Published online before print May 24, 2001, doi: 10.1161/hh1101.091190
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Clock Genes in the Heart
Characterization and Attenuation With Hypertrophy
From the Department of Internal Medicine, Division of Cardiology, University of Texas–Houston Medical School, Houston, Tex.
Correspondence to Heinrich Taegtmeyer, MD, DPhil, Department of Internal Medicine, Division of Cardiology, University of Texas–Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030. E-mail Heinrich.Taegtmeyer{at}uth.tmc.edu
Abstract
Abstract—We investigated whether the heart, like other mammalian organs, possesses internal clocks, and, if so, whether pressure overload–induced hypertrophy alters the clock mechanism. Clock genes are intrinsically maintained, as shown by rhythmic changes even in single cells. Clocks are believed to confer a selective advantage by priming the cell for the expected environmental stimulus. In this way, clocks allow anticipation, thereby synchronizing responsiveness of the cell with the timing of the stimulus. We have found that in rat heart all mammalian homologues of known Drosophila clock genes (bmal1, clock, cry1, cry2, per1, per2, per3, dbp, hlf, and tef) show circadian patterns of expression and that the induction of clock output genes (the PAR [rich in proline and acidic amino acid residues] transcription factors dbp, hlf, and tef) is attenuated in the pressure-overloaded hypertrophied heart. The results expose a new dynamic regulatory system in the heart, which is partially lost with hypertrophy. Although the target genes of these PAR transcription factors are not known in the heart, the results provide evidence for a diminished ability of the hypertrophied heart to anticipate and subsequently adapt to physiological alterations during the day.
Key Words: anticipation • circadian rhythms • rat
All mammalian cells investigated to date seem to possess internal circadian clocks.1 Such clocks are even present when isolated cells from peripheral tissues are cultured, suggesting that the basic clock mechanism is intrinsic and self-sustained, allowing the cell to anticipate and integrate changes in its normal environment.1 2 3 4 As the culture is maintained, the amplitude of the clock diminishes (followed by gene expression markers), as does the timekeeping of the duration of each cycle.3 However, reintroduction of serum to the culture normalizes the clock, suggesting that one or more humoral factors act as a zeitgeber (timekeeper).5 There are three major components of biological clocks. These are (1) input signals, such as light in the case of the mammalian eye or humoral factors for peripheral tissues; (2) the clock mechanism itself; and (3) the output genes.
The heart responds to both acute and chronic changes in its environment. Acutely, it responds to alterations in workload, hormones, and fuel supply, through allosteric, covalent, and topographical alterations in preexisting proteins.6 7 8 9 Chronically, the heart responds to sustained alterations in these same environmental factors, by the synthesis of new proteins, through both transcriptional and posttranscriptional mechanisms.10 11 For example, the heart responds to sustained pressure overload by increasing the size of the cardiomyocytes (hypertrophy), a process requiring changes in both gene expression and protein synthesis.12 How the responsiveness of the myocardium to physiological stimuli alters during the day has not previously been considered.
Intrinsic circadian rhythms have not been characterized in the heart at the transcriptional level. We therefore set out to investigate, in the rat heart, the level of gene expression of all of the major components involved in these rhythms over a 24-hour period. Because circadian rhythms are potentially involved in the daily adaptation of the heart, we hypothesized that the same factors might be involved in the development of hypertrophy (adaptation to sustained pressure overload). We therefore investigated whether circadian rhythms are altered in the hypertrophied heart and found that the rhythms of the major genes involved in the clock mechanism, with the exception of the period genes, were not affected by pressure overload–induced hypertrophy. In contrast, the output genes (and the period genes to a lesser extent) showed impairment in the level of induction during the light period. Pressure overload had no effect on the periodicity of the rhythms for any of the genes investigated. The results provide evidence for a diminished ability of the hypertrophied heart to anticipate and subsequently adapt to physiological alterations during the day.
Materials and Methods
Aortic Constriction
The care and use of animals followed the guidelines
of the Animal Welfare Committee at the University of Texas–Houston
Health Science Center. Cardiac hypertrophy was induced in
male Wistar rats (160 to 210 g initial weight) by constriction of
the ascending aorta, as described
previously.13 The diameter
of the lumen of the aorta after constriction was equivalent to that of
an 18-gauge needle. In control animals, sham operations were performed
without constriction of the aorta. As gene expression can be affected
by multiple factors, including physical activity, hormone levels, and
food intake, animals were allowed to recover from surgery for 7 weeks.
During this time, the initial modest elevation in pressure on the heart
using an 18-gauge needle for the banding procedure increased as the
animals grew (310 to 430 g final weight). This more gradual
increase in pressure on the heart with age is more akin to the
situation in humans, as opposed to an acute, dramatic pressure
overload. A total of 108 animals were studied, of which 55 were
controls and 53 were banded.
One week before they were euthanized, rats were housed in a separate environment-controlled room, in which a strict 12-hour light/12-hour dark cycle regimen was enforced (lights on at 7 AM; zeitgeber time [ZT] 0). On the day of the experiment, rats were killed every 3 hours from 7 AM to 7 AM the following day. To ensure the reproducibility of the gene expression cycles, at least two control and two banded animals were killed at each time point on 3 separate days. After administration of pentobarbital (100 mg/kg IP), hearts were rapidly isolated, freeze-clamped in liquid nitrogen, and stored at -80°C before RNA extraction.
RNA Extraction and Quantitative Reverse
Transcriptase–Polymerase Chain Reaction (RT-PCR)
RNA extraction and quantitative RT-PCR of samples was
performed using previously described
methods.10 14 15 16
Specific quantitative assays were designed from rat sequences available
in GenBank
(Table
),
except in the cases of cry1,
cry2,
per1, and
per3, for whom the rat
sequences were not available. In contrast, these genes have been cloned
in the mouse. Multiple assays were then designed using the mouse
sequence for these genes and subsequently tested for compatibility in
the rat. Primers and probes were designed from nonconserved sequences
of the genes (allowing for isoform specificity), spanning sites where
two exons join (splice sites) when such sites are known (preventing
recognition of the assay to any potential contaminating genomic DNA).
Standard RNA was made for all assays by the T7 polymerase method
(Ambion), using total RNA isolated from the rat
heart. The correlation between the
Ct (the
number of PCR cycles required for the fluorescent signal to
reach a detection threshold) and the amount of standard was linear over
at least a 5-log range of RNA for all assays (data not shown). The
level of transcripts for the constitutive housekeeping gene product
ß-actin was
quantitatively measured in each sample, to control for sample-to-sample
differences in RNA concentration. PCR data are reported as the number
of transcripts per number of
ß-actin
molecules.
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Statistical Analysis
Data are presented as the mean±SEM for
between six and eight hearts in each group. Statistically significant
differences between groups were calculated by the Student
t test between control and
hypertrophied hearts at the same zeitgeber time. A value of
P<0.05 was considered
significant.
Results
Aortic Constriction Induces Both Trophic and
Gene Expression Markers of Hypertrophy
Seven weeks after the initial surgery, the heart
weight–to–body weight (HW/BW) ratio was increased by 30%
(Figure 1A). This increase was due to increased heart weight
as opposed to fluctuations in body weight (data not shown). The HW/BW
ratio did not undergo obvious rhythmic changes during the day
(Figure 1A).
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) and banded (
) animals at the same zeitgeber time.
Pressure overload is known to cause the reexpression of
several fetal genes in the heart, including atrial
natriuretic factor
(anf). This genetic marker of
hypertrophy was elevated at all time points investigated in
pressure-overloaded hearts
(Figure 1B
). The average level of
anf expression throughout the
24-hour period was 4.6-fold greater in the pressure-overloaded hearts.
There was a trend for a steady increase in
anf expression during the dark
phase in control animals, with a subsequent decrease during the light
phase. This trend was not observed in the pressure-overloaded
hearts.
Expression of Clock Genes in Normal and
Hypertrophied Hearts
Mammalian homologues of known
Drosophila clock components
include CLOCK, BMAL1
(brain and
muscle
ARNT-like
protein 1), PER (periods 1, 2, and 3), and CRY (cryptochromes 1
and
2).1 2 17 18 19 20 21 22
Using real-time quantitative RT-PCR, we compared the rhythms of the
mRNA encoding for these proteins in control and hypertrophied hearts.
bmal1, a basic
helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) transcription factor,
undergoes striking circadian rhythms in the heart
(Figure 2A). There was a 19-fold induction of
bmal1 during the day (from peak
[ZT0] to trough [ZT12]). There were no differences in the level of
bmal1 expression between
control and hypertrophied hearts, when compared at the same time
points. BMAL1 heterodimerizes with a second bHLH/PAS transcription
factor, CLOCK. clock
expression, like that of bmal1,
undergoes a circadian rhythm in the heart, albeit less dramatically
(2-fold from peak [ZT0] to trough [ZT12];
Figure 2B). No significant difference in the level of
clock expression was observed
between control and hypertrophied hearts.
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We investigated the expression rhythms of
cry (1 and 2) and
per (1, 2, and 3) mRNA in
control and hypertrophied hearts. Unlike
bmal1 and
clock,
cry1 and
cry2 appear to have slightly
different rhythms compared with one another
(Figures 3A and 3B).
cry1 expression has a peak at
ZT21 and a trough at ZT9, whereas
cry2 has an earlier peak at
ZT15 and a trough at ZT3. The induction of both
cry1 and
cry2 was 3-fold. No significant
differences were observed between control and hypertrophied hearts. The
circadian rhythms of the 3 per
gene isoforms were very similar to one another, with peaks at ZT12/15
and troughs at ZT0/3
(Figures 4A through 4C). The levels of induction of
per1,
per2, and
per3 were 5-, 8-, and 10-fold,
respectively. In all three cases, there was a trend for decreased
induction of the per genes in
the hypertrophied heart, with no differences in expression at the basal
(trough) levels.
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Impaired Induction of Clock Output Genes in the
Hypertrophied Heart
Clock output genes include the PAR (rich in
proline and
acidic amino
acid residues)
transcription factors dbp
(albumin
D-element
binding
protein),
hlf
(hepatic
leukocyte
factor), and
tef
(thyrotrophic
embryonic
factor).23 24 25
The circadian rhythms of the genes for these three PAR factors are very
similar, with peaks at ZT12/15 and troughs at ZT0/3
(tef appears to have a slightly
later peak compared with dbp
and hlf;
Figures 5A through 5C). The levels of induction of
dbp,
hlf, and
tef were 41-, 6-, and 3- fold,
respectively. There was an impairment in the level of induction of
these transcription factors in the hypertrophied heart compared with
the control hearts. No differences were observed in the basal levels of
expression.
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) animals at the same zeitgeber time.Discussion
The present study exposes a new dynamic system of
transcriptional regulation in the heart, which is summarized in
Figure 6A. In a normal heart, the level of gene expression
changes dramatically within a 24-hour period. Many of the genes we
examined are transcription factors, with the potential to alter the
expression of a large number of downstream target proteins. These
transcription factors include BMAL1, CLOCK, DBP, HLF, and TEF. The
identity of the target gene promoters to which these
trans-acting factors bind
remains relatively unknown, particularly in the case of the latter
three PAR factors. In the heart, this family of transcription factors
has not been investigated previously.
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In addition to full characterization of the primary factors known to be involved in cellular clocks, we were interested in how such components, which are involved in the daily adaptation of the heart, might be altered in a chronically adapted heart, in the face of pressure overload. We found that the components of the clock mechanism itself were little affected in the hypertrophied heart. In contrast, known clock output genes (ie, the PAR transcription factors investigated) showed a significant impairment of induction during the 24-hour period. Whether this impairment of induction is amplified further in the target genes of these transcription factors, and whether these targets are directly involved in the development of hypertrophy, is unknown. Thus, in pressure overload–induced hypertrophy, the heart has partially lost its ability to anticipate environmental changes.
Cardiac Biological Clocks
Two key transcription factors involved in circadian
rhythms, BMAL1 and CLOCK, both undergo rhythms during the day in the
heart
(Figure 2). In the case of BMAL1, the present results are
consistent with previous
studies.26 However, whether
the clock gene undergoes
circadian rhythms in peripheral tissues is less
clear.1 Using real-time
quantitative RT-PCR, a highly sensitive technique, we have found that
clock expression in the heart
does indeed show a rhythmic pattern, in the same phase as
bmal1, albeit at lower
amplitude. Furthermore, the levels of expression of these two bHLH/PAS
transcription factors are similar at their maximal levels (ZT0). These
two factors form a transcriptionally active
heterodimer.19 The near
equal level of expression of these two factors at ZT0 will promote
effective dimerization. However, as the cycle progresses, the level of
bmal1 expression diminishes to
a greater extent than that of
clock and therefore becomes
limiting.
The BMAL1-CLOCK heterodimer binds to
cis-acting elements (E-boxes)
in the promoter region of various target genes, including
per,
cry, and
bmal1 itself
(Figure 6B).19 27
Once synthesized, PER and CRY isoforms translocate into the nucleus and
generally act as repressors of BMAL1-CLOCK heterodimer
function.28 Such repression
might be achieved by direct binding to the heterodimer components,
binding to the E-box sequence, or binding to as-yet-unidentified
cofactors. In the case of
Drosophila, a protein named
timeless (TIM) has been identified that binds to the PERs and may be
involved in the translocation and/or repression mechanism mentioned
above.29 30 A
mammalian TIM homologue was believed to be found, which has been shown
to be permanently located in the
nucleus.28 31
However, recent reports suggest that this gene is not the true
mammalian homologue to tim, and
therefore it was not investigated in the present
study.32 The
per and
cry gene products therefore
form a negative feedback loop, resulting in the downregulation of their
own expression
(Figure 6A). With the exception of
cry1, the phases of these
transcriptional modulators are very similar to one another and are
opposite to those of both bmal1
and clock. Thus, when the
expression of bmal1 and
clock is at a maximum, the
expression levels of per1,
per2,
per3, and
cry2 are at their lowest
(Figure 6A).
Cryptochromes form a second class of photoreceptor found in
the mammalian eye (the other being
rhodopsin).33 These proteins
are putative vitamin B2–based photopigments
that contain a pterin as a chromophore; cryptochromes have been
hypothesized to be involved in
photoentrainment.33 Indeed,
mutation of cry1 and
cry2 in mice results in a
complete loss of circadian rhythmicity in wheel-running behavior, in
addition to elevated arrhythmic levels of
per1 and
per2.34 35
However, the circadian rhythms of these mutant mice still have light
sensitivity, suggesting that cryptochromes are not the only protein
involved in
photoentrainment.35 The
heart of the semitransparent zebrafish responds directly to light,
through cryptochromes.4
Despite the fact that mammals are opaque and peripheral
tissues are not normally exposed to light, cryptochromes have been
shown to be expressed in multiple internal organs, including the heart
(although their circadian oscillations have not been
investigated previously in this
tissue).33 Period gene
products are believed to bind to and result in the translocation of
the cryptochromes into the nucleus, where the latter inhibit the
transcriptional activity of BMAL1-CLOCK (see above). The nature of the
signal for cryptochrome activation in peripheral tissues is
unknown. Evidence suggests that phosphorylation and/or
degradation of PER proteins might be involved in clock
modulation.1 2
Phosphorylation of PER is catalyzed by casein kinase I
(CKI), the mammalian homologue to
Drosophila DOUBLETIME, the
mutation of which results in
arrhythmia.36 We
speculate that factors (zeitgebers) affecting the activity of CKI
are involved in peripheral clock resetting.
The separation in the phases of cardiac
cry1 and
cry2 expression by 6 hours
gives rise to several exciting hypotheses. For example, whether CRY1
and CRY2 differentially suppress the binding of the BMAL1-CLOCK
heterodimer to different promoter regions is one possibility. As
cry2 expression is 10-fold
greater than that of cry1 in
the heart, and given that the periodicity of
cry2 expression is in rhythm
with the per genes, and
opposite to bmal1 and
clock, it is likely that CRY2
is involved more in the negative feedback loop mechanism in the heart
than is CRY1. Whether CRY1 is involved in the repression of other genes
or differentially modulates the PER isoforms is unknown. For example,
PER2 is believed to promote
bmal1 expression, as part of
the bmal1 reexpression stage of
the clock (positive loop;
Figure 6A).32 It
is possible that CRY1 preferentially binds to PER2, resulting in
subsequent translocation to the nucleus later than PER1 and PER3 and
allowing for delayed bmal1
induction. Alternatively, CRY1 and CRY2 may compete for PERs, and the
later induction of CRY1 may displace CRY2 from its PER partner,
altering its function at a specific moment in the cycle.
The BMAL1-CLOCK heterodimer can also activate the
transcription of a number of clock-controlled genes (or output genes;
Figure 6B). Clock-controlled genes identified to date
include vasopressin and the family of PAR transcription factors
dbp,
hlf, and
tef.23 24 25 31
The latter three showed an expression pattern similar to that of the
per genes and
cry2. The maximal levels of
expression of hlf and
tef were similar to one another
and were approximately one third that of
dbp. Very little is known
concerning the identity of the target genes of this family of PAR
transcription factors. In extracardiac tissues,
dbp has been shown to regulate
angiotensinogen, steroid 15
-hydroxylase, and coumarin
7-hydroxylase
expression.37 38
No targets have been identified in the heart as yet. Clearly these
transcription factors, of which the level of expression can change up
to 40-fold within 1 day, must have
physiologically relevant targets in
cardiomyocytes.
Adaptation of the Heart
The mechanisms by which the heart adapts to various
stimuli has been an intense area of research. Factors known to affect
the myocardium include humoral factors (such as substrate
availability and hormones), neurohormones, and mechanical stress. All
of these factors change during a single day. Food intake, hormones,
sympathetic tone, blood pressure, and physical activity are all known
to be altered at different times in the day. Despite this, very little
is known concerning the adaptation or responsiveness of the heart
during the 24-hour period to this assortment of
physiological stimuli. It can be hypothesized that
the normal heart adapts to pressure overload every day, but only
chronic exposure to these conditions causes a full phenotypic response
and the ensuing long-term complications. For example, blood pressure
undergoes a circadian rhythm, with a trough during the day and a peak
during the night (in the case of the
rodent).39 The phase of
increased blood pressure may initiate alterations in the heart
(biochemical and/or transcriptional, such as
anf induction
[Figure 1B]), similar to those observed in the hypertrophied
heart, but the full development of hypertrophy does not
occur because of the reduction of pressure later in the day. However,
in response to pressure overload, these normal mechanisms are
chronically activated (for example, induction of
anf;
Figure 1B).
Cardiomyocytes are not different from other cells. Its internal clock mechanism is important for the anticipation of the cell to changes in the environment within which it finds itself, thereby synchronizing responsiveness with the stimulus. These clocks are intrinsic within each cell and can continue even when the cells are in isolation.3 However, external factors are required for the resetting of these clocks, forming a communication between the cell and its environment.4 5 In the case of the suprachiasmatic nucleus of the brain (the central body clock), light sensing by the eyes appears to be the signal for the resetting of the clock (a process termed photoentrainment).40 In contrast, peripheral tissues are not always in direct contact with external light and rely on other factors to modulate the clock mechanism. No single factor has been ascribed to the zeitgeber of peripheral tissues. It is possible that more than one factor is involved in the regulation of peripheral clocks in mammals, allowing for multilevel adaptation. Additional factors might include hormones, substrate availability, and blood pressure.
Impaired Rhythms in the Hypertrophied
Heart
We compared the expression patterns of components of
the biological clocks in normal and pressure overload–induced
hypertrophied hearts for the following reasons. First, as blood
pressure undergoes circadian rhythms during the day, it is possible
that pressure acts as a zeitgeber for peripheral clocks.
Thus, chronic pressure overload occurring locally on the heart would be
expected to modulate the clock. Second, we wanted to know whether the
mechanisms involved in circadian rhythms were also involved in the
development of hypertrophy. These studies would also aid in
the understanding of how the hypertrophied heart anticipates and adapts
to additional environmental changes in the face of pressure overload.
If the hypertrophied heart is less flexible in terms of adaptation to
additional environmental factors, this "rigidity" may play a role
in the subsequent development of cardiac dysfunction and
failure.
In the rat, blood pressure, heart rate, and cardiac output are greatest during the dark phase, when the animal is most active and forages for food.39 In the light phase, these parameters decrease in the more sedentary animal.39 Aortic constriction increases the local pressure on the heart, which subsequently maintains cardiac output through the development of hypertrophy.13 Mechanical stress on cardiomyocytes, caused by increased blood pressure, for example, induces a cascade of intracellular signaling events resulting in alterations in gene expression.12 It could therefore be hypothesized that blood pressure acts as a zeitgeber for circadian rhythms in the heart through overlapping mechanisms compared with the mechanical stress–induced hypertrophic response. If this hypothesis were true, then aortic constriction should diminish the fluctuation of genes in the light phase, when pressure is normally decreased. Genes encoding for the PERs and the family of the PAR transcription factors are induced in the light. In the hypertrophied heart, the induction of these genes is blunted (with no effect on basal expression), suggesting that pressure may play a role in modulation of rhythms in the normal heart. However, as the amplitude of the rhythms is only partially attenuated, and no effect is seen on periodicity, it is likely that other factors in addition to pressure act as zeitgebers in the heart. This conclusion is consistent with other studies, both in vitro and in vivo.5 41
Assuming that internal clocks are important for the anticipation of cells to environmental stimuli, the present results suggest that the hypertrophied heart is less able to adapt to changes in physiological factors during the day. Previous studies have shown that the hypertrophied heart can maladapt (fail) either with sustained pressure overload over longer periods of time or when an additional stress is placed on the heart.42 43 For example, both pressure overload–induced and streptozotocin-induced diabetes mellitus result in specific programs of cardiac adaptation, allowing for maintenance of contractile function within their respective environments.11 13 However, when both stresses are placed on the heart simultaneously, the myocardium fails.43 Thus, the adapted heart appears less able to respond to additional stimuli. This is consistent with the present study in which the hypertrophied heart is unable to induce the expression of specific clock output genes (the PAR transcription factors). We have also found this to be true for the circadian rhythms of metabolic genes (authors’ unpublished observation, 2001), suggesting that the hypertrophied heart anticipates and adapts less well to fluctuations in substrate/fuel availability, hormones, and energy demand/workload. Whether the impairment of the hypertrophied heart to adapt worsens with prolonged or more severe pressure overload is presently unknown.
Conclusions
The present study has characterized fully the major
components of the intracellular clock in the heart, of which the level
of gene expression changes dramatically during a 24-hour period. Many
of the components of this system are transcription factors that have
the potential to alter the expression of a host of as-yet-unidentified
genes in the heart. In the case of the hypertrophied heart, the
circadian rhythms of the PAR transcription factors are attenuated.
Whether these circadian clocks are important for diurnal variation in
cardiac function or whether attenuation of this mechanism contributes
to the development of contractile dysfunction is currently
unknown.
>
Acknowledgments
This work was supported in part by NIH Grants HL-43133 and HL-61483 and by the American Heart Association National Center. We thank J. Ying and P. Guthrie for technical assistance.
Footnotes
Original received March 5, 2001; revision received April 6, 2001; accepted April 6, 2001.
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