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Introduction
The circadian clock system is responsible for the daily
timing of behavior and physiological
processes[1]. In mammals, the master circadian pacemaker controlling most
of these rhythms is located in the suprachiasmatic nuclei
(SCN) of the anterior hypothalamus, whose phase is mainly
light-entrained directly through the retinohypothalamic
tract[2]. Recent experiments have demonstrated an
oscillatory system including a transcriptional-translational
feedback loop of clock genes. These include 3 homologues of
the Drosophila gene period (Per1,
Per2, and Per3), 2 cryptochrome genes
(Cry1 and Cry2), and the transcriptional
activator genes: clock (the mammalian counterpart of the
Drosophila cycle) and Bmal1 (a member of the basic
helix-loop-helix/PER-ARNTSIM domain family of transcription
factors)[3,4]. These clock genes are expressed in a pattern
with circadian oscillations close to 24 h in the SCN. Among
these clock genes, Per2 and Bmal1 are most important and
were studied in the present work.
Intra-arterial blood pressure monitoring and
non-invasive ambulatory blood pressure monitoring had shown that
blood pressure exhibited variations consistent with
circadian rhythm[5,6]. Most normotensive and essential
hypertensive individuals show a diurnal pattern of blood
pressure with markedly higher values during the active phase and
lower values during the rest phase (dippers); however, a
number of subjects display abnormal circadian blood
pressure profiles characterized by an absence or even reversal in
a nocturnal fall in blood pressure (non-dippers). Cross-
sectional studies have indicated that target-organ damage is
more pronounced in "non-dipper" than in "dipper" patients
with comparable clinical blood pressure
levels[7]. The transgenic hypertensive TGR(mREN2)27 rats harboring the
murine Ren2 gene manifest an inverted circadian rhythm of
blood pressure similar to "non-dippers", while 24 h profiles
of motor activity and heart rate remain
undisturbed[8]. Moreover, the circadian rhythm of clock gene mRNA
expression in the SCN of TGR(mRen2)27 rats was
abolished[9].
Arterial baroreflex (ABR) is one of the most important
mechanisms in the regulation of cardiovascular activities,
especially in the stabilization of blood pressure. ABR
function is impaired in hypertension and closely related to
hypertensive organ damage[10]. In addition, the interruption
of ABR by sinoaortic denervation could lead to irreversible
organ damage such as cardiac lesion, vascular remodeling,
and renal damage in rats[11]. Furthermore, it was found that
sinoaortic denervation changed the rhythmicity of blood
pressure from a 24 h period to a 12-h
period[12]. However, it is not clear whether the function of baroreflex influences the
circadian expression of clock genes in the central nervous system.
The present work was therefore designed to observe the
possible effects of sinoaortic denervation on the mRNA and
protein expression of 2 main clock genes:
Per2 and Bmal1 in the rat SCN.
Angiotensin II (Ang II), a key molecule for
cardiovascular regulation, influences the activity of brain areas involved
in the determination of circadian-dependent variations of
blood pressure[13]. Therefore, the expression of Ang II type 1
(AT1) receptors in the same brain area of rats was also studied.
Materials and methods
Preparation of sinoaortic-denervated (SAD)
rats Male Sprague-Dawley (SD) rats were provided by Sino-British
SIPPR/BK Lab Animal Ltd (Shanghai, China). At the age of
12 weeks, sinoaortic denervation was performed with a
previously used method[14,15]. Briefly, the rats were
anesthetized with a mixture of ketamine (50 mg/kg) and diazepam
(5 mg/kg), intraperitoneally, and were then medicated with
atropine sulfate (0.5 mg/kg, intraperitoneally) and procaine
benzylpenicillin (60000 U, intramuscularly). After a midline
cervical incision and bilateral isolation of the neck muscles,
aortic baroreceptor denervation was carried out bilaterally
by cutting the superior laryngeal nerves near the vagi,
removing the superior cervical ganglia, and sectioning the
aortic depressor nerves. The carotid sinus baroreceptors
were denervated bilaterally by stripping the carotid
bifurcation and its branches followed by the application of 10%
phenol (in 95% ethanol) to the external, internal, and
common carotid arteries and the occipital artery. In the control
rats, the sham operation was performed under the same
conditions, without cutting and removing the nerves and
carotid adventitia. The animals were allowed to recover
spontaneously after the denervation. All procedures were in
accordance with institutional animal care guidelines, and
approved by the local institutional committee.
Blood pressure measurement Blood pressure and heart
period were continuously recorded using previously
described techniques[16,17]. Briefly, the rats were
anesthetized with a combination of ketamine (50 mg/kg) and
diazepam (5 mg/kg). A floating polyethylene catheter was
inserted into the lower abdominal aorta via the left femoral
artery for blood pressure measurement. The catheters were
exteriorized through the interscapular skin. After a 2 d
recovery period, the animals were placed for blood pressure
recording in individual cylindrical cages containing food and
water. The aortic catheter was connected to a blood
pressure transducer via a rotating swivel that allowed the
animals to move freely in the cage. After about 4 h of habituation,
the blood pressure signal was digitized by a microcomputer.
Blood pressure and heart period values from every
heartbeat were determined online. The mean values of these
parameters during a period of 4 h were calculated. The
standard deviation of the mean values was also calculated and
served as the variabilities of blood pressure or heart period.
Experimental protocol Four weeks after the operation,
blood pressure and heart period were measured in the
conscious state in a group of sham-operated
(n=10) and SAD rats (n=9). The remaining 72 (36 SAD rats and 36
sham-operated) rats were brought up in a separate,
environmentally-controlled room (temperature 23_25 °C) with ventilated
chambers under a 12/12 h light/dark cycle (8:00-20:00 light,
20:00-8:00 dark) for at least 10 d with food and water
available ad libitum. The light was provided by white
fluorescent bulbs (220 mW/cm2). Under entrained conditions, lights
on was defined as Zeitgeber time (ZT) 0. Six SAD rats and 6
sham-operated rats were killed every 4 h from ZT0 to ZT20
by ether anesthesia. The mRNA and protein expression of
the clock genes (Per2, Bmal1) and AT1 receptors were
examined by RT-PCR and Western blotting, respectively. The
brain was removed from the skull and snap-frozen in liquid
nitrogen until use. The SCN region was punched out with a
2 mm diameter needle that was inserted to a 1 mm depth into
the surface of the coronal plane, an area that included the
SCN[17,18]. The animals were killed at each time point on 3
separate days to ensure reproducibility of the gene
expression cycle.
RNA extraction and RT-PCR Total RNA was extracted
from the SCN using TRIzol reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer's instructions.
Total RNA concentrations were determined by
spectrophotometry at 260 nm, and total RNA quality was assessed by
electrophoresis on 1.3% denaturing formaldehyde agarose
gels. Total RNA (<1 µg) was reverse transcribed using an RT
system kit from Promega (Promega Corporation, Madison,
WI, USA) according to the manufacturer's protocols.
Primers were designed according to cDNA sequences reported
in the GenBank database and by computer analysis using
the Primer premier 5.0. (PREMIER Biosoft International, Palo
Alto, CA, USA) and synthesized at the SBS Genetech Co
(Beijing, China). The sequences of primers are described in
Table 1. PCR was performed with the 2×Taq
PCR MasterMix (Tianwei Biotech, Beijing, China) using a Peltier Thermal
Cycler 200 (MJ Research Inc, Watertown, MA, USA). The PCR
protocol for Per2 was as follows: initial incubation at 94 °C
for 2 min, 35 cycles of denaturation at 94 °C for 40 s,
annealing at 54 °C for 1 min, and extension 72 °C for 1 min, and
finally a prolonged extension step at 72 °C for 10 min. The
PCR protocol for Bmal1 and AT1 differed slightly from that
of Per2; annealing was performed at 58 °C or 56 °C,
respec-tively. The relative amount of each mRNA was normalized to
the housekeeping gene, β-actin, mRNA that the expression
levels remained constant throughout the day. The PCR
products were resolved by electrophoresis on a 2.0
% agarose gel, stained with ethidium bromide (EB), and their relative
quantities were determined using the Gel-pro Analyzer 4.5
software (Media Cybernetics, Silver Spring, MD, USA).
Western blotting Microdissected SCN tissue was
homogenized in lysis buffer [50 mmol/L Tris-HCl (pH 7.5)], 150
mmol/L NaCl, 5 mmol/L EDTA, 1% NP-40, 1 mmol/L PMSF,
plus a protease inhibitor cocktail (pH 7.4) for 30 min on ice.
After removal of tissue debris by centrifugation (12
000×g, 5 min), the protein concentration of each lysate sample was
determined using the BCA-100 protein assay kit (Boshide
Biotech, Wuhan, China) according to the manufacturer's
instructions. The samples were boiled in ×2 SDS sample
buffer and loaded with 50 µg proteins per lane onto 8%
SDS-polyacrylamide gels. Following separation at 125 V for 120
min, the proteins were transferred onto nitrocellulose
membranes, which were then blocked with 5% nonfat dried
milk in TBS-Tween (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L
NaCl, 0.25% Tween-20) for 4 h at room temperature. After
brief washes, the membranes were incubated with polyclonal
anti-mouse Per2 (1:100) and Bmal1 (1:200) antibodies (Abcam
Inc, Cambridge, MA, USA) at 4 °C overnight.
Immunoreactive bands were visualized using anti-rabbit IgG-HRP
antibody and an ECL chemiluminescence system (Santa Cruz
Biotech. Inc, CA, USA). The exact quantities of
Per2 and Bmal1 were normalized against actin as a constitutively
expressed internal control using optical density determined
by the Gel-pro Analyzer 4.5 software (Media Cybernetics,
USA).
Statistical analysis The statistical analyses were
conducted directly with SPSS11.5 software. All data are
express-ed as mean±SD. Multi-group mean values were compared
by using ANOVA followed by Student Newman-Keuls test,
and two-group mean values were compared by using an
unpaired Student's t-test. A significant difference among mean
values was assigned at P<0.05.
Results
Blood pressure and heart period in SAD rats
It was found that blood pressure levels in the SAD rats were similar
to those of the sham-operated rats. However, blood
pressure variabilities significantly increased in the SAD rats
compared with the sham-operated rats (Figure 1). Heart period
and heart period variability were not modified by sinoaortic
denervation operation.
Circadian expression of Per2 mRNA in the SCN of SAD
rats The Per2 gene exhibited a robust circadian expression
pattern in the SCN of the sham-operated SD rats, as
previously reported in Wistar rats[18]. By visual inspection of gel
electropherogram, high intensity DNA bands were observed
at ZT12, ZT16, and ZT20, and weak, but still recognized bands,
at ZT0, ZT4, and ZT8 (Figure 2A). Clear circadian
transcription profiles of Per2 mRNA were detected by the relative
quantitative analyses, with higher transcription levels around
the light-to-dark transition (ZT12) and lower levels during
the early daytime (ZT0-4). The peak expression was
observed at ZT12 and was 2.19-fold stronger than the trough
value at ZT0 (Figure 2B).
In the SAD rats, the Per2 gene showed a similar, but
attenuated, circadian oscillation. The intensity of the DNA
bands was weaker throughout the day than in the
sham-operated rats (Figure 2A). The Per2 mRNA levels in the SCN
were significantly lower in the SAD than in the
sham-operated rats at most time points, except for ZT4 and ZT8 (Figure
2B).
Circadian expression of Bmal1 mRNA in the SCN of
SAD rats A clear circadian expression of the
Bmal1 gene was observed in the SCN of the sham-operated rats, which is
in accordance with the finding in Wistar
rats[19]. By visual inspection of gel electropherogram, high intensity DNA bands
were detected at ZT16, whereas moderate intensity bands
were detected at ZT0, ZT4, and ZT20. The intensity of the
bands was faint at ZT8 and ZT12 (Figure 3A). The relative
quantitative analyses displaying Bmal1 mRNA expression
reached its peak early at night at ZT16 and descended to a
trough late in the day at ZT8. The expression pattern was
anti-phase to that observed for Per2 mRNA (Figure 3B).
In the SAD group, the Bmal1 gene also presented
syn-chronous, but subdued, circadian oscillation. There was
scarcely any difference among the intensity of DNA bands
at most time points by visual inspection of
electropherogram (Figure 3A). Bmal1 mRNA expression in SAD rats
significantly decreased throughout the day compared with the
sham-operated rats, except at ZT8 (Figure 3B).
Circadian expression of the Per2 and Bmal1 proteins in
the SCN of SAD rats The proteins extracted from the
microdissected SCN were probed to detect Per2 and Bmal1.
The anti-Per2 antibody consistently identified a major band
of -130 kDa in the sham-operated and SAD rats, in
accordance with the predicted mass of 136 kDa (Figure 4A). The
intensity of the Per2-immunoreactivity (ir) bands exhibited a
robust circadian oscillation in the sham-operated groups,
with high expression during the early dark phase
(ZT12-16), and low levels at the early light phase
(ZT0-4), as previously reported in
mice[20]. In the SAD rats, the intensity of
the Per2-ir bands was significantly reduced at every time
point compared with the controls, but a circadian pattern
persisted. The quantitative analysis of normalizing the
Per2-ir bands against actin-ir bands demonstrated that levels
peaked at ZT16 and declined to a trough at ZT4 in both
groups. There was a trend for decreased Per2 levels in the
SCN of the SAD rats at most time points; the significant
difference was only observed at ZT12 (Figure 4B).
The anti-Bmal1 antibody identified a band around 68 kDa
in the same SCN samples of the sham-operated and SAD
rats (Figure 5A). The intensity of the Bmal1-ir band varied
with a circadian pattern synchronized in both groups, with
peak expression during late night at ZT16-20 and a nadir
early in the subjective day at ZT0-4, accorded with the
findings in Wistar rats[21]. The quantitative analysis of
normalizing the Bmal1-ir bands against actin-ir bands showed that
Bmal1 expression reached its peak at ZT20 and descended
to a trough at ZT4. The Bmal1 levels significantly decreased
at ZT8 and ZT20 in the SCN of the SAD rats compared with
the sham-operated groups (Figure 5B).
These results reinforced the findings that the circadian
rhythm of some clock gene cycles in the SCN were
attenuated after interruption of the arterial baroreflex.
Circadian expression of AT1 receptor mRNA in the SCN
of SAD rats The circadian expression pattern of AT1
receptor mRNA expression in the SCN of the sham-operated rats
was obvious. High-intensity bands were detected during
the dark phase (ZT12-20), while weak intensity bands were
detected in the light phase (ZT0-8, Figure 6A). AT1
receptor expression peaked at ZT16 and descended to a trough at
ZT8 in the sham-operated rats (Figure 6B).
In contrast, the circadian expression profile of AT1
receptor mRNA changed to a bimodal pattern in the SCN of
the SAD rats. High-intensity bands were detected
abnormally at ZT4 (Figure 6A). AT1 receptor mRNA levels in the
SAD group significantly increased in the light phase, greater
than in the sham-operated group (Figure 6B).
Discussion
The present work shows, for the first time, that arterial
baroreflex dysfunction may dramatically attenuate the
circadian variation of clock gene expression in the SCN. The
main findings of this study are as follows: (i) although
Per2 and Bmal1 mRNA oscillated synchronously in the SCN of
the SAD and sham-operated rats, the expression of mRNA
levels and amplitude of oscillation were remarkably depressed
in the SAD rats; (ii) the contents of the
Per2 and Bmal1 proteins and the extent of the circadian variations were
distinctly weakened in the SCN of the SAD rats compared with
the controls; and (iii) AT1 receptor mRNA expressions in the
SCN were abnormally upregulated in the light phase and
altered the circadian rhythm of AT1 receptors in the SAD
rats.
Circadian clock is normally entrained by periodic
environmental cues, with the daily light-dark cycle being the most
potent entraining signal in mammals. The clock showed phase
delay and phase advance shifts in response to light
exposure in early and late night, respectively.
Per1 and Per2 have been demonstrated to be light-inducible in different
rodent species[22,23]. In the present study,
Per2 and Bmal1 genes oscillated synchronously in the SCN of the SAD and
sham-operated rats. The peak level of Per2 mRNA was
observed at ZT12 and at the trough level at ZT0, while
Bmal1 mRNA transcription reached a peak at ZT16 and a nadir at
ZT8. However, the Per2 and Bmal1mRNA levels and the
amplitude of oscillation were dramatically reduced in the SCN
of the SAD rats. The phenomena were confirmed by a
Western blot analysis of Per2 and Bmal1 proteins in the same
samples. Circadian profiles of the Per2 protein exhibited
evident oscillation with a peak at ZT16; the Bmal1 protein
showed a nocturnal peak at ZT20, indicating that the protein
cycles were delayed by about 4_6 h, relative to the mRNA
cycles under the light-dark conditions in the SCN. Similarly,
the contents of Per2 and Bmal1 proteins at certain time points
and the extent of circadian variations were distinctly
weakened in the SCN of the SAD rats compared with the controls.
The post-transcriptional regulation mechanisms may
implicate the difference between the levels of mRNA and proteins
of the clock genes. Our findings suggested that interruption
of the arterial baroreflex could attenuate the circadian
expression and rhythmicity of some central clock genes.
The ABR is most important in the regulation of heart rate
and the stabilization of blood pressure. When ABR function
is destroyed by sinoaortic denervation operation, blood
pressure becomes unstable and blood pressure variability is great.
However, the blood pressure level remains
unchanged[14,15]. ABR function is impaired in hypertension, heart failure, and
diabetes and has been found to be a crucial determinant of
sudden death after acute myocardial
infarction[24]. In hypertensive rats, the target organ damage correlates negatively
with ABR function[10,25]. Interruption of ABR by sinoaortic
denervation induced aortic and cardiac hypertrophy
independent of the mean level of blood
pressure[26_28]. In the present study, it was found that dysfunction of the ABR
attenuated the circadian expression of clock genes in the
SCN. It has been reported that mean blood pressure was
significantly elevated during the light period in the SAD
rats, thus the 24 h rhythmicity in mean blood pressure was
suppressed and a bimodal pattern was observed under
light_dark cycles, whereas the heart rate and locomotor activity
were not dramatically affected in the same
rats[12]. Although the precise mechanisms responsible for the selective
elimination of the circadian rhythm of mean blood pressure in the
SAD rats have not yet been satisfactorily clarified, the
alteration of central clock genes induced by the disruption of
ABR may be related with the phenomenon. These results
further support the finding that ABR is not only important in
maintaining the stability of blood pressure in the short term,
but also in regulating the diurnal variation in blood pressure
during a 24 h cycle.
It is well established that the central nervous system plays
an important role in controlling ABR function. The major
baroreceptor reflex pathway exists in the brain stem, such as
the nucleus tractus solitarii and the caudal and rostral
ventrolateral medulla[29]. Although these brain regions do not
project directly to the SCN, the multisynaptic connections
between them are much more extensive. Using the
retrograde transneuronal tracer, the pseudorabies virus (PRV),
which localizes the afferent inputs to the SCN in SD rats, has
demonstrated that cells in the nucleus tractus solitarii, C3
catecholamine region, rostral ventrolateral medulla,
peria-queductal gray matter, and lamina I and dorsomedial area of
the spinal trigeminal nucleus are higher order sites in
pathways which affect the SCN[30]. The presence of PRV-infected
neurons in the brain stem indicates a pathway associated
with regulatory mechanisms of arterial pressure which can
influence SCN function and even clock gene expression.
However, there is insufficient evidence showing a direct
action of ABR on gene expression in SCN.
The renin-angiotensin system (RAS) is one of the major
humoral mechanisms regulating cardiovascular function.
Ang II plays a major role in the modulation of blood pressure,
stimulation of vasopressin and aldosterone release, sodium
appetite, and sympathetic facilitation. Most of these effects
show a day_night variation and are mediated via AT1
receptors. Furthermore, the SCN contains not only Ang
II-immunoreactive cells and fibers, but also a high density of
Ang II receptors[31]. It was recently described that Ang II
induces oscillatory expression of clock genes in vascular
smooth muscle cells through the AT1 receptor
subtype[32] and alters the ABR through peripheral (vascular and renal)
and central effects[33]. Impaired ABR function is usually
accompanied by overactivation of tissue RAS. Previous
studies have confirmed that plasma Ang II levels did not increase
in the chronic phase of sinoaortic denervation; in contrast,
cardiac, aortic, and renal Ang II contents increased and AT1
receptor mRNA expression in the left ventricle and aorta was
upregulated after sinoaortic
denervation[34,35]. In the present work, the SCN AT1 receptor mRNA expression was
abnormally upregulated in the light phase, resulting in the
circadian rhythm of AT1 receptors from a 24 h cycle to a 12 h cycle
in the SAD rats. This is in accordance with the changes of
blood pressure rhythm induced by the interruption of ABR.
It is reasonable to hypothesize that ABR dysfunction
may activate tissue RAS, including the SCN. Ang II acts as
a candidate entraining signal, altering circadian expression
of central clock genes through AT1 receptors. However, the
cause_effect relationship between the changes in clock genes
and RAS is not clear at this stage of study.
In conclusion, the circadian variation of the 2 central clock
genes was attenuated in the SAD rats. ABR dysfunction
also induced a disturbance in the expression of AT1
receptors in the SCN. The precise mechanisms underlying ABR
dysfunction inducing an attenuated expression of the 2 clock
genes remain to be elucidated in the future.
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