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Introduction
Intermittent hypoxia, or periodic exposure to hypoxia interrupted
by a return to normoxia or less hypoxic conditions, is encountered
more frequently in life than sustained hypoxia[1, 2].
Many studies have shown that intermittent hypoxia adaptation might
have cardioprotective effects similar to those observed in ischemic
preconditioning (IPC)[3-6]. To date, several potential
factors have been proposed to be involved in the protective mechanisms
afforded by IH[4-10]; however, the precise mechanisms
in which IH increases resistance to myocardial ischemia remain far
from clear.
Beall et al reported that exhalation of nitric oxide (NO)
by chronically hypoxic populations of Tibetans and Bolivian Aymara
is unexpectedly increased compared with low-altitude populations[11].
The similar response of these two geographically separate high-altitude
populations underlines the importance of NO for life under hypoxic
stress. Beall et al speculated that one possible adaptation
to maintaining high-output NO synthesis under hypoxia included increased
expression of the synthase enzymes themselves. Nitric oxide synthase
(NOS) are a family of three isozymes responsible for the production
of NO: the constitutive endothelial (eNOS) and neuronal (nNOS) isozymes
and the inducible isozyme. It is known that iNOS is expressed
in a wide variety of cell types, including cardiac myocytes. iNOS
is usually expressed in response to various physiological and pathophysiological
stimuli, such as intense exercise and hypoxia. Recent studies have
shown that NO plays an important role in protecting myocardium from
I/R injury[12-14]. In addition, studies have indicated
that the cardioprotective effects of late preconditioning
observed after 24 h resulted from the upregulation of NOS and, more
specifically, of iNOS[15,16]. Neckar et al[17]
reported that the cardioprotective effects of chronic hypoxia and
IPC were not additive, suggesting that the mechanisms of protection
conferred by chronic hypoxia and preconditioning may share some
common signaling pathways. At present, little is known about the
role of iNOS-derived NO in the cardioprotection of IH and there
is no evidence about the changes in iNOS mRNA and protein in IH
rat hearts subjected to ischemia and reperfusion. The aim of the
present study was to evaluate: (i) the effect of aminoguanidine
(AG) on the post-ischemic recovery of left ventricular function
in IH rat hearts, thereby determining the role of iNOS-derived NO
in the cardioprotection afforded by IH; and (ii) the effect of IH
on iNOS mRNA and protein expression in rat hearts during baseline
perfusion, ischemia and reperfusion period.
Materials and methods
Animal preparation Adult male Sprague-Dawley rats (Clean
grade, Shanghai Experimental Animal Center, Chinese Academy of Sciences,
Shanghai, China), initially weighing 100-130 g and finally weighing
280-330 g, were exposed to intermittent high-altitude hypoxia of
5000 m in a hypobaric chamber for 6 h/day. Barometric pressure (pB)
was lowered to the level equivalent to an altitude of 5000 m (pB=54
kPa; po2=11.3 kPa). The total number of exposures
was 42-day. The temperature in the chamber was maintained at 22-24
oC. The animals were examined the day after
the last hypoxic exposure. The control group of animals was kept
under normoxic environmental conditions. All animals were maintained
with a natural light-dark cycle (12 h Light: 12 h Dark).
Isolated rat heart perfusion The rats were anesthetized
with sodium pentobarbital (60 mg/kg, ip) as previously described
[9]. Hearts were quickly excised and mounted on a Langendorff
apparatus for a retrograde perfusion with Krebs-Henseleit
solution (K-H buffer solution) at a constant pressure of 80 mmHg.
K-H buffer solution contains (mmol/L): NaCl 118.0, KCl 4.7, CaCl2
2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4
1.2, glucose 11.0, and sodium pyruvate 2.0. The medium was
continuously gassed with 95% O2 and 5% CO2 (pH
7.4) and maintained at 37 oC. A water-filled latex
balloon connected to a pressure transducer (Gould P23Db) was
introduced into the left ventricle via the mitral valve to
record isovolumic left ventricular pressure. The balloon volume
was adjusted to achieve a stable left ventricular end-diastolic
pressure (LVEDP) of 5-10 mmHg during the initial equilibration.
Heart rate (HR), left ventricular peak systolic pressure (LVPSP),
LVEDP, left ventricular developed pressure (LVDP), coronary flow
(CF), and the peak rate of pressure developed (±dp/dtmax)
were monitored using a PowerLab system (AD Instrument
Ltd, Castle Hill, Australia). LVDP=LVPSP-LVEDP. Pressure-rate product
(PRP) was calculated, PRP = HR×LVDP.
Experimental protocol and groups We chose AG, an iNOS-specific
inhibitor[18], to detect its effect on the cardioprotection
of IH. Rats were divided into four groups: (1) corresponding control
(CON) group; (2) CON+AG group; (3) IH group; and (4) IH+AG group.
In the present study, we used 20 min baseline perfusion, 30 min
no-flow global ischemia, followed by 30 min reperfusion protocol.
The hearts of rats in the drug group were treated with AG 100 µmol/L
for 5 min before ischemia and maintained during 30 min ischemia,
followed by 30 min reperfusion with K-H buffer solution. To obtain
samples for biochemical, RT-PCR, and Western-blot experiments, each
group was further divided into three subgroups: (1) baseline perfusion
group: 20 min baseline perfusion; (2) ischemia group: 20 min baseline
perfusion followed by 30 min no-flow global ischemia; and (3) ischemia/reperfusion
group: 20 min baseline perfusion, 30 min no-flow global ischemia,
followed by 30 min reperfusion. At the end of baseline perfusion,
ischemia, and reperfusion, respec-tively, the hearts were dissected
into left and right ventricles, frozen in liquid nitrogen, and stored
at -80 oC.
Nitrate plus nitrite (NOx) measurement NOx, the stable
end product of NO, was assessed as nitrite concentration after conversion
of nitrate to nitrite with nitrate reductase and measured using
a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing,
China). NOx was designated as mmol/g protein. Its concentration
was determined at an optical density of 550 nm in a spectrophotometric
method. Protein determination was carried out according to the Bradford
method using bovine serum albumin as the standard.
RNA isolation and semi-quantitative determination of iNOS using
RT-PCR Total RNA was extracted from frozen ventricular
tissue with Trizol reagent and quantified by absorption at 260 nm.
Reverse transcription from 0.5 µg total RNA was incubated in
a 20-µL mixture containing 40 U reverse transcriptase, 500
µmol/L of each dNTP, 500 ng oligo(dT) and reaction buffer at
42 °C for 50 min. The sequences of the iNOS primers were 5'-ACTGCTGGTGGTGA-CAAG-3'
(forward) and 5'-CGTTGGAAGTGTA-GCGTT-3' (reverse), allowing the
amplification of a 333-bp fragment; the sequences of the M28S primers
were 5' AGCAGCCGA-CTTAGAACTGG-3' (forward) and 5'-TAGGGACAGTGGGA-ATCTCG-3'
(reverse), allowing the amplification of a 250-bp fragment. The
PCR contained 0.1 µmol/L of each primer, 200 µmol/L of
each dNTP, 2.5 mmol/L MgCl2 reaction buffer and 1 U Taq
DNA polymerase in a final reaction volume of 20 µL. The reaction
mixture was incubated in a thermocycler (Eppendorf Mastercycler
gradient, Germany) programmed to predenature at 94 °C for 5
min, denature at 94 °C for 40 s, anneal at 60 °C for 40
s, and extend at 72 °C for 40 s for a total of 38 cycles. The
last cycle was followed by a final elongation at 72 °C for
5 min and cooled to 4 °C. A pilot experiment had shown that
this cycle number allowed product detection within the linear phase
of amplification.
The amplified products were electrophoresced on a 1.0 % agarose
gel stained with ethidium bromide, visualized under ultraviolet
light, and scanned using Gel doc 2000 (Bio-Rad, Richmond, CA, USA).
The results were expressed as the relative intensity of bands for
iNOS PCR product normalized by the intensity of the band for M28S.
Preparation of protein extracts and Western blot analysis of
iNOS isozyme The protein extracts were prepared by homogenizing
the left ventricles in isotonic sucrose buffer A (mmol/L): Tris-HCl
20.0, sucrose 250.0, Na3VO4 0.03, MgCl2
2.0, edetic acid 2.0, egtazic acid 0.5, PMSF 2.0, DTT 1.0, protease
inhibitor cocktail 0.02 % (v/v), pH 7.4. The homogenates were centrifuged
at 100 000×g for 60 min at 4 °C to separate the
particulate fraction from the cytosolic fraction. The supernatant
containing soluble NOS, designated as cytosol, was used for the
Western blot analysis of iNOS.
After boiling for 10 min, equivalent amounts of cytosolic protein
(40 µg) were separated by 8% denaturing sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE), electroblotted
onto a nitrocellulose membrane and immunoreacted with iNOS primary
antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at
4 °C, followed by a 2-h incubation at room temperature with
the second antibody (goat anti-rabbit IgG, Sigma Chemical Company,
St Louis, MO, USA) conjugated with horseradish peroxidase. The iNOS
isozyme was detected using enhanced chemiluminescence (ECL, Amersham
Biosciences, UK) as 130-kDa bands. Each iNOS isozyme signal was
normalized to the standard signal of the normoxic control heart.
The scanned image was imported into Adobe Photoshop software; scanning
densitometry was used for quantitative analysis of the data.
Materials and reagents AG was purchased from the Sigma
Chemical Company; Trizol isolation reagent was from Invitrogen Life
Technologies (San Diego, CA, USA); primers of iNOS and M28S were
synthesized by Sangon Bioengineering Company (Shanghai, China);
and the reverse transcription system was obtained from Promega (Madison,
WI, USA).
Statistical analysis All data are expressed as mean±SD.
Statistical analysis were carried out using one-way ANOVA or Student's
t tests when appropriate. Differences were considered significant
at P<0.05.
Results
Effects of AG on recovery of CF and ventricular function after
I/R in IH and normoxic rats Our previous study showed that
the ratio of whole ventricle weight to body weight of rats in IH
groups was not significantly different from normoxic control animals,
which meant that intermittent hypoxia in this experimental condition
did not result in heart hypertrophy[6,19]. The present
study demonstrated that CF was slightly but significantly higher
in IH hearts during baseline perfusion (Table 1). CF dramatically
decreased during reperfusion in all groups, but the improvement
of CF during reperfusion was greater in the IH group compared with
the normoxic control group. The addition of AG 100 µmol/L did
not significantly change either pre-ischemic CF or LVDP, but inhibited
pre-ischemic ±dp/dtmax in normoxic
and IH hearts (data not shown). The addition of AG 100 µmol/L
inhibited the improvement of CF during reperfusion in the IH group,
but had no influence on normoxic hearts.
As shown in Table 1, HR did not change in any group and was not
affected by AG 100 µmol/L. Baseline values of ventricular function
variables did not differ in any group during baseline perfusion,
except for CF, but the values of LVPSP, ±dp/dtmax,
and PRP were greatly decreased, whereas LVEDP significantly increased
during reperfusion. During reperfusion, values of ±dp/dtmax,
LVEDP, and PRP in the IH group were superior to the normoxic group,
suggesting that left ventricular functional recovery was modestly
facilitated by IH adaptation. AG significantly inhibited the recovery
of IH hearts, whereas it had no effect on normoxic hearts during
reperfusion (Figure 1).
Effects of AG on the nitrate/nitrite content of IH and normoxic
rat hearts The biochemical experiment demonstrated that the
baseline content of NOx in IH hearts was 30.4% higher than that
in normoxic hearts (P<0.01). After 30 min ischemia, the
NOx level in normoxic heart tissue increased from 0.608±0.060
to 0.747±0.062 µmol/g protein, whereas in IH heart tissue
there was no significant change (P>0.05, 0.793±0.075
vs 0.837±0.054 µmol/g protein). The addition of
AG 100 µmol/L significantly diminished the content of NOx in
IH hearts from 0.837±0.054 to 0.606±0.070 µmol/g
protein and from 0.747±0.062 to 0.541±0.062 µmol/g
protein in normoxic hearts during the ischemia period. AG also significantly
decreased the content of NOx in IH hearts from 0.742±0.062
to 0.535±0.072 µmol/g protein and from 0.617± 0.072
to 0.519±0.059 µmol/g protein in normoxic hearts during
the reperfusion period (Figure 2).
Effects of AG on iNOS mRNA expression in IH and normoxic rat
hearts The expression of iNOS mRNA is shown in Figure 3A,3B.
The baseline level of iNOS mRNA in IH hearts was higher than that
in normoxic hearts by 50.2% (P<0.01). After reperfusion,
iNOS mRNA level in normoxic hearts increased from 0.470±0.051
to 0.590±0.092 (P<0.05); however, the iNOS mRNA level
in IH hearts decreased from 0.706±0.061 to 0.549±0.066
after reperfusion (P<0.05). The addition of 100 µmol/L
AG significantly diminished iNOS mRNA level in normoxic and IH hearts
after reperfusion; however, the reduced extent of IH hearts was
higher than that of normoxic hearts (P<0.01).
Effects of AG on iNOS protein expression in IH and normoxic
rat hearts The expression of iNOS protein is demonstrated in
Figure 4A, 4B. In accordance with the changes at mRNA level, the
baseline level of iNOS protein in IH hearts was 33.8% higher than
that of normoxic hearts (P<0.05). After reperfusion, iNOS
protein level in the normoxic hearts increased by 18.4% (from 100%
± 0.0% to 118.4%±8.4%, P<0.05); however, iNOS
protein level in IH hearts decreased by 31.9% (from 133.8%±16.6%
to 91.1%± 7.0%, P<0.01). After reperfusion, the level
of iNOS protein between normoxic and IH hearts was significantly
different (P<0.05). The addition of 100 µmol/L AG
significantly decreased iNOS protein level in IH hearts; however,
AG had no influence on iNOS protein expression in normoxic hearts
after reperfusion. Ischemia significantly decreased iNOS protein
expression in the different groups (P<0.05).
Discussion
The present study showed that IH increased the tolerance of hearts
to I/R injury, determined by improved recovery of post-ischemic
ventricular function. This protective effect was abolished by AG,
suggesting that iNOS-derived NO may participate in the cardioprotection
of IH. Our results also showed that the baseline level and the recovery
of CF after ischemia were higher in IH hearts compared with normoxic
hearts. Zhong et al's study revealed that capillary
densities were increased in IH hearts, which contributed to better
functional recovery when isolated rat hearts were subjected to an
I/R injury[6]. The recovery of post-ischemic CF in IH
hearts was abolished by AG, suggesting that iNOS-derived NO may
be involved in the improvement of CF by IH.
The level of NOS expression relates directly to the quantity of
NOx production. To examine the effect of IH on iNOS expression,
we investigated NOx content, iNOS mRNA, and protein expression in
normoxic and IH rat hearts during baseline perfusion. Providing
important additional insights into IH against I/R injury, we also
examined NOx content, iNOS mRNA and protein expression in IH and
normoxic rat hearts subjected to ischemia/reperfusion. This may
help us to understand why IH could improve the post-ischemic recovery
of left ventricular function. Our results showed that iNOS mRNA
and protein were unregulated and that NOx content increased after
IH adaptation in rat hearts. Palmer et al showed that
hypoxia induced iNOS expression in cardiac myocytes and vascular
endothelial cells[20,21]. Rouet-Benzineb et al
also indicated that after 15 days of hypoxia there was a two-fold
increase in iNOS protein abundance in rat left ventricles[22].
However, Baker et al demonstrated that increased tolerance
to ischemia in rabbit hearts adapted to chronic hypoxia was associated
with increased expression of eNOS isozymes, which led to an increase
in myocardial nitrite/nitrate content and cGMP level[23].
The signaling functions of NO begin with its binding to protein
receptors, such as guanylyl cyclase, which generates cGMP. In the
present study, the elevated expression of myocardial iNOS in IH
hearts compared to control hearts was also consistent with Zhong
et al's report[6], which showed that the
myocardial cGMP level in IH rats increased compared to normoxia
control rats. Baker et al suggested that a small increase
in NO levels appears to be cardioprotective, whereas a large increase
in NO production may be detrimental, resulting in vasodilation,
decreased blood pressure and, perhaps, vascular leakage[23].
Importantly, in our study the magnitude of iNOS upregulation caused
by IH was mild. This quantitative induction of iNOS may be critical
in explaining the protective effects of iNOS following IH adaptation.
The discrepancies between Baker et al's study and ours
lie in the age of the animals (adult vs neonatal), the species
(rabbit vs rat), the experimental model, and the
training duration and/or intensity.
In the present study, iNOS-derived NO was assessed by measuring
the AG-inhibitable nitrite/nitrate content in IH and normoxia rat
hearts. The addition of 100 µmol/L AG significantly decreased
NOx content, iNOS mRNA and protein levels in IH hearts after reperfusion
and abolished the protective effect of IH; AG also decreased NOx
content and iNOS mRNA expression in normoxic hearts; however, the
inhibitory extent was lower in normoxic hearts compared with IH
hearts. This result suggests that iNOS is tonically higher in IH
hearts compared with normoxic hearts and that iNOS may play an important
role in the cardioprotection of IH.
Studies examining the role of NO in modulating ischemia/reperfusion
injury are complicated. There are conflicting reports about the
changes in NO content during ischemia/reperfusion period. A number
of studies have demonstrated that NO content increased in hearts
subjected to ischemia[24,25]. Studies have also shown
that short-term ischemia leads to an increase in iNOS activity and
expression; however, iNOS activity and expression decreased with
prolonged ischemia. This may be related to a deficiency in essential
co-factors for protein synthesis and stability (eg, haem, FAD, FMN,
calmodulin). In addition to NO production by specific NOS, Zweier
et al also demonstrated that NO could be generated in tissues
by either direct disproportionation or reduction of nitrite to NO
under the acidic and highly reduced conditions that occur in disease
states, such as ischemia[26]. Thus, we considered that
the basal level of nitrite and nitrate was a better index of nitric
oxide production from the aerobically perfused heart.
In addition, we found that iNOS mRNA and protein level in normoxic
hearts increased after reperfusion; however, iNOS mRNA and protein
level decreased in IH hearts. The results from Zingarelli et
al's study differed from our result in that iNOS mRNA in wild-type
mice was significantly increased after 30 min reperfusion compared
to the basal level [27]. In the present study, the level
of iNOS protein in normoxic hearts was higher than the level in
IH hearts after I/R. It is known that induction of high-output iNOS
usually occurs in an oxidative environment, and thus high levels
of NO have the opportunity to react with superoxide anion (O2-)
leading to peroxynitrite (ONOO-) formation and cell toxicity[28].
The generation of ONOO- can account for the toxicity
of NO in biological systems. Yasmin et al and Wang and Zweier's
studies indicated that generation of ONOO- at reperfusion
contributed to the I/R injury in isolated rat hearts[29,30].
In general, O2- formation increases during
the early period of reperfusion and reacts with NO to form ONOO-,
which results in amino acid nitration and cellular injury[30].
Our previous study suggested that an increase in antioxidant capacity
might play an important role in the effect of IH reducing I/R injury[5].
Based on the above investigations, we propose that during reperfusion,
normoxic hearts may be predisposed to produce more O2-
and ONOO- than IH hearts, and thus produce more potent
toxicity than IH hearts. Our results also suggest that during reperfusion
after sustained ischemia, NO may have bidirectional effects on myocardium
because of the coexistence of NO and O2-.
AG significantly diminished NOx content, iNOS mRNA and protein level
in IH hearts after reperfusion and led to the inhibitory effect
of AG on the recovery of left ventricular function of IH rat hearts.
As far as we know, NO is considered to play a pivotal role in numerous
physiological and pathophysiological processes, with effects arising
from both a lack and a surfeit of this chemically reactive molecule.
This seemingly paradoxical behavior may be explained by the amount
of NO generated, temporal-spatial intracellular compartmentalization
of NO, and the intracellular redox environment.
The mechanisms leading to upregulation of iNOS after IH adaptation
and the mechanism whereby iNOS-derived NO plays a role in the protection
of IH have not been clarified and need to be further investigated.
Mechanisms by which hypoxia induces gene expression include transcriptional
and posttranscriptional regulation. The molecular mechanisms of
hypoxia adaptations are centered on the activation of hypoxia-inducible
factor 1 (HIF-1). During hypoxia, however, HIF-1¦Á is stabilized,
leading to accumulation of the active HIF-1¦Á, HIF-1¦Â heterodimer,
which binds to specific recognition elements within promoter/enhancer
regions of many target genes, the induction of which generates this
cytoprotective process. The set of genes induced in this manner
include iNOS[20,21], which drives cytoprotective events
mediated by NO. At the same time, complex mechanisms may exist and
interplay in the regulation of iNOS expression in the heart.
NO is an important endogenous regulatory molecule involved in a
variety of biological functions. For example, it maintains coronary
vasodilator tone, inhibits platelet aggregation and the adhesion
of neutrophils to vascular endothelium[31]. It can also
regulate myocardial contractile function. Studies have suggested
that NO could also regulate oxygen demand and the delivery of oxygen[32].
Therefore, moderate increases in NO during intermittent hypoxia
might be involved in the regulation of the above effects.
We concluded that IH upregulated the baseline level of iNOS mRNA
and protein expression leading to an increase in NO production,
which may play an important role in the cardiac protection of IH
against I/R injury.
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