Hill and Beverly A. Rothermel
Sikder, Victoria Copeland, Misook Oh, Erik Bush, John M. Shelton, James A. Bibb, Joseph A.
Nita Sachan, Asim Dey, David Rotter, D. Bennett Grinsfelder, Pavan K. Battiprolu, Devanjan
Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart
Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in
Print ISSN: 0009-7330. Online ISSN: 1524-4571
Copyright © 2011 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research
doi: 10.1161/CIRCRESAHA.110.235309
2011;108:437-445; originally published online January 13, 2011;Circ Res.
http://circres.ahajournals.org/content/108/4/437
World Wide Web at:
The online version of this article, along with updated information and services, is located on the
http://circres.ahajournals.org/content/suppl/2011/01/13/CIRCRESAHA.110.235309.DC1.html
Data Supplement (unedited) at:
http://circres.ahajournals.org//subscriptions/
is online at: Circulation Research Information about subscribing to Subscriptions:
http://www.lww.com/reprints
Information about reprints can be found online at: Reprints:
document. Permissions and Rights Question and Answer about this process is available in the
located, click Request Permissions in the middle column of the Web page under Services. Further information
Editorial Office. Once the online version of the published article for which permission is being requested is
can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin
Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:
by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from
Sustained Hemodynamic Stress Disrupts Normal Circadian
Rhythms in Calcineurin-Dependent Signaling and Protein
Phosphorylation in the Heart
Nita Sachan,* Asim Dey,* David Rotter, D. Bennett Grinsfelder, Pavan K. Battiprolu,
Devanjan Sikder, Victoria Copeland, Misook Oh, Erik Bush, John M. Shelton, James A. Bibb,
Joseph A. Hill, Beverly A. Rothermel
Rationale: Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and
disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac
function and remodeling.
Objective: To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to
changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts.
Methods and Results: We found evidence of large circadian oscillations in calcineurin-dependent activities in
the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the
NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day,
peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor
1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with
calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although
calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained,
whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal
gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these
may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was
significantly lower in the morning than in the evening when assessed by echocardiography.
Conclusions: Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of
proteins that regulate contractility are disrupted in heart failure. (Circ Res. 2011;108:437-445.)
Key Words: calcineurin � circadian rhythms � heart failure � RCAN1/MCIP1
Circadian rhythms are self-sustaining, 24-hour cycles inmolecular, biochemical, and behavioral parameters that
help an organism prepare for anticipated changes in physio-
logical demand. Many important cardiovascular factors, in-
cluding metabolism, heart rate, blood pressure, and hormone
release, oscillate over a 24-hour period.1 In humans, the
incidence of adverse cardiac events, such as myocardial
infarction, ventricular tachycardia, and death from ischemic
heart disease, vary according to the time of day.2 Despite
overwhelming evidence of the importance of circadian
rhythms in cardiovascular health and disease, little is known
regarding the circadian regulation of intracellular signaling
pathways in the heart.
The molecular basis of the circadian clock consists of cell-
autonomous, positive and negative transcriptional and posttran-
scriptional feedback loops.3 The “master clock,” located in the
suprachiasmatic nucleus within the hypothalamus, influences the
phase of independent molecular clocks found in peripheral
organs, including the heart. Many cells and tissues also display
circadian fluctuations in cytoplasmic Ca2� levels, although the
source of these Ca2� oscillations and their relationship to the
transcriptional clock mechanism is not fully understood.4
Dysregulation of Ca2� handling is a hallmark of heart disease.
Several Ca2�-responsive signaling pathways, including the pro-
tein phosphatase calcineurin, have been causally linked to the
progression of heart failure.5 Sustained activation of calcineurin
Original received October 30, 2008; resubmission received October 26, 2010; revised resubmission received December 27, 2010; accepted January 3,
2011. In November 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2
days.
From the Departments of Internal Medicine (N.S., A.D., D.R., D.B.G., P.K.B., M.O., J.M.S., J.A.H., B.A.R.), Molecular Biology (J.A.H.), and
Psychiatry, University of Texas Southwestern Medical Center (J.A.B.), Dallas; Sanford-Burnham Medical Research Institute (D.S.), Orlando, FL; GlycoFi
(V.C.), Lebanon, NH; and Thermo Fisher Scientific (E.B.), Lafayette, CO.
*Both authors contributed equally to this work.
Correspondence to Beverly A. Rothermel, PhD, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8573.
E-mail beverly.rothermel@utsouthWestern.edu
© 2011 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.235309
437 by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from
is sufficient to drive pathological hypertrophic remodeling of the
myocardium with subsequent heart failure and premature death.6
Calcineurin is activated by an elevation in intracellular Ca2� and
binding of a Ca2�/calmodulin complex. In a healthy heart,
calcineurin is thought to be inactive and unresponsive to high-
amplitude, high-frequency waves of Ca2� that drive contraction.
Calcineurin is activated in response to stress presumably when
either diastolic resting Ca2� or Ca2� in subcellular domains
exceed a required threshold.
Calcineurin has numerous substrates including the tran-
scription factor NFAT (nuclear factor of activated T cells)
through which calcineurin influences long-term changes in
gene expression associated with pathological cardiac remod-
eling.6 When NFAT is dephosphorylated, it translocates to
the nucleus where it binds to and activates calcineurin-
responsive genes. Among target genes is the exon 4 isoform
of the Rcan1.4 (regulator of calcineurin 1), previously called
MCIP1, DSCR1, or calcipressin. RCAN1 proteins are potent
inhibitors of calcineurin activity.7 Expression of the mouse
Rcan1.4 gene is extremely responsive to changes in cal-
cineurin activity in vivo8; thus, altered Rcan1.4 transcript
levels have been used as a sensitive indicator of changes in
calcineurin activity in the heart and other tissues.
Both Ca2� handling and cardiac myocyte contractility are
regulated by changes in phosphorylation of key proteins.9
�-adrenergic stimulation activates the cAMP-dependent protein
kinase (PKA), which increases cardiac output by phosphorylat-
ing a number of proteins including phospholamban (PLB). This
releases inhibition of the sarcoplasmic reticulum Ca2� ATPase
(SERCA)2, thereby enhancing relaxation rate and contractility.
PLB and other regulatory proteins are dephosphorylated by the
protein phosphatase 1 (PP1). Phosphorylation of the PP1 inhib-
itor-1 (I-1) by PKA at threonine 35 (I-1Thr35) prolongs
�-adrenergic responses by inhibiting PP1, thus slowing dephos-
phorylation of PLB.10 Changes in I-1 levels and/or phosphory-
lation have been implicated in human heart failure and chronic
atrial fibrillation.9,11 I-1 can be phosphorylated at other sites
including Ser67.12 The in vivo consequence of phosphorylation
at these various sites remains controversial, however, calcineurin
can dephosphorylate both I-1Ser67 and I-1Thr35.12,13 In vitro and in
vivo studies suggest that calcineurin activity can promote de-
phosphorylation of PLB via regulation of I-1.14,15
Given the need for the heart to adapt to daily changes in
cardiac demand and the potentially antagonistic roles of PKA
and calcineurin, we asked whether calcineurin activity and/or
PLB phosphorylation change over the course of 24 hours in a
healthy heart. We found circadian oscillations in both these
parameters that were directly out of phase with each other.
We then tested what happens to these rhythms when both
�-adrenergic activity and calcineurin activity increase in the
pressure stressed myocardium.
Methods
An expanded Methods section is available in the Online Data Supple-
ment at http://circres.ahajournals.org and provides expanded details for
in situ hybridization, immunohistochemistry, chromatin immunopre-
cipitation, quantitative RT-PCR, and Western blot analysis.
Animal Procedures
Male C57BL/6 mice were housed and fed under standard laboratory
conditions with a strict 12:12 hour light:dark cycle with lights
turning on at 6:00 AM, circadian time 0 (CT0), and off at 6:00 PM
(CT12). For pressure-overload experiments, mice were subjected to
thoracic aortic constriction (TAC) or severe (s)TAC for 3 weeks as
described previously.16 Mice were shifted to constant darkness at the
end of the normal light cycle for 24 hours before harvesting. Hearts
were removed and the ventricles flash frozen within 30 seconds of
euthanasia to preserve phosphorylation. A minimum of 3 animals
was analyzed for each time point. A VisualSonics Vevo 770 imaging
system was used to assess cardiac function in unanesthetized
animals. The �MHC-RCAN1 mice were described previously,17 and
wild-type littermates were used as controls. Surgically implanted
miniosmotic pumps (Alzet, Palo Alto, CA) were used to deliver
cyclosporine at a rate of 50 �g per hour per 25 kg of body weight.
Results
Changes in RCAN1.4 Protein and mRNA Levels
Display Circadian Rhythmicity
Biochemical assays of calcineurin activity are limited to mea-
suring the potential activity of the entire cellular pool of
calcineurin, rather than the fraction of the pool that was active in
vivo. We therefore used multiple indirect methods to assess
calcineurin activity. Initially, we quantified changes in both
protein and mRNA levels of the Rcan1.4 gene, a direct target of
calcineurin/NFAT. Male C57BL/6 mice were entrained to a
12:12 light:dark cycle then shifted to constant darkness at
circadian time 12 (CT12) the day before samples were harvested
for analysis. RCAN1.4 protein levels were highest in the heart at
the beginning of the day (CT1 to CT3) and lowest at the end of
the day (CT11 to CT13) (Figure 1A). In comparison, there were
no significant changes in either the level of the exon 1 isoform
of RCAN1 (RCAN1.1) or tubulin. A similar circadian pattern in
RCAN1.4 protein levels was found in the hearts of 129/Sv and
C3H/He inbred lines demonstrating that the oscillations were not
strain dependent (data not shown). These findings are consistent
with genome-wide microarray analysis identifying Rcan1 as
having a circadian pattern of mRNA expression in the mouse
heart.18 We found a 20-fold oscillation in Rcan1.4 mRNA levels
with a peak at CT23 to CT1 and a trough at CT11 (Figure 1B)
Non-standard Abbreviations and Acronyms
ANF atrial natriuretic factor
CT circadian time
Glut4 high-affinity glucose transporter
GSK glycogen synthase kinase
I-1 protein phosphatase 1 inhibitor-1
I/R ischemia/reperfusion
MHC myosin heavy chain
NFAT nuclear factor of activated T cells
Per2 period 2
PKA cAMP-dependent protein kinase
PLB phospholamban
PP1 protein phosphatase 1
RCAN1 regulator of calcineurin 1
SERCA sarcoplasmic reticulum Ca2� ATPase
sTAC severe transverse aortic constriction
TAC transverse aortic constriction
438 Circulation Research February 18, 2011
by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from
directly preceding the peak and trough in RCAN1.4 protein
levels. In contrast, there were no significant circadian changes in
the transcript levels of either Rcan1.1 or I-1 (Figure 1C and 1D).
Thus, circadian regulation of Rcan1 expression is unique to the
Rcan1.4 isoform and controlled at the level of transcript abun-
dance. Transcription of the circadian clock gene Period 2 (Per2)
oscillated with 24-hour periodicity (Figure 1E) verifying the
presence of a functional clock in these samples.
Calcineurin-Dependent Signaling Is Most Active in
a Mouse Heart as the Animal Enters a Period of
Decreased Physical Activity
Immunohistochemical analysis for NFATc1 in the left ven-
tricle revealed nuclear staining at 6:00 AM (CT0) (Figure 2A)
but not at 6:00 PM (CT12) (Figure 2B). Although only a
modest number of nuclei stained positive for NFATc1 even at
the peak of activity, these positive nuclei were always
Figure 1. Rcan1.4 mRNA and protein
levels oscillate with 24-hour periodic-
ity in the hearts of healthy mice. Sam-
ples were pooled from 3 mice for each
time point. A, Immunoblot analysis of
RCAN1.4, RCAN1.1, and tubulin are
shown. B through E, Real-time RT-PCR
for Rcan1.4 (B), Rcan1.1 (C), I-1 (D), and
Per-2 (E) mRNA levels were normalized
to 18S rRNA. Trough values for each
gene were set at a value of 1 (n�3 each
time point in B through E).
Figure 2. More NFATc1 is located in the nucleus and bound to chromatin at CT0 than at CT12. Left ventricular free-wall harvested
at 6:00 AM (CT0) (A, C, and D) and 6 PM (CT12) (B) were stained with a FITC-labeled NFATc1 antibody (yellow/green) and propidium
iodide (PI) (red). Cardiac myocytes have a high level of autofluorescence because of the abundance of mitochondrial flavins and fla-
voproteins, which emit in a broad band overlapping the FITC-NFATc1 signal. In image C, the gain on the green channel has been
turned down to obtain a clear outline of the nuclear PI signal. In D, the intensity of the green overlay has been restored, so that the
autofluorescence of the sarcomere now obscures the myocyte-localized NFATc1-positive nuclei marked with arrows. A nonmyocyte
nucleus is marked with an asterisk (*) and is not obscured. NFATc1 occupancy of the RCAN1.4 promoter was determined by chromatin
immunoprecipitation from ventricular lysates using either preimmune IgG or NFATc1 antibodies (E) (n�4 each time point). In situ
hybridization was carried out using an Rcan1.4-specific probe on the left ventricular wall harvested at 6:00 AM (CT12). Antisense probe
(F) and sense probe (G) are shown. White bars denote 20 �m (A, B, F, and G) or 10 �m (C and D).
Sachan et al Calcineurin Circadian Rhythms in the Heart 439
by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from
embedded within sarcomere-positive cells and never ob-
served in nonmyocyte nuclei (Figure 2C and 2D).
NFAT binding to the Rcan1.4 promoter was assessed by
chromatin immunoprecipitation studies. A six-fold increase in
NFAT occupancy of the Rcan1.4 promoter was detected at CT0
compared with at CT12 (Figure 2E), verifying that the circadian
expression pattern of Rcan1.4 was driven by changes in NFAT
nuclear translocation. An in situ hybridization specific for
Rcan1.4 indicated that transcription was elevated uniformly
across the wall of the myocardium (Figure 2F and 2G). Peak
Rcan1.4 transcript levels were blunted in the hearts of mice with
cardiomyocyte-specific expression of a transgene encoding
RCAN1 to inhibit calcineurin (Figure 3A) or treated with the
calcineurin inhibitor cyclosporine (Figure 3B). Taken as a
whole, these results suggest that activation of the calcineurin/
NFAT signaling pathway occurs throughout the left ventricular
myocardium and is greatest when the animal is entering its rest
phase and cardiac demand decreases.
Phosphorylation of I-1 and PLB Oscillates Out of
Phase With Calcineurin Activity
Immunoblot analyses were conducted to assess phosphoryla-
tion of the calcineurin substrate I-1 at Thr35 and Ser67.
Unfortunately, we were not able to detect phosphorylation of
Thr35 in either heart extracts or forskolin-treated cells trans-
fected with an I-1 expression construct (data not shown).
There were, however, pronounced circadian changes in Ser67
phosphorylation. Phospho–I-1Ser67 was lowest in the morning
(CT1 to CT8), increased notably at CT11 as the animals
became active, and peaked at CT14 directly opposed to
circadian changes in calcineurin activity (Figure 3C and 3D).
In contrast, total I-1 protein (Figure 3C) and transcript levels
(Figure 1D) did not change.
Cardiac contractility and �-adrenergic drive are both
higher at night in the hearts of nocturnal rodents.19,20 Anti-
bodies specific for phospho-PLBSer16 showed a peak at CT14,
coincident with the peak in I-1Ser67 phosphorylation (Figure
3C). The change in phosphorylation was even more pro-
nounced when protein extracts were run such that PLB was
maintained as a pentameric complex. Phospho-PLBSer16 and
phospho-PLBThr17 were both elevated at CT14 compared
with CT2 (Figure 4A, 4B, 4D, and 4E). This was evident in
the slower electrophoretic migration of total PLB complexes
from CT14 lysates (Figure 4C). Thus, the overall phosphor-
ylation state of PLB was elevated during the period when
calcineurin-dependent activities were lowest.
Although we were not able to detect phosphorylation of I-1 at
Thr35 we predict that phosphorylation of this site by PKA
should parallel PLBSer16 phosphorylation. The kinase responsi-
ble for I-1Ser67 phosphorylation in the heart has not yet been
identified definitively. To test whether I-1Ser67 is phosphorylated
Figure 3. Calcineurin-dependent tran-
scription oscillates out of phase with
protein phosphorylation. Rcan1.4 tran-
script levels were quantified in the hearts
of wild-type (WT) and �MHC-Rcan1 (TG)
mice (A) or wild-type mice receiving
either vehicle (V) or cyclosporine A (CsA)
via a mini-osmotic pump (B) (n�4 each).
Ventricular lysates collected from 3 dif-
ferent wild-type hearts at the times indi-
cated were pooled and probed for
phospho–I-1Ser67, total I-1, phospho-
PLBSer16, total PLB, phospho-
GSK3Ser9, and total ERK1/2 (C). Signals
from 3 experiments were quantified by
densitometry (D and E).
Figure 4. Increased PLB and I-1 phosphorylation occurs dur-
ing times of increased adrenergic activity. Ventricular lysates
isolated at CT2 and CT14 were probed for phospho-PLBSer16
(2 �g of protein per lane) (A), phospho-PLBThr17 (0.5 �g of
protein) (B), or total PLB (0.5 �g of protein) (C) per lane.
Phospho-PLBSer16 and phospho-PLBThr17 were quantified by
densitometry (n�6 each) (D and E). In F and G, wild-type mice
were injected with 8 mg/kg body weight of isoproterenol at
CT10. Hearts were harvested at the indicated time points after
injection.
440 Circ