the day/night proteome in the murine heart · resis (2d-dige) and liquid chromatography mass...

The day/night proteome in the murine heart

Peter Podobed,1 W. Glen Pyle,1 Suzanne Ackloo,2 Faisal J. Alibhai,1 Elena V. Tsimakouridze,1

William F. Ratcliffe,4 Allison Mackay,1 Jeremy Simpson,3 David C. Wright,3 Gordon M. Kirby,1

Martin E. Young,4 and Tami A. Martino1

1Cardiovascular Research Group, Biomedical Sciences, University of Guelph, Ontario, Canada; 2Center for Biologic Timingand Cognition Toronto, Ontario, Canada; 3Human Health and Nutritional Sciences, University of Guelph, Ontario, Canada;and 4University of Alabama at Birmingham, Birmingham, Alabama

Submitted 9 January 2014; accepted in final form 24 April 2014

Podobed P, Pyle WG, Ackloo S, Alibhai FJ, Tsimakouridze EV,Ratcliffe WF, Mackay A, Simpson J, Wright DC, Kirby GM, YoungME, Martino TA. The day/night proteome in the murine heart. Am J PhysiolRegul Integr Comp Physiol 307: R121–R137, 2014. First published April 30,2014; doi:10.1152/ajpregu.00011.2014.—Circadian rhythms are essentialto cardiovascular health and disease. Temporal coordination of car-diac structure and function has focused primarily at the physiologicaland gene expression levels, but these analyses are invariably incom-plete, not the least because proteins underlie many biological pro-cesses. The purpose of this study was to reveal the diurnal cardiacproteome and important contributions to cardiac function. The 24-hday-night murine cardiac proteome was assessed by two-dimensionaldifference in gel electrophoresis (2D-DIGE) and liquid chromatogra-phy-mass spectrometry. Daily variation was considerable, as �7.8%(90/1,147) of spots exhibited statistical changes at paired times acrossthe 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to inves-tigate underlying diurnal rhythms in corresponding mRNA. We nextrevealed that disruption of the L:D cycle altered protein profiles anddiurnal variation in cardiac function in Langendorff-perfused hearts,relative to the L:D cycle. To investigate the role of the circadian clockmechanism, we used cardiomyocyte clock mutant (CCM) mice. CCMmyofilaments exhibited a loss of time-of-day-dependent maximalcalcium-dependent ATP consumption, and altered phosphorylationrhythms. Moreover, the cardiac proteome was significantly altered inCCM hearts, especially enzymes regulating vital metabolic pathways.Lastly, we used a model of pressure overload cardiac hypertrophy todemonstrate the temporal proteome during heart disease. Our studiesdemonstrate that time of day plays a direct role in cardiac proteinabundance and indicate a novel mechanistic contribution of circadianbiology to cardiovascular structure and function.

cardiovascular; circadian; diurnal; proteomics; two-dimensional dif-ference in gel electrophoresis

CIRCADIAN RHYTHMS ARE INTRINSIC 24-h rhythms that underliebehavior and physiology, as reviewed previously (52, 62, 65).They are crucial to many major processes in the cardiovascularsystem in humans and other mammals, including heart rate(51) and blood pressure (69), consistent with the sympathova-gal balance of the autonomic nervous system (reviewed inRefs. 19, 22, 34, 40, and 74). Circadian rhythms are alsoassociated with the timing of onset of adverse cardiac events[e.g., myocardial infarction (16, 47, 48, 61), ventricular tachy-arrhythmia (79), and sudden cardiac death (88)], which peakearly in the day. Rhythm disruptions occur in shift work andsleep disorders and are associated with increased prevalence ofadverse cardiac events, exacerbation of heart disease, and

metabolic disorders linked with heart disease, as evidenced bymany clinical (5, 20, 21) and experimental studies (6, 39, 41,56). Circadian rhythms are of central relevance to the healthycardiovascular system, and their disruption contributes signif-icantly to cardiovascular morbidity and mortality.

Recent studies demonstrate circadian variations not only incardiac physiology, but also at the molecular level in the heart(95). Global mRNA profiling studies reveal that �13% of genetranscripts are rhythmic across 24 h light (L) and dark (D)diurnal (38, 41, 82) and circadian cycles (76); undoubtedly, theheart is transcriptionally a different organ in the day vs. thenight. These rhythms are necessary to coordinate biologicaland biochemical processes crucial for maintaining the normalstructure and function of the heart, and remodeling in cardio-vascular disease (as reviewed in Refs. 7, 19, 40, 53, 74).However, our current understanding is incomplete. For exam-ple, the relative contribution of factors that are extrinsic (e.g.,neurohormonal factors) vs. intrinsic (e.g., the cardiomyocytecircadian clock), which drive these rhythms are not fullyknown. Moreover, although many studies have investigateddiurnal gene expression underlying time-of-day cardiovascular(and other organ) physiology, it is only recently that investi-gations have turned toward the proteins. Proteins are funda-mentally important as they underlie many biological processes.Moreover, levels of mRNA and proteins do not always corre-late with each other because of posttranscriptional mechanismscontrolling translation rates, half-lives, and posttranslationalmodifications, for example (23, 50); thus, the proteome war-rants independent investigation. This concept was demon-strated on a global scale by Reddy et al. (60), who reported thatonly �50% of fluctuations in the hepatic proteome could beaccounted for by changes in mRNA levels. Notably, research-ers are now turning their focus toward proteomics approachesapplied to the circadian field, where large-scale quantitativeprotein abundance measurements will increase understandingof how protein rhythms underlie circadian physiology (24, 42,46, 54, 60, 64, 83).

Proteomics has been established as a powerful tool foranalyzing biological phenomena. Most recently, we and othershave begun using large-scale proteomics approaches to dem-onstrate de novo time-of-day changes in the proteome (12, 14,24, 42, 46, 60, 83); however, to date, no studies have investi-gated the global day-night cardiovascular proteome, despite itscritical importance underlying cardiac structure and function.Here, we used two-dimensional difference in gel electropho-resis (2D-DIGE) and liquid chromatography mass spectrome-try (LC/MS/MS) to discover that up to �7.8% (90/1,147 spotson 2D-DIGE) change in abundance in the normal heart over24-h daily periods and that protein spots identified with LC/

Address for reprint requests and other correspondence: T. A. Martino,Cardiovascular Research Group, Biomedical Sciences 1646B, Univ. ofGuelph, Ontario, Canada, N1G 2W1 (e-mail: [email protected]).

Am J Physiol Regul Integr Comp Physiol 307: R121–R137, 2014.First published April 30, 2014; doi:10.1152/ajpregu.00011.2014.

0363-6119/14 Copyright © 2014 the American Physiological Societyhttp://www.ajpregu.org R121

mailto:[email protected]

MS/MS and Western blot may have a corresponding cyclingtranscript by JTK_CYCLE gene expression analyses. In addi-tion, we demonstrated that shortening the 24-h photoperiod to20 h (to which the animals cannot entrain) alters the relation-ship between the L:D cycle and their endogenous rhythms, byusing the Comprehensive Lab Animal Monitoring System(CLAMS), along with functional consequences in left ventric-ular developed pressure on Langendorff. Moreover, we alsoexamined the cardiac proteome mechanistically using car-diomyocyte-specific clock mutant (CCM) mice. We detectedchanges in CCM vs. wild-type (WT) mice in myofilamentATPase activity, myofilament phosphorylation, and abundanceof cardiac metabolic enzymes, revealing the importance of theintact molecular clock for the temporal cardiac proteome.Finally, we demonstrate important contributions of proteomicprogramming in a model of cardiovascular disease.

MATERIALS AND METHODS

Animals. All animal work was conducted under the guidelines ofthe Canadian Council on Animal Care and were approved by theAnimal Care Committee of the University of Guelph. The experimen-tal approach is in Fig. 1, and the rationale for these approaches is inthe Fig. 1 legend. For detection of the diurnal cardiac proteome (Fig.1A), male C57BL/6 mice (8 wk old; Charles River Laboratories) wereentrained to a 12:12-h light-dark cycle (12:12 L:D) for 2 wk at theUniversity of Guelph. Animals were euthanized by isoflurane andcervical dislocation. Tissues were collected from animals killed every4 h starting 1 h before lights on [Zeitgeber Time (ZT) 23] for 1day/night cycle (n � 3/time). For the L:D-disrupted protocol (Fig.1B), animals were housed under controlled conditions in a 12:12 L:Dcycle for 2 wk, then on either their normal 12:12 L:D or an establisheddiurnal disruption protocol that shortens the photoperiod (10:10 L:D)(32, 39). Animals were euthanized (n � 3/time point, 4 times; n �12/group) for tissue collection. To investigate the role of the car-diomyocyte-specific clock (Fig. 1C), we used male CCM mice (FVBbackground, University of Alabama at Birmingham) and WT litter-mates. Animals were euthanized (n � 8/time point, 4 times; n � 32per group), and ventricular tissues were collected. To investigatediurnal rhythms in heart disease (Fig. 1D), we used the transverseaortic constriction (TAC) model of cardiac hypertrophy in maleC57BL/6 mice, as described previously (41). Briefly, cardiac hyper-trophy was induced in mice anesthetized with isoflurane, and intu-bated and ventilated (0.3 l/min O2, 140 respirations/min; HarvardApparatus 687). A thoracotomy was performed in the second leftintercostal space, aorta distal to the subclavian artery cleared, and asilk suture (Ethicon 7–0) that was placed around a 27-gauge needlewas used to constrict the arch. Sham-operated animals underwent thesame surgical procedure, except the ligature was not tightened. Micewere administered buprenorphine (0.1 mg/kg) upon awakening and at8 h and 24 h postoperation. At 1 wk postsurgery, animals wereeuthanized by isoflurane and cervical dislocation every 4 h starting 1h before lights on (ZT23) for 1 diurnal cycle (n � 3/time point, 18TAC, 18 sham), and serum and ventricular tissue was collected.Samples were frozen in liquid nitrogen and stored at �80°C until use.

Protein purification. Soluble heart proteins were collected follow-ing tissue homogenization in urea/CHAPs lysis buffer (10 mM TrispH 8, 8 M urea, 4% wt/vol 3-[(3-cholamidopropyl) dimethylammo-nio]-1-propanesulfonate), with protease inhibitors (Roche; completeMini EDTA-free). Tissue was homogenized on ice using Potter-Elvehjem grinder and centrifuged at 12,000 g, the supernatant wascollected, and the protein concentration was measured by the Bradfordassay (Bio-Rad).

2D-DIGE. Soluble cardiac protein extracts (50 �g) were incubatedin the dark with 200 pmol CyDye Fluors (GE Healthcare) for 30 minat 0°C, and the reaction quenched with 10 mM lysine, as described

previously (26). For detection of the diurnal cardiac proteome (Fig.1A), individual hearts collected 12 h apart were analyzed as 1) ZT23(Cy5) vs. ZT11 (Cy3), or 2) ZT15 (Cy5) vs. ZT03 (Cy3), or 3) ZT07(Cy5) vs. ZT19 (Cy3) (n � 3 gels/time point, 9 gels total). Therationale for pairing time points that were 12 h apart is in relation tomathematical modeling of circadian expression by cosine function,where peak and trough are �12 h apart (for example, Refs. 3, 28, 60,77, 80). For studies investigating the clock mechanism (Fig. 1C),CCM samples were labeled with Cy3, and WT controls were labeledwith Cy5. Gels were labeled as 1) ZT23, 2) ZT03, 3) ZT11, or4) ZT15 (n � 3 gels/time, 12 gels total). For TAC heart disease (Fig.1D), samples were labeled as ZT23 (Cy5) vs. ZT11 (Cy3), ZT15(Cy5) vs. ZT03 (Cy3), or ZT07 (Cy3) vs. ZT19 (Cy5) (n � 3gels/time, 9 gels total). For all studies, the internal standard waslabeled with Cy2 and consisted of the pooled protein extracts used ineach study, in accordance with field standards (1, 37, 81). For the firstdimension, Cy-dye-labeled proteins were mixed with rehydrationbuffer (8 M urea, 1% CHAPS, 65 mM DTT, 0.5% vol/vol pharma-lytes pH 3–10) and were added to 13-cm nonlinear immobilized pHgradient dry strips (pH 3–10) (GE Healthcare). Isoelectric focusing(IEF) was performed using a Protean cell (GE Healthcare). For thesecond dimension, IEF strips were equilibrated [75 mM Tris (pH 8.8),6 M urea, 30% glycerol, 2% SDS, 1% wt/vol DTT, 2.5% wt/voliodoacetamide], overlaid on 12% SDS-polyacrylamide gels, and elec-trophoresed using a DALT 6 apparatus (GE Healthcare). Gels werescanned with a Typhoon 9410 using the excitation/emission filters: 532nm/580 nm for Cy3, 633 nm/670 nm for Cy5, and 488 nm/520 nm forCy2. The photomultiplier tube was set at 550 and pixel size at 100 �m.

Bioinformatics. 2D-DIGE gels were analyzed with DeCyder 6.5software (GE Healthcare). Difference in gel analysis was used todetect and match differentially labeled proteins within the gels.Protein abundance was calculated after background subtraction andnormalization to the internal Cy2 control. Biological variance analysiswas used to match multiple 2D-DIGE gels and calculate fold change(FC). Spots present in �75% of gels with volumes � 2 � 104 wereincluded in the analyses. Normalized protein abundances were visu-alized in DeCyder where peak height and volume correlated with spotintensity. Normalized protein abundance and FC were visualizedusing graph view, and dashed lines represent individual FC, and thesolid line is average FC.

In-gel digestion. Spots were excised from 2D-DIGE SYPRO Rubygels containing 300-�g pooled protein, using the Ettan Spot Picker(GE Healthcare). Trypsin digestion was carried out using in-gel digestkit (Thermo Scientific). Gel pieces were destained (25 mM ammo-nium bicarbonate, 50% vol/vol acetonitrile), reduced (50 mM Tris[2-carboxyethyl]phosphine in 25 mM ammonium bicarbonate), alkylated(100 mM iodoacetamide), then dried (acetonitrile), as per manufac-turer’s directions. Gel spots were rehydrated in 10 ng/�l trypsindigestion buffer and incubated at 30°C overnight. Solution containingtryptic peptide fragments was collected for LC/MS/MS.

LC/MS/MS analysis. Peptide fragments were identified by nanoliq-uid chromatography (LC)-1D� coupled to a hybrid linear ion trap/triple quadrupole mass spectrometer (QTRAP4000, ABSciex). Sepa-ration was performed using a linear binary gradient starting at 5%solvent B and ramping to 30% solvent B in 60 min; then, the gradientreached 100% B in 5 min and was held for 15 min before returning tothe initial conditions. Solvent A contained 2% acetonitrile in waterwith 0.1% formic acid; solvent B contained 2% water in acetonitrilewith 0.1% formic acid. Separation was performed at a flow-rate of 500nl/min. The MS was set up in independent data acquisition modewhere an enhanced mass scan triggered an enhanced product ion scanfor ions between m/z 400 and m/z 1,000, with charge state 2 and 4,which exceeded 3,000 counts per second (cps). Mascot version 2.4,ProteinPilot v2.0.1, X!Tandem and Mouse Swiss Prot database wereused for searching. Search parameters were trypsin, mouse, fixed mod-ifications (carbamidomethyl), partial modifications [dehydration, deami-dated (NQ), dioxidation (M), Gln¡pyro-Glu (N-term Q), Gln¡pyro-

R122 DAY/NIGHT CARDIAC PROTEOME

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00011.2014 • www.ajpregu.org

C57Bl/6 miceNormal 24h (12L:12D)

C57Bl/6 miceDisrupted (10L:10D)

Detection of Diurnal Cardiac

Proteome

CCM vs. WT miceNormal 24h (12L:12D)

Role ofClock Mechanism

NORMAL HEART PROTEOME: 2D-DIGE, day vs. night- ZT03 vs. ZT15- ZT07 vs. ZT19- ZT11 vs. ZT23• DeCyder Bioinformatics• SYPRO Ruby• Spot picking • LC/MS/MS• Mascot

VALIDATION:•Western blot

DIURNAL PROTEOME vs. mRNA•Microarrays•JTK_CYCLE

Heart tissue collection time points

CLOCK MUTANT PROTEOME:2D-DIGE, CCM vs. WT- ZT11- ZT15- ZT23- ZT03• DeCyder • SYPRO Ruby• Spot picking • LC/MS/MS• Mascot


DIURNAL MYOFILAMENT ACTIVITY:•Actomyosin MgATPase •Phosphorylation patterns

L:D Disrupted Protocol

10:10 L:D:• shortened diurnal photoperiod (20h) ivs. Normal (24h) •Protein abundance•Western blot

DIURNAL FUNCTION:• L:D disrupted (20h) vs. normal (24h) •CLAMS•Langendorff



B C

TAC miceNormal 24h (12L:12D)

Heart Disease(TAC)

TAC MODEL of CARDIAC HYPERTROPHY PROTEOME: 2D-DIGE, day vs. night- ZT03 vs. ZT15- ZT07 vs. ZT19- ZT11 vs. ZT23• DeCyder • SYPRO Ruby• Spot picking• LC/MS/MS• Mascot


DIURNAL MYOFILAMENT ACTIVITY:•Actomyosin MgATPase

DIURNAL CYTOKINE RHYTHMS


DA

Light DarkLight Dark Light Dark Light Dark

Fig. 1. Experimental design. Four approaches were used to investigate circadian rhythms relevant to cardiovascular health and disease. A: model 1, detection ofthe diurnal cardiac proteome in male C57BL/6 mice housed in a normal 12:12-h light-dark (L:D) environment. This first classic approach investigates the cardiacproteome under conventional diurnal conditions. B: model 2, L:D disrupted protocol and cardiac protein abundance and heart function. Experimental andepidemiologic studies demonstrated that altered L:D increased risk of heart disease in humans and animals and exacerbated underlying heart disease, for example(21, 39, 41, 56). This second approach interrogates what happened when the L:D cycle was shortened. C: model 3, role of the cardiomyocyte-specific circadianclock (CCM) mechanism on the cardiac proteome and cardiac myofilament activity. Critical to circadian clock function is the transcription factor CLOCK(reviewed in Refs. 19, 40, 52, 62, 65, and many others). In this third model, CCM mice (17) were used to determine the importance of CLOCK (circadian clockmechanism) in the heart on the cardiac proteome. D: model 4, diurnal proteome in cardiovascular disease. Circadian rhythms play an important role in timingof onset of adverse cardiac events (for example, Refs. 45, 48, 49, 79, 89). In addition, blood pressure exhibits a 24-h cycling pattern; a major risk factor forcardiovascular disease occurs in hypertensive subjects that do not exhibit a dip during the night (87). Moreover, diurnal timing for cardiovascular therapies maybenefit cardiac remodeling (43). Thus this fourth model begins to investigate what happens to the diurnal proteome in heart disease, using the pressure-overloadinduced cardiac hypertrophy (TAC) model in mice at 1 wk post TAC.

R123DAY/NIGHT CARDIAC PROTEOME


Glu (N-term E), oxidation (M)], and three missed cleavages. Falsepositives were controlled by comparing molecular mass of the target spoton 2D-DIGE with the molecular weight (MW) of the identified proteinand estimated to be 1%.

Western blot analysis. Proteins identified by LC/MS/MS wereindependently validated at their 2D-DIGE identified time points (12-hpaired time points) using pooled protein extracts (n � 3). In addition,time-of-day profiles (all 6 ZT time points) for proteins were indepen-dently determined from n � 3 individual samples taken at each timepoint across the diurnal cycle. Proteins were visualized by 12%SDS-PAGE [4–20% (Lonza) for PER2] and transferred to polyvinylfluoride membrane (Bio-Rad). The following antisera were used:1:10,000 mouse monoclonal STIP1 (Stressgen; SRA1500), 1:2,500rabbit polyclonal IGF2 (Abcam; Ab9574), 1:100 rabbit polyclonalALDH4A1 (Santa Cruz Biotechnology; sc130948), 1:1,000 rabbitpolyclonal PER2 (Millipore, AB5428P), 1:1,000 goat polyclonalALDH2 (Santa Cruz Biotechnology; sc48837), 1:2,000 rabbit poly-clonal ECHS1 (ProteinTech; 11305–1-AP), 1:3,000 goat polyclonalLDHB (Abcam; ab2101), 1:1,500 rabbit polyclonal DLST (AvivaSystems Biology; OAAB01666), 1:1,500 rabbit polyclonal GOT2(Aviva Systems Biology; ARP43518T100), 1:2,000 mouse monoclo-nal PDHE1a (Abcam; ab110330), 1:5,000 rabbit polyclonal CA2(Santa Cruz Biotechnology; sc25596), 1:100 goat polyclonal FGB(Santa Cruz Biotechnology; sc18029), 1:200 rabbit polyclonalPSMA6 (Santa Cruz Biotechnology; sc67343), 1:200 goat polyclonalPCCA (Santa Cruz Biotechnology; sc68997), 1:1,000 rabbit poly-clonal PDIA3 (Enzo; ADISPA585), and 1:1,000 rabbit polyclonalHSPB1 (Enzo; ADISPA801). Mouse monoclonal to -actin at1:25,000 (Millipore clone C4; MAB1501) was used as a loadingcontrol. Immunoreactive bands were visualized with ECLplus (GEHealthcare) and 1:5,000 HRP-conjugated secondary antibodies asapplicable [goat anti-mouse (Sigma; A2304), rabbit anti-goat (Sigma;A5420), and goat anti-rabbit (Sigma; A6667).

mRNA expression and JTK analysis. mRNA expression profileswere derived from our Affymetrix MOE430A GeneChip microarrays(GEO accession no. GSE36407) (41, 82). Briefly, a separate set ofhearts were collected in the same diurnal manner as those used forprotein studies, that is, every 4 h over 24 h starting at 1 h before lightson (ZT23) (ZT23, ZT03, ZT07, ZT11, ZT15, ZT19; n � 3 per timepoint, 18 samples total). Total RNA was isolated by TRIzol (Invitro-gen) and assessed for high quality using a Nanodrop ND1000(Thermo Scientific) and the Agilent Bioanalyzer 2100 (Agilent Tech-nologies). Hybridization and scanning were in accordance with Af-fymetrix specifications, as described previously (82).The raw data setcovered 22,690 transcripts (based on UniGene database build 107,June 2002) and were analyzed for diurnal rhythms in gene expressionusing the JTK_CYCLE nonparametric algorithm (28), as described onthe CircaDB database (http://bioinf.itmat.upenn.edu/circa) (57).

Real-time PCR. The microarray mRNA expression profiles corre-sponding to our proteins of interest were validated at peak and troughtimes by real-time PCR. The criteria for correlating the proteinidentifiers were as follows. Protein identifiers and correspondingtryptic peptide sequences provided by Mascot were used to retrieveprotein identification data from SwissProt, including the protein fullname, short name, identifier, references, and other cross-referencingmaterial (see Supplemental Data S1). These were then used to retrievethe protein and mRNA sequences from the NCBI database. TheNM_sequence gene reference identifier was used to search the Af-fymetrix microarray data.

For real-time PCR, total RNA from an independent set of normalC57BL/6 hearts collected every 4 h over 24 h was prepared usingTRIzol (Invitrogen) as described previously (8, 82) and quality-assessed by Nanodrop (ThermoScientific). Amplification was per-formed on a VIIA7 real-time PCR system (Life Technologies) usingthe Power SYBR Green RNA-to-Ct one-step PCR kit (Life Technol-ogies) under the following protocol: reverse transcription, 48°C for 30min, 95°C for 10 min for 1 cycle, followed by amplification at 95°C

for 15 s, 60°C for 1 min for 40 cycles. All primers were validated bytwofold dilutions from 200 ng to 12.5 ng of mRNA and are listed inSupplemental Data S2. Real-time PCR samples were run in triplicate,and all values were normalized to histone using the delta delta CTmethod.

In silico circadian motif search. The 2,000 base pair region up-stream of the coding sequence for genes of interest (http://genome.ucsc.edu/cgi-bin/hgTables) were searched for putative circadian E/E=-boxsequences (CANNTG, CACGTG, CACGTT), RORE motifs (AAAG-TAGGTCA), and D-boxes ([A/G]T[G/T]A[T/C]GTAA[T/C]). Wealso searched the circadian mammalian promoter/enhancer database(PEDB, http://promoter.cdb.riken.jp/circadian.html) for E-boxes, D-boxes, and RREs that are predicted conserved in human vs. mouse,and searched circadian motifs identified in published reporter assays(35), and published ChIP and deep-sequencing analyses (63), andtime-dependent patterns of circadian transcription (33).

CLAMS. To investigate whole body behavioral and metabolicanalyses, mice were housed in a CLAMS (Columbus Instruments).Recordings were first taken under a 12:12 L:D cycle (normal envi-ronment) for 5 days, after which the photoperiod was shortened to a10:10 L:D for 5 days (6 L:D cycles). Daily patterns of respiratoryexchange ratio (RER), energy expenditure, food intake, and physicalactivity were measured, as described previously(2).

Langendorff. For ex vivo functional studies, hearts were excised,mounted, and perfused with Krebs-Henseleit buffer (118 mM NaCl,25 mM NaHCO3, 1.2 mM KH2PO4, 4.7 mM KCl, 2.5 mM CaCl2, 1.2mM MgSO4, 11 mM glucose, 0.2 mM EDTA, 0.5 mM Na pyruvate(95% O2-5% CO2, 37°C and 80-mmHg perfusion pressure). A balloonattached to a pressure transducer (AdInstruments) was inserted intothe left ventricle and inflated to give end-diastolic pressures of 1–5mmHg. Left ventricular developed pressure (LVDP) was determinedafter 30 min of stabilization (ADInstrument PowerLab, Chart Soft-ware v5.5.5, Colorado Creeks, USA). Function was assessed in heartscollected in the light period (murine sleep time) at 3 h after lights on,and in the dark period (murine wake time) at 3 h after lights off.

Myofilaments. Cardiac myofilaments were isolated as describedpreviously (90). Ventricular tissue was homogenized in ice-coldstandard buffer (30 mM imidazole (pH 7.0), 60 mM KCl, 2 mMMgCl2, 0.2 mM PMSF, 0.01 mM leupeptin, 0.1 mM benzamidine).Tissue pellets were dissolved in skinning buffer (14.4 mM KCl, 60mM imidazole (pH 7.0), 10 mM EGTA, 8.2 mM MgCl2, 5.5 mMATP, 12 mM creatine phosphate, 10 U/ml bovine creatine phospho-kinase, 0.2 mM PMSF, 0.01 mM leupeptin, 0.1 mM benzamidine, 1%Triton X100) for 45 min at 4°C. Resulting pellets were washed threetimes in ice-cold standard buffer, and protein concentrations weredetermined by the Bradford assay.

Actomyosin MgATPase. We used an actomyosin Mg2� ATPaseassay to assess myofilament function. Myofilaments (50 �g) wereincubated at 32°C for 10 min in mixtures of activating and relaxingbuffer to create a range of free Ca2�. Activating buffer contained (inmM) 23.5 KCl, 20 imidazole (pH 7.0), 5 MgCl2, 3.2 ATP, 2 EGTA,2.2 CaCl2, 0.2 PMSF, and 0.01 leupeptin; 0.1 benzamidine relaxingbuffer consisted of 26 mM KCl, 5.1 mM MgCl2, 3.2 mM ATP, 2 mMEGTA, 20 mM imidazole (pH 7.0), 4.9 �M CaCl2. Free calcium wascalculated using MaxChelator software (55). Reactions werequenched by adding an equal volume of ice-cold 10% trichloroaceticacid (TCA). Proteins were removed by centrifugation, and an equalvolume of developing solution (0.5% FeSO4, 1% ammonium molyb-date in 1 M H2SO4) was added to the supernatant. Absorbance wasmeasured at 630 nm. ATPase values were plotted as the amount ofphosphate released (nmol Pi·min�1·mg protein�1) vs. free Ca2�

(�M). Sigmoidal actomyosin Mg2� ATPase activity-calcium rela-tions were fit by a nonlinear procedure to a modified Hill equation:P � max·[Ca2�]H/([Ca2�]H � EC50

H), where P is actomyosinMgATPase activity, max is the maximum value at saturating calcium,EC50 is the calcium concentration at which 50% of maximum isreached, and H is the slope of the relationship (Hill coefficient) (59).



http://bioinf.itmat.upenn.edu/circa

http://genome.ucsc.edu/cgi-bin/hgTables


http://promoter.cdb.riken.jp/circadian.html

Myofilament phosphorylation. Myofilament isolates from flash-frozen isolates as were used for the actomyosin MgATPase assay (10�g) were resolved using SDS-PAGE, and ProQ Diamond stain toevaluate protein phosphorylation. Load was determined using Coo-massie blue stain. Gels were scanned with a Typhoon 9410.

Blood serum cytokines. Serum cytokines were quantified using themurine Th1/Th2/Th17 cytometric bead array (BD BioSciences), ac-cording to the manufacturer’s specifications.

Statistical analyses. Values are expressed as means � SE. Statis-tical comparisons were done using DeCyder values for normalizedprotein abundance (2D-DIGE) and either an unpaired Student’s t-testor one-way ANOVA followed by the Tukey test for multiple groupsin GraphPad Prism (GraphPad Software). Values of P 0.05 wereconsidered statistically significant.

RESULTS

We first used a conventional diurnal environment to showthat hearts from mice housed in the normal 24-h L:D cycle hadevidence of a diurnal cardiac proteome, as indicated by thedifference in spot abundance on 2D-DIGE (Fig. 2). The 2D-DIGE approach is a variant of two-dimensional (2D) gelelectrophoresis that specifically addresses the variation be-tween gels (which used to occur with 2D gels). Moreover, thistechnique has the advantage of running an internal standard aspart of each separation (Cy2-labeled pools of all proteins runon the gel). It also allows for running up to two test samplessimultaneously (thus, the paired time points) with more accu-rate and reliable results. Gels were run for each 12-h time pointcomparison (ZT11 vs ZT23, ZT07 vs. ZT19, and ZT03 vs.ZT15), using the soluble cardiac proteome from individual murinehearts (three individual murine hearts per time point, all gels runin triplicate), and a total of 1,147 protein spots were reliablydetected on the gels (�75% of gels, spot volume �2 � 104) (Fig.2A). Of these consistently detected spots, there were 382uniquely identified as exhibiting altered abundance at pairedtime points, and 90 (7.8%) were significant (P 0.05) byANOVA (DeCyder software). Of these 90 spots, 38 exhibitedstatistically (P 0.05) increased abundance in the light period(murine sleep time), and 52 were increased (P 0.05) in thedark period (murine wake time).The DeCyder software illus-tration of abundance (top image), area under the peak (middleimage), and relative expression (bottom graph) for six of thesespots at paired time points is shown in Fig. 2B. Thus, overall,we estimated that about 7.8% of the soluble cardiac proteomevaries in abundance over 24-h diurnal cycles.

To independently demonstrate time-of-day changes and con-firm that these spots correlated with proteins that vary inabundance in the heart over the 24-h diurnal cycle, we selected10 high-stringency spots identified with 1.4-fold or greaterdifference in abundance at pairwise time points (ZT23 vs.ZT11; ZT03 vs. ZT15; ZT07 vs. ZT19) and significant (P 0.05) by ANOVA (Fig. 2A arrows, Supplemental Data S3).The spots were identified by LC/MS/MS as �-1-pyrrolinie-5-carboxylate dehydrogenase, mitochondrial (ALDH4A1)(2.4 � 0.5 FC), cAMP-dependent protein kinase- (PRKACA)(1.7 � 0.4 FC), ATP synthase- , mitochondrial (ATP5A1)(2.8 � 0.3 FC), stress-induced phosphoprotein-1 (STIP1)(1.5 � 0.1 FC), aconitate hydratase, mitochondrial (ACO2)(1.5 � 0.1 FC), peroxiredoxin-1 (PRDX1) (1.95 � 0.04 FC),trifunctional enzyme subunit-, mitochondrial (HADHB)(1.6 � 0.1 FC), insulin-like growth factor II (IGF2) (2.0 � 0.3FC), inner membrane protein, mitochondrial (IMMT) (1.5 �

0.1 FC), and 60-kDa heat shock protein (HSPD1) (2.5 � 0.5FC). See Supplemental Data S1 for protein name, replicategels, accession numbers, MW, Mascot search and query scores,other database information, number of peptides, tryptic se-quences, measured/predicted peptide mass, and specificity. Weindependently validated proteins by Western blot analysis (Fig.2C). Our choice of targets was determined by unique trypticpeptide matches per protein and the availability of commer-cial antisera. Moreover, we examined protein abundanceover the 24-h diurnal cycle using samples from all six timepoints by Western blot analysis (Fig. 2C). Protein abun-dance (n � 3 hearts per time point) varied across the L:Dcycle, as illustrated by densitometry analyses of the Westernblots (Fig. 2D). The core circadian clock protein Period2(PER2) was used as a positive control for rhythmic proteinabundance and exhibited a 24-h cycle consistent with itsknown mRNA cycling pattern (38).

To determine whether proteins exhibiting diurnal profileshad an underlying rhythmic mRNA complement in the heart,we compared the protein profiles with our earlier diurnal (L:D)Affymetrix microarray gene expression data collected fromindependent sets of hearts over the 24-h day-night cycle (38,41, 82). Diurnal mRNA rhythms were determined using thewell-established JTK_CYCLE algorithm (28, 57). A total of3,875 out of 22,690 (17%) rhythmic mRNA transcripts weredetected using JTK (ADJ, P 0.05), and, moreover, 599/22,690 (2.6%) were detected at highest stringency using JTK(BH, Q 0.05). We found diurnal microarray data for allcorresponding proteins of interest (Fig. 3A). The significantJTK P values were for Igf2 (P 0.006) and Stip1 (P 0.022),indicative of rhythmic expression and consistent with thepositive control Per2 (P 2.5e-05) (Supplemental Data S2).All the microarray data in Fig. 3A were further validated byusing an independent set of hearts and examining the expres-sion at the peak vs. trough time points and real-time PCR (Fig.3B, Supplemental Data S2). Of the genes that significantly(P 0.05) peaked in the dark phase, Igf2 exhibited a 1.3 � 0.1FC (ZT19 vs. ZT07), and Hspd1 had a 1.2 � 0.1 FC (ZT23 vs.ZT07). For the genes that significantly (P 0.05) peaked inthe light phase, Aco2 showed a 1.5 � 0.04 FC (ZT07 vs.ZT03). Aldh4a1, Stip1, and Prdx1 mRNA did not show asignificant (P � 0.05) peak-trough change by real-time PCR.Per2 was used as a positive control for circadian cycling andexhibited a significant (P 0.05) peak at ZT15 and a trough atZT03, as anticipated. Moreover, the Per2 mRNA exhibited adelay between message and protein, as anticipated; however,interestingly, Igf2, Aldh4a1, and Aco2 were relatively coinci-dent, and Stip1 appeared to exhibit a delay.

We explored mechanisms that could account for underlyingmRNA patterns, to investigate whether they might be under clockmechanism control. To do this, we searched promoter sequences(retrieved from http://genome.ucsc.edu/cgi-bin/hgTables) for cir-cadian elements (E/E= box), and the (http://promoter.cdb.riken.jp/circadian.html) circadian mammalian promoter/enhancer databasePEDB (35), and previously published ChIP assays with deepsequencing and reporter assays investigating circadian elements(33, 63). We detected elements in all mRNA that exhibited adiurnal rhythm, as well as those mRNA that were not rhythmicacross the diurnal cycle by microarray and JTK_CYCLE or PCRanalyses (Supplemental Data S2), suggesting that there are alsoother mechanisms underlying diurnal translational control.






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Fig. 2. Detection of diurnal cardiac proteome with two-dimensional difference in gel electrophoresis (2D-DIGE). A: cardiac protein samples collected 12 h apartwere analyzed by 2D-DIGE, at L (light, ZT0–ZT12, murine sleep time) vs. D (dark, ZT12-ZT24, murine wake time) points. Left to right, ZT11 (Cy3) vs. ZT23(Cy5), ZT07 (Cy5) vs. ZT19 (Cy3), and ZT03 (Cy3) vs. ZT15 (Cy5). Spots with FC � 1.4 (P 0.05) were identified by LC/MS/MS (arrows). B: representativeDeCyder bioinformatics revealing single channel Cy3/Cy5 spot images (top) and 3D peaks (middle), and graphs (bottom) for proteins subsequently validated byWestern blot. For all 2D-DIGE studies, an internal standard was labeled with Cy2, and consisted of the pooled protein extracts used in each study, in accordancewith field standards. C) Independent validation by Western blot analysis, representative blots for ALDH4A1, HSPD1, PRDX1, ACO2, STIP1, and IGF2. PER2is a positive control for rhythmic protein abundance, and -actin is a loading control. D: densitometry of the proteins by triplicate Western blots (n � 3 samplesper time point) for these proteins. See Supplemental Data S1 for protein data (names, replicate gel runs, molecular weight, Mascot search and query scores, otherdatabase information, peptides used for identification, measured/predicted peptide mass, specificity). Supplemental Data S3 provides DeCyder statistical analyses.



Next, we examined what happens when the diurnal cycle isaltered to 10:10 L:D. The animals cannot entrain to thisshortened photoperiod, and thus, it uncouples the L:D environ-ment from their circadian rhythms (32). The rationale foraltering L:D is that numerous experimental and epidemiologicstudies have previously demonstrated that altered L:D cyclescan affect cardiovascular health and disease in humans androdents, for example (21, 39, 41, 56). Moreover, altering the24-h L:D cycle affects molecular cardiac gene expression (40).Thus, we tested whether altering the L:D cycle also impactedprotein abundance in the heart. Mice were subjected to anestablished L:D disruption protocol (10:10 L:D) (32, 41), and

hearts were collected after 5 days. As a control, independentpooled sets of hearts were taken from mice under normaldiurnal 12:12-h L:D, and they exhibited the same relativeabundance of the sentinel proteins ALD4a1, STIP1, IGF2, andPER2 at D:L (transition to sleep time, Fig. 4, A and B) and L:D(transition to wake, Fig. 4, C and D) time points. However,profiles were significantly (P 0.05) altered in hearts fromdiurnally disrupted mice (Fig. 4, A–D, Supplemental Data S4).To further investigate the effect of the shortened photoperiodon endogenous rhythms, we used a CLAMS. As shown in Fig.4E (left, black lines), in the normal 12:12-h L:D environment,RER (a measure of substrate utilization, where lower RER in

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Fig. 3. The diurnal cardiac proteome and transcriptome. A: diurnal profiles of microarray gene expression data (dashed line), compared with proteins identifiedby 2D-DIGE and LC/MS/MS, then validated by triplicate Western blot across all diurnal time points (solid line). Microarray gene expression values are providedas log-normalized mRNA abundance (f.u. � fluorescence units) using GeneSpring software. Rhythmic gene expression was assessed by JTK_CYCLE algorithm,and values are provided in Supplemental Data S2. For protein expression, values are provided as triplicate densitometry from Western blots (see Fig. 2D). Forall graphs, the first (left) y-axis is normalized protein abundance (a.u., arbitrary units) and the second (right) y-axis is log-normalized mRNA abundance (f.u. �fluorescence units), and x-axis is time of day in zeitgebers as denoted in the legend key box inset. Values are expressed as means � SE; n � 3/time point.B: quantitative real-time PCR was performed to validate the peak and trough mRNA expression time points from the microarrays. real-time PCR samples wererun in triplicate, and all values were normalized to histone using the delta delta CT method. *P 0.05, diurnal rhythm significant by JTK_CYCLE. §P 0.05peak:trough ratio validated by real-time PCR. See Supplemental Data S2 for mRNA analyses, including JTK_CYCLE, mRNA vs. protein ZT peaks, fold change,in silico data for the corresponding circadian promoter binding elements (canonical E box, E=box, D box, RORE), searches against published CLOCK/BMALChIP assays, and PCR primer sequences.



the light phase is indicative of increased fatty acid oxidation)exhibits a predictable diurnal profile that is lowest during thelight phase (rodent sleep time) and highest in the dark (rodentwake time). The animals cannot entrain to the shortened 20-hphotoperiod, and as shown in Fig. 4E (right, red lines), theycontinued to exhibit a clear endogenous rhythm in RER thatdid not follow the L:D cycle. The mice showed a similarpattern for the other CLAMS variables as well, includingenergy expenditure, food intake, and physical activity (Supple-mental Data S4). These data were consistent with the findingsof Karatsoreos et al. (32), which showed that the endogenous

circadian rhythm in body temperature was maintained in the10:10 L:D cycle. The times at which heart tissue was collectedfor protein analyses, in reference to both the L:D cycle and theendogenous rhythms, are illustrated in Fig. 4F. To pursuefurther the functional impact of L:D disruption, we assessedcontractile function in Langendorff perfused hearts. Functionwas assessed in hearts collected in the light period at 3 h afterlights on, and the dark period at 3 h after lights off. Consistentwith LVDP as a measure of cardiac function ex vivo, heartsfrom animals maintained in the normal (12:12-h L:D) environ-ment exhibited diurnal variation (P 0.05) (light period,

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Fig. 4. Diurnal disruption. Mice subjected to ashortened L:D environment (10:10 h) exhib-ited altered abundance of cardiac proteinsALDH4A1, STIP1, IGF2, PER2 relative to theL:D cycle, vs. mice housed in the normal12:12-h environment. A: Western blot, show-ing dark (1 h before lights on) to light (3 hafter lights on) transition times. B: densitom-etry relative protein abundance (in a.u.).C: Western blot, showing light (1 h beforelights off) to dark (3 h after lights off) transi-tion times. D: densitometry relative proteinabundance (in a.u.). Images in A and C repre-sent Western blots using pooled samples (n �3 per time point). Graphs in B and D are fromtriplicate Western blots using individual sam-ples (n � 3/time point) and scanned by den-sitometry (values are in Supplemental DataS4). Values are expressed as means � SE;§P 0.05 represents a statistical differenceunder normal 12:12-h L:D conditions, and*P 0.05 represents a statistical difference in10:10-h LD. PER2 is used as a positive cyclingcontrol. Bands are normalized to -actin to con-trol for loading. The lower case letters a,b,c,d inparts A and C denote the time points wheresamples were taken (red letters denote 10:10,while black letters denote 12:12 environment).These sampling times are relative to the L:Dcycle, as illustrated in F. E: whole body meta-bolic substrate utilization rhythms (respiratoryexchange ratio, RER) measured in a CLAMSover 12:12-h L:D cycle exhibited a diurnalrhythm that peaked in the dark (animals awake)and troughed in the light (sleep time) (left, blacklines), and they continued to exhibit a clearendogenous rhythm in RER that did not followthe L:D cycle under the shortened 10:10-h L:Dphotoperiod (right, red lines). F: RER rhythmson the day of collection showing that samplingtimes are relative to the L:D cycle but differ inregards to the endogenous rhythm. n � 5/group,metabolic values are in Supplemental Data S4.The lower case letters a,b,c,d denote the timeswhen samples were taken (red letters denote10:10, while black letters denote 12:12 environ-ment). G: there is normal diurnal variation incardiac function on Langendorff (left ventriculardeveloped pressure, LVDP) under 12:12-h L:D(left, lower during the light/animal sleep time,while higher during the dark phase/wake time);however, this difference is altered with respect tothe L:D cycle in the 10:10 L:D diurnal disruptedgroup (right). Function was assessed in heartscollected in the light period (murine sleep time)at 3 h after lights on, and the dark period (murinewake time) at 3 h after lights off. Values areexpressed as means � SE. *P 0.05 light vs.dark; n � 5 hearts/group.



66.81 � 2.02 mmHg vs. dark, 96.85 � 6.71 mmHg; Fig. 4G),revealing differences in cardiac function day vs. night, asanticipated (94). However, hearts from the 10:10 mice lackedthis diurnal variation in LVDP relative to the L:D cycle (light:72.91 � 2.44 mmHg vs. dark: 72.37 � 2.36 mmHg; Fig. 4G).

Critical to circadian clock function is the transcription factorCLOCK (reviewed in Refs. 19, 40, 52, 62, 65, and manyothers). To investigate the relative contribution of the cardio-myocyte clock on cardiac protein abundance, we next usedCCM mice (17). We performed 2D-DIGE to investigate globalchanges in CCM vs. WT hearts across the diurnal cycle.Triplicate mouse hearts (n � 3) were examined at each timepoint. As shown in Fig. 5A, 2D-DIGE revealed that CCMhearts had 56 spots that differed (P 0.05) in abundance (of1,471 spots identified on these gels) vs. WT. A subset wasidentified by LC/MS/MS and Mascot (Fig. 5A, arrows), thenindependently validated by DeCyder (Fig. 5B), and Westernblot analysis (Fig. 5C). Proteins with increased (P 0.05)abundance in CCM hearts included pyruvate dehydrogenaseE1a, mitochondrial (PDHE1a) (1.4 � 0.1 FC), aspartate ami-notransferase, mitochondrial (GOT2) (1.4 � 0.02 FC), anddihydrolipoyllysine succinyltransferase of 2-oxoglutarate de-hydrogenase, mitochondrial (DLST) (1.5 � 0.1 FC), while

proteins with decreased (P 0.05) abundance were aldehydedehydrogenase, mitochondrial (ALDH2) (1.4 � 0.01 FC),L-lactate dehydrogenase B (LDHB) (1.3 � 0.01 FC), enoyl-CoA hydratase, mitochondrial (ECHS1) (1.6 � 0.1 FC), andD--hydroxybutyrate dehydrogenase, mitochondrial (BDH1)(2.1 � 0.1 FC) (see Supplemental Data S1, S3 for details).Taken together, we estimated that 3.8% of soluble cardiacproteome exhibited differences in abundance in CCM, provid-ing further support for a role for the circadian clock in regu-lating protein abundance in the heart.

In addition to modulation of total protein levels, the cardio-myocyte clock may influence the proteome through posttrans-lational modifications. Our 2D-DIGE revealed multiple iso-forms of proteins in the heart; it has been noted by others thatisoforms carry different charges and, thus, are segregated byisoelectric focusing (60). For example, three distinct spots werepresent for 60-kDa heat shock protein, mitochondrial (HSPD1)in normal C57BL/6 hearts (Fig. 5D). Moreover, we found sixdistinct PDHE1a spots, which predominated in CCM vs. WTsat different times of day or night (Fig. 5E). PDHE1a protein atspot i exhibits a 1.88 increase at ZT03 (P 0.05) in CCM vs.WT (1.70 � 0.26 au vs. 0.90 � 0.04 au) heart, and at spot iia 1.57 decrease (P 0.005) in CCM vs. WT (0.67 � 0.03 au

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Fig. 5. The proteome in cardiomyocyte clockmutant (CCM) heart. Detection of de novo pro-teins with altered abundance in CCM (Cy3) vs.wild-type (WT; Cy5) hearts at four different timepoints (ZT23, ZT03, ZT11, ZT15) by 2D-DIGE(A) and DeCyder bioinformatics analyses (B).C: Western blots were performed to independentlyvalidate spots with FC � 1.4 (P 0.05) andselected for identification by LC/MS/MS. SeeSupplemental Data S1, S3 for tryptic peptide hitsfrom mass spectrometry, protein identification,and DeCyder data. D: posttranslational modifica-tions also occur at different times of day or nightin the heart, with multiple rhythmic isoforms re-vealed for HSPD1 (upper, 2D-DIGE; lower,SYPRO ruby stain) in normal C57BL/6 hearts(D), and PDHE1a (upper, 2D-DIGE; lower, De-Cyder peak views) in CCM hearts For all inves-tigations, n � 3 per group.



vs. 1.05 � 0.01 au) heart. One likely posttranslational modi-fication that expresses in a circadian manner is phosphoryla-tion, as described by Reddy et al. (60); other possibilities mayalso exist.

To examine whether the cardiomyocyte clock influenceddiurnal myofilament function, we next purified cardiac myo-filaments at different times across the diurnal cycle for WT andCCM hearts, and measured actomyosin MgATPase activity.The subcellular fraction was enriched for myofilaments andactinomyosin ATPase evaluated using established protocols(55, 59, 90). Compared with hearts from WT littermates, theCCM hearts lacked rhythmic myofilament calcium sensitivity(Fig. 6, A and B, Supplemental Data S5). For example, myo-filament calcium sensitivity as measured by EC50 lacked arhythm in CCM hearts, but was variable across the diurnalcycle in WT hearts (Fig. 6B). We examined the expression ofmyosin in the myofilament preparations and found no evidenceof isoform shifts contributing to this effect. To further inves-tigate diurnal changes in cardiac myofilaments, we examinedphosphorylation patterns by SDS-PAGE and Pro-Q Diamondstain (Fig. 6, C and D). The myofilament phosphorylationsamples were taken from the same flash-frozen tubes as theATPase assay and run simultaneously. Consistent with thefunctional measurements across the diurnal cycle, CCM heartsexhibited altered (P 0.05) myofilament protein phosphory-lation at times across the diurnal cycle for cardiac myosinbinding protein-C, desmin, troponins T and I, and tropomyosincompared with WT (Fig. 6E, Supplemental Data S5). Thesefindings illustrate time-dependent posttranslational modifica-tions of cardiac myofilament proteins, which are dependent onthe cardiomyocyte circadian clock.

Finally, we examined what happens in heart disease. Circa-dian rhythms play an important role in timing of onset ofadverse cardiovascular events, for example (45, 48, 49, 79, 89).In addition, blood pressure exhibits a 24-h cycling pattern; amajor risk factor for cardiovascular disease occurs in hyper-tensive subjects that do not exhibit a dip during the night (87).Moreover, diurnal timing for cardiovascular therapies maybenefit cardiac remodeling (43). Also, diurnal gene rhythmscan be useful biomarkers of heart disease (82). Thus in thisfourth model, we began to investigate what happened to thediurnal proteome in heart disease, using an established murinemodel of pressure overload-induced cardiac hypertrophy(TAC). As anticipated, increased (P 0.01) heart weight-to-body weight ratio was observed in sham vs. TAC (4.9 � 0.1mg/g vs. 6.4 � 0.4 mg/g) mice at 1 wk postsurgery (Fig. 7A,also Supplemental Data S6 for pathophysiology). Moreover,analysis by 2D-DIGE revealed that TAC hearts had 70 spotsthat differed (P 0.05) in abundance across the diurnal cycleon 2D-DIGE (of the 997 identified). We selected six (arrows)with differences at pairwise times (ZT23 vs. ZT11, ZT03 vs.ZT15, and ZT07 vs. ZT19) for identification by LC/MS/MS(see Supplemental Data S1 and S3). One representative gel isshown in Fig. 7B, with the identified spots noted. Differencesin abundance were also illustrated by DeCyder single channel(Fig. 7C, upper), graphs (lower), and validated by Western blotanalyses across the diurnal time points (Fig. 7D). Proteins withincreased (P 0.05) abundance in TAC hearts in the lightphase were fibrinogen beta chain (FGB) (ZT07 vs. ZT19,1.6 � 0.2 FC), heat shock protein b1 (HSPB1) (ZT03 vs.ZT15, 2.0 � 0.4 FC), proteasome subunit 6a (PSMA6) (ZT11

vs. ZT23, 1.4 � 0.1 FC), and propionyl-CoA carboxylase- (PCCA) (ZT07 vs. ZT19, 3.7 � 0.9 FC), and protein disulfideisomerase A3 (PDIA3) (ZT11 vs. ZT23, 1.5 � 0.3 FC).Carbonic anhydrase 2 (CA2) (ZT03 vs. ZT15, 1.5 � 0.2 FC)was increased (P 0.05) during the dark. Moreover, wepurified cardiac myofilaments and measured actomyosinMgATPase activity (Fig. 7E, Supplemental Data S7), revealingthat TAC hearts exhibited a diurnal pattern of myofilamentsensitivity (dark, 217.06 � 14.24 vs. light, 181.31 � 4.93 nmolPi·min�1·mg�1); however, levels were reduced (P 0.05)compared with sham hearts (dark, 242.69 � 17.18 vs. light,198.70 � 11.82 nmol Pi·min�1·mg�1). We also investigatedthe peripheral cytokine proteome revealing that TAC alters(P 0.05) diurnal cycling of innate serum cytokines IL-6,TNF- , and IL-17 , but not Th1/Th2 cytokines implicated inother types of responses (Fig. 7F, Supplemental Data S8).Taken together, these studies confirm that coordination ofcardiac function in health and disease is more complex thanpreviously anticipated and that circadian regulation occurringacross multiple levels, including transcriptional, translational,and posttranslational, can play a role in the disease process.

DISCUSSION

In this study, global proteomic approaches were used to testthe hypothesis that the cardiac proteome differs in abundanceover the 24-h day/night period, that temporal changes inproteins can be dependent on the cardiomyocyte circadianclock mechanism and that maintaining these rhythms is impor-tant for cardiac function. We found differences in abundance in�7.8% of the normal soluble cardiac proteome as analyzed by2D-DIGE and mass spectrometry at 12-h paired time pointsacross the diurnal cycle. This estimate is conservatively withinthe range in other murine tissues and other organisms (14, 29,46, 60, 75, 83), albeit slightly lower than murine liver (up to20%) (60). Daily oscillations in cellular function are due to acombination of fluctuations in the neurohormonal milieu (i.e.,extrinsic factors) and cell autonomous rhythms (driven bycircadian clocks). Manipulation of the L:D cycle affects bothextrinsic and intrinsic factors, and, accordingly, disruptsrhythms in both the cardiac proteome and contractility relativeto the diurnal cycle. Selective genetic disruption of the cardi-omyocyte circadian clock attenuates rhythms in the subset ofthe cardiac proteome. There is also a temporal cardiac pro-teome underlying heart disease.

In our first study in the normal heart, we identified severalmitochondrial metabolic proteins involved in substrate gener-ation for the citric acid cycle [HADHB (84), ALDH4a1(85)],the citric acid cycle itself (ACO2), and ATP production[ATP5A1(31)]. HADHB, a component of the trifunctionalprotein complex responsible for fatty acid -oxidation, wasmost abundant during the sleep phase, which corresponds withtimes of greater reliance on fatty acids by the heart (92).Conversely, ALDH4a1 was most abundant during the L:Dtransition times, making it tempting to speculate on alternativesubstrates for energy production during this transitional meta-bolic period. Moreover, ACO2, which catalyzes the conversionof citrate to isocitrate in the citric acid cycle was least abundantduring ALD4a1 peak abundance, thus providing further sup-port for time-of-day activity. Decreased isocitrate productionmay be compensated for by increased -ketogluturate produc-



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Fig. 6. Diurnal variation in myofilament function and phosphorylation. To examine whether temporal posttranslational modifications were associated withmyofilament function, cardiac myofilaments were purified at different times of day or night. Compared with normal WT hearts, the CCM hearts lacked diurnalvariation in myofilament activity as measured by actomyosin MgATPase (A), and EC50 (B). Time-of-day myofilament phosphorylation patterns across the diurnalcycle revealed by SDS-PAGE and Pro-Q Diamond stain (C), and normalization by Coomassie blue stain (D), and densitometry values plotted (E). MyofilamentATPase and phosphorylation data are in Supplemental Data S5. Values are expressed as means � SE. Letters a,b,c,d denote statistical significance, P 0.05;for time points, see Supplemental Data S5. *P 0.05; n � 8 per time point.



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Fig. 7. Altered day/night protein profiles in TAC heart. To demonstrate a time-of-day proteome underlying heart disease, we used a model of compensated heart disease(transverse aortic constriction; TAC). A: representative hearts from mice at 1 wk post-TAC were enlarged (left, top) vs. sham (left, bottom), and characterized by cardiachypertrophy (right). TAC, black, sham, white. *P 0.05; n � 36 (18 TAC, 18 sham). HW, heart weight, mg; BW, body weight, g. See Supplemental Data S6 forpathophysiology data (morphometrics, echocardiography, and hemodynamics). B: proteins with altered abundance in hearts at 1 wk post-TAC at different time pointsacross the diurnal cycle were identified by 2D-DIGE. and C: DeCyder bioinformatics (top, single-channel spot images; bottom, graph views). D: independent validationof DeCyder bioinformatics by Western blot, using -ACTIN as a loading control. One-week TAC profiles were further compared with the sham profiles on Westernblots. See Supplemental Data S1 and S3 for tryptic peptide hits from LC/MS/MS, protein identification, and DeCyder data. E: hearts at 1 wk post-TAC were maintainedwith diurnal variation in myofilament activity, as measured by actomyosin MgATPase, but the diurnal calcium profiles were attenuated; also see Supplemental Data S7.F: compared with shams, the 1 wk post-TAC mice had altered diurnal profiles in serum cytokines important for early remodeling, as measured by multiplex ELISA (alsosee Supplemental Data S8). Values are expressed as means � SE. *P 0.05; n � 6 samples per time point, 6 time points total.



tion, allowing the TCA cycle to continue. Since our studiesfocused on soluble proteins, many of the diurnal proteinsidentified included enzymes predominantly associated withvital cardiac metabolic pathways. Indeed, it is tempting tospeculate that mitochondrial content, in general, exhibits acircadian pattern. This would be consistent with the notion ofdiurnal metabolism, and moreover, it was recently demon-strated that mitochondrial proteins involved in metabolic path-ways are influenced by circadian clock-driven acetylation (44).A more detailed look at the day-night cardiac mitochondrialproteome is likely worthy of further investigation, althoughbeyond the scope of the current study.

We also investigated the underlying diurnal mRNA expres-sion by JTK_CYCLE, which revealed statistically significantrhythms for up to 17% of the cardiac transcriptome, which isconsistent with reports by others (38, 41, 76, 82). Theseincluded several of our genes of interest, supporting the notionthat transcription can underlie rhythmic protein abundance.Moreover, the Per2 mRNA exhibited a delay between messageand protein as anticipated, and interestingly, Igf2, Aldh4a1, andAco2 were relatively coincident and Stip1 appeared to exhibita delay. Our in silico analyses revealed the presence of E-boxesin many additional genes, suggesting that translational controlthrough promoter activity of known circadian elements alone isnot sufficient to explain these corresponding mRNA profiles.Additional mechanisms likely are also involved in regulatingdiurnal protein abundance in the heart. These findings areconsistent with reports in other tissues such as liver (60).Collectively, these studies promote new understanding of car-diac processes, and diurnal regulation provides a means forpromoting protein abundance and flexibility over a 24-h pe-riod.

Next, we examined what happens to the cardiac proteomewhen L:D rhythms were altered. We and others have previ-ously demonstrated that altered L:D adversely affects theexpression of diurnal gene rhythms in the heart and cardiacfunction (18, 39–41, 56, 93); however, effects on the cardiacproteome have not been similarly investigated. This studyrevealed altered cardiac protein profiles with altered L:D. ACLAMS was used to demonstrate that the mice do not syn-chronize to the shortened L:D cycle, consistent with previousstudies (32). Therefore, heart proteins were sampled in refer-ence to the L:D cycle, but there was still a clear endogenousrhythm in body physiology that likely contributed to alteredprotein abundance. Moreover, there was loss of diurnal varia-tion in LVDP on Langendorff analyses in the hearts of diur-nally disrupted mice, relative to the L:D cycle. Thus, thealtered diurnal environment can impact cardiac protein abun-dance and heart function relative to the L:D cycle. Thesefindings have implications for cardiovascular health and dis-ease of individuals subjected to rhythm disruptions, such asshift workers, patients with sleep disorders (including sleepapnea), nondipping hypertensives, as well as others associatedwith the 24/7 demands of society.

Our findings also suggest that protein abundance in the heartis regulated, in part, by the cardiomyocyte clock. For example,we found significantly altered abundance of GOT2 and BDH1in CCM hearts. Similarly, previous microarray studies revealedaltered got2 and bdh1 gene expression in CCM vs. WT hearts(7), consistent with our findings. Also, regulation of PDHE1aand LDHB by the circadian clock may be at the translational

level contributing to the observed rhythms in glucose andlactate metabolism and are supported by previous observationsthat rhythmic cardiac glucose and lactate metabolism are dis-rupted in CCM mice (15). Lastly, it is tempting to speculatethat the posttranslational modification of PDH observed inCCM hearts is potentially phosphorylation. PDH, when phos-phorylated, is inactivated. Decreased PDH activity, plus de-creased LDHB, ECHS1, and BDH1 could limit acetyl-CoAproduction in CCM hearts. A mechanistic illustration of themetabolic proteins with altered abundance in CCM hearts is inFig. 8A.

The heart responds to diurnal variations in drive, alteringmetabolic expression, oxidative metabolism, and energy han-dling. While significant research has been undertaken to un-derstand the circadian variances in these areas, relatively littleis known about how myofilaments—the central contractileelement and largest consumer of energy in cardiac myocytes—are affected by circadian rhythms. Recently, investigators iden-tified a link between cardiac myofilaments and the circadianclock when they reported the movement of CLOCK proteinbetween cardiac Z-discs and nuclei of cells under stress (4);however, subsequent work failed to repeat these findings(86).Interestingly, recent work by Collins et al. (11) reports noveltime-of-day variation in myocardial contractility that could notbe fully explained by differences in calcium handling. More-over, a comprehensive cardiac myofilament subproteome hasbeen described with potential for studying dynamic changesimportant for myofilament contraction (91). Roles for myofila-ments have been hypothesized; however, no previous study hasexamined diurnal variations in myofilament function or regu-lation. Thus, despite these and other studies suggesting thatcardiac myofilaments are targeted by the circadian clock, to ourknowledge, the current study is the first investigation of circa-dian variations in cardiac myofilament function.

A novel finding of our experiments is that myofilamentfunction exhibits a temporal oscillation in the heart. A mech-anistic illustration of these myofilament proteins is provided(Fig. 8B).The observed rhythms may be due in part to dailyvariations in cAMP levels (58), a molecule known to regulatebioavailability of the cAMP-dependent protein kinase A,which is crucial for phosphorylating myofilament proteins(72).Consistent with this notion is that we found rhythms inPRKACA, the major catalytic subunit of cAMP protein-depen-dent kinase (PKA) responsible for regulation of cardiac struc-ture and function via posttranslational modification of numer-ous intracellular targets. PRKACA was most abundant innormal hearts during the early waking hours, which may allowfor increased PKA activity and modulation of cardiac functionvia myofilament phosphorylation. Moreover, we found alteredATPase activity in CCM hearts, further indicating a role for theclock mechanism in myofilament structure and function. Thesediurnal differences in myofilament function were associatedwith observable alterations in phosphorylation of several keymyofilament proteins important for sarcomere function (72,73). Consistent with these findings of a role for the circadianmechanism in sarcomeric structure/function is that GSK3bexhibits diurnal activity(30) and is mechanistically tied to thecardiac circadian clock mechanism (16) and has been shownrecently to phosphorylate cMyBP-C (36). A critical implica-tion of these findings relates to cardiac hypertrophy (TACmice), in which we observed a reduction in myofilament



sensitivity to Ca2�. Moreover, these findings are consistentwith recent reports of circadian oscillations in calcineurinactivity and protein phosphorylation in normal vs. TAC hyper-trophy and failing hearts (67). There are also diurnal variationsin myocellular excitation-contraction coupling that relate tocalcium homeostasis (10). Other avenues could also be worthyof future study. For example, we enriched for myofilamentsconsistent with our established protocols (55, 59, 90); however,future studies could use 2D-DIGE to examine preparationsenriched for sarcomeric proteins, including alpha-myosin andbeta-myosin heavy-chain changes in disease, and circadian

changes in phosphorylation of sarcomeric proteins identifiedby isoelectric focusing. In summary, these findings indicatenovel time-dependent posttranslational mechanisms withincardiac sarcomeres that influence myofilament contractility andrelaxation over the course of the day.

Perspectives and Significance

Clinical and experimental studies have revealed that circa-dian rhythms play an important role in the pathophysiology ofheart disease [e.g., timing of onset of myocardial infarction

Fig. 8. Circadian control of critical cardiac pro-cesses. A: protein abundance differs in CCM vs. WTheart at different times of day or night, includingrate-limiting enzymes important for vital metabolicpathways that provide substrate for the trichloro-acetic acid (TCA) cycle. Identified proteins are cir-cled. B: schematic diagram of the sarcomere, thefunctional unit of the cardiomyocyte. Contraction isachieved through complex interactions between thethin and thick filaments and the Z-disc. The thinfilaments comprise proteins, including actin sub-units, -helix tropomyosin (Tm), and the troponincomplex [troponin T (TnT), troponin I (TnT), andtroponin C (TnC)]. Thick filaments are composed ofa number of proteins, including myosin bindingprotein C (MyBP-C), and myosin heads consistingof myosin heavy chain (MHC) and myosin lightchain (MLC) and magnesium-dependent ATPase(MgATPase). Z-discs consist of demsin, -actinin,titin, and actin-capping protein (capZ) and otherproteins to anchor filaments. Calcium binding to thetroponin complex allows results in thin-filamentconformational changes, permitting the myosin headof the thick filament to bind actin, leading to sarco-mere contraction. When the myosin head is re-leased, ATP is used by the Mg-dependent ATPase tohydrolyze ATP to ADP � Pi, and return the myosinhead back to its starting conformation.



(MI) (9, 27, 48), severity of MI (16, 61, 78), time of day ofventricular tachyarrhythmias (79), temporal nocturnal hyper-tensive profiles (25, 70), and response to angiotensin-convert-ing enzyme inhibitors (43, 71)]. Furthermore, a flurry of recentstudies has associated cardiovascular structure and functionwith an underlying rhythmic genome by microarray and bioin-formatics analyses (38, 66, 76). There are altered diurnalgenomic expression profiles in TAC heart disease (41, 82).Moreover, diurnal gene expression profiles in the heart areregulated by both the diurnal environment (40, 41) and thecardiomyocyte clock mechanism (17, 19). However, the diur-nal proteome has not been similarly investigated. Althoughgenes encode proteins, it is the proteins that actually performmany important cellular functions. Here, we use proteomicapproaches to reveal a temporal cardiac proteome, regulationby both the environment and the cardiac clock, and altereddiurnal protein abundance in heart disease. These findingssupport the notion that temporal regulation of substrate utili-zation optimizes metabolic efficiency and cardiac function in anormal diurnal environment, and elucidates an important yetrelatively unrecognized role in heart disease. The implicationsare that de novo studies, such as these, investigating the diurnalcardiac metabolic, sarcomeric, and myocellular proteome, canlead to new approaches to understand and treat heart disease.

ACKNOWLEDGMENTS

The authors sincerely thank Dr. John Hogenesch at the Institute forBiomedical Informatics, Institute for Translation Medicine and Therapeutics,University of Pennsylvania Perelman School of Medicine, Philadelphia PA, foranalysis of the microarray data using JTK_CYCLE, and for his thoughtfulcomments and helpful advice.

GRANTS

This work was supported by grants from the Canadian Institutes of HealthResearch (MOP119518 to T. A. Martino) and the National Heart, Lung, andBlood Institute (HL-074259, M. Young).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.P., W.G.P., S.A., F.J.A., E.V.T., W.F.R., A.M., J.A.S., D.C.W., M.E.Y.,and T.A.M. performed experiments; P.P., W.G.P., S.A., F.J.A., E.V.T., A.M.,J.A.S., D.C.W., and T.A.M. analyzed data; P.P., W.G.P., F.J.A., E.V.T., andT.A.M. prepared figures; P.P., W.G.P., S.A., F.J.A., E.V.T., W.F.R., A.M.,J.A.S., G.K., D.C.W., M.E.Y., and T.A.M. approved final version of manu-script; W.G.P., J.A.S., G.K., M.E.Y., and T.A.M. conception and design ofresearch; P.P., W.G.P., S.A., F.J.A., E.V.T., D.C.W., and T.A.M. interpretedresults of experiments; W.G.P., M.E.Y., and T.A.M. edited and revisedmanuscript; T.A.M. drafted manuscript.

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