Differential effects of REV-ERBαβ agonism on cardiac…
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Research Article Integrative Cardiovascular
Physiology and Pathophysiology
Differential effects of REV-ERBα/β
agonism on cardiac gene expression,
metabolism, and contractile function
in a mouse model of circadian
disruption
Sobuj Mia, Mariame S. Kane, Mary N. Latimer,
Cristine J. Reitz, Ravi Sonkar, Gloria A. Benavides,
Samuel R. Smith, Stuart J. Frank, Tami A. Martino,
Published
Online: 28
MAYM.
2020
//
Jianhua Zhang,
Victor
Darley-Usmar,
and
https://doi.org/10.1152/ajpheart.00709.2019
Martin E. Young
This is the final version - click for previous version
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Abstract
Cell-autonomous circadian clocks have
emerged as temporal orchestrators of
numerous biological processes. For example,
the cardiomyocyte circadian clock modulates
transcription, translation, posttranslational
modifications, ion homeostasis, signaling
cascades, metabolism, and contractility of the
heart over the course of the day. Circadian
clocks are composed of more than 10
interconnected transcriptional modulators, all
of which have the potential to influence the
cardiac transcriptome (and ultimately cardiac
processes). These transcriptional modulators
include BMAL1 and REV-ERBα/β; BMAL1
induces REV-ERBα/β, which in turn feeds
back to inhibit BMAL1. Previous studies
indicate that cardiomyocyte-specific BMAL1knockout (CBK) mice exhibit a dysfunctional
circadian clock (including decreased REVERBα/β expression) in the heart associated
with abnormalities in cardiac mitochondrial
function, metabolism, signaling, and
contractile function. Here, we hypothesized
that decreased REV-ERBα/β activity is
responsible for distinct phenotypical
alterations observed in CBK hearts. To test
this hypothesis, CBK (and littermate control)
mice were administered with the selective
REV-ERBα/β agonist SR-9009 (100
mg·kg−1·day−1 for 8 days). SR-9009
administration was sufficient to normalize
cardiac glycogen synthesis rates,
cardiomyocyte size, interstitial fibrosis, and
contractility in CBK hearts (without
influencing mitochondrial complex activities,
nor normalizing substrate oxidation and
Akt/mTOR/GSK3β signaling). Collectively,
these observations highlight a role for REVERBα/β as a mediator of a subset of circadian
clock-controlled processes in the heart.
NEW & NOTEWORTHY Circadian clocks are
composed of more than 10 interconnected
transcriptional modulators, all of which have
the potential to influence the cardiac
transcriptome (and ultimately cardiac
processes). Previous studies indicate that
cardiomyocyte-specific BMAL1 knockout
(CBK) mice exhibit a dysfunctional circadian
clock (including decreased REV-ERBα/β
expression) in the heart, associated with
abnormalities in cardiac mitochondrial
function, metabolism, signaling, and
contractile function. Here we highlight
decreased REV-ERBα/β as a mediator of
glycogen synthesis, cardiomyocyte size,
interstitial fibrosis, and contractile function
abnormalities observed in CBK hearts.
INTRODUCTION
Virtually all biological processes are
influenced by time of day. Day/night
differences have been reported at whole
body (e.g., behavior), organ (e.g., endocrine),
and cellular (e.g., transcription) levels (45).
The heart must contend with dramatic
fluctuations in workload and neurohumoral
stimuli over the course of the day, many of
which are associated with sleep/wake and
fasting/feeding cycles (11, 15). Therefore, it is
not surprising that cardiac signaling [e.g.,
phosphorylation status of signal transduction
kinases, such as Akt, AMP-activated protein
kinase (AMPK), and mammalian target of
rapamycin (mTOR)], electrophysiology (e.g.,
R-R and QTc intervals, as well as heart rate
variability), metabolism (e.g., substrate
reliance), and contractility (e.g., diastolic
function) change over a 24-h period (53).
Classically, daily fluctuations in many of these
cardiac processes have been attributed to
extra-cardiac stimuli/stresses, such as shear
stress, autonomic/sympathetic tone, and
various endocrine factors (33, 37, 48). More
various endocrine factors (33, 37, 48). More
recently, circadian clocks have emerged as
cell-autonomous molecular timekeeping
mechanisms that temporally govern
biological processes, many of which are
critical for normal cardiac function (45). This
is underscored by observations that genetic
disruption of the murine circadian clock,
either germline or cardiomyocyte specific,
results in an age-onset cardiomyopathy and
reduced lifespan (27, 52).
Circadian clocks are transcriptionally based
molecular mechanisms composed of a series
of positive and negative feedback loops (45).
At the core of the mammalian mechanism are
two transcription factors, BMAL1 and CLOCK
(it is noteworthy that NPAS2 appears to have
functional redundancy with CLOCK) (16, 21,
35). Upon binding to E-boxes, BMAL1:CLOCK
heterodimers typically induce target genes,
including a number of core clock components
that form negative feedback loops, such as
period (PER1/2/3), cryptochrome (CRY1/2),
and REV-ERB (REV-ERBα/β) isoforms (26, 32,
40). More specifically, PER:CRY heterodimers
bind directly to BMAL1:CLOCK, forming an
inactive complex (26, 40). In contrast, REVERBα/β binds to the BMAL1 promoter,
resulting in transcriptional repression (32).
Importantly, oscillations in the activity/levels
of clock components have a periodicity of ∼24
h, and positive components (BMAL1 and
CLOCK) are essentially antiphase to negative
components (PER, CRY, and REV-ERBα/β)
(45). Circadian clock components not only
target expression/activity of each other but
also modulate expression of genes whose
protein products do not directly feed back on
the circadian clock mechanism. These are
termed clock-controlled genes (CCGs).
Between 3% and 10% of an organ’s
transcriptome appears to be circadian
regulated, and proteins encoded by CCGs
impact a wide variety of critical cellular
functions, ranging from transcription and
translation to ion homeostasis, signal
transduction, and metabolism (55).
Because of functional redundancy between
many core circadian clock components,
complete disruption of the mechanism
consequent to single gene manipulations is
possible only when specific components are
targeted. For example, genetic manipulation
of the BMAL1:CLOCK heterodimer typically
results in disruption of the entire circadian
clock mechanism (16, 21). Phenotypical
characterization of cardiomyocyte-specific
BMAL1-knockout (CBK) and cardiomyocytespecific CLOCK mutant [CCM; involves
overexpression of a dominant negative
CLOCK mutant, thus overcoming neuronal
PAS domain protein 2 (NPAS2) redundancy]
mice has revealed that the circadian clock
mechanism in the heart governs
fundamentally important processes, including
insulin signaling, substrate use, and Na+/K+
channel activity (5, 9, 29, 38, 39, 46).
Moreover, unbiased transcriptomic analyses
suggest that the cardiomyocyte circadian
clock influences between 5 and 10% of the
cardiac transcriptome (5, 52). What remains
less clear are the molecular links between the
clock mechanism and the processes that it
governs. Genetic manipulation of BMAL1
and/or CLOCK alters expression of virtually all
clock components, making it difficult to define
which clock component serves as a
mechanistic link to a distinct gene target
and/or biological function (5, 52). Examples
include REV-ERBα and REV-ERBβ, which are
chronically repressed in the heart following
disruption of the cardiomyocyte circadian
clock (5, 52). Here, we hypothesized that
decreased REV-ERBα/β activity observed in
CBK hearts is responsible for distinct
phenotypic alterations. We report that the
selective REV-ERBα/β agonist SR-9009
attenuates abnormalities in glycogen
synthesis, cardiomyocyte size, interstitial
fibrosis, and contractile function that are
observed in CBK hearts in the absence of
effects on mitochondrial complex activities
nor normalization of substrate oxidation and
Akt/mTOR/GSK3β signaling. Collectively,
these observations highlight REV-ERBα/β as
an important mechanistic link between the
cardiomyocyte circadian clock and distinct
cardiac processes.
MATERIALS AND METHODS
Mice.
The present study used cardiomyocytespecific Bmal1-knockout (CBK;
Bmal1flox/flox/MHCαCre+/−) and littermate
control (CON; Bmal1flox/flox/MHCαCre−/−) mice,
as described previously (10). All experimental
mice were male and were housed at the
Center for Comparative Medicine at the
University of Alabama at Birmingham (UAB),
under temperature-, humidity-, and lightcontrolled conditions. A strict 12-h:12-h lightdark cycle regime was enforced [lights on at
6 AM; zeitgeber time (ZT) 0]; the light-dark
cycle was maintained throughout these
studies, facilitating investigation of diurnal
variations (as opposed to circadian rhythms).
Mice were housed in standard microisolator
cages and received food and water ad
libitum. All animal experiments were
approved by the Institutional Animal Care
and Use Committee of the University of
Alabama at Birmingham.
SR-9009 administration.
Mice were administered with the REV-ERBα/β
dual agonist SR-9009 (Cayman) at 100
mg·kg−1·day−1 ip. The agonist was dissolved
in a DMSO/Kolliphor/water vehicle (10:15:75;
% by volume). Mice received either SR-9009
or vehicle at a volume of 10 mg·kg−1·day−1 ip.
For the acute (“disruption”) study, SR-9009 or
vehicle was administered once at ZT0,
followed by heart isolation 3 h later (i.e., ZT3).
For the chronic (“normalization”) study, SR9009 or vehicle was administered at ZT9 for
a total of 8 days, followed by heart isolation 6
h after the last injection (i.e., ZT15 on day 8).
Quantitative RT-PCR.
RNA was extracted from hearts using
standard procedures (8). Candidate gene
expression analysis was performed by
quantitative RT-PCR, using previously
described methods (18, 20). For quantitative
RT-PCR, specific Taqman assays were
designed for each gene from mouse
sequences available in GenBank or
purchased from Applied Biosystems. All
quantitative RT-PCR data are presented as
fold change from an indicated control group.
Western blot analysis.
Qualitative analysis of protein expression and
phosphorylation status was performed via
standard Western blotting procedures as
described previously (9). Briefly, 10–30 µg
protein lysate was separated on
polyacrylamide gels and transferred to PVDF
membranes. Membranes were probed for the
following targets: REV-ERBα (Cell Signaling,
13418) REV-ERBβ (Proteintech, 13906-1AP), p-GSSer461 (Cell Signaling, 3891), GS
(Cell Signaling, 3886), GP (Agrisera
AS09455), p-GSK3βSer9 (Cell Signaling,
9336), GSK3α/β (Santa Crutz 7291), pAKTSer473 (Cell Signaling, 9271), AKT (Cell
Signaling, 9272), p-mTORSer2448 (Cell
Signaling, 2971), mTOR (Cell Signaling 2983),
LC3I/II (Cell Signaling, 12741), p62 (Novus
Biologicals, H00008878-M01), STBD1
(Proteintech, 11842-1-AP), pERK1/2Thr202/Tyr204 (Cell Signaling, 9101), pSMAD3(Ser423/Ser425) (Cell Signaling, 9520),
and total OXPHOS Complexes (Abcam,
ab110413). Rabbit and mouse HRPconjugated secondary antibodies (Cell
Signaling, 7074 and Santa Cruz sc-2005
respectively) were used for chemiluminescent
detection with Luminata Forte Western
Blotting substrate (Millipore, WBLUF0100).
All densitometry data were normalized to
amido black staining. Importantly, due to the
nature of time course studies, to minimize the
contribution that position on the gel might
have on outcomes, samples were randomized
on gels; samples were reordered
postimaging, only for the sake of illustration
of representative images (note, all bands for
representative images for an individual
experiment were from the same gel; original
images are presented in Supplemental Fig. S1
(available online at
10.6084/m9.figshare.12030411).
RNA sequencing.
Transcriptomic analysis was performed in
biventricular samples through the use of RNA
sequencing in the UAB Genomics Core
facility. The quality of the RNA samples was
tested using the Agilent BioAnalyzer, and
RNA with RIN values >7.0 were used in
downstream library preparation. The RNA
was DNAse treated before library
preparation. The RNA-sequencing libraries
were generated using the NEBNext Ultra II
RNA kit (NEB, Ipswich, MA), following the
manufacturer’s protocol. The resulting
libraries were sequenced on the Illumina
NextSeq 500 (Illumina, Inc., San Diego, CA)
using paired end 75-bp sequencing reads per
standard methods.
Mitochondrial complex activities.
Mitochondrial complex activities were
assessed as recently described (1). Briefly,
snap-frozen heart tissues were pulverized in
a liquid nitrogen and subsequently
homogenized in MAS buffer (70 mM sucrose,
220 mM mannitol, 5 mM KH2PO4, 5 mM
MgCl2, 1 mM EGTA, and 2 mM HEPES, pH 7.4;
10 μL/mg tissue) using a glass-glass dounce
homogenizer. Homogenates were centrifuged
at 1,000 g for 10 min at 4°C, followed by
supernatant collection. Protein concentration
was determined by DC Protein Assay (BioRad). Homogenates were diluted and loaded
into Seahorse XF96 microplate (Agilent,
Santa Clara, CA) in 20 µL of MAS (final
concentration of 1 µg/well). The loaded plate
was centrifuged at 2,000 g for 20 min at 4°C.
After centrifugation, 160 µL of MAS prepared
with cytochrome c (10 µM), and alamethicin
(10 µg/ml) was added to each well. Substrate
concentrations to measure complex activities
were as follows: 1 mM NADH (C-I), 10 mM
succinate with 2 μM rotenone (C-II), 0.5 mM
duroquinol (C-III), or 2 mM ascorbate with 0.5
mM TMPD (C-IV). Complex inhibitors were
used at the following concentrations: 2 µM
rotenone (C-I), 10 µM antimycin A (AA) (C-II
and C-III), or 20 mM azide (C-IV). Citrate
synthase activity was assessed as described
previously (1).
Working mouse heart perfusions.
Myocardial substrate use was measured ex
vivo through isolated working mouse heart
perfusions, as described previously (5, 9, 46,
47). All hearts were perfused in the working
mode (nonrecirculating manner) for 40 min
with a preload of 12.5 mmHg and an
afterload of 50 mmHg. Standard KrebsHenseleit buffer was supplemented with 8
mM glucose, 0.4 mM oleate conjugated to 3%
BSA (fraction V, fatty acid-free; dialyzed), 10
μU/ml insulin (basal/fasting concentration), 2
mM β-hydroxybutyrate, 0.2 mM acetoacetate,
0.05 mM L-carnitine, and 0.13mM glycerol.
Metabolic fluxes were assessed through the
use of distinct radiolabeled tracers: 1) [U14C]glucose
(0.12mCi/L; glycolysis, glucose
oxidation); and 2) [9,10-3H]oleate (0.067
mCi/L; β-oxidation). Measurements of cardiac
metabolism (e.g., oxygen consumption) and
function (e.g., cardiac power) were monitored
as described previously (5, 9, 46, 47). At the
end of the perfusion period, hearts were
snap-frozen in liquid nitrogen and stored at
−80°C before analysis. Data are presented as
steady-state values (i.e., values during the
last 10 min of the perfusion protocol). Heart
perfusion conditions were chosen for
consistency with a prior study describing the
metabolic phenotype of CBK hearts (52).
Glycogen content.
Glycogen content was assessed using a
spectrophotometric-based assay, as
described previously (31).
Histologic assessment.
Cross-sections from the medial heart were
taken immediately upon removal of heart and
fixed in formalin for 24 h. Wheat germ
agglutinin (WGA) staining was used for
measurement of myocyte cross-sectional
area; at least 45 myocytes were assessed per
heart using ImageJ software (NIH), as
described previously (23). Picrosirius Red
staining of collagen fibers was used for
semiquantitative measurement of left
ventricular fibrosis, using ImagePro Plus
software (Media Cybernetics, Inc., Rockville,
MD), as described previously (10).
Statistical analysis.
Statistical analyses were performed using
two-way ANOVA, as described previously (5,
6). Briefly, analyses were performed on Prism
statistical software to investigate main
effects of time, genotype, and/or treatment,
followed by Bonferroni post hoc analyses for
pairwise comparisons (indicated in the
figures). In all analyses, the null hypothesis of
no model effects was rejected at P < 0.05.
RNA-seq data from each experimental group
(CBK ± SR-9009; CON ± SR-9009) were
curated into an Excel file list. This list was
imported into GeneSpring Version 14.9-Build
11939 (Agilent Technologies, Inc.), generating
expression data from 26,988 entities. First, a
new experiment was launched using NGS
analyses, on a gene expression experiment
type, with input parameters set as mouse
and linear scale. File format validation was
set as tab separator, with no text qualifier, no
missing value indicators, and no comment
indicators. Data were annotated by Ensembl
ID using the Genes and Transcript Model
annotation source, Mouse mm10 (UCSC)
build, and Ensembl Genes Annotation
(2015.10.05). All original annotations were
included with the import. Experiment
parameters of genotype (CON or CBK) and
treatment (SR-9009 or vehicle) were defined
for each sample. Preprocess baseline options
were set to the median of all samples. Next,
an interpretation was created based on
experimental parameters (genotype and
treatment), and the profile plot display mode
was set to categorical, with the conditions
defined to include both comparator
conditions, and such that we could view the
chip averages or individual data points. We
then performed a statistical analysis using all
entities and genotype-treatment
interpretation by two-way analysis of
variance (ANOVA) with an asymptotic P
value computation, no multiple testing
corrections, and across four condition pairs.
We selected entities at P < 0.01 for
treatment, treatment/genotype, and
genotype. From these lists, the entity IDs,
normalized data values, fold change, and
statistical P values from GeneSpring were
exported into a Microsoft Excel file. The
Ensembl gene IDs were imported into DAVID
(Database for Annotation, Visualization, and
Integrated Discovery) version 6.8 for Gene
Ontology (GO) biological processes analyses
(22). For cell type-specific analyses, we
performed in silico digital cytometry using
CIBERSORTx, a machine learning platform
designed to infer single-cell abundance and
gene expression profiles from bulk tissue
samples (30). First, a reference data set was
obtained from open access fluorescenceactivated cell sorting (FACS) and RNA-seq
data of single cell populations isolated from
male C57Bl/6 mouse left ventricular tissue
(GSE109774) (44). Raw gene expression data
from this reference population was then
imported into CIBERSORTx to generate a
signature matrix consisting of barcode genes
that discriminate individual cell types of
interest (CM = cardiomyocyte, ET = endothelial
cell, F = fibroblast). In silico cell enumeration
and transcriptional analyses were performed
from our bulk tissue samples from each of our
four experimental groups (CON ± SR-9009;
CBK ± SR-9009) using the impute cell fraction
and impute group level gene expression
modules in CIBERSORTx. Cells from the
reference data set were visualized using twodimensional t-distributed Stochastic Neighbor
Embedding (RStudio version 1.2.5001). For
this cell-specific gene expression from whole
heart RNA-seq, data were imported into
GeneSpring and differentially expressed
genes plotted using heat maps and fold
change scatter plots.
RESULTS
Aberrant temporal expression of
circadian clock genes in CBK hearts.
We initially assessed gene expression of 10
core circadian clock components (bmal1,
clock, npas2, per1, per2, per3, cry1, cry2, reverbα, and rev-erbβ) and two established
direct clock-controlled genes (dbp and e4bp4)
in hearts isolated from CBK and littermate
control (CON) mice at 3-h intervals across a
24-h period. Cosinor analysis of the data
revealed significant 24-h oscillations in all
genes investigated in CON hearts (Fig. 1A
and Table 1) in a temporal pattern that is
consistent with operation of a functional
circadian clock. Importantly, CBK hearts
exhibit either a significant attenuation (i.e.,
decreased amplitude) or loss of oscillation in
all clock components (Fig. 1A and Table 1).
Moreover, daily average values for bmal1,
per1, per3, rev-erbα, rev-erbβ, and dbp are
significantly decreased in CBK hearts (CONto-CBK ratio of 2.6, 1.4, 5.4, 2.3, 3.0, and 2.8,
respectively), whereas clock, npas2, cry1,
cry2, and e4bp4 are significantly increased
(CBK-to-CON ratio of 1.5, 2.6, 1.9, 1.2, and
2.2, respectively; Fig. 1A and Table 1). When
differential expression is averaged for core
clock components within a redundant group
(based on established transcriptional
targets), the period (per1/2/3) and rev-erb
(rev-erbα/β) isoforms are decreased by an
average of 2.7- and 2.7-fold, respectively,
whereas clock/npas2 and cryptochrome
(cry1/2) isoforms are increased by an average
of 2.0- and 1.5-fold respectively. As such, of
the core circadian clock components
investigated, period and rev-erb isoforms
were differentially expressed to the greatest
extent in CBK hearts (relative to CON hearts)
and were at a similar magnitude of change
compared with bmal1 (the gene specifically
targeted in CBK hearts). It is noteworthy that
per1 and per2 differential expression is
relatively minor (1.4- and 1.1-fold,
respectively) compared with per3 (5.4-fold).
For these reasons, REV-ERBα and -β were
considered the most consistently differentially
expressed clock components in CBK hearts
and, therefore, were the subjects of
subsequent investigation.
Caption
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
Table 1. Cosinor analysis of
circadian clock genes and
proteins in hearts isolated
from CBK and littermate
CON mice
Enlarge table
Although BMAL1 protein levels have been
investigated previously in CBK and CON
hearts (52), REV-ERBα and/or REV-ERBβ
protein levels have not. Accordingly, we
assessed protein levels of REV-ERBα and
REV-ERBβ in CBK and CON hearts isolated at
4-h intervals across a 24-h period. Time-ofday-dependent variations in REV-ERBα, but
not REV-ERBβ, significantly fit a cosine curve
(with a periodicity of 24 h) in CON hearts; this
oscillation was significantly attenuated (i.e.,
39% amplitude decrease) in CBK hearts (Fig.
1B and Table 1). Moreover, time-of-dayindependent protein levels for REV-ERBα and
REV-ERBβ were significantly decreased in
CBK hearts (relative to CON hearts) by 36%
and 18%, respectively (Fig. 1B). Collectively,
these data are consistent with disruption of
the circadian clock in CBK hearts, which is
associated with significant repression of
REV-ERBα/β protein levels.
Pharmacologic activation of REVERBα/β at ZT0 modulates clock genes
in the heart.
Given that both REV-ERBα and REV-ERBβ
are repressed in CBK hearts (Fig. 1B), we
reasoned that lower activity of these
transcription factors may contribute toward
distinct phenotypical changes described
previously in this model of cardiomyocyte
circadian clock disruption. To address this
possibility, we employed the use of the REVERBα/β dual agonist SR-9009 as a way to
reactivate these nuclear receptors in CBK
hearts. As an initial proof-of-principle study,
CON and CBK mice were administered with
SR-9009 at the beginning of the light phase
(i.e., ZT0), when REV-ERBα/β activity is
predicted to normally be low, based on 1) low
REV-ERBα/β protein levels in CON hearts at
ZT0 (Fig. 1B) and 2) high levels of e4bp4
mRNA at ZT0 [which is repressed by REVERBα/β (49); Fig. 1A]. Three hours after CBK
and CON littermates were treated with SR9009 or vehicle (i.e., ZT3), hearts were
isolated for subsequent gene expression
analysis. In both CON and CBK hearts, SR9009 administration significantly increased
expression of pdk4 [a predicted REV-ERBα/β
target gene (54); Fig. 2A]. Interrogation of
distinct core circadian clock components and
clock-controlled genes revealed anticipated
genotype main effects (Fig. 2B). Moreover,
SR-9009 administration repressed clock and
e4bp4 expression and concomitantly induced
per1 (i.e., SR-9009 main effect; Fig. 2B).
Collectively, these data indicate that SR-9009
treatment at ZT0 (when REV-ERBα/β activity
is normally low) acutely alters circadian clock
genes in the heart (i.e., perturbs the clock).
Caption
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Minimal effects of pharmacological
activation of REV-ERBα/β at ZT9 on
clock gene expression in the heart.
Consistent with the primary goal of the study
(to investigate the effects of REV-ERBα/β
reactivation/normalization in CBK hearts),
CON and CBK mice were treated with SR9009 toward the end of the light phase (ZT9),
when REV-ERBα/β activity is normally
predicted to be high, given that 1) cosinor
analysis indicates peak protein levels of REVERBα around ZT10 in CON hearts (Table 1)
and 2) e4bp4 mRNA, which is repressed by
REV-ERBα/β (49), exhibits the lowest
expression levels in CON hearts around ZT10
(Table 1). Accordingly, mice were treated with
SR-9009 or vehicle once daily at ZT9 (for 8
consecutive days); 6 h after the last treatment
(i.e., ZT15), hearts were isolated for
subsequent gene expression analysis. This
intervention induced pdk4 in the heart,
independent of genotype (i.e., SR-9009 main
effect; Fig. 3A). In contrast, this treatment
regime had minimal effects on expression of
circadian clock components/output genes in
the heart [with the exception of e4bp4, which
was slightly increased; SR-9009 main effect
(Fig. 3B)]. Collectively, these data indicate
that SR-9009 treatment at ZT9 (when REVERBα/β activity is usually high) does not
perturb the circadian clock in the heart.
Caption
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Transcriptome-wide effects of REVERBα/β agonist in the heart.
Given that BMAL1, REV-ERBα, and REVERBβ are transcription factors, we reasoned
that defining the transcriptome-wide effects
of genetic deletion of BMAL1 (i.e., CBK) and
pharmacological activation of REV-ERBα/β
(i.e., SR-9009) may provide insight regarding
the importance of these nuclear receptors in
clock control of distinct cardiac processes.
Accordingly, RNA-seq was performed for
hearts isolated from CON and CBK mice that
had been administered with SR-9009 or
vehicle at ZT9 for 8 days (i.e., the
“normalization” protocol). A two-way ANOVA
analysis revealed main effects of genotype
[of the 3,266 differentially expressed genes,
1,746 genes were induced in CBK hearts, and
1,520 genes were repressed in CBK hearts;
Supplemental Table S1 (available online at
10.6084/m9.figshare.11306999)] and SR9009 administration [of the 242 differentially
expressed genes, 115 genes were induced by
SR-9009, and 120 genes were repressed by
SR-9009; Supplemental Table S2 (available
online at 10.6084/m9.figshare.11306993)].
Consistent with prior studies (52), gene
ontology analysis indicated that genetic
deletion of BMAL1 in cardiomyocytes
influenced biological processes such as cell
signaling, growth/remodeling, transport, and
metabolism, whereas SR-9009
administration influenced inflammation, cell
signaling, metabolism, and transcription (Fig.
4A). The two-way ANOVA also revealed that
91 genes exhibited a significant genotypetreatment interaction [Supplemental Table S3
(available online at
10.6084/m9.figshare.11306996)]; these
genes cluster in processes such as
growth/remodeling, cell signaling,
transcription, and transport (Fig. 4A).
Comparison of differentially expressed genes
based on genotype and SR-9009
administration main effects revealed that of
the 3,487 total genes that were affected,
1,236 were either partially or fully normalized
in CBK hearts in response to SR-9009
administration (i.e., if a gene was induced in
CBK hearts, then SR-9009 decreased
expression; or if a gene was repressed in CBK
hearts, then SR-9009 increased expression).
When stringent twofold cutoffs were applied,
26 “normalized” genes were identified (Table
2); examples of these genes include slc1a7,
rgs1, cd27, and trim40 (Fig. 4B).

Table 2. Expression of
genes that were partially
normalized in CBK hearts
following SR-9009
administration for 8 days at
ZT9
Enlarge table
Caption
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To infer cell-specific changes from the whole
heart RNA-Seq data, we used in silico digital
cytometry analyses. The workflow is shown
in Fig. 4Ci. Based on these analyses, we
found distinct gene clustering attributable to
cardiomyocytes (CM), endothelial cells (ET),
and fibroblasts (F) (Fig. 4Cii). We next
identified which cell type-enriched genes
were differentially expressed in CON versus
CBK hearts; 326 differentially expressed
genes were identified in cardiomyocytes, 938
differentially expressed genes were identified
in endothelial cells, and 1,500 differentially
expressed genes were identified in fibroblasts
(Fig. 4Ciii, Supplemental Table S4; available
online at 10.6084/m9.figshare.12003210).
Moreover, 158 genes (out of the 326
differentially expressed genes) were
normalized in CBK cardiomyocytes following
SR-9009 treatment, 421 genes (out of the
938 differentially expressed genes) were
normalized in endothelial cells, and 922 (out
of the 1,500 differentially expressed genes)
were normalized in fibroblasts (Fig. 4Civ and
Supplemental Table S4). Thus these RNAseq
results suggest that gene expression changes
in CBK hearts occur within different cell
types.
REV-ERBα/β activation in CBK hearts
selectively influences glycogen
synthesis in CBK hearts.
Prior studies suggest that CBK hearts exhibit
impairments in multiple metabolism-related
parameters, including mitochondrial complex
activities and substrate selection (19, 24, 29,
52). Moreover, SR-9009 increases
mitochondrial biogenesis in skeletal muscle
(50) and has previously been suggested to
influence cardiac metabolism (based on
transcriptional changes) (54). Indeed, our
RNA-seq studies suggest that SR-9009
administration influences a number of
metabolism-related genes in the heart,
including partial restoration of cox6b2 mRNA
(a complex IV subunit; Table 2). Collectively,
these observations led us to hypothesize that
SR-9009 may influence mitochondrial
function in CBK hearts. Initial investigation of
mitochondrial complex protein levels revealed
decreased complex II levels in CBK hearts
(i.e., genotype main effect; Fig. 5A).
Somewhat surprisingly, complex IV levels
were significantly higher in CBK hearts (i.e.,
genotype main effect), and complex III levels
were significantly decreased by SR-9009
administration (i.e., SR-9009 main effect),
whereas complex I levels were not influenced
by either genotype or SR-9009
administration (Fig. 5A). Assessment of
mitochondrial complex activities revealed
increased complex IV activity in CBK hearts
(i.e., genotype main effect) in the absence of
significant differences in activity of complexes
I, II, or III (Fig. 5B). Finally, citrate synthase
activity was assessed, revealing no
significant effects of either genotype or SR9009 [although a trend for genotype main
effect was observed (P = 0.052); Fig. 5C].
Importantly, no significant genotype-SR9009 interactions were observed for complex
activities/levels or citrate synthase activity
(Fig. 5, A–C). Collectively, these data suggest
that SR-9009 (and therefore REV-ERBα/β)
has minimal impact on mitochondrial
complexes in the heart.
Caption

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Consistent with our prior studies (29, 52),
CBK hearts exhibit increased rates of fatty
acid oxidation, concomitant with decreased
rates of glucose oxidation, 14C-lactate
release, and triglyceride synthesis (genotype
main effects; Fig. 6, A and B). When substrate
reliance is calculated, CBK hearts exhibit
increased fatty acid oxidation reliance,
whereas both glucose and other substrate
[combination of unlabeled exogenous (i.e., βhydroxybutyrate and acetoacetate) and
endogenous (e.g., triglyceride and glycogen)
substrates] oxidation reliance is decreased
(genotype main effects; Fig. 6A). SR-9009
administration had no significant effect on
rates of fatty acid oxidation, glucose
oxidation, 14C-lactate release, or triglyceride
synthesis in either CON or CBK hearts (Fig. 6,
A and B). In contrast, a significant genotypeSR-9009 interaction was observed for
glycogen synthesis; post hoc analysis
revealed decreased glycogen synthesis in
CBK vehicle hearts (compared with CON
vehicle hearts) which, was reversed by SR9009 administration (Fig. 6B,iii). Collectively,
these observations suggest that REV-ERBα/β
may influence cardiac glycogen metabolism.
Caption

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We next decided to investigate in greater
depth the extent to which SR-9009 influences
cardiac glycogen metabolism. First, we
assessed glycogen content in freshly isolated
hearts (i.e., hearts that were not subjected to
ex vivo perfusions) from the four
experimental groups. Contrary to expectation,
we observed that SR-9009 significantly
decreased glycogen content independent of
genotype (i.e., SR-9009 main effect; Fig. 7A).
Moreover, although not statistically
significant, glycogen content tended to be
higher in CBK hearts (relative to CON hearts;
Fig. 7A). To interrogate the latter further,
glycogen content was assessed in CON and
CBK hearts collected at 3-h intervals over the
course of the day. This analysis revealed main
effects for both time and genotype; in the
latter case, glycogen levels were statistically
increased in CBK hearts (Fig. 7B). The net
synthesis of glycogen is determined not only
by the rate of synthesis (via glycogen
synthase) but also by its degradation (via
glycogen phosphorylase and glycophagy).
Accordingly, we next investigated key
components of these pathways at protein
and posttranslational levels. Neither glycogen
synthase nor phosphorylase total protein
levels differed between the four experimental
groups (Fig. 7, C and D). In contrast, the
phosphorylation status of glycogen synthase
at Ser461 (inhibitory site) tended to be
decreased in CBK hearts (P = 0.084,
genotype main effect when normalized to
amido black; genotype main effect P value
was 0.086 when normalized to total glycogen
synthase levels; Fig. 7C). We have previously
suggested that the cardiomyocyte circadian
clock modulates cardiac glucose use via the
Akt/mTOR/GSK3β signaling axis (29).
Consistent with prior reports (29), Akt and
mTOR phosphorylation (at Ser473 and
Ser2448, respectively) was higher in CBK
hearts, whereas GSK3β phosphorylation (at
Ser9) was decreased (genotype main effect;
Fig. 7, E–G). Interestingly, SR-9009 increased
p-Akt levels (SR9009 main effect; Fig. 7E). In
contrast, SR-9009 had no significant effect
on either mTOR or GSK3β phosphorylation
(Fig. 7, F and G). mTOR is an established
repressor of autophagy, and we have
previously reported attenuated autophagy in
CBK hearts (29). Given that glycophagy
impacts glycogen turnover, we next
investigated autophagy/glycophagy
components. Consistent with prior
observations (29), p62 levels were increased
in CBK hearts (genotype main effects) in the
absence of alterations in LC3II (Fig. 7, H and
I). In contrast, STBD1 levels were not altered
in CBK hearts (Fig. 7J). SR-9009 did not
significantly influence LC3II, p62, or STBD1
levels in the heart (Fig. 7, H–J). Collectively,
these observations suggest that SR-9009 is
unable to normalize perturbations in the
Akt/mTOR/GSK3β signaling axis observed in
CBK hearts.
Caption

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SR-9009 influences adverse cardiac
remodeling in CBK Mice.
CBK mice exhibit age-onset adverse cardiac
remodeling, precipitating in development of a
severe hypertrophic cardiomyopathy and
reduced lifespan (52). We hypothesized that
decreased REV-ERBα/β activity in CBK hearts
potentially contributes toward this
pathological phenotype. Although CBK mice
do not exhibit contractile dysfunction in vivo
(as assessed by echocardiography) at 16 wk
of age (the age at which mice were
investigated in the current study), adverse
remodeling is observed at histological and
molecular levels as well as during ex vivo
assessment of contractility (52). Consistent
with these previous reports, CBK hearts
exhibit decreased rate pressure product
(assessed ex vivo), increased cardiomyocyte
size, and hypertrophic markers (anf, mhcβ),
as well as increased interstitial fibrosis (Fig. 8,
A–D). Interestingly, SR-9009 administration
for 8 days (at ZT9) normalized rate pressure
product in CBK mice and concomitantly
decreased cardiomyocyte size and interstitial
fibrosis (Fig. 8, A–D). SR-9009 did not affect
these parameters in CON hearts (Fig. 8, A–D).
Somewhat surprisingly, SR-9009
administration increased anf and mhcβ
expression (genotype main effect; Fig. 8C).
Neither genotype nor SR-9009 significantly
influenced the phosphorylation status of
ERK1/2 or SMAD3 (Figs. 8, E and F).
Collectively, these observations are consistent
with the concept that decreased REV-ERBα/β
activity in CBK hearts contributes, at least
partially, toward adverse cardiac remodeling.
Caption

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DISCUSSION
The purpose of the present study was to
investigate whether pharmacological
activation of REV-ERBα/β normalizes distinct
phenotypical alterations observed in the
heart following cardiomyocyte-specific
BMAL1 deletion (i.e., CBK mice). In doing so,
we hypothesized that the contribution of
REV-ERBα/β as a mediator of clock control of
cardiac processes might be unmasked.
Consistent with previously published
observations, we report that CBK hearts
exhibit profound transcriptomic alterations
(including decreased rev-erbα/β expression)
associated with perturbations in metabolism,
cellular signaling, cardiomyocyte size,
interstitial fibrosis, and contractility. Here, we
report that administration of CBK mice with a
REV-ERBα/β dual agonist (SR-9009) for only
8 days significantly attenuates abnormalities
in glycogen synthesis, cardiomyocyte size,
interstitial fibrosis, and contractile function in
the absence of effects on mitochondrial
function, substrate reliance, and the
Akt/mTOR/GSK3β signaling axis. Collectively,
these observations highlight REV-ERBα/β as
a potential mechanistic link between the
cardiomyocyte circadian clock and distinct
cardiac processes.
Cell-autonomous circadian clocks have
emerged as critical regulators of numerous
biological functions (45). Being composed of
more than 10 transcriptional modulators, the
mammalian mechanism has the capability of
modulating expression of thousands of genes
in a temporally orchestrated manner (55).
Although much progress has been made
regarding the processes under circadian
governance in the heart, unanswered
questions remain regarding the mechanistic
links involved. Here, we focus on REV-ERBα/β
as putative mediators between the
cardiomyocyte circadian clock and cardiac
processes. Previous studies investigating the
roles of these nuclear receptors have focused
primarily on extracardiac tissues (12). The
importance of REV-ERBα for temporal
governance over the hepatic transcriptome
was demonstrated through transgenic
overexpression of REV-ERBα in the liver,
which inactivated time-of-day-dependent
oscillations of essentially all mRNA species
(with the exception of only 31 genes, which
still oscillated) (25). In addition to established
circadian clock gene targets, genome-wide
ChIPseq studies reveal that REV-ERBα binds
to promoter/enhancer regions for various
genes involved in lipid metabolism (13).
Indeed, genetic deletion of REV-ERBα leads
to hepatic steatosis, which is exacerbated
when REV-ERBβ is also knocked down in
these mice (consistent with some redundancy
between α- and β-isoforms) (7). In other
tissues, REV-ERBα has been suggested to
play a role in oxidative metabolism. For
example, REV-ERBα modulates brown
adipose tissue-mediated thermogenesis (17),
whereas it promotes mitochondrial
biogenesis and function in skeletal muscle
(50). Such observations, coupled with
knowledge that heme serves as the natural
ligand for REV-ERBα/β (51), are consistent
with this nuclear receptor playing a
prominent role in energy metabolism. In
contrast, only a few studies have investigated
the role of REV-ERBα/β in the heart. Zhang et
al. (54) have recently reported that the REVERBα/β dual agonist SR-9009 influences
expression of a number of fatty acid
metabolism genes in cardiomyocytes,
particularly in the presence of
prohypertrophic stimuli. However, these
studies did not observe any effects of the
agonist on mitochondrial function; substrate
oxidation was not investigated. Interestingly,
SR-9009 attenuates pressure overload
induced hypertrophy, interstitial fibrosis, and
contractile dysfunction (54). Similarly, Alibhai
et al. (2) have reported that SR-9009
attenuates age-onset cardiac hypertrophy.
Collectively, such studies suggest that REVERBα/β plays important roles in cardiac
physiology and pathophysiology.
The present study investigated to what
extent REV-ERBα/β serves as a mechanistic
link between the cardiomyocyte circadian
clock and cardiac processes. Here, we
employed a mouse model of cardiomyocytespecific circadian clock disruption (namely
CBK mice). Similar to previously published
reports (52), circadian clock gene oscillations
are severely attenuated or completely
abolished in CBK hearts (Fig. 1A and Table 1).
Importantly, protein levels of both REV-ERBα
and REV-ERBβ are decreased in CBK hearts
(consistent with decreased levels of their
corresponding mRNAs; Fig. 1, A and B).
These studies also revealed striking time-ofday-dependent oscillations in REV-ERBα
levels in control hearts, which peak ∼10 h into
the light phase (i.e., ZT10); oscillations in
REV-ERBβ levels did not reach statistical
significance, although maximal proteins levels
tended to be observed around ZT12 (i.e., the
light-to-dark phase transition). These
observations are similar to those reported
previously in the liver, wherein both REVERBα and REV-ERBβ protein levels peak
around ZT10, with greater oscillations
observed for REV-ERBα (compared with
REV-ERBβ) (7). Indeed, liver ChIPseq studies
indicate very low occupancy of REV-ERBα on
target gene promoters at ZT22 (i.e., the end of
the dark phase, when REV-ERBα levels are
lowest) (14). We hypothesized that
pharmacological activation of REV-ERBα/β
around this time would have dramatic effects
on target genes, given that baseline activity is
usually low. Indeed, SR-9009 administration
at ZT0 markedly altered expression of
circadian clock components in the heart (Fig.
2). In contrast, SR-9009 administration at
ZT9 (when REV-ERBα/β activity is already
high) had essentially no effect on these target
genes (Fig. 3). Such observations are
consistent with prior studies indicating that
SR-9009 treatment at the beginning of the
light phase disrupts sleep cycles in mice (3).
Thus, SR-9009 administration toward the
end of the light phase (e.g., ZT9) is predicted
to augment normal REV-ERBα/β activity
rhythms.
BMAL1, REV-ERBα, and REV-ERBβ are all
transcription factors. Therefore, we
hypothesized that genetic deletion of BMAL1
from cardiomyocytes and/or pharmacological
activation of REV-ERBα/β would impact
cardiac processes primarily through
transcriptional alterations. Accordingly, RNAseq studies were performed. Consistent with
previously published studies, CBK hearts
exhibit >3,000 differentially expressed genes
(compared with control hearts), which cluster
in processes such as cellular signaling,
growth/repair, and metabolism. SR-9009 also
influenced the expression of 242 genes
(compared with vehicle hearts) with known
functions in processes such as inflammation,
cellular signaling, and metabolism. Given that
REV-ERBα/β levels are low in CBK hearts and
that our 8-day SR-9009 administration
strategy was designed to augment REVERBα/β activity at the correct time of the day
(i.e., ZT9), we next looked for genes whose
levels were (partially or fully) normalized in
CBK hearts by SR-9009 administration. We
also inferred from the RNA-seq data that
distinct gene expression alterations following
SR-9009 administration appear to have
occurred in cell-specific populations within
the heart, including many known to influence
cellular signaling and metabolism. Also of
note is that mitochondrial genes such as
cox6b2 (a cytochrome c oxidase subunit;
Table 2) were influenced by both BMAL1
deletion and SR-9009 administration.
Interestingly, previously published studies
suggest that genetic deletion of BMAL1 leads
to mitochondrial dysfunction in the heart (24).
Although SR-9009 did not appear to affect
mitochondrial function in normal hearts (54),
this agonist does promote mitochondrial
biogenesis in skeletal muscle (50). Contrary to
studies by Kohsaka et al. (24), the current
study did not observe a signature of overt
mitochondrial dysfunction in CBK hearts; the
only genotype-specific alterations observed
included decreased complex II levels and
increased complex IV activity/levels in CBK
hearts (Fig. 5, A and B). Similarly, 8 days of
SR-9009 administration had only minimal
effects these parameters (i.e., only a modest
decrease in complex III levels; Fig. 5, A and B).
Collectively, these studies are not consistent
with a major effect of REV-ERBα/β on overall
mitochondrial complex levels/activities in the
heart.
Circadian clocks dramatically influence
metabolism in multiple tissues, including the
heart. For example, the cardiomyocyte
circadian clock increases cardiac glucose use
(oxidation, glycolysis, and glycogen synthesis)
and triglyceride synthesis during the active
period (5, 9, 46). CBK hearts exhibit
chronically decreased ketone body and
glucose oxidation rates and concomitant
increased fatty acid oxidation (52). Here, we
report that SR-9009 is unable to normalize
perturbations in substrate reliance observed
in CBK hearts (Fig. 6A). We have previously
postulated that elevated fatty acid oxidation
rates observed in CBK hearts are secondary
to diminished rates of both ketone body (due
to BMAL1-mediated regulation of BDH1; see
Ref. 52) and glucose (due in part to BMAL1mediated regulation of AS160 activation and
glucose uptake; see Ref. 29) oxidation.
Consistent with these concepts, SR-9009
administration did not normalize either
decreased BDH1 or phospho-AS160 levels in
CBK hearts (data not shown). Similarly, SR9009 treatment failed to normalize either
glycolytic flux (using 14C-labeled lactate
release as an indirect marker) or triglyceride
synthesis in CBK hearts (Fig. 6B). We have
previously suggested that decreased
triglyceride synthesis in cardiomyocyte
circadian clock disruption is due in part to
repression of dgat2 (46); we found that SR9009 administration did not influence dgat2
expression (data not shown). However, SR9009 administration did normalize glycogen
synthesis rates in CBK hearts (Fig. 6B), an
effect that appears to be independent of
changes in proteins involved in glycogen
turnover (glycogen synthase and
phosphorylase) and glycophagy (LC3II, p62,
and STBD1) (Fig. 7, C–J). Interestingly, we
found that glycogen content is increased in
freshly isolated CBK hearts (Fig. 7B) and that
SR-9009 decreases cardiac glycogen content
(Fig. 7A), leading to speculation that low
glycogen reserves following SR-9009
administration may prime the heart for
glycogen repletion during an ex vivo heart
perfusion. It is also important to note that
abnormalities in Akt/mTOR/GSK3β signaling
reported previously in CBK hearts persist
after SR-9009 administration (Fig. 7B),
suggesting that changes occurring in this
signaling cascade following BMAL1 loss are
independent of REV-ERBα/β (29).
Circadian disruption is associated with
increased risk of cardiovascular diseases,
including hypertension, atherosclerosis,
myocardial infarction, and stroke (28). In
animal models, genetic deletion of BMAL1 (in
either a whole body or cardiomyocytespecific manner) leads to severe adverse
cardiac remodeling associated with ageonset dilated cardiomyopathy and decreased
longevity (27, 52). Recent animal-based
studies suggest that the REV-ERBα/β agonist
SR-9009 exerts cardiovascular benefits. For
example, SR-9009 increases tolerance of the
heart to both ischemia-reperfusion and
myocardial infarction, attenuates
atherosclerosis progression, ameliorates
pressure overload induced heart failure, and
improves cardiac function during aging (2, 36,
41–43, 54). Therefore, we hypothesized that
SR-9009 might reverse the adverse cardiac
remodeling observed in CBK mice. Consistent
with this concept, SR-9009 decreased both
cardiomyocyte size and interstitial fibrosis, as
well as increased contractility of CBK hearts
(as assessed ex vivo; Fig. 8, A–D). However,
somewhat surprisingly, SR-9009 increased
molecular markers of cardiac hypertrophy
(anf and mhcβ) in both control and CBK
hearts (Fig. 8B); these observations are in
contrast to those of Zhang et al. (54), which
indicate that SR-9009 decreases anf
expression in neonatal cardiomyocytes
stimulated with phenylephrine. Collectively,
these data reveal that SR-9009 reverses
adverse cardiac remodeling in CBK hearts.
Although the current study has a several
important strengths (e.g., highlighting REVERBα/β as an important contributing factor
toward adverse remodeling in CBK hearts), a
number of distinct weaknesses should be
highlighted. First, a pharmacological
approach was employed to activate REVERBα/β. Although both translational and
transient in nature (the latter being important
to activate REV-ERBα/β only at specific times
of the day), such a strategy can limit
mechanistic insight. More specifically, SR9009 will activate REV-ERBα/β in a variety of
cells/organs, leading to concern that
extracardiac influences may contribute
toward the outcomes observed. Indeed,
evidence exists suggesting that CBK hearts
are in a proinflammatory state (23), whereas
REV-ERBα/β have anti-inflammatory
properties (34). Interrogation of the RNA-seq
data suggests that SR-9009 does influence
inflammatory markers in the heart (Fig. 4A). A
second concern regarding mechanism is that
the current study is unable to establish
causality between parameters measured. For
example, which SR-9009-mediated gene
expression changes contributed toward
glycogen synthesis normalization and/or
reversal of adverse cardiac remodeling in
CBK hearts is uncertain. Moreover, recent
studies suggest that REV-ERBα/β may exert
some functions in a transcriptionindependent fashion (e.g., direct interaction
with O-GlcNAc transferase and subsequent
modulation of protein O-GlcNAcylation; see
Ref. 4). The current study also primarily
assessed end points at single time of the day
(i.e., ZT15), leading to the possibility that
genotype and/or SR-9009 effects may
become evident at other distinct times.
Moreover, mitochondrial activity
measurements were performed in frozen
tissues; use of fresh preparations may have
revealed perturbations in efficiency. It is also
noteworthy that Cre has been reported to
exert phenotypical effects in the heart;
although Cre-positive controls were not
included in the present study, the impact of
Cre-induced cardiotoxicity was minimized
through investigation of mice at 16 wk of age
(52). Finally, the current study did not assess
contractile function in vivo (e.g., through the
use of echocardiography); this is because
contractile dysfunction is not observed in CBK
mice in vivo at the current study target age
(i.e., 16 wk). Future studies are required to
determine whether SR-9009 attenuates
diastolic dysfunction in CBK mice or whether
prolonged SR-9009 administration prevents
age-onset systolic dysfunction in CBK mice.
In summary, we report that acute (8 days)
pharmacological activation of REV-ERBα/β is
sufficient to normalize glycogen synthesis
and ameliorate adverse cardiac remodeling in
a genetic model of cardiomyocyte circadian
clock disruption. In contrast, this
pharmacological intervention did not
normalize mitochondrial function, substrate
oxidation, or the Akt/mTOR/GSK3β signaling
axis. These observations suggest that REVERBα/β likely plays an important role in
mediating clock control of a subset of cardiac
processes (Fig. 9). These studies highlight
further the importance of normal circadian
clock function for the maintenance of cardiac
function.
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GRANTS
This work was supported by the National
Heart, Lung, and Blood Institute Grants HL123574 and HL-142216.
DISCLOSURES
No conflicts of interest, financial or otherwise,
are declared by the authors.
AUTHOR CONTRIBUTIONS
M.E.Y. conceived and designed research; S.M.,
M.S.K., M.N.L., R.S., G.A.B., S.R.S., and M.E.Y.
performed experiments; S.M., M.S.K., M.N.L.,
C.J.R., R.S., G.A.B., S.R.S., T.A.M., V.D.-U., and
M.E.Y. analyzed data; S.M., M.S.K., M.N.L.,
C.J.R., S.R.S., S.J.F., T.A.M., J.Z., V.D.-U., and
M.E.Y. interpreted results of experiments;
C.J.R., T.A.M., and M.E.Y. prepared figures;
M.E.Y. drafted manuscript; S.M., M.S.K., M.N.L.,
C.J.R., R.S., G.A.B., S.R.S., S.J.F., T.A.M., J.Z.,
V.D.-U., and M.E.Y. edited and revised
manuscript; S.M., M.S.K., M.N.L., C.J.R., R.S.,
G.A.B., S.R.S., S.J.F., T.A.M., J.Z., V.D.-U., and
M.E.Y. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Maximiliano Grenett and Stephanie
Reed for technical assistance.
AUTHOR NOTES
Correspondence: M. E. Young
(meyoung@uab.edu).
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