease: a report of the American College of
Cardiology/American Heart Association Task Force on Practice
Guidelines (Writing Committee to Revise the 1998 Guidelines for
the Management of Patients with Valvular Heart Disease):
endorsed by the Society of Cardiovascular Anesthesiologists,
Society for Cardiovascular Angiography and Interventions, and
Society of Thoracic Surgeons. Circulation 118, e523–e661
Ho, K. K., Pinsky, J. L., Kannel, W. B. and Levy, D. (1993) The
epidemiology of heart failure: the Framingham Study. J. Am. Coll.
Cardiol. 22, 6A–13A
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Paulus, W. J., Tschope, C., Sanderson, J. E., Rusconi, C.,
Flachskampf, F. A., Rademakers, F. E., Marino, P., Smiseth, O. A.,
De Keulenaer, G., Leite-Moreira, A. F. et al. (2007) How to
diagnose diastolic heart failure: a consensus statement on the
diagnosis of heart failure with normal left ventricular ejection
fraction by the Heart Failure and Echocardiography Associations
of the European Society of Cardiology. Eur. Heart J. 28,
2539–2550
Sahn, D. J., DeMaria, A., Kisslo, J. and Weyman, A. (1978)
Recommendations regarding quantitation in M-mode
echocardiography: results of a survey of echocardiographic
measurements. Circulation 58, 1072–1083
Lang, R. M., Bierig, M., Devereux, R. B., Flachskampf, F. A.,
Foster, E., Pellikka, P. A., Picard, M. H., Roman, M. J., Seward, J.,
Shanewise, J. et al. (2006) Recommendations for chamber
quantification. Eur. J. Echocardiogr. 7, 79–108
Quinones, M. A., Pickering, E. and Alexander, J. K. (1978)
Percentage of shortening of the echocardiographic left
ventricular dimension. Its use in determining ejection fraction
and stroke volume. Chest 74, 59–65
López, B., González, A., Querejeta, R., Larman, M. and Dı́ez, J.
(2006) Alterations in the pattern of collagen deposition may
contribute to the deterioration of systolic function in hypertensive
patients with heart failure. J. Am. Coll. Cardiol. 48, 89–96
González, A., Lóez, B., Ravassa, S., Querejeta, R., Larman, M.,
Dı́ez, J. and Fortuño, M. A. (2002) Stimulation of cardiac
apoptosis in essential hypertension: potential role of angiotensin
II. Hypertension 39, 75–80
González, A., López, B., Querejeta, R., Zubillaga, E., Echeverrı́a,
T. and Dı́ez, J. (2010) Filling pressures and collagen metabolism
in hypertensive patients with heart failure and normal ejection
fraction. Hypertension 55, 1418–1424
Anilkumar, N., Weber, R., Zhang, M., Brewer, A. and Shah, A. M.
(2008) Nox4 and Nox2 NADPH oxidases mediate distinct cellular
redox signaling responses to agonist stimulation. Arterioscler.
Thromb. Vasc. Biol. 28, 1347–1354
Craige, S. M., Chen, K., Pei, Y., Li, C., Huang, X., Chen, C.,
Shibata, R., Sato, K., Walsh, K. and Keaney, Jr, J. F. (2011)
NADPH Oxidase 4 promotes endothelial angiogenesis through
endothelial nitric oxide synthase activation. Circulation 124,
731–740
Schröder, K., Zhang, M., Benkhoff, S., Mieth, A., Pliquett, R.,
Kosowski, J., Kruse, C., Ludike, P., Michaelis, U. R., Weissmann,
N. et al. (2012) Nox4 is a protective reactive oxygen species
generating vascular NADPH oxidase. Circ. Res. 110, 1217–1225
Lassegue, B., San Martin, A. and Griendling, K. K. (2012)
Biochemistry, physiology, and pathophysiology of NADPH
oxidases in the cardiovascular system. Circ. Res. 110,
1364–1390
Kuroda, J., Nakagawa, K., Yamasaki, T., Nakamura, K., Takeya,
R., Kuribayashi, F., Imajoh-Ohmi, S., Igarashi, K., Shibata, Y.,
Sueishi, K. et al. (2005) The superoxide-producing NAD(P)H
oxidase Nox4 in the nucleus of human vascular endothelial cells.
Genes Cells 10, 1139–1151
Zhang, L., Nguyen, M. V., Lardy, B., Jesaitis, A. J., Grichine, A.,
Rousset, F., Talbot, M., Paclet, M. H., Qian, G. and Morel, F.
(2011) New insight into the Nox4 subcellular localization in
HEK293 cells: first monoclonal antibodies against Nox4.
Biochimie 93, 457–468
Yamazaki, T. and Yazaki, Y. (1999) Role of tissue angiotensin II in
myocardial remodelling induced by mechanical stress. J. Hum.
Hypertens. 13 (Suppl. 1), S43–S47
Lassegue, B., Sorescu, D., Szocs, K., Yin, Q., Akers, M., Zhang,
Y., Grant, S. L., Lambeth, J. D. and Griendling, K. K. (2001) Novel
gp91phox homologues in vascular smooth muscle cells: nox1
mediates angiotensin II-induced superoxide formation and
redox-sensitive signaling pathways. Circ. Res. 88, 888–894
www.clinsci.org
299
M. U. Moreno and others
35
36
37
38
39
40
41
Wosniak, Jr, J., Santos, C. X., Kowaltowski, A. J. and Laurindo, F.
R. (2009) Cross-talk between mitochondria and NADPH oxidase:
effects of mild mitochondrial dysfunction on angiotensin
II-mediated increase in Nox isoform expression and activity in
vascular smooth muscle cells. Antioxid. Redox Signaling 11,
1265–1278
Wingler, K., Wunsch, S., Kreutz, R., Rothermund, L., Paul, M. and
Schmidt, H. H. (2001) Upregulation of the vascular
NAD(P)H-oxidase isoforms Nox1 and Nox4 by the
renin-angiotensin system in vitro and in vivo. Free Radical Biol.
Med. 31, 1456–1464
Alvarez, E., Rodino-Janeiro, B. K., Ucieda-Somoza, R. and
Gonzalez-Juanatey, J. R. (2010) Pravastatin counteracts
angiotensin II-induced upregulation and activation of NADPH
oxidase at plasma membrane of human endothelial cells. J.
Cardiovasc. Pharmacol. 55, 203–212
Hwang, J., Ing, M. H., Salazar, A., Lassegue, B., Griendling, K.,
Navab, M., Sevanian, A. and Hsiai, T. K. (2003) Pulsatile versus
oscillatory shear stress regulates NADPH oxidase subunit
expression: implication for native LDL oxidation. Circ. Res. 93,
1225–1232
Goettsch, C., Goettsch, W., Arsov, A., Hofbauer, L. C., Bornstein,
S. R. and Morawietz, H. (2009) Long-term cyclic strain
downregulates endothelial Nox4. Antioxid. Redox Signaling 11,
2385–2397
Seenarain, V., Viola, H. M., Ravenscroft, G., Casey, T. M.,
Lipscombe, R. J., Ingley, E., Laing, N. G., Bringans, S. D. and
Hool, L. C. (2010) Evidence of altered guinea pig ventricular
cardiomyocyte protein expression and growth in response to a
5 min in vitro exposure to H2 O2 . J. Proteome Res. 9, 1985–1994
Oyama, K., Takahashi, K. and Sakurai, K. (2009) Cardiomyocyte
H9c2 cells exhibit differential sensitivity to intracellular reactive
oxygen species generation with regard to their hypertrophic vs
death responses to exogenously added hydrogen peroxide. J.
Clin. Biochem. Nutr. 45, 361–369
42
43
44
45
46
47
48
Kwon, S. H., Pimentel, D. R., Remondino, A., Sawyer, D. B. and
Colucci, W. S. (2003) H2 O0 regulates cardiac myocyte
phenotype via concentration-dependent activation of
distinct kinase pathways. J. Mol. Cell. Cardiol. 35,
615–621
Gupta, M. K., Neelakantan, T. V., Sanghamitra, M., Tyagi, R. K.,
Dinda, A., Maulik, S., Mukhopadhyay, C. K. and Goswami, S. K.
(2006) An assessment of the role of reactive oxygen species
and redox signaling in norepinephrine-induced apoptosis and
hypertrophy of H9c2 cardiac myoblasts. Antioxid. Redox
Signaling 8, 1081–1093
Brewer, A. C., Murray, T. V., Arno, M., Zhang, M., Anilkumar, N. P.,
Mann, G. E. and Shah, A. M. (2011) Nox4 regulates Nrf2 and
glutathione redox in cardiomyocytes in vivo. Free Radical Biol.
Med. 51, 205–215
Rakusan, K., Flanagan, M. F., Geva, T., Southern, J. and Van
Praagh, R. (1992) Morphometry of human coronary capillaries
during normal growth and the effect of age in left ventricular
pressure-overload hypertrophy. Circulation 86, 38–46
Rajappan, K., Rimoldi, O. E., Dutka, D. P., Ariff, B., Pennell, D. J.,
Sheridan, D. J. and Camici, P. G. (2002) Mechanisms of coronary
microcirculatory dysfunction in patients with aortic stenosis and
angiographically normal coronary arteries. Circulation 105,
470–476
Tsagalou, E. P., Anastasiou-Nana, M., Agapitos, E., Gika, A.,
Drakos, S. G., Terrovitis, J. V., Ntalianis, A. and Nanas, J. N.
(2008) Depressed coronary flow reserve is associated with
decreased myocardial capillary density in patients with heart
failure due to idiopathic dilated cardiomyopathy. J. Am. Coll.
Cardiol. 52, 1391–1398
Gaudino, M., Anselmi, A., Abbate, A., Galiuto, L., Luciani, N.,
Glieca, F. and Possati, G. (2007) Myocardial apoptosis predicts
postoperative course after aortic valve replacement in patients
with severe left ventricular hypertrophy. J. Heart Valve Dis. 16,
344–348
Received 13 November 2012/27 March 2013; accepted 4 April 2013
Published as Immediate Publication 4 April 2013, doi: 10.1042/CS20120612
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