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Different Effects of Phase Advance and Delay in Rotating Light-Dark Regimens on Clock and Natriuretic Peptide Gene Expression in the Rat Heart


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Under physiological conditions the mammalian circadian system is synchronized to a cyclic environment. The central oscillator in the suprachiasmatic nuclei (SCN) responds predominantly to an external light (L) dark (D) cycle. Peripheral oscillators are more efficiently synchronized by metabolic cues. When the circadian system is exposed to opposing synchronizing cues, peripheral oscillators uncouple from the SCN. To consider influence of phase advances and delays in light regimens mimicking shift work, we analyzed the expression of clock genes (per2, bmal1) and natriuretic peptides (anp, bnp) in the heart of male rats. Experimental groups were exposed to a rotating LD regimen with either 8 h phase advance or delay for 11 weeks. Samples were taken for a 24 h cycle in 4 h intervals. Peripheral oscillators responded to rotating phase advance by decreasing rhythm robustness, while phase delay mostly influenced the phase angle between the acrophase of rhythmic gene expression and the external LD cycle. The expression of anp was arrhythmic in the heart of control rats and was not influenced by rotating LD regimens. The expression of bnp showed a daily rhythm with a nadir during the active phase. The daily rhythm in bnp expression diminished under rotating LD regimen conditions.
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PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)
2014 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
Fax +420 241 062 164, e-mail:,
Physiol. Res. 63 (Suppl. 4): S573-S584, 2014
Different Effects of Phase Advance and Delay in Rotating Light-Dark
Regimens on Clock and Natriuretic Peptide Gene Expression in the
Rat Heart
1Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius
University in Bratislava, Bratislava, Slovak Republic
Received March 14, 2014
Accepted August 10, 2014
Under physiological conditions the mammalian circadian system
is synchronized to a cyclic environment. The central oscillator in
the suprachiasmatic nuclei (SCN) responds predominantly to an
external light (L) dark (D) cycle. Peripheral oscillators are more
efficiently synchronized by metabolic cues. When the circadian
system is exposed to opposing synchronizing cues, peripheral
oscillators uncouple from the SCN. To consider influence of phase
advances and delays in light regimens mimicking shift work, we
analyzed the expression of clock genes (
) and
natriuretic peptides (
) in the heart of male rats.
Experimental groups were exposed to a rotating LD regimen with
either 8 h phase advance or delay for 11 weeks. Samples were
taken for a 24 h cycle in 4 h intervals. Peripheral oscillators
responded to rotating phase advance by decreasing rhythm
robustness, while phase delay mostly influenced the phase angle
between the acrophase of rhythmic gene expression and the
external LD cycle. The expression of
was arrhythmic in the
heart of control rats and was not influenced by rotating LD
regimens. The expression of
showed a daily rhythm with a
nadir during the active phase. The daily rhythm in
diminished under rotating LD regimen conditions.
Key words
Shift work
Corresponding author
I. Herichova, Department of Animal Physiology and Ethology,
Faculty of Natural Sciences, Comenius University Bratislava,
Mlynska dolina B-2, 842 15 Bratislava, Slovak Republic.
Fax: 00 421 2 654 29 064. E-mail:
The circadian system developed during
evolution as a genetically conserved adaptation to the
regular cycle of the Earth’s rotation around its axis
(Aschoff et al. 1981). It generates and synchronizes
endogenous circadian rhythms to external cycles to
predict and better accommodate to expected changes. The
circadian system in mammals is hierarchically organized.
The central oscillator is located in the suprachiasmatic
nuclei of the hypothalamus (SCN) and is synchronized
predominantly by external L (light) D (dark) cycles via
the retinohypothalamic tract from the retina (Rusak and
Zucker 1979). Peripheral oscillators are localized in all
other tissues, including other brain tissues, and can be
synchronized by neural and humoral inputs from the SCN
(Guo et al. 2005).
Both central and peripheral components of the
circadian system are composed from autonomous
unicellular circadian oscillators. The molecular basis of the
generation of mammalian circadian rhythms is a feedback
loop created by positive and negative components that
influence the transcription of clock genes. Clock genes per
and cry possess the capacity to inhibit their own
transcription and therefore represent a negative component
of the molecular loop. There are three homologous per
genes (1, 2 and 3) and two homologous cry genes (1 and
2). The deletion of these genes from the genome causes
arrhythmicity under constant conditions. Transcriptional
factors BMAL1, CLOCK and NPAS2 represent positive
regulators and induce the expression of per and cry genes
S574 Herichová et al. Vol. 63
via the E-box regulatory region (Lowrey and Takahashi
2011). In addition to this set of genes, several others were
proposed to play an important role in adjusting the basic
molecular feedback loop. The nuclear receptor reverse
erythroblastosis virus (REV-ERB) is activated by the
heterodimer BMAL1:CLOCK via the E-box and feeds
back via the REV-ERB/ROR response element (RORE).
REV-ERB competes with the stimulatory factor ROR for
binding on the RORE element in the promoter region of
BMAL1 and NPAS2, and inhibits their expression
(Ripperger et al. 2011). Details regarding the function of
the core feedback loop, additional modulatory loops and
many newly revealed post-transcriptional and post-
translational modifications have been described elsewhere
(Albrecht 2012).
Circadian regulation is mediated downstream
from the basic molecular feedback loop via clock
controlled genes. There are hundreds of rhythmically
expressed genes in peripheral tissues (Panda et al. 2002,
Bray et al. 2008). In many cases, this rhythmic
transcription is mediated via E-box, as it is for per and
cry genes. In some cases, the regulation of rhythmic
compounds is controlled by other or unknown way.
Atrial natriuretic peptide (ANP) and brain
natriuretic peptide (BNP) are produced by the heart and
their production increases under conditions of heart
failure, myocardial infarction, hypertension, left
ventricular hypertrophy and pulmonary hypertension
(Nishikimi et al. 2011). Their function is to increase
electrolyte and water excretion in the kidney, regulate
permeability of the systemic vasculature, and influence
proliferation and cardiac hypertrophy. ANP shows a
distinct daily rhythm in the plasma of humans (Portaluppi
et al. 1990, Goetze et al. 2012), while BNP was not
proved to exhibit a daily rhythm in healthy human
volunteers (Goetze et al. 2012). On the other hand,
expression of bnp mRNA shows a daily rhythm under
synchronized (Herichova et al. 2013) and constant
darkness conditions (Goetze et al. 2010) in the left
ventricle of normotensive rats. Rhythmicity in expression
of anp in the rat atria and ventricles showed a borderline
significance or arrhythmicity (Goetze et al. 2010, Young
et al. 2001). Upon the appropriate signal, ANF and BNP
are rapidly released from pre-stored granules. Release of
ANP and BNP in response to heart pathology occurs in
parallel to a large extent (Sergeeva and Christoffels
2013), and is regulated predominantly via mechanical
stress, growth factors and G-protein-coupled receptors
(e.g. catecholamines and angiotensin II) (Ma et al. 2005,
Sergeeva and Christoffels 2013). The circadian regulation
of anp and bnp expression has not been studied
extensively. To our knowledge, functional E-box with a
palindromic canonical sequence of CACGTG has not yet
been described in the sequences of anp and bnp, although
information regarding the presence of CANNTG E-boxes
does exist (Thattaliyath et al. 2002, Luo et al. 2006,
Wilhide and Jones 2006).
Recent industrial lifestyle changes together with
significant light contamination have brought about
substantial changes in the organization of a 24-h day. The
capacity of peripheral and central oscillators for
entrainment by synchronizing cues differs. Light
synchronizes the SCN effectively within a day but its
influence on clock gene expression in the periphery is
rather small (Dibner et al. 2010). Peripheral oscillators can
disconnect from central oscillators under conditions of
food restriction cycle, which is a weak Zeitgeber for the
SCN under synchronized conditions (Damiola et al. 2000).
Cycles of food restriction can influence central oscillators
under very specific conditions (Nováková et al. 2011,
Challet and Mendoza 2010). Food reward is also able to
influence the functioning of the circadian system, but this
influence is less pronounced compared with food
restriction (Challet and Mendoza 2010). All these factors
(and many others) influence humans under conditions of
shift work and signals to the circadian system become
confusing. Deregulation of the circadian system after long-
term exposure to irregular LD cycles (10 years) can
promote the progression of some diseases. Epidemiological
studies implicate an increased incidence of metabolic
disturbances, cardiovascular pathologies and facilitation of
carcinogenesis (Haus and Smolensky 2006).
Industrialization has become an indispensable part
of life and its risks are understood. Optimization of signals
to the circadian system is vital to ensure this evolutionary
adaption remains beneficial, or at the very least, to ensure
consequences of internal desynchronization are prevented
(Saksvik et al. 2011, Herichova 2013). Understanding of
entrainment by light regimen and food intake provides a
good experimental basis for studying which schedules
should be used in shift work and identifying schedules less
deleterious for the functioning of the circadian system.
Therefore, the aim of this study was to analyze
the effects of LD schedules used during rotating shift work
with phase advances or phase delays on clock genes and
ANP and BNP mRNA in the rat heart, and compare the
effects of these schedules on peripheral oscillators in the
2014 Effect of Advance or Delay in LD Regimen on Heart S575
Fig. 1. Scheme of rotating light:dark regimen with phase advances (upper panel) or phase delays (lower panel). ZT – Zeitgeber time.
Black bars indicate the dark part of the 24 h cycle. Gray bars indicate delay in rotating regimen.
Male Wistar rats (n=145) were obtained from
Anlab Praha (Czech Republic) at 5 weeks of age with an
initial weight of 139.4±2.1 g. Animals were housed in
temperature-controlled rooms (21±2 °C) under a 12:12
LD cycle. Food and water were available ad libitum. The
study was performed in two consecutive phases with two
independent control groups. The effects of phase advance
were investigated in ‘Experiment 1’, and the influence of
phase delay was analyzed in ‘Experiment 2’. After an
acclimatization period, experimental groups were
exposed to a LD regimen mimicking shift work with 8-h
phase advances (the dark phase of the 24 h cycle was
shortened by 8 h three times per week over a period of
12 weeks) or delays (the dark phase of the 24 h cycle was
lengthened by 8 h every second day over a period of
11 weeks) (Fig. 1). Blood pressure was measured during
the whole experiment using the tail-cuff plethysmography
method (AD Instruments, Germany).
Locomotor activity was measured in seven rats
from Experiment 1 and in four rats from Experiment 2 by
radiotelemetry as described previously (Molcan et al.
2013). From 15-min averaged segments of acquired data,
double-plot actograms were created using Chronos-Fit
(Zuther et al. 2009).
At the end of the experiment sampling was
performed in 4 h intervals during a 24 h cycle when the
LD cycles of the control and experimental groups were
the same for 1 day (Fig. 1). Prior to sampling rats were
anaesthetized by carbon dioxide and subsequently
decapitated (n=4-8). The heart was weighed and samples
were taken within a few minutes, snap frozen in liquid
nitrogen and stored under 80 °C until RNA isolation.
The experimental protocol was approved by the Ethical
Committee for the Care and Use of Laboratory Animals
at Comenius University Bratislava (Slovakia) and the
State Veterinary Authority.
Total RNA from the heart was isolated with the
use of TRI Reagent® (Total RNA Isolation reagent,
MRC, USA). RNA integrity and contamination with
DNA were examined using 1.5 % agarose/10.7 M
formaldehyde gel (Herichova et al. 2001). First-strand
cDNA synthesis was carried out with the use of the
ImProm-II™ Reverse Transcription System (Promega,
USA) according to the manufacturer’s instruction.
Quantification of cDNA was performed by real-time PCR
using the QuantiTect SYBR® Green PCR kit (Qiagen,
Germany) and StepOne™ System (Applied Biosystems,
USA). The primer pairs used for the amplification of
per2, bmal1, rev-erba, bnp, gapdh and rplp1 and real-
time PCR conditions were as described in previous
reports (Szántóová et al. 2011, Herichova et al. 2013).
The other primer pairs used for the amplification were:
anp (NM_012612) sense 5'-TCA AGA ACC TGC TAG
ACC A-3', antisense 5'-TCT GAG ACG GGT TGA CTT
CC-3' (annelation 49 °C). Gene expression was
normalized to rplp1 or gadph. The specificity of the PCR
reaction was validated by melting curve analysis. The
fluorescence dye ROX served as an internal reference for
normalization of the SYBR Green I fluorescent signal.
Statistical evaluation
To compare gene expression between two
groups an unpaired t-test was used, and ANOVA
followed by Tukey‘s post-hoc test was used to compare
gene expression between more than two groups. To
analyze the daily profiles of clock genes, expression data
were fitted into a cosinor curve 24 h period (Nelson et al.
1979, Klemfus and Clopton 1993). When experimental
data significantly matched the cosinor curve, the
following parameters were calculated with a confidence
limit of 95 %: mesor (the time series mean), amplitude
(one half of the peak-trough difference expressed herein
relative to the mesor) and acrophase (peak time
referenced to the time of lights on in the animal facility).
Goodness of fit (R value – correlation coefficient) of the
approximated curve was estimated by ANOVA.
S576 Herichová et al. Vol. 63
Confidence limits of rhythm parameters were calculated
by SigmaPlot (USA). Time is expressed in Zeitgeber time
(ZT), when ZT0 is defined as the beginning of the light
part of the 24 h cycle. Circadian and ultradian rhythm
analysis of the individual locomotor activity was
performed with a Lomb-Scargle periodogram using
Chronos-Fit software (Zuther et al. 2009, Molcan et al.
2013). In the actograms, the black bars indicate animal
activity, each line represents 1 day of experiment and
recordings are double plotted. Data in graphs displaying
gene expression are presented as the arithmetic mean and
standard error of the mean.
Exposure of rats to rotating LD regimens for
11 weeks did not cause an increase in blood pressure or
heart hypertrophy in the experimental groups compared
with the corresponding control group. Furthermore, we
did not observe a significant change in body weight
compared with the control (data not shown).
Fig. 2. Effect of rotating light:dark regimen with phase advances on
(B) and
(C) mRNA expression in rat
hearts. The solid line represents the control group and the broken line shows data from the experimental group (n=4-8). The black bar
on the bottom of the graph represents the dark part of the 24 h cycle. Time scale is given in relative units: Zeitgeber time
(ZT0 = beginning of the light part of the light:dark cycle).
2014 Effect of Advance or Delay in LD Regimen on Heart S577
Fig. 3. Effect of direction of light:dark regimen rotation on the amplitude and acrophase of rhythmic profiles of
mRNA expression in the heart. The white circles and columns represent the control groups. The black circles and gray columns
correspond to data measured in rats exposed to rotating light:dark regimens. Amplitude is expressed as a relative value assuming
calculated amplitude of the control group to be 100 % (control of Experiment 1 or 2 depending on regimen). Differences in acrophase
and amplitude induced by rotating phase delay were calculated from a previous study (Szántóová
et al.
2011). * P<0.05 cosinor
(Table 1).
Rotating regimen with phase advances caused a
significant decrease in mesor and amplitude of per2
expression and the per2 acrophase was shifted by 2 h
compared with the control (Figs 2A, 3; Table 1).
The rotating phase advance LD regimen strongly
influenced the daily profile of bmal1 expression. The
expression of bmal1 showed a significant decrease in
mesor and amplitude, and the acrophase shifted by 4 h
compared with the control (Figs 2B, 3; Table 1).
Expression of rev-erba was significantly
suppressed in the group exposed to the LD regimen with
rotating advances compared with the control. The daily
profile of rev-erba showed a decreased mesor and
amplitude with no significant influence on the acrophase
(Figs 2C, 3; Table 1) compared with the control group.
The effects of phase delays and advances on
rhythmic clock genes in the heart were compared, and the
effect of rotating LD regimen with phase delays on clock
gene expression was calculated from our previous work
(Szántóová et al. 2011). The effects of phase advance and
phase delay on per2, bmal1 and rev-erba expression were
very different. The LD regimen with rotating phase
advance had a much stronger effect on amplitude and less
pronounced effect on acrophase compared with the LD
regimen with rotating phase delays (Fig. 3).
The locomotor activity of animals was
monitored by individual actograms (Fig. 4). During the
first week of monitoring under a standard 12:12 LD
regimen, all animals displayed a typical circadian pattern
(period ~24 h, P<0.0001). The effect of a rotating LD
regimen with phase advance was analyzed in seven rats
(Experiment 1). At the end of the experiment four
animals still maintained a periodicity of ~24 h period
(P<0.001), although with decreased significance in three
of them. Two animals displayed only periodicities shorter
than 24 h, and one animal acquired several shorter and
one longer period compared with the control profile.
There was an increase in ultradian components
occurrence and activity during the light part of LD
regimen and in six animals. Under conditions of a
rotating LD regimen with phase delays we observed an
increase in periodicity (27-29 h, P<0.001) in all four
S578 Herichová et al. Vol. 63
Table 1. Cosinor analysis of the daily pattern of gene expression.
SEM Amplitude Amplitude
SEM Mesor Mesor
per2 control 14:46 0:32 0.856 0.131 1.094 0.089 0.0001 Advance in acrophase, decrease
in amplitude and mesor
advance 16:56 1:06 0.327 0.098 0.927 0.068 0.0086
bmal1 control 0:53 0:18 1.254 0.108 1.187 0.073 0.0001
Advance in acrophase, decrease
in amplitude and mesor
advance 5:28 0:51 0.504 0.117 0.901 0.081 0.0007
rev-erba control 8:52 0:22 1.210 0.106 1.263 0.078 0.0001
Decrease in amplitude and mesor
advance 9:44 0:53 0.523 0.120 0.890 0.085 0.0006
anp control ns ns ns ns 1.144 0.229 0.3432
ns ns ns ns 0.795 0.135 0.6277
bnp 5:31 1:37 0.497 0.201 2.034 0.145 0.0590
advance ns ns ns ns 1.817 0.139 0.1663 Diminishing of rhythm
anp control ns ns ns ns 1.291 0.212 0.0801
delay ns ns ns ns 0.957 0.188 0.5420
bnp control 5:32 0:48 1.128 0.227 1.796 0.163 0.0003
delay ns ns ns ns 1.345 0.115 0.6032 Diminishing of rhythm
A cosine curve with a 24 h period was approximated to the time series data of control and experimental groups of rats. Mesor is the mean value of the fitted curve; amplitude is one-half of the peak-
trough difference; and acrophase is the time of curve peak from the time point of Zeitgeber time zero (dark to light transition). Mesor and amplitude are given in relative units. Amplitude and mesor are
given in relative units. P values indicate the statistical significance of the fitted cosine curve. Parameters of rhythms were compared when the fitted curve for both groups significantly corresponded
with experimental data. Gray fields indicate statistical significance of the difference between control and corresponding experimental groups revealed by cosinor analysis (P<0.05). SEM – standard error
of the mean, ns – non-significant
2014 Effect of Advance or Delay in LD Regimen on Heart S579
animals and the appearance of several periodicities with a
shorter duration (Experiment 2). According to our results
the response of animals was more homogenous in group
exposed to rotating delays compared with backward
Fig. 4. Representative double-plotted actograms showing rat locomotor activity rhythm during 1 week of light:dark conditions followed
by 11 weeks of phase advance shifts of light or 8 weeks of phase delay shifts of light. LD – regular cycle 12 light:12 dark; S – regimen
with rotating light:dark cycle. Number indicates the duration of experiment at the time of recording.
To evaluate the effect of rotating LD regimens
on the heart, we measured the expression of natriuretic
peptides anp and bnp that closely parallel many
pathological states of the cardiovascular system.
A significant rhythm in anp expression was not observed
in the left ventricle of the control and experimental
groups (Fig. 5A,C; Table 1). A decreasing trend in anp
expression between control and experimental groups was
observed in both experiments but did not reach a level of
significance (t-test: P=0.136 Experiment 1; P=0.282
Experiment 2). ANOVA did not show significant
differences between anp mRNA expression at six time
points of a 24 h cycle in control groups (Experiment 1
anp control F(5,28)=0.468, P=0.797; Experiment 2 anp
control F(5,18)=1.446, P=0.256). Differences in the
expression of anp in the heart of rats exposed to phase
shifts at six time points of Experiment 1 (anp advance
F(5,29)=0.218, P=0.952) and Experiment 2 (anp delay
F(5,17)=0.279, P=0.918) were not detected. Taken
together, no significant relationship between the LD
regimens and anp expression was observed under these
experimental conditions.
The significance of rhythmicity in bnp
expression was borderline in the control group of
Experiment 1 (Fig. 5D; Table 1). A very distinct daily
pattern was observed in the control group of
Experiment 2 (Fig. 5B; Table 1). In both cases a daily
rhythm peaked at ZT5 during the passive phase of the
24 h rhythm. ANOVA confirmed the differences between
time points in the control group of Experiment 2 (bnp
control F(5,17)=5.231, P=0.004) and the trend in
Experiment 1 (bnp control F(5,28)=2.063, P=0.100).
A rhythmic pattern in bnp expression was not observed in
experimental animals exposed to either rotating advances
or delays (Table 1). ANOVA did not indicate any
differences among time points in the experimental groups
(Experiment 1 bnp advance F(5,29)=0.787, P=0.567;
Experiment 2 bnp delay F(5,17)=1.711, P=0.186).
The mRNA expression of natriuretic peptides
did not correlate with blood pressure under normotensive
conditions in either the control or experimental groups.
Furthermore, no significant correlation between
natriuretic peptide mRNA expression and heart/body
weight index was observed in the control or experimental
groups (data not shown).
In both control groups, the expression of anp and
bnp correlated together during the light part of the 24 h
cycle (control Experiment 1: y = 1.7083x 2.3936,
R=0.852, n=12; control Experiment 2: y = 1.3897x
344.48, R=0.607, n=16). anp and bnp expression did not
correlate during the light part of the 24 h cycle in animals
exposed to rotating LD regimens.
S580 Herichová et al. Vol. 63
Fig. 5. Effect of rotating light:dark regimen with phase advances or phase delays on
(A, C) and
(B, D) mRNA expression in
the hearts of rats. The solid line represents the control group and the broken line shows data from the experimental group (n=3-8).
The black bar on the bottom of the graph represents the dark part of the 24 h cycle. Time scale is given in relative units Zeitgeber time
(ZT0 = beginning of the light part of the light:dark cycle).
The aim of this study was to elucidate the
physiological principles of uncoupling of peripheral
oscillators from the central oscillator under conditions of
rotating LD regimens. According to the classical rules of
entrainment, the strength of Zeitgeber depends upon its
robustness and angle difference (Aschoff et al. 1981).
This study indicates that under conditions of
rotating LD regimen with phase delays robustness of the
molecular oscillator is preserved, but the oscillator is out
of phase with the ambient LD cycle. Under conditions of
rotating LD regimen with phase advances oscillations of
rhythmic clock gene expression are maintained and
acrophase is similar to the control group but with less
robustness. Both phase advances and phase delays
resulted in weakened regulation of clock controlled genes
by the central loop, but via different way.
The daily profiles of per2, bmal1 and rev-erba
mRNA observed in the control groups showed the same
pattern as it was published previously (Young et al. 2001,
Szántóová et al. 2011, Herichova et al. 2013). The
application of phase-advanced rotating LD cycles
resulted in a pronounced decrease in the amplitude of
per2 and bmal1 rhythmic expression (down to 40 %),
while phase-delayed rotating LD cycles caused a phase
2014 Effect of Advance or Delay in LD Regimen on Heart S581
delay in the acrophase (by 8-9 h) but the change in
amplitude was much smaller.
Rev-erba (NR1D1) encodes an orphan member
of the nuclear receptor superfamily and is a clock
controlled gene. The basic molecular loop influences rev-
erba via E-boxes in its promoter region and the rhythmic
pattern of rev-erba expression is impaired in clock mutant
mice (Triqueneaux et al. 2004). Rhythmic expression of
rev-erba mRNA showed a significant decrease in
amplitude under both LD regimens, but the difference
was more pronounced in animals exposed to the LD
regimen with rotating advances. The acrophase of the
rhythmic expression of rev-erba was significantly
influenced by rotating phase delays. Rotating phase
advances did not cause a significant change in rev-erba
acrophase. This suggests that the rhythmic expression of
rev-erba is less disturbed in animals exposed to phase
advance rotating LD regimen since under these
conditions it corresponds better to actual LD regimen. It
is possible that lower robustness of per2 and bmal1
expression results in weakened circadian regulation of
rev-erba and that other regulatory regions of the rev-erba
gene or protein contribute to its regulation to a greater
extent (Duez and Staels 2009).
The activity records of three of the four animals
exposed to rotating phase delays showed a substantial and
significant increase in period length. The fourth rat also
displayed a periodicity of ~29 h, but component was the
second most powerful. The response of animals exposed
to rotating LD regimen with phase delays was more
homogenous compared with those exposed to phase
advances. In the animals exposed to backward shifts, four
animals maintained a significant periodicity of ~24 h. All
animals in Experiment 1 displayed an increase in the
presence of ultradian, and in two cases prolonged
periodicities with high individual variability.
Actograms and data obtained at the level of gene
expression cannot be linked directly. Some parallel can
be seen in Experiment 1 where the amplitude of rhythm
in locomotor activity as well as gene expression is
lowered. The circadian regulation of locomotor activity
and gene expression seem to more persistently take after
rotating LD regimen with phase delays compared with
phase advances.
During shift work many environmental and
individual factors play a role in the final response of the
organism (Saksvik et al. 2011). Together with changes in
LD regimen also a food intake is changed and this factor
is particularly important in the synchronization of
peripheral oscillators (Damiola et al. 2000). Peripheral
oscillators in the heart can be synchronized to food
regimen when the LD cycle is reversed, although it is the
influence of both of these factors (LD cycle and food
cycle) that significantly accelerates this process (Wu et
al. 2008, 2010). It was showed that reversed LD cycle
lasting for 1 week does not significantly influence the
acrophase of bmal1, per1, cry1 and dec1 in the heart of
rats with no access to food during the dark phase (Wu et
al. 2008). However, reversed accessibility to food
inverted the acrophase of clock genes within 7 days, even
when the rat could only access food during the light
phase. If the effect of food availability is strengthened by
the LD cycle, synchronization occurs within 5 days.
Expression of rev-erba, which is proposed to play a role
as a metabolic sensor of the circadian system (Duez and
Staels 2009), is able to synchronize to reversed food
regimen strengthened by the LD cycle in 5 days (Wu et
al. 2010). The role of food availability was implicated in
a model of metabolic desynchrony in rats with forced
activity during the passive phase and food availability
during the active phase of the LD cycle (Salgado-
Delgado et al. 2010). The study demonstrated that the
time of food consumption was a more crucial factor in the
generation of metabolic disturbances than forced activity.
In our study, food intake to some extent corresponded
with the presented actograms as food was available ad
libitum. We suppose that food intake is important factor
in the generation of the final pattern of clock gene
expression in the heart, and it represents the most
appropriate tool for synchronizing peripheral oscillators
under conditions of irregular LD cycles.
In the experimental setups in this study, an
increase in blood pressure and heart/body weight index
was not observed. Under normotensive conditions, anp
shows an arrhythmic daily profile and hypertension
induces its expression (Young et al. 2001). Our data are
in accordance with previous reports. Expression of anp in
the heart did not show a daily rhythm and its expression
was not increased by rotating LD regimens, presumably
because these treatments did not cause hypertension.
Expression of bnp shows a daily profile in the heart, as
has previously been reported (Goetze et al. 2010,
Herichova et al. 2013). In the present study we detected
significant daily rhythm in one control group
(Experiment 2) and a borderline rhythmic pattern in the
control group of Experiment 1, indicating that the daily
pattern of bnp expression has a rather low amplitude.
Both rotating LD regimens resulted in loss of the
S582 Herichová et al. Vol. 63
rhythmic pattern in bnp expression in the heart of
experimental rats. This finding suggests that the rhythmic
influence of bnp on blood pressure is weakened under
conditions of disturbed circadian system. This is also
supported by our previous study showing that rats kept
under irregular LD conditions have suppressed circadian
control of heart rate and blood pressure, and rely more on
an acute response to the LD regimen (Molcan et al.
The question of which direction of rotation is
less deleterious for the individual can be elucidated by
this study only to a limited extent. Most previous
epidemiological and experimental studies indicated that
rotating LD regimen with phase advances are more
harmful to humans (Saksvik et al. 2011, Deacon and
Arendt 1996). In our study, rotating phase delays
synchronized peripheral oscillators in the heart to a
shifted acrophase and rev-erba expression remained
coupled to the basic loop, while bnp expression did not.
Backward rotation, which causes a decrease in the
amplitude and rhythmicity of all measured genes,
possibly allows the system to be more responsive to acute
To conclude, the observed significant changes
demonstrate the two ways by which the circadian system
can cope with fast rotating LD regimens. In case of
rotating delays, the predictive capacity was preserved but
this process was not well timed in respect to the actual
LD cycle and in case phase advances we observed a
lower robustness, perhaps leading to weaker circadian
coordination in the periphery.
Conflict of Interest
There is no conflict of interest.
Research was supported by grant VEGA 1/1262/12,
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... Also similarly to humans, cortisol, neurohormones, renin, and aldosterone cycle in a way to raise HR and blood in the active phase. 4,9,[26][27][28][29] Created with ...
... The effect of light on the rhythmic expression of cardiac clock genes is well established. [9][10][11][12] Stimulated by light, the SCN acts as the central clock and synchronizer of all peripheral clocks. The SCN induces melatonin secretion by the pineal gland which influences clock gene expression in the heart. ...
... 9,30 ANP (atrial natriuretic peptide) gene expression in rats was seen to be arrhythmic throughout the day. 9 However, ANP protein quantification in perfused rat hearts demonstrated rhythmic levels of expression. 114 ANP concentrations oscillate rhythmically in humans, increasing at night and peaking before morning in an opposite trend to blood pressure. ...
Driven by autonomous molecular clocks that are synchronized by a master pacemaker in the suprachiasmatic nucleus, cardiac physiology fluctuates in diurnal rhythms that can be partly or entirely circadian. Cardiac contractility, metabolism, and electrophysiology, all have diurnal rhythms, as does the neurohumoral control of cardiac and kidney function. In this review, we discuss the evidence that circadian biology regulates cardiac function, how molecular clocks may relate to the pathogenesis of heart failure, and how chronotherapeutics might be applied in heart failure. Disrupting molecular clocks can lead to heart failure in animal models, and the myocardial response to injury seems to be conditioned by the time of day. Human studies are consistent with these findings, and they implicate the clock and circadian rhythms in the pathogenesis of heart failure. Certain circadian rhythms are maintained in patients with heart failure, a factor that can guide optimal timing of therapy. Pharmacologic and nonpharmacologic manipulation of circadian rhythms and molecular clocks show promise in the prevention and treatment of heart failure.
... The present study extends our previous results, which demonstrated that chronic phase advance and delay shifts disturb circadian system functioning based on changed temporal organisation of locomotor activity, cardiovascular parameters and clock gene expression [23,25]. In the current study, we analysed effects of repeated phase shifts of LD cycle on behaviour and the activity of autonomic nervous system (ANS) in response to different behavioural tests. ...
... Recent studies have shown that rapid shifts of LD cycle may prevent entrainment to such light schedules and induce a free-running like pattern of activity rhythm in animals [18] suggesting that SHIFT rats could be tested in different circadian times. However, in our previous study we demonstrated that rats exposed to 8-h phase delay shifts every second day substantially lengthened their period of activity rhythm by about 4 h [25,35], which is out of the range of free-running periods in rats [36] Moreover, clock gene expression in peripheral organs exhibited robust rhythmicity [25,37] indicating that rats were synchronised among each other. Since all behavioural tests were performed during the second half of the light phase on the second day after prolonged dark phase, it is likely that both SHIFT and CTRL rats were tested during their subjective passive phase (Fig. 1, 2). ...
... Recent studies have shown that rapid shifts of LD cycle may prevent entrainment to such light schedules and induce a free-running like pattern of activity rhythm in animals [18] suggesting that SHIFT rats could be tested in different circadian times. However, in our previous study we demonstrated that rats exposed to 8-h phase delay shifts every second day substantially lengthened their period of activity rhythm by about 4 h [25,35], which is out of the range of free-running periods in rats [36] Moreover, clock gene expression in peripheral organs exhibited robust rhythmicity [25,37] indicating that rats were synchronised among each other. Since all behavioural tests were performed during the second half of the light phase on the second day after prolonged dark phase, it is likely that both SHIFT and CTRL rats were tested during their subjective passive phase (Fig. 1, 2). ...
... However, we thought it is important to investigate substances that shorten the circadian clock period as many humans show longer endogenous circadian periods than 24 h (Brown et al. 2008). In addition, mammals, including mice and humans, find it more difficult to adjust to a short period length than a long period length as delaying the onset of activity rhythms is easier than accelerating them (Herichov a et al. 2014;West et al. 2017). Therefore, in this study, we focussed on crude drug candidates capable of shortening clock gene expression rhythms. ...
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Context The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. Objective We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. Materials and methods We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. Results We found that Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. Discussion and conclusions Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.
... Insomnia is associated with a 68 % increased incidence of myocardial infarction and an 85 % increase in risk of stroke (Somers et al. 2008). In one experimental model, the circadian rhythm of natriuretic peptide was altered by rotating the light-dark regimen (Herichová et al. 2014). ...
Obesity is linked to a wide range of serious illnesses. In addition to the important impact on the health of the individual, obesity also has a substantial impact on the economy. Disruption of physiological day-night cycles could contribute to the increased incidence of obesity. According to the American National Sleep Federation, the percentage of the people who reported a sleep duration of six hours or less increased from 12 to 37 % over ten years. Insufficient sleep leads not only to an increase of the total calorie intake but changes the meal preference in favor of palatable foods and meals with high carbohydrate content. A decrease of leptin and increase of ghrelin levels caused by sleep deficiency can also play a role. In addition to the higher caloric intake, the timing of food consumption should be taken into account. The same meal eaten during the night versus the day is associated with increased postprandial glucose and triglyceride levels. The gut microbiome has also been recently understood as an endocrine system, with links between the gut microbiome and circadian rhythm changes possibly influencing increased obesity.
... Therefore, access to food at equal intervals throughout the light/ dark cycle, which abolishes the daily feeding rhythm, does not reset peripheral clock rhythms. It is well known that information about light (and darkness) is very important to set the circadian clocks in the SCN and periphery (Yamazaki et al. 2000;Wu et al. 2008;Herichova et al. 2014;Herrero et al. 2015). Recently, Ikeda et al. (2015) showed that the length of the light period during the light/dark cycle affects the acrophase of Per2 in kidney, liver, and salivary gland under the 6-meals-a-day feeding schedule. ...
The master clock in the hypothalamic suprachiasmatic nucleus (SCN) is assumed to synchronize the tissue-specific rhythms of the peripheral clocks with the environmental day/night changes via neural, humoral and/or behavioral connections. The feeding rhythm is considered an important Zeitgeber for peripheral clocks, as daytime feeding reverses (clock) gene rhythms in the periphery, but not in the SCN. In this study, we investigated the necessity of a daily feeding rhythm for maintaining gene expression rhythms in epididymal white adipose tissue (eWAT). We showed that 7 of 9 rhythmic metabolic/adipokine genes, but not clock genes, lost their rhythmicity upon exposure to 6-meals-a-day feeding. Previously, we showed comparable effects of adrenalectomy on eWAT; therefore, subsequently we investigated the effect of simultaneous disruption of these humoral and behavioral signaling pathways, by exposing adrenalectomized animals to 6-meals-a-day feeding. Interestingly, under these conditions, all the clock genes and 10 of 11 rhythmic metabolic/adipokine genes lost their rhythmicity. These data indicate that adrenal hormones and feeding rhythm are indispensable for maintaining daily rhythms in metabolic/adipokine gene, but not clock gene, expression in eWAT. In contrast, at least one of these two signals should be present in order for eWAT clock gene rhythms to be maintained.
... Such situation may occur in shift workers who are exposed to two independent time cues, the photic and food. It is well known that light act via the retina and entrains the master circadian oscillator in the SCN (Warren et al. 2006), while food acts mainly through the liver and synchronises peripheral oscillators (Escobar et al. 2009), which have been found also in the heart (Herichová et al. 2014) and vessels (Xie et al. 2015). Such dual synchronising effects can lead to internal desynchronisation and splitting of circadian rhythms in LA which is governed predominantly by the SCN and cardiovascular rhythm, which are controlled in a more complex way than the physical activity. ...
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Effects of phase delay shifts (PDS) of light in combination with moderately increased salt intake (SL) (2%) or time restriction of food (FR) during the light-time (passive phase) on heart rate (HR), blood pressure (BP) and locomotor activity (LA) in radiotelemetry-measured rats were evaluated. PDS decreased amplitude and spectral power of circadian oscillations of HR, BP and LA. Moderately increased SL did not interfere with the circadian rhythmicity of HR, BP or LA. A prominent decrease in amplitude and spectral power of circadian oscillations was observed if food was available during the lighttime. Combination of PDS with FR split cardiovascular and behavioural parameters. In conclusion, food availability during the light-time in combination with PDS decreased amplitude and spectral power of circadian oscillations of BP, HR and LA more than PDS only. Different response of cardiovascular and behavioural parameters to photic and non-photic stimuli can have consequences for shift workers.
Cardiovascular (CV) health is often expressed by changes in heart rate and blood pressure, the physiological record of which may be affected by moving, anaesthesia, handling, time of day and many other factors in rodents. Telemetry measurement minimises these modulations and enables more accurate physiological recording of heart rate and blood pressure than non-invasive methods. Measurement of arterial blood pressure by telemetry requires implanting a catheter tip into the artery. Telemetry enables us to sample physiological parameters with a high frequency continuously for several months. By measuring the pressure in the artery using telemetry, we can visualize pressure changes over a heart cycle as the pressure wave. From the pressure wave, we can subtract systolic, diastolic, mean and pulse pressure. From the beat-to-beat interval (pressure wave) and the RR' interval (electrocardiogram), we can derive the heart rate. From beat-to-beat variability, we can evaluate the autonomic nervous system's activity and spontaneous baroreflex sensitivity and their impact on CV activity. On a long-term scale, circadian variability of CV parameters is evident. Circadian variability is the result of the circadian system's activity, which synchronises and organises many activities in the body, such as autonomic and reflex modulation of the CV system and its response to load over the day. In the presented review, we aimed to discuss telemetry devices, their types, implantation, set-up, limitations, short-term and long-term variability of heart rate and blood pressure in CV research. Data collection by telemetry should be, despite some limitations, standard in modern experimental CV research.
Social jetlag (SJL) is defined as the discrepancy between social and biological rhythms and calculated by the difference between the midpoint of sleep time on working-days and free-days. Previous human and mouse studies showed SJL is positively related to evening chronotype and significantly related to smoking habit, cardiovascular risk, cognitive ability, and that SJL-mimicking conditions, simulating the real lifestyle situation of SJL in many humans, disrupt the regularity of estrous cycles of female animals. The effects of SJL-mimicking conditions on circadian rhythms and cognitive function and the reasons why the discrepancy between social and biological rhythms is involved in SJL have not yet been investigated. Therefore, in this study, we utilized a mouse model of SJL-mimicking conditions – 6-hour delayed-light/dark (LD) conditions for 2 days and normal-LD conditions for the following 5 days – applied for several weeks during which biological rhythms were monitored. Circadian rhythms of central and peripheral clocks and metabolism of the mice under the SJL-mimicking condition were always delayed for 2–3 hours compared with those under the normal-LD condition. Moreover, SJL-mimicking conditions impaired their cognitive function using a novel object recognition test. Only the delayed timing of either the light phase of the LD or of feeding for 2 days, comparable to the free-days situation of humans, delayed the circadian staging of rhythms the following 5 days. Furthermore, sleep deprivation during the early mornings for 5 days, which is comparable to early rise times experienced by humans on working-days and does affect the staging of circadian rhythms (circadian misalignment schedule), delayed the locomotor activity rhythms the next 2 days, comparable to free-days in humans, which is similar to the lifestyle rhythm of the evening chronotype. Our results demonstrated that the circadian misalignment schedule for 5 days changed the locomotor activity rhythms the following 2 days to the evening chronotype, that light- and/or feeding-shift conditions for 2 days exacerbate SJL, and that SJL-mimicking conditions delay the metabolic rhythm and cause cognitive impairment.
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The incidence of colorectal cancer (CRC) shows a sex-dependent difference in humans. The aim of this study was to analyze estrogen receptor beta mRNA (ERbeta) expression in patients with CRC with respect to their gender and clinicopathological features. Since cancer progression is accompanied by tumor vascularization, VEGF-A (vascular endothelial growth factor A) transcription was analyzed along with ERbeta mRNA. ERbeta mRNA was also correlated with the expression of clock genes, which are known to influence the cell cycle. ERbeta mRNA expression in females with CRC showed an inverse association with increasing tumor staging that was not observed in males. Lower levels of ERbeta mRNA were observed in females with a higher clinical stage compared with those with earlier-stage tumors. ERbeta mRNA expression showed a significant positive correlation with mRNA of clock genes period 2 and cryptochrome 2 in healthy but not in cancerous tissue in males. Expression of VEGF-A mRNA showed a negative correlation with ERbeta mRNA after splitting of the cohort according to gender and nodus involvement. We propose that gender differences in ERbeta mRNA expression in tumors during the early stages of CRC can partially explain the lower occurrence of CRC in females compared with males.
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Exercise intolerance is the first symptom of heart disease. Yet an objective and standardised method in canine cardiology to assess exercise capacity in a clinical setting is lacking. In contrast, exercise testing is a powerful diagnostic tool in humans, providing valuable information on prognosis and impact of therapeutic intervention. To investigate whether an exercise test reveals differences between dogs with early stage mitral regurgitation (MR) and dogs without cardiac disease, 12 healthy beagles (healthy group, HG) and 12 dogs with presymptomatic MR (CHIEF B1 / B2, patient group, PG) underwent a six-stage submaximal exercise test (ET) on a motorised treadmill. They trotted in their individual comfort speed for three minutes per stage, first without incline, afterwards increasing it by 4% for every subsequent stage. Blood samples were taken at rest and during two 3-minute breaks in the course of the test. Further samples were taken after the completion of the exercise test and again after a 3-hour recovery period. Measured parameters included heart rate, lactate and the cardiac biomarkers N-terminal pro-B-Type natriuretic peptide and cardiac Troponin I. The test was performed again under the same conditions in the same dogs three weeks after the first trial to evaluate individual repeatability. Cardiac biomarkers increased significantly in both HG and PG in the course of the test. The increase was more pronounced in CHIEF B1 / B2 dogs than in the HG. N-terminal pro-B-Type natriuretic peptide increased from 435 ± 195 to 523 ± 239 pmol/L (HG) and from 690 to 815 pmol/L (PG). cTnI increased from 0.020 to 0.024 ng/mL (HG) and from 0.06 to 0.08 ng/ml (PG). The present study provides a method to assess exercise-induced changes in cardiac biomarkers under clinical conditions. The increase of NT-proBNP and cTnI is more pronounced in dogs with early-stage MR than in healthy dogs. Results indicate that measuring the parameters before and after exercise is adequate and taking blood samples between the different stages of the ET does not provide additional information. Also, stress echocardiography was inconclusive. It can be concluded that exercise testing, especially in combination with measuring cardiac biomarkers, could be a helpful diagnostic tool in canine cardiology.
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Cardiovascular parameters, such as blood pressure and heart rate, exhibit both circadian and ultradian rhythms which are important for the adequate functioning of the system. For a better understanding of possible negative effects of chronodisruption on the cardiovascular system we studied circadian and ultradian rhythms of blood pressure and heart rate in rats exposed to repeated 8 h phase advance shifts of photoperiod. The experiment lasted 12 weeks, with three shifts per week. Spectral power as a function of frequency for both circadian and harmonic ultradian rhythms was expressed as the circadian-ultradian power ratio. The circadian rhythms of blood pressure, heart rate and locomotor activity were recorded during the control light:dark (LD) regimen with higher values during the D in comparison with the L. Phase advance shifts resulted in a diminished circadian-ultradian power ratio for blood pressure, heart rate and locomotor activity indicating suppressed circadian control of these traits greater in heart rate than blood pressure. In conclusion, rats kept under irregular LD conditions have suppressed circadian control of heart rate, blood pressure and locomotor activity and rely more on an acute response to the LD regime. Their ability to anticipate regular loads can be weakened and in this way chronodisruption can contribute to the pathogenesis of cardiovascular diseases.
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Shift work was positively associated with higher incidence of metabolic syndrome, obesity, cardiovascular disease, sleep disturbances, decreased immune functions, and cancer. Observed disorders were manifested usually after longer time of shift work (more than 10 years). On the other hand, disturbed daily profile of melatonin and cortisol during shift work were detected even in human self reporting well tolerated shift work. Similarly, changes in thyroid stimulating hormone, prolactin, growth hormone, insulin, and ghrelin were demonstrated. Changes in hormone concentrations are influenced by shift work, sleep or circadian system or combinations of above mentioned regulatory factors. The circadian system consists of the central part localized in the hypothalamus and peripheral oscillators located in all tissues of the body. The central oscillator is predominantly synchronized by light and peripheral oscillators are more responsive to metabolic signals. Under conditions of shift work, central and peripheral oscillators dissociate that causes misalignment of daily rhythms in physiological functions. Synchronization during shift work can be improved by melatonin supplementation and manipulation with light:dark cycles and food regimens. Shift work tolerance is individual. Partial positive selection can be achieved on the basis of several psychological traits. Appropriate schedule can be estimated on the basis of chronotype. Keywords: melatonin, glucocorticoids, insulin, SIRT1, PER, chronotype, synchronization.
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Endogenous daily rhythms are generated by the hierarchically organized circadian system predominantly synchronized by the external light (L): dark (D) cycle. During recent years several humoral signals have been found to influence the generation and manifestation of daily rhythm. Since most studies have been performed under in vitro conditions, the mechanisms employed under in vivo conditions need to be investigated. Our study focused on angiotensin II (angII)-mediated regulation of Per2 expression in the suprachiasmatic nuclei (SCN) and heart and spontaneous locomotor activity in Wistar rats under synchronized conditions. Angiotensin II was infused (100 ng/kg/min) via subcutaneously implanted osmotic minipumps for 7 or 28 days. Samples were taken in 4-h intervals during a 24h cycle and after a light pulse applied in the first and second part of the dark phase. Gene expression was measured using real time PCR. Locomotor activity was monitored using an infrared camera with a remote control installed in the animal facility. Seven days of angII infusion caused an increase in blood pressure and heart/body weight index and 28 days of angII infusion also increased water intake in comparison with controls. We observed a distinct daily rhythm in Per2 expression in the SCN and heart of control rats and infused rats. Seven days of angII infusion did not influence Per2 expression in the heart. 28 days of angII treatment caused significant phase advance and a decrease in nighttime expression of Per2 and influenced expression of clock controlled genes Rev-erb alpha and Dbp in the heart compared to the control. Four weeks of angII infusion decreased the responsiveness of Per2 expression in the SCN to a light pulse at the end of the dark phase of the 24h cycle. Expression of mRNA coding angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) showed a daily rhythm in the heart of control rats. Four weeks of angII infusion caused a decrease in amplitude of rhythmic expression of Ace, the disappearance of rhythm and an increase in Ace2 expression. The Ace/Ace2 ratio showed a rhythmic pattern in the heart of control rats with peak levels during the dark phase. Angiotensin II infusion decreased the mean Ace/Ace2 mRNA ratio in the heart. We observed a significant daily rhythm in expression of brain natriuretic peptide (BNP) in the heart of control rats. In hypertensive rats mean value of Bnp expression increased. Locomotor activity showed a distinct daily rhythm in both groups. Angiotensin II time dependently decreased ratio of locomotor activity in active versus passive phase of 24h cycle. To conclude, 28 days of subcutaneous infusion of angII modulates the functioning of the central and peripheral circadian system measured at the level of Per2 expression and locomotor activity.
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The mammalian circadian system, which is comprised of multiple cellular clocks located in the organs and tissues, orchestrates their regulation in a hierarchical manner throughout the 24 hr of the day. At the top of the hierarchy are the suprachiasmatic nuclei, which synchronize subordinate organ and tissue clocks using electrical, endocrine, and metabolic signaling pathways that impact the molecular mechanisms of cellular clocks. The interplay between the central neural and peripheral tissue clocks is not fully understood and remains a major challenge in determining how neurological and metabolic homeostasis is achieved across the sleep-wake cycle. Disturbances in the communication between the plethora of body clocks can desynchronize the circadian system, which is believed to contribute to the development of diseases such as obesity and neuropsychiatric disorders. This review will highlight the relationship between clocks and metabolism, and describe how cues such as light, food, and reward mediate entrainment of the circadian system.
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Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5-7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.
B. D. Thattaliyath, B. A. Firulli and A. B. Firulli. The Basic-Helix-Loop-Helix Transcription Factor HAND2 Directly Regulates Transcription of the Atrial Naturetic Peptide Gene.Journal of Molecular and Cellular Cardiology (2002) 34, 1335–1344. The HAND basic Helix-Loop-Helix (bHLH) transcription factors are essential for the development of heart and extra embryonic structures. Although essential for embryonic development, the molecular pathways in which HAND factors participate are poorly understood. In efforts to identify downstream transcriptional targets, we have determined that HAND2 regulates the transcription of the Atrial Naturetic Peptide (ANP) gene. Results show that ANP expression is reduced in HAND2 null mice. Transactivation assays show significant transcriptional upregulation of ANP by HAND2 and cotransfection experiments using HAND2 and E12 suggest that an E-protein/HAND heterodimer is the likely trans -acting complex. The required cis -elements reside within a 258 bp proximal region that contains three evolutionarily conserved Ebox consensus sites. Surprisingly, mutations in these three sites suggest HAND2 activity is DNA-binding independent. In addition, HAND2 and the homeobox factor Nkx2.5 exhibit transcriptional synergy in the regulation of ANP. Taken together, this data shows that HAND2 is an upstream transcriptional regulator of ANP expression, and furthermore HAND2 can synergistically interact with Nkx2.5, showing a functional relationship between HAND2 and Nkx2.5 supporting the genetic observation, that mice null for both HAND2 and Nkx2.5 lack ventricle specification.
The mammalian heart expresses two closely related natriuretic peptide (NP) hormones, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). The excretion of the NPs and the expression of their genes strongly respond to a variety of cardiovascular disorders. NPs act to increase natriuresis and decrease vascular resistance, thereby decreasing blood volume, systemic blood pressure and afterload. Plasma levels of BNP are used as diagnostic and prognostic markers for hypertrophy and heart failure (HF), and both ANF and BNP are widely used in biomedical research to assess the hypertrophic response in cell culture or the development of HF related diseases in animal models. Moreover, ANF and BNP are used as specific markers for the differentiating working myocardium in the developing heart, and the ANF promoter serves as platform to investigate gene regulatory networks during heart development and disease. However, despite decades of research, the mechanisms regulating the NP genes during development and disease are not well understood. Here we review current knowledge on the regulation of expression of the genes for ANF and BNP and their role as biomarkers, and give future directions to identify the in vivo regulatory mechanisms. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.
Six methods for measuring the period of circadian rhythms (tau) have been compared using real and artificial data. The modified periodogram technique estimates tau by fitting a data‐specific form estimate to time series data with a period that minimizes the variance of data around the form estimate. The onset periodogram converts raw data to new values that reflect rapid increases of data magnitude, then fits a form estimate to these new values. Iterative cosinor determines the period of the single cosine curve that best describes the data. The iterative harmonic derives a form estimate, consisting of a cosine function plus a series of true harmonics, which most closely describes the data. Other methods measure the slope of the line fitting each day's acrophase (regressive cosinor) or subjectively selected phase markers (eyefitting).Our analyses indicate that the estimated value of tau can vary depending on the method used. Periodogram and iterative harmonic methods provided the most consistently satisfactory estimates of tau from all sets of data examined. Eyefitting and onset periodogram methods accurately estimated tau from artificial data based on hamster activity rhythms, but were less useful for estimating temperature tau. The eyefitting procedure was less replicable than the automated methods for estimating tau. The choice of analysis method ultimately depends upon the character of the data to be analyzed and the nature of the hypotheses to be tested.