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Introduction
3-Hydroxy-3-methylglutaryl-coenzyme A reductase
inhibitors, or statins, are widely used clinically as a lipid
lowering agent. Many studies have demonstrated that
statins prevent the development of heart failure in ischemic
heart disease or after myocardial
infarction[1_3], but those effects of statins are always considered beneficial because
of its role on atherosclerotic plaque, such as decreasing
plasma cholesterol levels, inhibiting platelet activation,
stabilizing plaque, and preventing cell proliferation and
migration. However whether statins also inhibit heart failure
in non-ischemic heart disease and its mechanism for it is still
controversial. Recently, some clinical and basic studies
indicated that statins improved cardiac function and survival
in non-ischemic heart failure[4_8]. Concerns have also been
raised about the potential adverse effects of statins on heart
failure, such as decreased serum cholesterol, which causes
the worst outcomes in heart
failure[9] and reduced ubiquinone (coenzyme
Q-10)[10_12], which may adversely affect
mitochondrial and cardiac muscle function.
Matrix metalloproteinases (MMP) are a family of
zinc-dependent enzymes that play an important role in the
degradation of extracellular matrixes. Changes in expression and
activity of several MMP have been identified in failing
myocardium, and pharmacological inhibition of some MMP
have resulted in the suppression of heart
failure[13_17]. However, MMP species are not uniformly upregulated in
heart failure; it is dependent on different heart failure
etiologies and different stimulus. For example, MMP-2 and 3 were
only upregulated in human non-ischemic dilated
cardiomyopathy (DCM); MMP-9 was upregulated while MMP-1 was
downregulated in both non-ischemic and ischemic
DCM[18].
Some animal strains and heart failure models have been
used to demonstrate the mechanisms of statins on heart
failure, such as spontaneously hypertensive rats, Dahl
salt-sensitive rats[19], the aortic stenosis model, and the
rapid-pacing-induced model. However, all of the models have their
shortcomings and some do not closely mimic the alterations
observed clinically in most patients. In the present study,
we planned to use the arteriovenous (AV) fistula-induced
heart failure model, a well-established
and thoroughly characterized model of volume-overload in rats, to evaluate the
effect of atorvastatin on heart failure and its role on
myocardial MMP-2 and 9 expression and activity.
Materials and methods
Surgical preparation and experimental protocol
This study was approved by our institutional animal research
committee (Zhejiang University, Hangzhou, China) and
conformed to the Guide for the Care and Use of Laboratory
Animals published by the United States National Institute of
Health (NIH publication, No 85-23, revised 1996). Four-week
old, male Sprague-Dawley rats, weighing 70 g, were obtained
from the Medical Laboratory Animal Center (Zhejiang
University, China). Chronic volume overload was induced
using an infrarenal AV fistula model. A
ventral abdominal laparotomy was performed to expose a 20.0 mm
portion of the aorta and inferior vena cava at a level -3.0 mm below
the renal arteries. Both vessels were occluded with finger
pressure above and below the fistula site, and an 18 gauge
short-bevel needle was passed through the
exposed abdominal aorta and advanced into the vena cava. The rats were randomly
divided into groups as follows: controls (CON), untreated
fistula rats (FIS), treated fistula rats (ATO) and treated with 3
mg·kg-1·d-1 a dosage of atorvastatin. The dosage we chose
was based on some of our pilot studies and previous
published research[20]. The atorvastatin was dissolved in 0.9%
saline and administered by daily gavages. The rats were
weighed before the initial dosing and weekly thereafter
during treatment to ensure constant dosing. Treated fistula rats
received the drug beginning 1 week after fistula and until the
completion of the study at 18 weeks after surgery.
Echo study The rats were anesthetized with ketamine
HCl (50 mg/kg), and transthoracic echocardiography was
conducted at 18 weeks. All rats with the HP Sonos 100
(Hewlett-Packard Co, Stockton, CA, USA) with a 10 MHz
imaging linear scan probe transducer. The heart was imaged
at the level of the papillary muscles to obtain left ventricle
(LV) wall thickness and fractional shortening. Three beats
were averaged for each measurement.
We determined the LV end diastolic diameter (LVEDd) as
the widest and the end systolic diameter (LVEDs) as the
narrowest dimension in the M-mode recordings. LV fractioning shortening (FS) was calculated according to the
following formula:
LV FS (%)=(LVEDd_LVEDs)/LVEDd×100
Zymography of MMP activity LV myocardial samples
were homogenized in 2 mL of an ice-cold extraction buffer
containing cacodylic acid (10 mmol/L), NaCl (0.15 mol/L),
ZnCl (20 mmol/L), NaN3 (1.5 mmol/L), and 0.01% Triton
X-100 (pH 5.0). The homogenate was then centrifuged (4 °C, 10
min, 12 000×g) and the supernatant was saved and stored at
-80 °C.
The myocardial extracts were loaded onto SDS-PAGE gel
containing 1 mg/mL gelatin. The gelatin was stirred and
heated at 50 °C for 1 h before adding to the gel. The
myocardial extracts, at a final protein content of 4 µg, were loaded
onto the gels using a 3:1 sample buffer (10% SDS, 4% sucrose,
0.25 mol/L Tris-HCl, and 0.1% bromophenol blue, pH 6.8).
The gels were run at 20 mA/gel, maintaining a running buffer
temperature of 4 °C. After that, the gels were washed twice
with 2.5% Triton X-100 for 30 min each on ice and incubated
with substrate buffer (50 mmol/L Tris-Cl,
5 mmol/L CaCl2,
0.02% NaN3, and 1% Triton X-100, pH 8) at 37 °C for 18 h.
After incubation, the gels were stained with 0.05% Brilliant
Blue R-250 (Sigma, St Louis, MO, USA), destained with 10%
acetic acid and 20% methanol (v/v), and
digitized. The gelatinolytic activity was tested as specific MMP activity
by adding EDTA and phenylmethylsulphonyl fluoride PMSF.
EDTA inhibited gelatinolytic activity, but PMSF did not.
Immunoblot analysis Proteins were isolated from the LV
tissue, and 50 µg of total proteins were analyzed on 10%
SDS-PAGE under the reducing condition. The proteins were
blotted onto Polyvinylidene fluoride PVDF membranes and
incubated in 5% non-fat milk in PBS for at least 1 h. After
incubation, the PVDF membranes were subjected to
immuno-blot analysis with rabbit polyclonal antibodies to MMP-2 or
MMP-9 (1:200 dilutions; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) or actin (1:400 dilutions, Santa Cruz
Biote-chnology, USA) Immune complexes were detected with
appropriate horseradish peroxidase-conjugated secondary
antibodies and quantitated with Quantity One Image
software (Bio-Rad, Hercules, CA, USA).
RNA isolation and RT-PCR analysis Total RNA was
extracted from the LV tissue using TRIzol reagent. Briefly,
total RNA (2 µg) was converted to single stranded cDNA
using a reverse transcription system (Promega, Madison,
WI, USA). The target cDNA was amplified using the
following sense primer and antisense primers for rat brain
natriuretic peptide (BNP). Sense: 5'-GGA AAT GGC TCA GAG
ACA GCT C-3'; antisense: 5'-AAG TCT CTC CTG GAT CCG
GAA G-3'. For GAPDH, sense: 5'-AAG GTC GGA GTC AAC
GGA TTT-3'; and antisense: 5'-AGA TGA TGA CCC TTT TGG CTC-3'. The amplification cycles were 95 °C for 1 min,
55 °C for 1 min, and 72 °C for 1 min. After 30 cycles, the PCR
products were separated by electrophoresis on 1.8%
agarose (106 bp for BNP and 352 bp for GADPH).
Morphological measurement The LV was fixed with 4%
paraformaldehyde, embedded in paraffin, sectioned at a
thickness of 6 μm, and stained with hematoxylin-eosin and van
Gieson staining. The degree of collagen fiber accumulation
was quantified blindly on 6 sections per animal (12 randomly
selected fields per section), and the ratio of the van Gieson
staining fibrosis area to the total myocardium area was
calculated.
Statistical analysis Statistical analyses were performed
with SPSS 10.0 software (SPSS Inc, Chicago, IL, USA). All
grouped data were expressed as mean±SD.
Grouped data comparisons were made by one-way ANOVA followed by
Bonferroni post-hoc testing. A P value of <0.05 was
considered statistically significant.
Results
Left ventricular dilation and hypertrophy
The FIS rats developed remarkable left ventricular dilation and
hypertrophy compared with the CON rats at 18 weeks. LVEDd, LVEDs,
and heart weight HW/body weight (BW) increased
significantly in the FIS rats compared with the CON rats. FS in the
FIS rats (41.4%±4.5%) decreased significantly compared with
the CON rats (55.1%±4.5%, P<0.01). Atorvastatin treatment
suppressed left ventricular dilation and hypertrophy
significantly. LVEDd, LVEDs, and HW/BW in the ATO rats
decreased remarkably compared with the FIS rats.
Atorvas-tatin treatment also improved FS from 41.4%±4.5% to
52.7%±4.2% (P<0.01; Table 1).
Marked left ventricular dilation and interstitial fibrosis
were observed in the FIS group compared with the CON
group. Atorvastatin treatment inhibited the left ventricular
dilation and interstitial fibrosis significantly (Figures 1, 2).
Atorvastatin reduced the ratio of fibrosis from 8.6%±1.4% in
the FIS group to 6.4%±1.2% in the ATO group
(P<0.05; Figure 2B).
Left ventricular BNP mRNA level The mRNA levels of
BNP increased about 2-fold in the FIS group compared with
the CON group. Atorvastatin treatment reduced the mRNA
levels of BNP significantly, and the ratio of BNP/GAPDH in
the FIS group decreased from 0.43±0.03 to 0.27±0.03 in the
ATO group (P<0.05, Figure 3A, 3B).
Effect of atorvastatin on myocardial MMP-9 and MMP-2
protein level and activity In the FIS rats, the MMP-9 protein
level at 18 weeks after surgery was significant higher
compared with the CON rats. Although the protein level of
MMP-9 in the ATO rats was still significantly higher than
the CON group, treatment with atorvastatin 17 week after
surgery markedly inhibited the MMP-9 protein expression
compared with the FIS rats (P<0.01, Figure 4A, 4B).
Ator-vastatin treatment slightly, but still significantly, decreased
the protein level of MMP-2 in the ATO rats compared with
the FIS rats (P<0.05; Figure 4A, 4B). There were no
significantly differences in the MMP-2 protein level between the
ATO rats and the CON rats. Changes of MMP-9 and
MMP-2 activity measured by zymography were consistent with the
changes of MMP-9 and MMP-2 protein levels (Figure 5).
Discussion
The rats with an AV fistula can develop heart failure with
normal sodium balance. This heart failure experimental model
is characterized by the hemodynamic and neurohormonal
changes, which closely mimic the alterations observed
clinically in patients with heart
failure[21,22]. Previous reports indicated that cardiac hypertrophy occurred within 1 week
after AV fistula operation, while decompensate heart failure
developed 8_16 weeks after the creation of AV fistula,
characterized by circulatory congestion and decreased cardiac
function[23]. The present study demonstrates that long-term
administration of atorvastatin could prevent
volume-overload-induced heart failure and left ventricular hypertrophy.
More and more research has indicated that statins have
therapeutic properties that are of potential benefit to heart failure
with non-ischemic etiologies, irrespective of lipid levels.
Rainer et al[24] demonstrated that simvastatin normalized
autonomic neural control and reduced plasma norepinephrine
in rapid-pacing-induced heart failure rabbits. Chen
et al[20] demonstrated that simvastatin, initiated after hypertrophy,
inhibited oxidative stress and prevented heart failure in rats
with aortic stenosis. Inflammatory cytokines may also be
involved in the role of statins on heart failure. Inflammatory
cytokines, which are produced by activated macrophages,
vascular wall cells, and cardiac myocytes, were elevated in
heart failure. These cytokines, especially TNF-α and
inter-leukin-6 (IL-6), exert negative inotropic effects and induce
apoptosis in cardiac myocytes. Short-term simvastatin
therapy has been tested to improve cardiac function and
symptoms in patients with idiopathic dilated
cardiomyopathy by reducing plasma concentrations of
TNF-α and IL-6[4]. Failing myocardium of patients with DCM was characterized
by upregulation of NADPH oxidase-mediated ROS release
associated with increased Rac1 activity. Oral atorvastatin or
pravastatin treatment could inhibit myocardial Rac1-GTPase
activity[5]. These data suggest that antioxidative effects of
statins may also be beneficial for heart failure. Other
mechanisms of statins, such as promoting
angiogenesis[25], increasing endothelial NO production, improving endothelial
function and endothelial progenitor cell
mobilization[3], have been demonstrated to take effect in heart failure after myocardial
infarction, but whether these effects are also beneficial to
heart failure with non-ischemia etiologies is still unknown.
Interstitial collagenases (such as MMP-1 and MMP-13),
stromelysins (such as MMP-3), and gelatinases (such as
MMP-2 and MMP-9) are expressed in mammalian myo-cardium. The relationship between MMP and the LV
remodeling process has been demonstrated through the use of
animal models of developing chronic heart failure, transgenic
models, and the use of pharmacological MMP inhibition
studies. Clinical research has also shown that MMP are
involved in the process of LV remodeling and heart failure.
The Framingham Heart Study demonstrated that plasma
MMP-9 levels were associated with increased LV diastolic
dimensions and increased wall thickness in
men[26]. Levels of plasma MMP-2 were tested for an association with
neurohormonal activation and levels of
noradrenaline in human heart
failure[27]. In ischemic heart failure, MMP-2 and
MMP-13 levels increased, and statins were demonstrated to
ameliorate ventricular remolding through inhibiting
them[28]. Fibrosis during the progression of heart failure was also
associated with increased MMP[29]. However, the types of
MMP expressed in normal and congestive heart failure states
remains unclear. The molecular basis for a selective
portfolio of MMP to be increased within the failing human
myocardium is likely due to the type, degree, and duration of the
specific extracellular stimuli that are presented. Statins have
been claimed to inhibit MMP in
atherosclerosis[30_32], but its role on MMP in non-ischemic heart failure is not clear. This
study found that atorvastatin prevented heart failure and LV
hypertrophy, and these effects were associated with
decreas-ed MMP-2 and MMP-9 protein expression and activity.
Several possible mechanisms may be involved in the role of
statins on the MMP system. Decreasing the secretion of
inflammatory factor by statins is one of the possible
mechanisms. For example, TNF-α, an important MMP
regulator which activates MMP through the MAPK, NF-kappaB,
and AP-1 pathways[33_35], may be involved in the role of statins
on MMP. Turner NA et al[36] also demonstrated that
simvastatin reduces MMP-9 secretion from human
saphenous vein, smooth muscle cells by inhibiting isoprenoid
formation and RhoA kinase. But whether it is the same in
myocardium is unknown. More research needs to be done about
the role of statins on different MMP species in heart failure.
In summery, our study demonstrated that atorvastatin
may be beneficial for non-ischemic heart failure and may
inhibit MMP-2 and 9 expression and activity.
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