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Am J Physiol Heart Circ Physiol 290: H1–H16, 2006;
doi:10.1152/ajpheart.00582.2005.
Invited Review
The circadian clock within the heart: potential influence on myocardial gene
expression, metabolism, and function
Martin E. Young
United States Department of Agriculture/Agricultural Research Service Children’s Nutrition
Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas
anticipation; cardiovascular; diurnal variations; zeitgeber
SINCE THE INITIATION OF LIFE on Earth, one selective pressure that
has been imposed continuously on terrestrial organisms is the
time of day. Over the course of a normal 24 h, the environment
of, and the demands placed on, an organism fluctuate dramatically. Despite environmental influences, biological processes
must remain within specific physiological boundaries. Intuitively, homeostatic mechanisms would function more efficiently if the organism possessed a means for anticipation of
daily routine changes in the environment. It is therefore not
surprising that virtually every organism (with the exception of
certain prokaryotes) has evolved specific mechanisms allowing
for anticipation of environmental fluctuations over the course
of the day. These mechanisms, collectively known as circadian
clocks, allow individual cells to perceive the time of day (34).
In doing so, circadian clocks confer the selective advantage of
anticipation, conditioning the cell to changes in its environment before they occur.
Circadian clocks have been identified in every mammalian
cell investigated to date, including key components of the
cardiovascular system, such as cardiomyocytes and vascular
smooth muscle cells (VSMCs) (26, 33, 34, 82, 90, 94). Given
Address for reprint requests and other correspondence: M. E. Young, United
States Dept. of Agriculture/Agricultural Research Service Children’s Nutrition
Research Center, Dept. of Pediatrics, Baylor College of Medicine, 1100 Bates
St., Houston, TX 77030 (e-mail: [email protected]).
http://www.ajpheart.org
the universal appreciation for diurnal variations in both physiological and pathophysiological cardiovascular events, the
recent exposure of a molecular machinery within the individual
cells of the cardiovascular system that has the potential of
modulating an array of cellular processes has sparked increasing interest. Historically, diurnal variations in blood pressure,
heart rate, and cardiac output, as well fatal cardiovascular
events, have been attributed primarily to diurnal variations in
environmental stimuli, such as a sudden increase in sympathetic activity (89, 102, 105, 138). However, the ability of the
cardiovascular system to respond in an appropriate and timely
manner to neurohumoral stimuli is likely of equal importance.
Intracellular circadian clocks provide a means by which the
heart and vasculature can anticipate diurnal variations in stimuli, such as autonomic and sympathetic nervous activity, ensuring an optimal response. Attenuation of this molecular
mechanism would therefore impair the ability of the heart
and/or vasculature to respond appropriately to environmental
stimuli, which in turn may contribute toward the development
of cardiovascular disease.
The purpose of this review is to summarize our current
knowledge regarding circadian clocks within the cardiovascular system (with special emphasis on the heart), the biological
processes they influence, how they are regulated, and the
factors that lead to their impairment. What should become
apparent is that although circadian rhythmicity of cardiovasH1
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Young, Martin E. The circadian clock within the heart: potential influence on
myocardial gene expression, metabolism, and function. Am J Physiol Heart Circ
Physiol 290: H1–H16, 2006; doi:10.1152/ajpheart.00582.2005.—It is becoming
increasingly clear that the intrinsic properties of both the heart and vasculature
exhibit dramatic oscillations over the course of the day. Diurnal variations in the
responsiveness of the cardiovascular system to environmental stimuli are mediated
by a complex interplay between extracellular (i.e., neurohumoral factors) and
intracellular (i.e., circadian clock) influences. The intracellular circadian clock is
composed of a series of transcriptional modulators that together allow the cell to
perceive the time of day, thereby enabling preparation for an anticipated stimulus.
These molecular timepieces have been characterized recently within both vascular
smooth muscle cells and cardiomyocytes, giving rise to a multitude of hypotheses
relating to the potential role(s) of the circadian clock as a modulator of physiological and pathophysiological cardiovascular events. For example, evidence strongly
supports the hypothesis that the circadian clock within the heart modulates myocardial metabolism, which in turn facilitates anticipation of diurnal variations in
workload, substrate availability, and/or the energy supply-to-demand ratio. The
purpose of this review is therefore to summarize our current understanding of the
molecular events governing diurnal variations in the intrinsic properties of the
heart, with special emphasis on the intramyocardial circadian clock. Whether
impairment of this molecular mechanism contributes toward cardiovascular disease
associated with hypertension, diabetes mellitus, shift work, sleep apnea, and/or
obesity will be discussed.
Invited Review
H2
MYOCARDIAL CIRCADIAN CLOCK
cular function is firmly established, our understanding of the
role of the circadian clock as a mediator of this phenomenon
remains in its infancy. Recent advances linking circadian
clocks with cardiovascular function suggest it is timely to
highlight the potential role(s) of this molecular mechanism
within the heart.
EXISTENCE OF CENTRAL AND PERIPHERAL
CIRCADIAN CLOCKS
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One of the first experiments suggesting the existence of an
intrinsic mechanism allowing organisms to perceive the time of
day also exemplifies the primary role of circadian clocks. In
1729, a French astronomer named Jean Jacques d’Ortous de
Marian investigated whether plants that unfolded their leaves
during the day did so in response to sunlight. He noted that
plants kept in constant darkness still unfolded their leaves
during daylight hours, despite never being exposed to the light,
and concluded that the observed rhythm was not driven by the
environment but was an intrinsic property of the organism (34).
This rhythm provides a selective advantage by allowing the
plant to anticipate when the sun will rise, thereby opening its
leaves to increase photosynthetic potential at the correct time
of day. By possessing an internal clock mechanism, cells/
organisms are able to anticipate temporal changes in their
environment, optimizing biological processes so that they occur at an advantageous time in the day. It has been suggested
that primordial clocks may have allowed noncomplex cells to
anticipate periods of high ionizing radiation from the sun (due
to a lack of an ozone layer), so that DNA synthesis would
occur only at night, thereby minimizing the occurrence of
DNA mutations (99).
Circadian clocks can be defined as a set of proteins that
generate self-sustained transcriptional positive and negative
feedback loops with a free-running period of 24 h (32, 34, 49,
50). These circadian clocks are intrinsic to the cell, persisting
when tissues and cells are isolated and cultured in vitro (4, 33,
82, 90, 152). To maintain their selective advantage, circadian
clocks are reset (or entrained) by environmental cues (the most
apparent being light); factors that influence the timing of this
molecular mechanism are known as zeitgebers (or timekeepers) (34, 55). Given that the clock mechanism is transcriptionally based, initial output manifests at the level of altered gene
expression. Those genes that are regulated directly by the
circadian clock, but are not integral components of the clock
mechanism, are termed clock-controlled, or output, genes.
After posttranscriptional events, output from the clock ultimately modulates protein expression, metabolism, and/or function, depending on the cell or organ (39).
Through the use of sophisticated molecular and genetic
approaches, considerable progress as been made within the last
decade toward elucidation of the inner workings of eukaryotic
circadian clocks. Initial studies in the fungus Neurospora and
in the fruit fly Drosophila melanogaster led investigators to
suggest that the circadian clock is composed of a single protein
that inhibits its own synthesis, thereby creating an oscillator
with a simple transcriptional loop (130). Although it is attractive, subsequent studies have dispelled this hypothesis, revealing a complex set of positive and negative feedback loops of
which the molecular machinery still awaits full elucidation (32,
55). Mammalian orthologs of these circadian clock compo-
nents are emerging (40, 132, 145, 162). Genetically manipulated mouse models have greatly accelerated characterization
of the mammalian circadian clock, exposing an interplay
among at least three negative loops and one positive loop that
require a multitude of transcriptional, translational, and posttranslational events (39). One of the negative loops of the
mammalian circadian clock that is understood to the greatest
extent involves the period (PER, of which three isoforms have
been identified, PER1, PER2, and PER3) and the cryptochrome
(CRY, of which two isoforms are known, CRY1 and CRY2)
proteins (85, 162). On heterodimerization, the basic helixloop-helix/PER-arylhydrocarbon receptor nuclear translocator
(ARNT)-SIM (bHLH/PAS) transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle
ARNT-like protein 1 (BMAL1, also known as MOP3) recognize E-boxes located within the promoters of various target
genes, including the per and cry genes (Fig. 1) (40, 57). Once
translated, the PER and CRY proteins form heterodimers,
translocate into the nucleus, and inhibit CLOCK/BMAL1
transactivation (potentially through interaction with the histone
acetyltransferase p300, an essential coactivator for CLOCK/
BMAL1-mediated transcription) (23, 36, 39, 73, 119). A key
facet of the PER/CRY-mediated negative loop is a specific
delay in the accumulation of the PER proteins by ⬃6 h, relative
to their mRNA (35, 39, 49, 106). The PER proteins are targets
for ubiquitin-mediated degradation, an event that is blocked by
serine phosphorylation. Thus, once in the phosphorylated state
[likely mediated by casein kinase 1⑀ (CK1⑀)], PER proteins
accumulate within the cytosol, allowing heterodimerization
with the CRY proteins (67). Subsequent inhibition of CLOCK/
BMAL1-mediated transcription decreases expression of the
per and cry genes, ultimately relieving inhibition on CLOCK/
BMAL1 (Fig. 1).
Two additional negative loops of the mammalian circadian
clock involve the bHLH transcription factor differentially expressed in chondrocytes (DEC, of which two isoforms have
been identified, DEC1 and DEC2) and the nuclear receptor
reverse strand c-ERBA␣ (REV-ERBA␣) (58, 101). Both the
dec1/2 and rev-erba␣ genes are under direct transcriptional
control by the CLOCK/BMAL1 heterodimer (Fig. 1). After
translation of the corresponding proteins and translocation
into the nucleus, both DEC1/2 and REV-ERBA␣ attenuate
CLOCK/BMAL1-mediated transcription; DEC1/2 appear to
associate with the CLOCK/BMAL1 heterodimer, impairing
transactivational capacity, whereas REV-ERBA␣ specifically
represses bmal1 transcription (potentially through recruitment
of the N-CoR/histone deacetylase 3 corepressor) (112, 153). In
contrast, the CLOCK/BMAL1- and/or PER2-mediated induction of bmal1 expression constitutes the positive loop in the
mammalian circadian clock (Fig. 1) (41, 119).
Although by definition the circadian clock involves an interplay of transcriptional positive and negative feedback loops,
multiple posttranscriptional events are essential for complete
clock function, including protein synthesis/degradation, reversible phosphorylation, and nucleocytosolic translocations (35,
55, 150). Each of these events represents a potential site of
regulation, enabling “fine-tuning” of this molecular clock by
extracellular factors (i.e., zeitgebers). CLOCK, BMAL1, and
CRY, as well as the PER proteins, have all been shown to
undergo phosphorylation in a circadian-like fashion; in addition to its influence on protein stability, increasing evidence
Invited Review
H3
MYOCARDIAL CIRCADIAN CLOCK
suggests that phosphorylation status is closely associated with
nucleocytosolic trafficking of specific circadian clock components (e.g., PER and CLOCK proteins) (71, 111, 144). Multiple
protein kinases likely play significant roles in the mammalian
clock mechanism, including CK1⑀, MAPK, GSK-3␤, and PKG
(type II), thereby providing avenues for modulation of the
circadian clock by an array of extracellular stimuli (79, 111,
134, 144).
Mammalian circadian clocks can be divided into two major
classes, depending on the cell type within which they are
found: the central and peripheral circadian clocks (18, 55, 106).
The central circadian clock (often termed the master clock) is
located within the suprachiasmatic nucleus (SCN; located in
the hypothalamus) of the brain, whereas peripheral clocks are
those clocks found within all non-SCN cells of the organism,
including other regions of the central nervous system. Zeitgebers are factors that reset or entrain central and/or peripheral
circadian clocks. The central clock is reset by light (via
electrical signals transmitted along the retinohypothalamic
tract), whereas peripheral circadian clocks are influenced by
multiple neurohumoral factors (as well as diurnal variations in
temperature) (7, 14, 25, 55). One of the strongest entrainers of
peripheral circadian clocks appears to be feeding (25, 124). It
is believed that the central clock entrains peripheral clocks via
modulation of neurohumoral stimuli, either directly (i.e., innervation between the SCN and specific peripheral tissues)
and/or indirectly (e.g., through alterations in feeding behavior).
The CLOCK/BMAL1 heterodimer binds to E-boxes in the
promoters of various genes that are not believed to be an
integral component of the clock mechanism. These clockcontrolled genes include lactate dehydrogenase A (ldha), vasopressin, wee1, prokineticin2, and the rich in proline and
acidic amino acid residues (PAR) transcription factors Delement binding protein (dbp), hepatic leukemia factor (hlf),
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and thyrotrophic embryonic factor (tef) (21, 37, 38, 60, 81, 107,
108). The latter family of transcription factors, which in turn
have the potential to modulate expression of a host of target
genes, are antagonized by another bHLH transcription factor,
E4BP4; e4bp4 is an additional example of a clock-controlled
gene, of which the expression is likely induced by REVERBA␣ (84, 139). Consistent with their reciprocal function,
oscillations in PAR transcription factors and E4BP4 have been
reported to be antiphase to one another, in various tissues,
including the heart (see CIRCADIAN CLOCKS WITHIN THE CARDIOVASCULAR SYSTEM).
CIRCADIAN CLOCKS WITHIN THE
CARDIOVASCULAR SYSTEM
Oscillations in circadian clock gene expression have been
reported by numerous investigators, for various cardiovascular
components. Gene expression measurements of hearts and
blood vessels (e.g., aorta) isolated from rodents at specific
times of the day have revealed dramatic rhythmicity in the
expression of genes encoding for both core circadian clock
components and output genes (23, 33, 48, 69, 80, 82, 86,
92–95, 101, 109, 110, 122, 125, 154, 159, 160). Figure 2
provides precise quantitative 24-h expression profiles for circadian clock genes in the intact hearts of rats kept in a normal
12:12-h light-dark cycle [lights on at zeitgeber time (ZT) 0].
Oscillations in bmal1, clock, and neuronal pas2 (npas2) (a
redox-sensitive clock homologue, also known as mop4) mRNA
molecules are essentially identical to one another, in terms of
both peak (i.e., zenith) level of expression and phase (Fig. 2A)
(104). However, trough (i.e., nadir) expression levels of bmal1
and npas2 are significantly lower than those of clock, suggesting that the former transcription factors likely become limiting
at the light-to-dark phase transition. The heart expresses all
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Fig. 1. Mammalian circadian clock. At the
heart of the clock mechanism, circadian locomotor output cycles kaput (CLOCK) and
brain and muscle arylhydrocarbon receptor
nuclear translocator (ARNT)-like protein 1
(BMAL1) induce multiple target genes, including components of the 3 characterized
negative loops [cryptochrome 1/2 (cry1/2),
period 1/2/3 (per1/2/3), dec1/2, and reverse
strand of c-erba␣ gene (rev-erba␣)], and
bmal1 itself (positive loop), as well as numerous clock output genes [e.g., the proline and
acidic amino acid residues (PAR) transcription factors D-element binding protein (dbp),
hepatic leukemia factor (hlf), and thyrotrophic embryonic factor (tef)]. REV-ERBA␣
also acts in a negative manner toward the
PAR transcription factors through induction
of E4BP4. Several posttranscriptional events
(e.g., reversible phosphorylation, nucleocytosolic translocation) are required for complete
function of the circadian clock. *Nucleocytosol translocation events putatively regulated by reversible phosphorylation; kinases
such as MAPK, PKG type II, and GKS-3␤
have been implicated as potential mediators
of these events. CK1⑀, casein kinase 1⑀.
Invited Review
H4
MYOCARDIAL CIRCADIAN CLOCK
known isoforms of the cry and per genes; cry2 is the predominant cryptochrome in the heart, whereas per1 and per2 are
expressed to greater extents than per3 (Fig. 2B). Peak levels of
cry2, per1, and per2 expression are observed at the light-todark phase transition in the rat heart, with similar absolute
levels of expression relative to one another. However, unlike
cry2 expression, per1 and per2 expression decreases dramatically at the dark-to-light phase transition, consistent with the
known clock mechanism, wherein PER expression becomes
limiting (relative to the CRY proteins). It is also worthy to note
that the antiphase nature of bmal1/clock/npas2 rhythms versus
cry2/per1/per2 rhythms is consistent with the latter antagonizing the former (Fig. 2, A and B). Additional negative loops of
the mammalian circadian clock include those governed by
REV-ERBA␣ and DEC1/2. Messenger RNA encoding for
rev-erba␣ exhibits dramatic oscillations in expression in the rat
heart, peaking in the middle of the light phase, consistent with
the idea that this nuclear receptor represses bmal1 expression
(Fig. 2C). Oscillations in dec1 (also known as stra13, sharp2,
and bhlhb2) are less striking in the rat heart, in comparison to
components of the other two negative loops (i.e., per1/2 and
rev-erba␣), but are again antiphase to bmal1/clock/npas2
rhythms, consistent with the notion that dec1 forms a negative
loop (Fig. 2, A and C). The rat heart expresses all three PAR
transcription factor family members, with dbp predominating;
myocardial expression of dbp is of a similar order of magnitude
in comparison to cardiac-enriched transcription factors [e.g.,
peroxisome proliferator-activated receptor-␣ (ppar␣), retinoic
acid receptor-␣ (rxr␣), myocyte enhancer factor 2 (mef2)]
(unpublished observations). Despite dramatic circadian oscillations and high absolute levels of expression, no target genes
are known for DBP in the heart. Identification of such target
genes will undoubtedly improve our understanding of the role
of the circadian clock within the heart. It is also noteworthy
that opposing circadian rhythmicity in e4bp4 expression should
antagonize DBP activity (as well as the activity of the other
PAR transcription factor family members) at the dark-to-light
phase transition.
AJP-Heart Circ Physiol • VOL
Taken together, the data presented in Fig. 2 are consistent
with the operation of a fully functional circadian clock within
the heart. However, one could hypothesize that oscillations in
circadian clock components observed in intact organs in vivo
are the result of diurnal variations in one or more neurohumoral
factors that directly influence expression of these genes. By
definition, the circadian clock is intrinsic to the cell. Therefore,
to conclusively demonstrate that the components of the cardiovascular system possess an intrinsic circadian clock mechanism, one must expose the cell autonomous nature through the
use of cultured organs or cells. The latter has been performed
for cultured vessels, VSMCs, and cardiomyocytes. Davidson et
al. (26) have recently cultured both arteries and veins isolated
from transgenic mice overexpressing the luciferase reporter
gene under control of the per1 promoter. They report that
oscillations in per1-driven bioluminescence persist in cultured
vessels, for between 3 and 12 days. McNamara et al. (82)
reported that exposure of human VSMCs to media containing
50% serum for 2 h (serum shock, a strategy utilized in numerous studies for the reestablishment of circadian clock gene
oscillations in cultured cells) induced rhythmic expression of
bmal1. Furthermore, this rhythm was phase advanced by dexamethasone and phase delayed by retinoic acid (82). Similarly,
Nonaka et al. (94) have shown that angiotensin II induces
significant oscillations in bmal1, per2, and dbp in VSMCs,
with periodicities close to 24 h. Together, these observations
expose an intrinsic circadian clock mechanism within VSMCs,
of which the timing is influenced by humoral factors such as
corticosterone, retinoic acid, and angiotensin II.
We (33) have recently examined the circadian clock within
the cardiomyocyte. Through the use of adult rat cardiomyocytes, we (33) found that the presence of 2.5% FCS within the
culture medium is sufficient to maintain oscillations in circadian clock (bmal1, rev-erba␣, and per2) and output (dbp)
genes; in serum-free media, these oscillations are either severely attenuated (bmal1 and dbp) or completely abolished
(rev-erba␣ and per2). Studies by Welch et al. (148) and
Nagoshi et al. (91) provide a plausible explanation for the
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Fig. 2. Quantitative circadian expression levels
for clock (A), bmal1 (A), neuronal PER-arylhydrocarbon receptor nuclear translocatorSIM 2 (npas2) (A), cry1 (B), cry2 (B), per1
(B), per2 (B), per3 (B), rev-erba␣ (C), dec1
(C), dbp (D), hlf (D), tef (D), and e4bp4 (D)
in intact rat hearts. Circadian rhythms in
expression were determined by quantitative
RT-PCR for total RNA prepared from hearts
that were isolated from rats at 3-h intervals.
Values are shown as means ⫾ SE for 12
separate observations at each time point.
Data are represented as number of mRNA
molecules per nanograms total RNA.
Invited Review
MYOCARDIAL CIRCADIAN CLOCK
ENVIRONMENTAL VERSUS INTRINSIC INFLUENCES ON
CARDIOVASCULAR FUNCTION
Intracellular circadian clocks clearly exist within at least two
major cell types of the cardiovascular system, namely, cardiomyocytes and VSMCs. This molecular mechanism undoubtedly exists within all mammalian cell types, although characterization has yet to be reported in, for example, endothelial
cells. Because of our recent appreciation for peripheral and
central circadian clocks, new hypotheses have arisen regarding
potential mechanisms responsible for diurnal variations in
cardiovascular physiology, as well as pathophysiology. Without question, circadian rhythms in environmental factors (e.g.,
locomotion, feeding) influence cardiovascular function. However, circadian clocks may contribute to normal diurnal variations in cardiovascular function in a number of different ways.
For example, through the use of the retrograde pseudorabies
virus, the presence of multisynaptic autonomic connections
from the SCN to the heart have been reported (113), suggesting
that the central clock may modulate cardiac function through
direct (autonomic) nervous stimulation. Circadian clocks
within individual cells of the cardiovascular system potentially
influence cardiovascular function by allowing anticipation of
the onset of neurohumoral factors (e.g., increased sympathetic
nervous stimulation before awakening), thereby ensuring an
appropriately rapid response. Thus, in the in vivo setting, a
complex interplay between environmental influences and intrinsic mechanisms (i.e., central and peripheral circadian
clocks) likely contributes to changes in cardiovascular function
over the course of the day. Here we provide a brief overview
of specific evidence supporting roles for environmental versus
intrinsic influences on cardiovascular function.
Circadian rhythms in blood pressure have been investigated
intensively (28, 29, 54, 64, 83, 141, 143, 161). In humans,
blood pressure is lowest in the night, reaching a trough at
around 3:00 AM, and peaks soon after waking (9:00 AM). A
second peak in blood pressure is often seen early in the evening
(7:00 PM) (29). The opposite is true for nocturnal animals,
such as rodents, that exhibit elevated blood pressure levels at
night, when the animal is most active (141). Day-to-night
differences in physical and mental activity appear to be major
determinates of blood pressure circadian rhythms (22, 28). In
humans, shift workers show an essentially complete resynchronization of blood pressure rhythms within the first 24 h of the
shift rotation (19, 128). Similarly, alterations in the light-dark
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cycles and/or restricted feeding induces rapid phase shifting of
blood pressure, heart rate, and behavioral activity circadian
rhythms in the rat (141). It has been postulated that changes in
the conscious state of the animal primarily influence blood
pressure through changes in sympathetic activity, suggesting
that environmental factors serve as the major influence on
blood pressure (76). Indeed, a strong correlation exists between
diurnal variations in this cardiovascular parameter and plasma
norepinephrine and epinephrine levels, suggesting that the
sympathetic system drives blood pressure circadian rhythms
(105, 123, 136). It should also be noted that additional cardiovascular reactive peptides (e.g., atrial natriuretic peptide, vasopressin, and components of the renin-angiotensin system)
exhibit considerable diurnal variations and therefore potentially influence blood pressure circadian rhythms (43, 62, 63,
100). Whether the circadian clock within the cells of the
cardiovascular system influences responsiveness to these neurohumoral factors, thereby contributing to circadian rhythmicity in blood pressure, is an attractive possibility that awaits
investigation.
Although blood pressure and heart rate circadian rhythms
are normally paralleled, several lines of evidence suggest that
the circadian rhythms of these two cardiovascular parameters
might be differentially regulated. Rhythms in heart rate appear
to be more intrinsic, driven in large part by diurnal variations
in autonomic nervous system activity (72, 114). Furthermore,
Hu et al. (59) have recently reported that despite controlling for
sleep-wake and behavior cycles in humans, circadian rhythmicity in heart rate variability persists, peaking in the early
hours of the morning. Such studies expose an intrinsic component influencing normal cardiovascular function. One could
hypothesize that SCN-driven diurnal variations in autonomic
stimulation, coupled to the cardiomyocyte circadian clockdriven diurnal variations in responsiveness of the heart to
autonomic stimulation, together act as major determinants of
heart rate circadian rhythms. Whether environmental modulation of synchronization between peripheral and central clocks
contributes to cardiovascular disease development remains
unknown; such a loss of synchronization is possible through
changes in feeding and sleep patterns, as occurs during diabetes mellitus, obesity, sleep apnea, and shift work, all of which
are associated with elevated risk for cardiovascular disease (see
MODULATION OF CIRCADIAN CLOCK DURING PHYSIOLOGICAL AND
PATHOPHYSIOLOGICAL STATES).
CIRCADIAN RHYTHMS IN THE INTRINSIC PROPERTIES OF
THE CARDIOVASCULAR SYSTEM
An important question that remains unanswered is whether
circadian rhythms in cardiovascular function are mediated
solely through changes in the level of neurohumoral stimuli or
whether diurnal variations in the intrinsic properties of the
cardiovascular system also play a significant role. Increasing
evidence suggests that specific components of the cardiovascular system respond differentially to neurohumoral stimuli at
specific times of the day. For example, healthy human endothelium exhibits a marked circadian rhythm, with decreased
function in the early hours of the morning (as assessed by
brachial artery flow-mediated, endothelium-dependent vasodilation) (97). This functional circadian rhythm persists ex vivo,
such that rat aortic rings isolated at distinct times of the day
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apparent loss of circadian clock function in cells cultured in the
absence of serum. Both sets of investigators report that culturing of fibroblasts for prolonged periods of time leads to
asynchrony between individual cell-autonomous clocks and
that serum reestablishes synchrony, as opposed to reactivation
of the clock mechanism itself (91, 148). We (33) have also
reported that challenge of isolated cardiomyocytes with norepinephrine for only 2 h (mimicking a burst of sympathetic
activity at the onset of consciousness) results in significant
oscillations in circadian clock genes, suggesting that norepinephrine acts as a putative zeitgeber for the circadian clock
within the heart. The latter observations are consistent with
those of Terazono et al. (133), who observed that adrenergic
stimulation influences circadian clock gene expression in the
liver.
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Invited Review
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MYOCARDIAL CIRCADIAN CLOCK
that contractile proteins are known to possess relatively long
half-lives, it is unlikely that significant changes in MHC
protein expression influence contractile function over the
course of a normal day.
Myocardial contractility and metabolism are inherently interlinked, such that changes in contractility affect metabolism,
and vice versa. We found that increased cardiac output observed for rat hearts excised during the dark phase was associated with increased carbohydrate oxidation and oxygen consumption (with no significant change in fatty acid oxidation)
(158). This is reminiscent of the observation that an acute
increase in myocardial workload results in increased rates of
carbohydrate oxidation, with lesser effects on fatty acid oxidation rates (42). Consistent with increased myocardial carbohydrate metabolism during the dark phase, rat hearts isolated at
this time of the day possess increased gene and protein expression of the contraction- and insulin-responsive glucose transporter GLUT4, as well as increased gene expression of GLUT1
and glycogen synthase (diurnal variations in protein levels of
the latter two metabolic proteins have not been reported) (158).
Furthermore, circadian rhythms in the tricarboxylic acid cycle
flux-generating enzymes citrate synthase and oxoglutarate dehydrogenase (at the level of gene expression) correlate with
increased oxygen consumption during the dark phase (158).
These observations have led to the suggestion that increased
myocardial oxidative capacity may promote increased contractile function during the active phase for the mammal.
Despite the reported lack of diurnal variation in fatty acid
oxidation rates for excised working rat hearts, we observe a
coordinated induction of genes known to promote fatty acid
oxidation during the dark phase (121, 122, 158). Table 1
summarizes observations regarding diurnal variations in 12
genes known to promote myocardial fatty acid oxidation, either
directly (e.g., component of the ␤-oxidation pathway) or indirectly (e.g., through modulation of intracellular malonyl-CoA
levels, a potent inhibitor of fatty acid oxidation). It could be
hypothesized that the apparent dissociation between diurnal
variations in fatty acid oxidation gene expression and rates of
exogenous fatty acid oxidation reflects a lack of significant
diurnal variation in myocardial fatty acid oxidation enzymes at
the protein level. However, it should be noted that the fatty acid
concentration (0.4 mM oleate) utilized in our previously pub-
Table 1. Diurnal variations in expression of genes promoting fatty acid oxidation in rat heart
Gene
Protein Function
Reference
lp1
cd36
fabp3
acs1
mcpt1
mcad
lcad
hadh␣
mte1
ucp3
mcd
pdk4
Cell surface triglyceride hydrolysis
Fatty acid uptake
Binding of intracellular fatty acids
Fatty acyl-CoA formation
Mitochondrial fatty acyl-CoA import
␤-Oxidation
␤-Oxidation
␤-Oxidation
Fatty acyl-CoA hydrolysis
Mitochondrial uncoupling (?)
Decarboxylation of malonyl-CoA
Phosphorylation of PDC
115
131
8
117
13
45,142
45
45
121
121,157
16,155
149
Time of Trough
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
3
9
3
9
6
6
6
9
9
9
6
9
Time of Peak
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
ZT
15
21
18
18
18
18
18
21
21
18
18
15
Trough-to-Peak
Fold Induction
2.0
1.4
1.4
1.8
1.6
1.5
1.6
1.3
1.4
4.2
1.4
2.5
Rats were housed in a standard 12:12-h light-dark cycle [lights on between zeitgeber time (ZT) 0 and ZT 12; lights off between ZT 12 and ZT 24] before
isolation of hearts at 3-intervals over course of day. Gene expression was measured by real-time quantitative RT-PCR for at least 12 separate observations. All
trough-to-peak inductions are statistically significant (P ⬍ 0.05). References provide information regarding protein function, gene regulation, and/or diurnal
variations. PDC, pyruvate dehydrogenase complex. See text for gene definitions.
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exhibit differences in endothelium-dependent and -independent vasodilation; diurnal variations in the responsiveness of
aortic rings to pharmacological stimulation (␤-adrenergic agonists, Ca2⫹ channel antagonists) also persist in vitro (46, 47).
Taken together, these studies reveal circadian rhythmicity in
the intrinsic properties of the vasculature.
Comparable observations have been made regarding diurnal
variations in the intrinsic properties of the heart. We (158) have
reported previously that diurnal variations in cardiac function
persist when rat hearts are isolated and perfused ex vivo; hearts
isolated in the middle of the dark phase (the active period for
the rat) exhibit greater cardiac power relative to those hearts
isolated in the middle of the light phase. Similarly, Yamashita
et al. (151) observed significant variations in the transient
outward and steady-state currents for ventricular myocytes
isolated at different times of the day, thereby exposing diurnal
variations in the intrinsic electrical properties of the cardiomyocyte. Additionally, Lapenna et al. (75) have reported
increased susceptibility of isolated perfused rat hearts to H2O2
during the dark phase, as assessed by release of thiobarbituric
acid-reactive substances (lipoperoxidation end products), exposing diurnal variations in oxidative stress tolerance of the
heart.
Given that ex vivo experiments inherently control for acute
influences of neurohumoral stimuli, diurnal variations in the
intrinsic properties of the heart (and vasculature) are likely
mediated by changes in gene and protein expression over the
course of the day. Both hypothesis-testing and hypothesisgenerating strategies have been adopted for identification of
mediators of diurnal variations in the intrinsic properties of the
heart. In the former case, candidate genes and proteins, as well
as metabolic and signaling pathways known to influence heart
function, have been investigated. For example, we (158) and
others (146) have observed that gene expression of myocardial
contractile proteins exhibits significant circadian rhythmicity
in the normal rodent heart. Increased contractility and cardiac
output during the dark phase for the rat, as observed both in
vivo and ex vivo, correlate with increased expression of both
myosin heavy chain (MHC) isoforms ␣ and ␤ (146, 158).
However, it should be noted that these studies investigated
gene expression only, and data concerning circadian rhythms
in MHC protein levels have not been reported. Indeed, given
Invited Review
MYOCARDIAL CIRCADIAN CLOCK
AJP-Heart Circ Physiol • VOL
a normal 12:12-h light-dark cycle exposed diverse and extensive circadian rhythms in myocardial gene expression (80).
Approximately 13% of all genes expressed in the murine heart
exhibit significant circadian rhythmicity. Clustering of rhythmic genes into biological pathways/processes exposed synchronous expression profiles for genes encoding proteins involved in mitochondrial oxidative metabolism, protein synthesis, calcium binding, and GTP-binding pathways. Consistent
with increased myocardial oxygen consumption during the
dark phase, a coordinated induction of mitochondrial oxidative
metabolism genes in the heart was observed at this time (80,
158). It was noted that for those genes identified as rhythmic in
the murine heart, a subset appeared to change in response to
light-dark cycle transitions. Storch et al. (125) performed a
similar microarray-based study, with the exception that mice
were placed in dim light for at least 42 h before isolation of the
heart. The study reports that despite exposure of the animals to
constant darkness, ⬃8% of myocardial genes exhibited rhythmicity in expression. Again, oscillations in gene expression
could be clustered in a biological process-specific manner, with
genes involved in metabolism, protein turnover, and G proteincoupled signaling pathways receiving particular attention (125).
It is apparent that the majority of studies investigating
diurnal variations in the intrinsic properties of the heart have,
to date, focused primarily on alterations in gene expression. An
important next step will require examination of the heart at the
proteomic level, enabling determination of the extent to which
myocardial proteins oscillate over the course of a normal 24 h.
The extent to which proteins exhibit a diurnal variation will
depend not only on oscillations for the encoding mRNA but
also on the turnover rate of the protein itself. Furthermore,
diurnal variations in the activity of a specific protein will also
be influenced by posttranslational modifications (e.g., reversible phosphorylation). Clearly, understanding the complex interplay between transcriptional, translational, and posttranslational events over the course of the day will improve our
understanding of the mechanisms mediating diurnal variations
in the intrinsic properties of the heart.
CIRCADIAN CLOCK VERSUS NEUROHUMORAL FACTORS AS
MEDIATORS OF DIURNAL VARIATIONS IN INTRINSIC
PROPERTIES OF HEARTS
Circadian rhythms in the intrinsic properties of the heart
(and vasculature) are potentially mediated by intracellular (i.e.,
circadian clock within the cardiomyocyte) and/or extracellular
(i.e., neurohumoral) influences (Fig. 3). It is reasonable to
hypothesize that diurnal variations in one or more circulating
hormones (e.g., thyroid hormone) mediate diurnal variations in
myocardial gene and protein expression (e.g., GLUT4) before
examination of the heart ex vivo, independent of the circadian
clock within the cardiomyocyte. Therefore, to distinguish between intracellular versus extracellular influences, one must
investigate each independently of the other. One strategy for
investigating the role of the intracellular circadian clock, in the
absence of confounding neurohumoral influences, is the utilization of cultured cells. Because of its intrinsic nature, the
circadian clock persists in cultured cells, whereas any potential
influences by diurnal variations of neurohumoral factors in the
in vivo setting are abolished (90). Such a strategy has been
utilized successfully in the identification of circadian clock-
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lished studies measures submaximal rates of fatty acid oxidation, as opposed to fatty acid oxidative capacity (158). Future
studies are therefore required to investigate whether diurnal
variations in myocardial fatty acid oxidation capacity exist at
the protein and metabolic flux levels.
A total of 14 different K⫹ channels were investigated by
Yamashita et al. (151) in the search for potential mediators of
observed diurnal variations in the intrinsic electrical properties
of the heart. Of the K⫹ channels investigated, circadian
rhythms in both mRNA and protein levels were observed for
Kv1.5 and Kv4.2 channels. These two K⫹ channels exhibit
opposing diurnal variations in expression relative to one another in the rat heart, with the Kv1.5 channel reaching a peak
level of expression during the dark phase, whereas expression
of the Kv4.2 channel peaked during the light phase (151).
Increased expression of Kv1.5 channels during the dark phase
is consistent with increased steady-state current of cardiomyocytes isolated at this time. In contrast, peak expression of
Kv4.2 channels during the light phase corresponded with
increased transient outward current. Pharmacological studies in
isolated perfused rat hearts further support the hypothesis that
circadian rhythms in the intrinsic electrical properties are
mediated, at least in part, by diurnal variations in myocardial
expression of Kv1.5 and Kv4.2 channels (151).
Reactive oxygen species (ROS) play a pivotal role in myocardial physiology and pathophysiology. Similar to other
highly reactive short-lived biological molecules [e.g., nitric
oxide (NO)], physiological concentrations act in an essential
signaling manner, whereas pathophysiological elevation (i.e.,
oxidative stress) is associated with multiple disease states,
including heart failure (51). Oxidative stress occurs when
production of ROS exceeds the antioxidant capacity of the cell.
ROS originate from multiple enzymatic and nonenzymatic
reactions. One potential site of ROS generation for an oxidative organ such as the heart is the mitochondrion; misdonation
of electrons to molecular oxygen at complexes I and III of the
electron transport chain (ETC) results in superoxide production
(12, 44). The latter occurs when components of the ETC
become highly reduced. Under conditions of normal oxygen
availability, the likelihood of superoxide production via the
ETC increases when ATP consumption is low (i.e., less active
state) and when fatty acid availability is high (this highly
reduced substrate supplies electrons to the ETC via both
FADH2 and NADH). Such conditions (i.e., decreased myocardial ATP consumption coupled with increased fatty acid availability) occur when the animal is resting (i.e., light phase for
the rat). Given this rationale, one would hypothesize that the
heart increases antioxidant capacity during the resting phase,
thereby minimizing oxidative stress. Antioxidant capacity has
been shown to exhibit marked diurnal variations in serum and
several peripheral tissues, although little is known for the heart
(6). Lapenna et al. (75) reported that glutathione peroxidase (a
major antioxidant enzyme in the heart) exhibits increased
activity in the rat heart during the light (resting) phase. Coincident with diurnal variations in myocardial antioxidant capacity, decreased susceptibility to H2O2-induced oxidative damage
was observed for hearts isolated during the light phase (75).
Putative mediators of diurnal variations in myocardial metabolism and contractile function have also been investigated
through hypothesis-generating approaches. Microarray analysis of hearts isolated at 3-h intervals from mice maintained in
H7
Invited Review
H8
MYOCARDIAL CIRCADIAN CLOCK
regulated genes in several cell types. For example, genes
encoding for insulin and ppar␣ exhibit significant 24-h oscillations in cultured pancreatic ␤-cells and hepatocytes, respectively, suggesting that these genes are regulated by the circadian clock (1, 96).
Through the use of adult rat cardiomyocytes, we (33) have
recently identified two metabolic genes as novel circadian
clock-regulated genes in the heart. We observed significant
circadian oscillations in the gene expression of pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3) in
isolated adult rat cardiomyocytes cultured under conditions
associated with maintenance of the circadian clock (i.e., 2.5%
FCS). In contrast, in the absence of serum, oscillations in both
circadian clock and metabolic genes were either severely
attenuated or abolished. The timing of metabolic gene oscillations was similar in vivo and in vitro when compared with
circadian clock gene oscillations. For example, oscillations in
pdk4 and ucp3 mRNAs were similar to one another, and
antiphase with respect to rev-erba␣ mRNA, both in vivo and in
vitro. Taken together, these observations strongly suggest that
the circadian clock within the heart regulates myocardial metabolism (33).
Experiments utilizing isolated cardiomyocytes, under conditions in which cell autonomous clocks remain synchronized,
will allow identification of multiple circadian clock-regulated
genes in the heart. Such a system will enable “follow-up”
investigation of putative circadian clock-regulated genes identified initially through either hypothesis-testing (i.e., candidate
gene) or hypothesis-generating (i.e., microarray) strategies. For
example, whether oscillations in Kv1.4 and/or Kv4.2 channels
persist in isolated cells has yet to be investigated; if these
oscillations persist, this will provide strong evidence in support
for the hypothesis that the circadian clock within the cardiomyocyte regulates diurnal variations in the electrical properties
of the heart. Similarly, if oscillations in antioxidant enzymes
persist in isolated cardiomyocytes, this would suggest that the
circadian clock mediates diurnal variations in susceptibility of
AJP-Heart Circ Physiol • VOL
POTENTIAL ROLES FOR CIRCADIAN CLOCK
WITHIN HEARTS
It is clear that the heart possesses an intrinsic, fully functional circadian clock. What is less apparent is the identity of
the processes that this mechanism influences. Given that circadian clocks confer the selective advantage of anticipation,
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Fig. 3. Potential roles of intramyocellular (i.e., circadian clock) versus extracellular (i.e., neurohumoral factors) on circadian rhythms in myocardial gene
expression and function. Schematic illustrates that certain humoral factors bind
to cell surface receptors (and utilize second messenger systems; e.g., angiotensin II), whereas others influence myocardial gene expression by binding to
nuclear receptors (e.g., fatty acids).
the heart to oxidative stress. Studies addressing such questions
currently await completion.
It is worthy to note that considerable evidence suggests
plasminogen activator inhibitor 1 (PAI-1; a key factor in
fibrolytic activity) is regulated directly by the circadian clock.
Expression of pai-1 exhibits striking circadian rhythmicity in
both the heart and vasculature, as does plasma PAI-1 activity
(86, 93). Maemura et al. (77) have shown that the bHLH/PAS
transcription factor cycle-like factor (CLIF) forms a heterodimer with CLOCK and binds to specific E-boxes within
the pai-1 promoter, subsequently inducing pai-1 gene expression. Such observations have led investigators to hypothesize
that the circadian clock plays a significant role in mediating the
onset of myocardial infarctions in the morning, through induction of pai-1 (78). Consistent with pai-1 being a circadian
clock-regulated gene, myocardial pai-1 expression peaks near
the light-to-dark phase transition in the rat, similar to that of
dbp, another clock output gene (86). However, pai-1 is undoubtedly regulated by an array of neurohumoral factors, in
addition to the circadian clock. These include glucose, insulin,
glucocorticoids, and transforming growth factor-␤ (15, 30, 65,
129). Indeed, a routine change of serum-free medium for
cultured adult rat cardiomyocytes causes an immediate induction in pai-1 expression (2.5-fold within 3 h; D. J. Durgan and
M. E. Young, unpublished observations). Such a high sensitivity of the pai-1 promoter to changes in the environment
severely impairs investigation of potential pai-1 gene oscillations in vitro.
As exemplified by pai-1, reliance on isolated adult rat
cardiomyocytes for identification of circadian clock-regulated
genes has limitations. The use of adult rat cardiomyocytes is
also somewhat hindered by the phenomenon of dedifferentiation that occurs as the culture is maintained. This process may
impair identification of specific circadian clock-regulated
genes. For these reasons, development of alternative models is
essential for identification of circadian clock-regulated genes
(and in turn circadian clock-regulated processes). One strategy
for dissociating between the influence of the circadian clock
versus that of neurohumoral factors is to eliminate the former.
A ubiquitous, constitutive clock null mouse exists, which has
proved useful in expanding our knowledge regarding the intricacies of the circadian clock mechanism (145). However, as
the circadian clock is impaired in all cells of clock mutant
mice, diurnal variations in neurohumoral factors are abnormal,
preventing dissociation of intracellular (i.e., circadian clock)
versus extracellular (i.e., neurohumoral) influences on circadian rhythms in myocardial gene expression and function. An
alternative strategy is therefore to manipulate the circadian
clock only within the cardiomyocyte. Such a heart-specific
circadian clock-impaired mouse model, which has recently
been generated in our laboratory, will undoubtedly improve
our understanding of the role(s) of this molecular mechanism
within the heart.
Invited Review
MYOCARDIAL CIRCADIAN CLOCK
phase for rodents), a sudden increase in sympathetic activity
and workload on the heart occurs (105, 123, 136, 141). By
increasing its capacity for carbohydrate oxidation, the heart
would anticipate the sudden increase in workload at this time.
The oxidative decarboxylation of pyruvate to acetyl-CoA is a
primary site of regulation for carbohydrate oxidation in the
heart, a reaction catalyzed by the pyruvate dehydrogenase
complex (PDC) (118, 127). PDC is regulated at multiple levels,
including reversible phosphorylation (126). Pyruvate dehydrogenase kinase (PDK, of which four isoforms have been identified in humans) phosphorylates and inactivates PDC, thereby
reducing carbohydrate oxidation. We (33, 122, 158) have
found that gene expression of the inducible isoform of PDK,
namely, pdk4, exhibits a trough in the rat heart just before the
onset of the dark phase and that these oscillations persist in
isolated cardiomyocytes, suggesting direct regulation by the
circadian clock within the heart. Consistent with the assumption that diurnal variations in PDK4 protein expression will
exhibit a slight temporal delay relative to those of pdk4 mRNA,
we have found that the percentage of PDC in the active form
is significantly higher at the light-to-dark phase transition,
compared with the dark-to-light phase transition (Fig. 4). The
circadian clock within the heart therefore likely increases
myocardial carbohydrate oxidative capacity in anticipation of
increased workload on awakening of the animal. Increased
circulating glucose levels at this time (due to a sudden increase
in hepatic glucose output, the so-called dawn phenomenon)
would provide an appropriate substrate for increased cardiac
output at the end of the overnight fast (9, 74). The dawn
phenomenon also appears to be mediated by intracellular
circadian clocks (74).
Dissociation between diurnal variations in circulating fatty
acids and the capacity for myocardial oxidative metabolism
exists in the ad libitum-fed laboratory rat (122, 158). Rates of
Fig. 4. Potential mechanism by which the
circadian clock within the cardiomyocyte influences myocardial metabolism in the rat.
After induction of myocardial pyruvate dehydrogenase kinase 4 (pdk4) expression during the dark phase, pyruvate dehydrogenase
complex (PDC) is less active at the dark-tolight phase transition, in comparison to the
light-to-dark phase transition (A); pdk4 gene
expression was measured by quantitative RTPCR, whereas PDC activity was measured by
following the oxidative decarboxylation of
[14C]pyruvate, as described previously (53).
Total PDC activity does not exhibit a diurnal
variation (data not shown). Because circadian
oscillations of pdk4 persist in isolated adult
rat cardiomyocytes, pdk4 is likely regulated
directly by the circadian clock within the
heart. As the dark phase progresses (B), circadian clock-mediated induction of pdk4 will
slow carbohydrate oxidation; the latter will
promote fatty acid oxidation at this time, as
will potential circadian clock-mediated induction of fatty acid oxidation (FAO) enzymes. In contrast, as the light phase
progresses (C), circadian clock-mediated repression of pdk4 will accelerate carbohydrate
oxidation; the latter will repress fatty acid
oxidation at this time, as will potential circadian clock-mediated repression of FAO enzymes. TCA, tricarboxylic acid.
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the question regarding the nature of the environmental stimuli/
events that the heart anticipates should be addressed. Anticipation of diurnal variations in cardiac workload would be a
logical candidate. The circadian clock within the heart could
potentially influence diurnal variations in cardiac output and
efficiency through a host of molecular mechanisms, including
modulation of myocardial 1) responsiveness to sympathetic
stimulation; 2) electrical properties; 3) calcium homeostasis; 4)
contractile protein composition; 5) antioxidant capacity; 6)
signaling cascades; and 7) metabolism (both oxidative and
nonoxidative). Diurnal variations in gene (and, in certain cases,
protein) expression of key components of all these processes
have been identified in the heart through hypothesis-testing and
hypothesis-generating approaches (75, 80, 121, 122, 125, 151,
158). However, to date, very few studies have linked the
circadian clock within the cardiomyocyte directly to diurnal
variations in myocardial processes. Clearly, with the identification of circadian clock-regulated genes in the heart, those
processes directly influenced by this molecular mechanism will
unfold.
We (33) have recently reported strong evidence that the
circadian clock within the cardiomyocyte influences myocardial metabolism (see discussion above). Our data support the
hypothesis that the circadian clock mediates increased myocardial carbohydrate metabolism after the light-to-dark phase
transition in the rat (and a reciprocal decrease in fatty acid
oxidative capacity at this time). This has led us to speculate
that the circadian clock within the heart allows anticipation of
diurnal variations in workload, substrate availability, and/or the
energy supply-to-demand ratio (154).
During a sudden increase in workload, the heart responds to
the increase in energetic demand by increasing carbohydrate
oxidation, with a lesser effect on fatty acid metabolism (42). At
the onset of the active phase (light phase for humans, dark
H9
Invited Review
H10
MYOCARDIAL CIRCADIAN CLOCK
AJP-Heart Circ Physiol • VOL
Fig. 5. Potential mechanism for circadian clock-mediated regulation of myocardial fatty acid responsiveness and oxidation capacity. We hypothesize that
a circadian clock-mediated coordinated induction of ppar␣, rxr␣, pgc1, and/or
p300, as well as a concomitant repression of rev-erba␣, results in increased
myocardial fatty acid responsiveness during the dark phase.
pression of PPAR␣-regulated genes (and responsiveness of the
heart to circulating fatty acids). This hypothesis is currently
under investigation.
Given that the primary role of the circadian clock is to
synchronize the cell/organ with environmental stimuli, it is not
obvious why a lack of synchronization exists between diurnal
variations in fatty acid availability and responsiveness of the
heart to fatty acids (as well as fatty acid oxidative capacity).
Multiple possibilities exist. One is that the laboratory rat fed a
standard rodent chow does not mimic the evolutionary selective pressures placed on the animal in the wild. Rodent chow is
typically low in fat (often 10% calories from fat, or less),
whereas the animal in the wild may consume a mixed diet
consisting of a greater proportion of calories from fat. Indeed,
when rats are fed a high-fat diet, circulating fatty acids spike
postprandially, such that diurnal variations in the responsiveness of the heart to fatty acids are synchronized with fatty acid
availability (i.e., both are elevated during the dark phase)
(122). An additional explanation for the apparent discrepancy
between fatty acid availability and responsiveness of the heart
to fatty acids in the ad libitum-fed rat stems from the previously described “thrifty gene” hypothesis. This hypothesis,
which states that organisms have evolved to anticipate periods
of prolonged fasting, was devised as an explanation for the
increasing development of obesity in Western society and
relates more to seasonal fluctuations in food availability, as
opposed to day/night fluctuations (24). However, such a hypothesis can be modified to accommodate shorter periods of
food deprivation (i.e., ⬍24 h). We have hypothesized that
diurnal variations in fatty acid responsiveness may be the result
of anticipation of prolongation of fasting, if the animal in the
wild is initially unsuccessful in its forage for food. Indeed, we
observe a rapid and dramatic induction of myocardial pdk4
expression at the light-to-dark transition in the fasted rat (122).
This would transiently promote utilization of fatty acids at the
initiation of the active phase, when availability of this substrate
is increased (as the forage for food continues). More recently,
we have found that manipulation of the light-dark cycle severely impairs the ability of the heart to adapt to fasting and
that the rate of resynchronization of the circadian clock within
the heart (after the light-dark cycle manipulation), correlates
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myocardial oxygen consumption and expression of mitochondrial oxidative metabolism genes, as well as other genes
encoding for proteins promoting fatty acid oxidation, peak
during the dark phase for the rat, a time at which circulating
fatty acids are present at their lowest level (122, 158). Responsiveness of the heart to fatty acids also appears to be out of
synchronization with circulating fatty acids levels (122). We
(122) have recently reported that the heart exhibits dramatic
diurnal variations in its responsiveness to fatty acids. Through
a series of in vivo models, we (122) found that fatty acids
induce the expression of myocardial fatty acid oxidation genes
to the greatest in the middle of the dark phase. Fatty acids
rapidly induce gene expression through direct activation of a
family of nuclear receptors known as peroxisome proliferatoractivated receptors, PPARs (116). All three PPAR family
members (␣, ␤/␦,and ␥) identified to date are expressed within
cardiomyocytes to varying extents, with expression of PPAR␣
and PPAR␤/␦ predominating over PPAR␥ (5, 20, 147). On
heterodimerization with RXR family members, the PPAR/
RXR dimer binds to fatty acid response elements (FAREs)
located within the promoter of various target genes (66).
Known PPAR␣ (and PPAR␤/␦) target genes include those
promoting fatty acid oxidation; indeed, all the genes listed in
Table 1 are known to be PPAR␣ regulated (20, 121, 156).
Similar to diurnal variations in the responsiveness of the heart
to fatty acids, the heart was more sensitive to the PPAR␣specific agonist WY-14,643 during the dark phase, suggesting
that sensitivity of the PPAR␣ system exhibits circadian rhythmicity (122). Consistent with these observations, we (122)
found a coordinated induction of transcriptional activators of
the PPAR␣ system (ppar␣, rxr␣, pgc1, and p300) during the
night. Interestingly, Kassam et al. (61) have shown that the
circadian clock component REV-ERBA␣ antagonizes PPAR␣
through binding of overlapping sequences in the promoter of
target genes. Consistent with its antagonistic function, oscillations in rev-erba␣ expression are antiphase to those of ppar␣
(122).
Diurnal variations in the responsiveness of the heart to fatty
acids are potentially mediated by changes in neurohumoral
influences and/or the circadian clock within the cardiomyocyte.
The in vivo studies performed to date cannot dissociate between these potential influences. It is possible that diurnal
variations in the sensitivity of the PPAR␣ system are due to
neurohumoral-mediated changes in gene and protein expression, and/or phosphorylation status, of any of the components
of the PPAR␣ system (e.g., PPAR␣, RXR␣, PGC1, P300),
independent of the intramyocellular circadian clock. However,
given that circadian rhythms in ppar␣ and rev-erba␣ are
antiphase to one another, that these rhythms are consistent with
diurnal variations in the responsiveness of the heart to fatty
acids, and that both these genes are directly regulated by the
circadian clock, it is tempting to hypothesize that the circadian
clock within the cardiomyocyte directly influences diurnal
variations in the responsiveness of the heart to fatty acids, as
depicted in Fig. 5. Here, increased expression of REV-ERBA␣
near the end of the light phase functions not only as an integral
component of the circadian clock but also to reduce the
expression of PPAR␣-regulated genes (and responsiveness of
the heart to circulating fatty acids). In contrast, repression of
REV-ERBA␣, and a concomitant induction of PPAR␣, RXR␣,
P300, and/or PGC1, during the dark phase will increase ex-
Invited Review
MYOCARDIAL CIRCADIAN CLOCK
MODULATION OF CIRCADIAN CLOCK DURING
PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL STATES
By definition, the role of intracellular circadian clocks is to
allow anticipation of alterations in the environment. If such a
mechanism were impaired, one would hypothesize that the cell
may not respond to its environment in an appropriate manner,
in terms of promptness and/or amplitude of adaptation. This
may manifest ultimately at the level of myocardial dysfunction.
For example, if the circadian clock within the cardiomyocyte
allows anticipation of increased fatty acid availability after
prolongation of an overnight fast, impairment of this molecular
mechanism may result in accumulation of intramyocardial fatty
acid derivatives; appreciable evidence suggests that the latter
contribute toward the development of contractile dysfunction
associated with dyslipidemia, as observed during diabetes
mellitus and obesity (140, 156).
Before the mechanisms involved in alteration of the circadian clock during disease states are addressed, those factors
known to influence this molecular mechanism during physiological conditions should be discussed. Multiple environmental
events modulate the timing of circadian clocks, such as diurnal
variations in light intensity, locomotion, and feeding (25, 34,
55, 87). In turn, these environmental influences affect intracellular circadian clocks via alterations in temperature, neurotransmitters (e.g., glutamate, norepinephrine), nutrients (e.g.,
glucose), ligands for nuclear receptors (e.g., retinoic acid), and
cellular redox state (i.e., NAD⫹/NADH ratio), as well as
AJP-Heart Circ Physiol • VOL
autocrine, paracrine, and endocrine factors (e.g., PGE2, glucocorticoids, adrenaline, angiotensin II) (3, 14, 31, 33, 56, 82,
94, 108, 133, 135). What is also becoming increasingly clear is
that zeitgebers likely act in cell type-specific manners.
Yamazaki et al. (152) have shown that manipulation of the
light-dark cycle is associated with tissue-specific rates of circadian clock resynchronization, with the most rapid rate of
resynchronization observed for the liver (cardiovascular system components were not investigated here). Furthermore,
parabiosis linkage of SCN-lesioned mice restores circadian
clock rhythmicity in the liver and kidney, but not the heart,
skeletal muscle, or spleen, suggesting that specific bloodborne cues are sufficient to reset some, but not all, peripheral circadian clocks (48). More recently, Davidson et al.
(26) have reported that the phase of per1 oscillations differed significantly between arteries and veins of distinct
anatomical location, suggesting that tissue-specific regulation of peripheral circadian clocks also occurs within the
cardiovascular system.
Given the suggestion that circadian clocks are differentially
regulated in a cell-specific manner, the question arises whether
certain physiological and/or pathological states result in an
asynchrony between peripheral circadian clocks. In the case of
the cardiovascular system, one could envisage situations in
which dyssychronization between circadian clocks located
within endothelial cells, VSMCs, and cardiomyocytes could
contribute toward disease states. For example, the circadian
clock within the endothelial cell may modulate diurnal variations in NO production, whereas the circadian clock within
VSMCs may influence diurnal variations in NO-mediated
vasodilation. Loss of synchronization between circadian clocks
within these two cell types would therefore attenuate blood
pressure diurnal variations (i.e., nondipping). If the circadian
clock within the cardiomyocyte allows anticipation of blood
pressure diurnal variations, then a dyssychronization between
the cardiomyocyte clock and this environmental factor may
prevent normal adaptation (i.e., increased cardiac output), ultimately contributing to contractile dysfunction and failure. A
loss of synchronization between circadian clocks within the
same cell type, but located within different regions of the same
organ, may also occur in pathological states. For example,
given that changes in redox state are known to influence the
timing of the circadian clock, myocardial infarction will likely
produce a phase shift in circadian clocks within the ischemic
region, relative to the nonischemic region. Whether such an
intraorgan asynchrony contributes toward cardiovascular disease progression awaits elucidation.
Circadian clocks are altered in various animal models of
cardiovascular disease. We have found that rhythmic expression of circadian clock-regulated genes (e.g., dbp, hlf,
tef, pdk4, and ucp3) is significantly attenuated in the rat
heart during pressure overload-induced hypertrophy (158,
159). Consistent with these observations, Mohri et al. (86)
report that oscillations in circadian clock genes are severely
attenuated in hypertrophied hearts isolated from high-saltfed Dahl rats (an animal model of hypertension). In contrast,
Naito et al. (92) report an augmentation of circadian clock
gene oscillations in aorta and hearts isolated from a different
rat model of hypertension (the spontaneously hypertensive
rat), which is associated with amplified rhythms in pai-1.
Diabetes mellitus, a major risk factor for the development of
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closely with recovery of the fasting response (N. Trexler, D. J.
Durgan, and M. E. Young, unpublished observations).
Diurnal variations in the responsiveness of the heart to fatty
acids can be viewed not only as increased sensitivity during the
dark phase but also as decreased sensitivity during the light
phase. More specifically, we (122) observe lowest responsiveness of the heart to fatty acids (as well as lowest fatty acid
oxidative capacity) 3 h before the light-to-dark phase transition
in the rodent (i.e., just before awakening of the animal). The
perception is therefore that the heart deliberately ignores fatty
acids at a time when they are most abundant in the circulation.
When the organism awakens, elevation of heart rate and
cardiac output increases the energetic demand on the heart.
However, at the same time, vascular resistance increases,
which has been attributed to an elevation in vasoconstrictive
factors (98). This paradox of increased energy demand of the
heart, at the same time as restrained coronary blood flow,
becomes clinically noticeable when the severity of ischemia
reaches a critical threshold, resulting in angina or myocardial
infarction, as is often observed in coronary artery disease
patients (27, 103). Given that the heart faces varying degrees of
ischemia during the day, it is therefore logical to hypothesize
that this organ has evolved mechanisms allowing anticipation
of such events, thereby minimizing impairment of cardiac
function. By decreasing responsiveness to fatty acids when the
animal awakens, the heart will increase reliance on carbohydrate oxidation. The latter is a more efficient fuel, in terms of
ATP generated per O2 molecules consumed, when compared
with fatty acid oxidation (120). Therefore, by modulating
myocardial fuel selection, the circadian clock may allow the
heart to anticipate diurnal variations in the energy supply-todemand ratio.
H11
Invited Review
H12
MYOCARDIAL CIRCADIAN CLOCK
AJP-Heart Circ Physiol • VOL
BENCHSIDE IMPLICATIONS
The existence of circadian clocks within components of the
cardiovascular system has far-reaching implications, which
extend beyond the clinical setting. Given the diversity of
diurnal variations in the intrinsic properties of the cardiovascular system, which manifest at multiple levels (gene expression, protein expression, and cellular and organ function),
extreme caution should be taken to consider time of day issues
during design of experimental strategies, for both in vivo and
in vitro studies. Performance of experiments at an inappropriate time of the day and/or omission of suitable time controls
may lead to erroneous conclusions being drawn. Such temporal
considerations will undoubtedly aid in reducing discrepancies
between studies performed in different laboratories and discrepancies between gene and protein expression measurements, as well as discrepancies between animal models and
humans. Indeed, the majority of clinical studies are undoubtedly performed during the daylight hours, when the subjects
are awake. For the sake of convenience, the majority of animal
studies are also performed during the daylight hours, although
this is a time at which rodents are asleep. Given the increased
reliance on genetically manipulated mouse models for studies
investigating the molecular mechanisms underlying cardiovascular disease in humans, it seems timely to reconsider the
standards for utilization of such models, to include the most
appropriate moment during the light-dark cycle at which such
studies should be performed. Undoubtedly, studies designed to
investigate myocardial gene expression in rodents are less
likely to generate erroneous conclusions when performed during the dark phase (122, 154). Simple manipulation of the
light-dark cycle within the housing facility can overcome
issues regarding convenience.
SUMMARY
In conclusion, circadian clocks exist within multiple components of the cardiovascular system, including cardiomyocytes and VSMCs. These clocks have the potential of impacting multiple cellular processes and therefore hold the promise
of modulating various aspects of cardiovascular function over
the course of the day. Future directions within this field
undoubtedly include identification of genes directly regulated
by the circadian clock within cardiomyocytes, VSMCs, and
endothelial cells. In doing so, those processes influenced by
this molecular mechanism will unfold. Indeed, the recent
identification of metabolic genes as circadian clock-regulated
genes in the heart suggests that the clock modulates myocardial
metabolism. Whether impairment of intracellular circadian
clocks contributes toward cardiovascular morbidity and mortality development is an attractive possibility that requires
further investigation.
ACKNOWLEDGMENTS
I thank Dr. Molly S. Bray for constructive comments before submission.
GRANTS
This work was supported by the American Heart Association Texas Affiliate (Beginning Grant-In-Aid Award 0365028Y) and by the National Heart,
Lung, and Blood Institute Grant HL-074259-01.
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heart disease in humans, is associated with a phase shift in
the circadian clock within the heart (95, 160). Shift workers
also exhibit increased incidence of cardiovascular disease
(52, 68, 70). Intuitively, alterations in behavior, such as
those exhibited in shift workers, would be expected to
influence intracellular circadian clocks. Kobayashi et al.
(69) reported that rat heart per2 gene expression oscillations
take at least 3 days to resynchronize after reversal of the
light-dark cycle; we find that at least 5 days are required for
complete resynchronization of the major circadian clock
components (bmal1, rev-erba␣, dbp; unpublished observations). In contrast, circadian rhythms in behavior and heart
rate resynchronize within 1–2 days, in both rodents and
humans, after reversal of the light-dark cycle (19, 128, 141).
These observations suggest that the circadian clock within
the heart is out of synchronization with the environment for
between 1 and 4 days after reversal of the light-dark cycle.
It is tempting to speculate that repetitive reversal of the
light-dark cycle in shift workers significantly attenuates
anticipation of the heart (as well as other components to the
cardiovascular system) to changes in its environment, ultimately contributing to the development of heart disease.
Sleep apnea, like shift work, is a cardiovascular risk factor
that would be expected to impair intracellular circadian
clocks (10, 11). Additionally, given that food intake appears
to be a primary zeitgeber for peripheral circadian clocks,
one would expect that development of obesity (an additional
cardiovascular disease risk factor) could also alter the timing of intracellular circadian clocks. Consistent with the
idea that impairment of the circadian clock likely increases
risk of cardiovascular disease incidence, Turek et al. (137)
have recently shown that the clock null mouse is predisposed toward the development of obesity and the metabolic
syndrome.
Circadian rhythms in pathophysiological cardiovascular
events are well documented. The onset of myocardial infarction and sudden death both show increased incidence in the
early hours of the morning (2, 17, 88, 89). The timing of a
cardiovascular event, such as myocardial infarction, also affects the severity of the insult; a greater incidence of heart
failure development has been observed for subjects that experience a myocardial infarction during the night, as opposed to
the morning (88). Similar to physiological cardiovascular circadian rhythms, the timing of pathological events has been
attributed primarily to a sudden rise in stimuli triggering
increased blood pressure and heart rate, such as sympathetic
and autonomic activity, in addition to increased prothrombotic
tendency and platelet aggregatability (89). As for studies investigating circadian variability in physiological cardiovascular parameters, those investigating pathological events have
largely ignored diurnal variations in the sensitivity of the
cardiovascular system to neurohumoral stimuli. We propose
that impairment in the ability of the cardiovascular system to
anticipate diurnal variations in neurohumoral stimuli contributes to the development of cardiovascular disease. If true,
future interventions for cardiovascular disease may target normalization of circadian clock timing. Clearly, substantial work
is required to elucidate fully the role of altered circadian clock
function in the progression of cardiovascular disease development.
Invited Review
MYOCARDIAL CIRCADIAN CLOCK
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