..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

Female ClockD19/D19 mice are protected from the

development of age-dependent cardiomyopathy

Faisal J. Alibhai1, Cristine J. Reitz1, Willem T. Peppler2, Poulami Basu2, Paul Sheppard3,

Elena Choleris3, Marica Bakovic2, and Tami A. Martino1*

1Department of Biomedical Sciences/OVC, Centre for Cardiovascular Investigations; 2Human Health and Nutritional Sciences; and 3Department of Psychology and NeuroscienceProgram, University of Guelph, Room 1646B, University of Guelph, Guelph, ON N1G2W1, Canada

Received 9 February 2017; revised 12 July 2017; editorial decision 12 August 2017; accepted 8 September 2017

Time for primary review: 37 days

Aims Circadian rhythms are important for healthy cardiovascular physiology and they are regulated by the molecular cir-cadian mechanism. Previously, we showed that disruption of the circadian mechanism factor CLOCK in maleClockD19/D19 mice led to development of age-dependent cardiomyopathy. Here, we investigate the role of biolo-gical sex in protecting against heart disease in aging female ClockD19/D19 mice.

....................................................................................................................................................................................................Methods andresults

Female ClockD19/D19 mice are protected from the development of cardiomyopathy with age, as heart structure andfunction are similar to 18 months of age vs. female WT mice. We show that female ClockD19/D19 mice maintain nor-mal glucose tolerance as compared with female WT. Tissue metabolic profiling revealed that aging femaleClockD19/D19 mice maintain normal cardiac glucose uptake, whereas the male ClockD19/D19 mice have increased car-diac glucose uptake consistent with pathological remodelling. Shotgun lipidomics revealed differences in phospho-lipids that were sex and genotype specific, including cardiolipin CL76:11 that was increased and CL72:8 that wasdecreased in male ClockD19/D19 mice. Additionally, female ClockD19/D19 mice show increased activation of AKT sig-nalling and preserved cytochrome c oxidase activity compared with male ClockD19/D19 mice, which can help to ex-plain why they are protected from heart disease. To determine how this protection occurs in females even withthe Clock mutation, we examined the effects of ovarian hormones. We show that ovarian hormones protect fe-male ClockD19/D19 mice from heart disease as ovariectomized female ClockD19/D19 mice develop cardiac dilation,glucose intolerance and reduced cardiac cytochrome c oxidase; this phenotype is consistent with the age-dependent decline observed in male ClockD19/D19 mice.

....................................................................................................................................................................................................Conclusions These data demonstrate that ovarian hormones protect female ClockD19/D19 mice from the development of age-

dependent cardiomyopathy even though Clock function is disturbed. Understanding the interaction of biological sexand the circadian mechanism in cardiac growth, renewal and remodelling opens new doors for understanding andtreating heart disease.

� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �

Keywords Cardiovascular • Circadian • Hypertrophy • Female • Aging • Lipidomics

1. Introduction

There is daily cyclic variation in our behaviour and physiology as humanshave adapted to be awake during the day and sleep at night. This is espe-cially relevant to our cardiovascular system, e.g. heart rate1 and bloodpressure2 which peak in the day when we are active and reach a nadir atnight when we are resting. Molecular circadian clocks in all cells, includingcardiomyocytes, allow us to entrain to the external 24-h day/night cycle,and prepare for the differing physiologic demands of daily events.3–7

The circadian clock mechanism at its most basic level consists of anauto-regulatory transcription and translation feedback loop which co-ordinates cellular function over the 24-h day/night cycle. Briefly, the posi-tive limb consists of two transcription factors termed CLOCK andBMAL1 which heterodimerize and bind to E-box enhancer elementswithin the promoter region of circadian output genes such as Period(Per) and Cryptochrome (Cry).8,9 This drives transcription of the negativelimb proteins PER and CRY. PER and CRY accumulate and in turn inhibitCLOCK: BMAL1 mediated transcription, thus repressing their own

* Corresponding author. Tel: þ519 824 4120 x54910, E-mail: tmartino@uoguelph.ca

Published on behalf of the European Society of Cardiology. All rights reserved. VC The Author 2017. For permissions, please email: journals.permissions@oup.com.

Cardiovascular Researchdoi:10.1093/cvr/cvx185

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.transcription.8–10 A number of excellent reviews on the circadian clockmechanism have been previously published in.11–13

The circadian mechanism is important for healthy cardiovascularphysiology as disruption is associated with adverse consequences for thecardiovascular system.14–16 Epidemiologic studies in humans suggest thatdisturbing rhythms is associated with increased risk of heart disease andworse outcome.3,4,7,17,18 Moreover, we and others previously showedexperimentally disturbing circadian rhythms in rodent models is an etio-logical cause of heart disease, and that rhythm disruption with pre-existing heart disease leads to worse outcomes.19–22 However, the ma-jority of these studies are done in males, and very little is known aboutthe effect of biological sex on circadian rhythms and heart disease withage. This is important as the prevalence of cardiovascular disease remainslower in females until �75 years of age at which point it is similar be-tween males and females.23

Here, we examined the role of CLOCK, a core component of the cir-cadian mechanism,24 and a key regulator of cardiac gene and proteinrhythms,25,26 in cardiac aging in females. We previously showed thatClockD19/D19 male mice develop profound cardiomyopathy with aging,showing that an intact circadian mechanism is crucial for cardiac aging.19

Remarkably we show here that female ClockD19/D19 mice are protectedfrom the development of age-dependent heart disease, despite disrup-tion of CLOCK function. We show that female biological sex and ovar-ian hormones play a role in mitigating the development of heart disease,even when there is a disturbed circadian mechanism. Conversely, loss ofovarian hormones in female ClockD19/D19 mice recapitulates the male car-diac phenotype of exacerbated cardiac aging. These findings are import-ant as they show, for the first time, how ovarian hormones and thecircadian mechanism interact to influence healthy cardiac aging.

2. Methods

2.1 AnimalsAll studies were approved by the University of Guelph InstitutionalAnimal Care and Use Committee and in accordance with the guidelinesof the Canadian Council on Animal Care. Female and male homozygoteClockD19/D19 heterozygote ClockD19/þ, and wild type (WT) littermates ona C57Bl/6 J background used for this study were obtained from ourbreeding colony at the University of Guelph, and genotyped as describedpreviously in.20,25 Mice were housed on a 12-h light: 12-h dark (12:12LD) cycle with food and water provided ad libitum throughout the study.All animals were sacrificed by isoflurane and cervical dislocation. Heartsfor histopathology were perfused with 1 M KCl and fixed in 10% neutralbuffered formalin. For all molecular experiments hearts were collected,snap frozen in liquid nitrogen and stored at -80 �C until use. For deter-mination of wheel running circadian period, individually housed micewere entrained to a diurnal 12:12 LD cycle for 2 weeks, followed bytransfer to constant darkness (DD) for 3 weeks. Data were analysedusing ClockLab (ActiMetrics).

2.2 Ovariectomy surgeryBilateral ovariectomy was performed using the method of Clipperton-Allen AE et al.27 Briefly, 6-week-old mice were administered carprofen(20 mg/kg; Rimadyl, Pfizer) 1 h prior to the procedure for analgesia. Micewere anesthetized at 4% isoflurane and maintained at 2.5% using a nose-cone. A small vertical midline dorsal incision of 2 cm was made in thelower back followed by two smaller bilateral lumbar incisions (<1 cm)through the muscles overlying the ovaries. The fallopian tubes and

ovarian arteries were clamped and the ovaries excised. The central inci-sion in the skin was closed with 1–2 MikRon Autoclip 9 mm wound clips(MikRon Precision Inc). Mice received a post-operative saline bolus(0.5 ml, subcutaneous) and recovered.

2.3 EchocardiographyEchocardiography was performed at 4, 8, 12, 18, and 21 months of ageusing a GE Vivid 7 Dimension ultrasound machine (GE Medical Systems)with the i13L 14 MHz linear-array transducer, as described in.19,20

Animals were maintained under light anesthesia (1.5% isoflurane) andbody temperature maintained at 37 �C during image acquisition. Dataanalyses were performed on an offline system using EchoPAC (GEMedical Systems).

2.4 In vivo haemodynamicsThe homozygote ClockD19/D19 heterozygote ClockD19/þ and WT mice at4 and 21 months of age were anesthetized using 4% isoflurane, intubated,and haemodynamic measurements were taken as previously described.19

LV contractile performance and blood pressure were measured using a1.4 Fr pressure catheter (Transonic) via the right carotid artery. Bodytemperature was continuously monitored and maintained at 37 �C. Datawere analyzed using Lab Chart 7 (Colorado Creeks, US). 30 mice wereused, n = 4–6 mice/group at 4 and 21 months of age.

2.5 HistologyFor histological analyses, formalin fixed hearts from 21-month-old micewere collected, paraffin embedded, and 5 mm sections were taken at themid papillary level. Sections were stained with Masson’s trichrome foranalysis of myocyte cross sectional area (MCSA), or picrosirius red forquantification of cardiac fibrosis. At least 100 myocytes were measuredper heart, over at least three sections, with n = 4 hearts/group, usingImage J v1.48 (NIH).

2.6 Comprehensive lab animal monitoringsystemThe comprehensive lab animal monitoring system (CLAMS) was used toexamine daily rhythms in metabolism and physical activity of 12-month-old female ClockD19/D19 and WT mice, using the methods described pre-viously in.20 Animals were acclimatized to the chambers for 48 h afterwhich metabolic parameters were measured for 24 h. Data wereanalysed using the Oxymax software (Columbus Instruments). A totalof 14 mice were used, n = 8 for female ClockD19/D19 mice, and n = 6 forfemale WT.

2.7 Glucose tolerance testAt 12 months of age glucose tolerance tests were performed in micefasted for 14 h. At either ZT02 or ZT14 glucose was injected at 2 g/kgbody weight (BW) (i.p.). Blood glucose concentrations following glucosechallenge were determined by tail vein sampling at baseline, 15, 30, 45,60, 90, and 120 min post-injection using a hand-held glucometer(Freestyle Lite, Abbot). A total of 24 mice were used, n = 6 for femaleClockD19/D19 and n¼ 6 for female WT, n = 6 for OVX female ClockD19/D19

and n = 6 OVX female WT. In addition, the day/night rhythm in ad libglucose levels was determined at ZT02 and ZT14 by drawing bloodfrom the saphenous vein and testing on the hand-held glucometer(Freestyle Lite, Abbot). n = 10 female ClockD19/D19 mice and n = 9 femaleWT mice were used.

2 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.2.8. Protein isolation and immunoblottingAnimals were sacrificed at ZT06 by isoflurane and cervical dislocation,and hearts were collected. Protein was isolated using the CelLytic MTCell Lysis reagent (Sigma) supplemented with cOmplete protease andPhosSTOP phosphatase inhibitor cocktails (Roche), using methods pre-viously described by our laboratory.25,26,28 Total protein concentrationwas determined using the Bradford assay (Bio-Rad). Lysates were sub-jected to SDS-PAGE and transferred to a PVDF membrane (Bio-Rad) at100 V for 2 h. Following transfer, membranes were blocked using 5%milk in tris-buffered saline with tween-20 (TBS-T) then incubated withthe primary antibodies overnight at 4 �C. Primary antibodies used werephospho-AKT (Ser473) (4060; 1:1000), or AKT (4691; 1:1000), or phos-pho-GSK-3b (Ser9) (9323; 1:1000), or GSK-3b (9315; 1:1000).Membranes were then washed in TBS-T and incubated with a goat anti-rabbit-HRP secondary antibody (1:5000) for 1 h at room temperature.All antibodies were purchased from Cell Signalling. Membranes werethen washed and proteins visualized by enhanced chemi-luminescenceon a ChemiDoc MP (BioRad) using the SignalFire Elite ECL reagent(12757, Cell Signalling). Loading was normalized to total protein usingthe Mem-code PVDF kit (Thermo Scientific). Images were analyzed usingImageLab (BioRad). A total of 14 mice were used, n = 7 female WT,n = 7 female ClockD19/D19.

2.9 2-Deoxyglucose and 2-bromopalmitateuptakeTo assess glucose and fatty acid uptake in tissues in vivo, 12-month oldmice were fasted for 14 h, then anesthetized at ZT06 with isoflurane,and injected retro-orbitally with 5 mCi 2-deoxyglucose in phosphate buf-fered saline (PBS) and 1.5 mCi 2-bromo[C14]palmitate complexed to bo-vine serum albumin in PBS. Hearts were collected 1 h after injection,homogenized in PBS, and radioactivity measured by liquid scintillationcounting (Tri-Carb 2900TR; Packard Instruments). Radioactivity in tissuehomogenates was normalized to the counts per minute (cpm) present in10 ml of serum collected 5 min following injection. A total of 19 micewere used, n = 5 male ClockD19/D19 n = 5 male WT, n = 4 femaleClockD19/D19 and n = 5 female WT.

2.10 LipidomicsCardiac lipids were extracted from hearts collected at ZT06 accordingthe Bligh and Dyer method,29 followed by shotgun lipidomics performedby the Analytical Facility for Bioactive Molecules, Hospital for SickChildren, Toronto, Canada. To do this, lipid extracts were reconstitutedin 900 ml of 1:1 Chloroform: Methanol containing 10 mM ammoniumacetate, followed by direct infusion-tandem mass spectrometry (DI-MS/MS) on a Sciex 6600 Triple time-of-flight (TOF) machine (ABSciex:Framingham, MA, USA). The MS/MSAll acquisition method was used inboth positive and negative electrospray ionization modes. Each samplewas individually infused for 7.5 min in each ionization mode at a flow rateof 15 ml/min. Quadrupole precursor ions were selected in the massrange of 200–2000 m/z and product ions at a TOF mass range of 100–2000 m/z. Qualitative data analyses were performed using LipidViewSoftware v1.3 beta (ABSciex: Framingham, MA, USA). MS/MS data werematched to the LipidView database and the corresponding TOF MSmass peak areas integrated in order to generate compositional lipid pro-files. Quantification is based on the peak area and expressed as a % ofeach lipid species count/total lipid class count. A total of 24 mice wereused, n = 6 hearts/group.

2.11 Cytochrome c activity assayAnimals were sacrificed at ZT06 by isoflurane and cervical dislocation,and hearts were collected. Mitochondria were isolated using theMitochondrial Isolation Kit, according to the manufacturer’s instructions(Sigma; MITOISO1). Total mitochondrial protein was determined usingthe Bradford assay (Bio-Rad). Cytochrome c Oxidase activity was deter-mined using the CYTOCOX1 Assay kit (Sigma) according the manufac-turer’s instructions. The change in absorbance at 550 nm was measuredin 1 ml cuvettes using a Helios Delta spectrophotometer (ThermoScientific). A total of 40 mice were used, n = 7 male ClockD19/D19 n = 7male WT, n = 7 female ClockD19/D19 n = 7 female WT, n = 6 OVX femaleClockD19/D19 and n = 6 OVX female WT.

2.12 Statistical analysisValues are expressed as mean ± SEM. All data were tested for normaldistribution and homogenous of variance. Statistical comparisons weredone using an unpaired Student’s t-test, or two-way analysis of variance(ANOVA) followed by Tukey post hoc for multiple comparisons as ap-plicable. Analyses were done using SigmaPlot 12 (Systat Software Inc.)and Prism 5 (Graph Pad). Values of *P < 0.05 are considered statisticallysignificant.

3. Results

3.1 Female ClockD19/D19 mice are protectedfrom cardiac agingThe female ClockD19/D19 mice have a disrupted circadian mechanism, yetremarkably, they are protected against heart disease with aging. In orderto first characterize the disrupted circadian mechanism, we used the clas-sical wheel running actigraphy methods. We show the day/night pheno-types of the homozygote ClockD19/D19 heterozygote ClockD19/þ and theWT mice. All animals entrain to the LD cycle under normal 12:12 LDconditions, as anticipated (Figure 1A). Moreover, under circadian condi-tions of constant darkness, where the animals receive their cues fromthe endogenous circadian mechanism instead of the light: dark cycle, theeffect of the Clock mutation is clearly evident in the female mice. TheClock mutation leads to a significant increase in wheel running period inhomozygote ClockD19/D19 mice (27.10 h ± 0.07) compared with hetero-zygote (24.27 h ± 0.05), and WT mice (23.83 h ± 0.03). This is very simi-lar to the phenotype that has been previously well established in themale Clock mice, by our group and others.19,24 Moreover, we found thatthe onset of activity in homozygous ClockD19/D19 mice was more variablecompared with the other genotypes, but there were no difference in thetotal amount of running wheel activity (see Supplementary material online, Figure S1A and B). Collectively these data show that the Clock muta-tion is functional in the female mice.

To carefully evaluate cardiac structure and function in the aging femaleClockD19/D19 ClockD19/þ, and WT mice, we used serial echocardiographyat 4, 8, 12, 18, and 21 months of age. Figure 1 and Table 1 show that fe-male ClockD19/D19 hearts are healthy to 18 months of age, as LV internaldimensions at diastole (LVIDd) (Figure 1B) and systole (LVIDs) (Figure1C), % ejection fraction (EF) (Figure 1D), and % fractional shortening (FS)(Figure 1E) are similar to heterozygotes and WTs. This is in contrast tothe male ClockD19/D19 mice which exhibit increased cardiac remodellingstarting at 12 months of age.19 Comparison of female cardiac morphom-etry at 12 months of age confirms that female mice show protectionfrom the adverse cardiovascular effects of CLOCK disruption, as female

Biological sex influences heart disease in Clock mice 3

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.ClockD19/D19 mice exhibit no difference in HW or HW/ tibia length (TL)compared with female WT littermates (see Supplementary material online, Figures S1C and D). In contrast, at 12 months the male ClockD19/D19

mice show a significant increase in heart weight (HW) and HW/TL vs.male WT littermates (see Supplementary material online, Figures S1Cand D). Thus biological sex appears to protect against heart disease inaging female ClockD19/D19 mice.

3.2 Cardiac aging in female ClockD19/D19

mice, by in vivo haemodynamicsThe female ClockD19/D19 are protected from heart disease for almost theirentire normal 2 year lifespan, and only at 21 months of age do they exhibitany changes in cardiac function by echocardiography (Figure 1B–E). Onlywith extreme age, at 21 months of age, do the female ClockD19/D19 micebegin to develop impaired heart function. These aged female ClockD19/D19

mice have decreased LV end systolic pressure (LVESP), decreased meanarterial pressure (MAP), and reduced dp/dtmax and dp/dtmin values byin vivo haemodynamics (Table 2). That is, the aged female ClockD19/D19

mice exhibit hypotension (reduced afterload) and a decrease in myocar-dial contractility indices. Thus these data show that the female ClockD19/D19

mice are protected from adverse changes in cardiac structure and func-tion for most of their lifespan, in contrast to the male ClockD19/D19

mice,19 and only begin to decompensate with old age.

3.3 Cardiac aging in female ClockD19/D19

mice, by histopathologyWe also examined additional cardiovascular parameters in the femaleClockD19/D19 mice at 4 and 21 months of age. The aging female mice ex-hibited no difference in HW, as compared with littermates (Figure 1F)suggesting that the hearts do not have increased hypertrophy with age.The HW:BW ratio was increased, but only because of the lower BW inthese aged animals (Figure 1G and Table 1). There was also no differencein MSCA, consistent with the lack of cardiac hypertrophy with age(Figure 1H). Histologically, however we did find greater cardiac fibrosis inthe female ClockD19/D19 mice by 21 months, consistent with the onset ofcontractile dysfunction at 21 months (Figure 1I). Thus the femaleClockD19/D19 mice do not develop the same classic cardiomyopathy asobserved previously with the male ClockD19/D19 mice,19 but they doshow signs of cardiac aging towards the end of their lifespan as comparedwith their littermates.

3.4 Diurnal metabolism in femaleClockD19/D19 miceSince female ClockD19/D19 mice show protection from the adverse effectsof CLOCK disruption on the heart by 12 months we further examineddifferences between male and female ClockD19/D19 and WT mice at thisage. We investigated diurnal metabolic parameters in the femaleClockD19/D19 vs. WT mice. Overall, the animals are nocturnal withincreased activity in the dark phase, as anticipated (Figure 2A). All animalsconsume the majority of their food in the dark (active) phase; however,female ClockD19/D19 mice also consume significantly more food during thelight (rest) phase than the female WTs (Figure 2B). Examination of wholebody substrate utilization in female ClockD19/D19 mice reveals significantlygreater respiratory exchange ratio (RER) during the light phase as com-pared with WTs (Figure 2C). There is no difference in oxygen consumption(Figure 2D), however energy expenditure is also significantly increased inClockD19/D19 mice during the light phase as compared with WTs (Figure2E). Collectively these data suggest that the female ClockD19/D19 mice are

......................................................................................................

Table 1 Cardiac aging in female ClockD19/D19 mice

ClockD19/D19 ClockD19/1 WT

4 month

BW (g) 25.16 ± 0.74* 23.13 ± 0.86 21.75 ± 0.89

HW (mg) 116.00 ± 2.58 105.83 ± 2.46 109.5 ± 3.86

HW/BW (mg/g) 4.64 ± 0.2 4.59 ± 0.09 5.06 ± 0.17

HW/TL (mm/g) 6.03 ± 0.13 5.47 ± 0.11 5.75 ± 0.19

LVIDd (mm) 3.88 ± 0.04 3.87 ± 0.03 3.83 ± 0.03

LVIDs (mm) 2.31 ± 0.02 2.35 ± 0.05 2.28 ± 0.03

EF (%) 77.30 ± 1.00 76.12 ± 1.20 77.74 ± 0.67

FS (%) 40.34 ± 0.98 39.27 ± 1.08 40.66 ± 0.60

HR (bpm) 429 ± 4 439 ± 15* 492 ± 7***

IVSd (mm) 0.69 ± 0.01 0.69 ± 0.01 0.68 ± 0.01

PWTd (mm) 0.68 ± 0.01 0.67 ± 0.01 0.66 ± 0.01

8 month

LVIDd (mm) 4.00 ± 0.07 3.92 ± 0.04 3.91 ± 0.02

LVIDs (mm) 2.49 ± 0.05 2.39 ± 0.04 2.36 ± 0.02

EF (%) 75.41 ± 0.81 75.73 ± 0.50 76.65 ± 0.76

FS (%) 38.76 ± 0.64 38.86 ± 0.42 39.65 ± 0.68

HR (bpm) 463 ± 15 490 ± 10 473 ± 12

IVSd (mm) 0.68 ± 0.01 0.69 ± 0.005 0.68 ± 0.01

PWTd (mm) 0.67 ± 001 0.67 ± 0.003 0.68 ± 0.01

12 month

LVIDd (mm) 4.12 ± 0.04 3.99 ± 0.05 4.05 ± 0.02

LVIDs (mm) 2.63 ± 0.07 2.45 ± 0.07 2.47 ± 0.01

EF (%) 72.52 ± 1.43 75.49 ± 1.53 75.84 ± 0.54

FS (%) 36.28 ± 1.13 38.70 ± 1.34 39.02 ± 0.49

HR (bpm) 429 ± 6 441 ± 11* 484 ± 14***

IVSd (mm) 0.73 ± 0.01 0.71 ± 0.01 0.71 ± 0.02

PWTd (mm) 0.72 ± 0.01 0.68 ± 0.01 0.68 ± 0.02

18 month

LVIDd (mm) 4.21 ± 0.05 4.18 ± 0.07 4.15 ± 0.04

LVIDs (mm) 2.78 ± 0.08 2.61 ± 0.06 2.56 ± 0.05

EF (%) 69.35 ± 1.89 74.07 ± 0.83 75.03 ± 1.03

FS (%) 34.03 ± 1.35 37.59 ± 0.70 38.40 ± 0.88

HR (bpm) 440 ± 12 434 ± 6 451 ± 8

IVSd (mm) 0.76 ± 0.01 0.75 ± 0.02 0.75 ± 0.03

PWTd (mm) 0.75 ± 0.02 0.72 ± 0.02 0.70 ± 0.02

21 month

BW (g) 26.60 ± 1.44** 32.62 ± 1.01 32.00 ± 1.64

HW (mg) 151.67 ± 8.56 141.50 ± 3.14 137.17 ± 2.90

HW/BW (mg/g) 5.73 ± 0.28** 4.36 ± 0.18 4.34 ± 0.22

HW/TL (mm/g) 7.88 ± 0.44 7.26 ± 0.17 7.13 ± 0.11

LVIDd (mm) 4.34 ± 0.05 4.18 ± 0.04 4.16 ± 0.10

LVIDs (mm) 2.98 ± 0.04** 2.64 ± 0.04 2.57 ± 0.07

EF (%) 65.77 ± 0.89** 73.32 ± 0.97 74.82 ± 0.59

FS (%) 31.24 ± 0.62** 36.98 ± 0.78 38.27 ± 0.46

HR (bpm) 431 ± 7 460 ± 9 500 ± 14***

IVSd (mm) 0.76 ± 0.01 0.77 ± 0.02 0.77 ± 0.02

PWTd (mm) 0.76 ± 0.01 0.72 ± 0.01 0.74 ± 0.02

BW, body weight; HW, heart weight; HW/BW, heart weight/body weight; TL,tibia length; LVIDd, left ventricle internal dimensions at diastole; LVIDs, LV in-ternal dimensions at systole; EF, ejection fraction; FS, fractional shortening; HR,heart rate; IVSd, septal wall thickness at diastole; PWTd, posterior wall thick-ness at diastole; n = 6 mice/group, *P < 0.05 vs. WT of same age, **P < 0.05ClockD19/D19 vs. all other groups of same age, ***P < 0.05 vs. ClockD19/D19 as calcu-lated by two-way ANOVA followed by Tukey post hoc. Values are mean ± SEM.

4 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.taking in more food, using more carbohydrates, but also expending moreenergy, as compared with their WT littermates. This is in contrast to maleClockD19/D19 mice which have been previously shown to exhibit increasedfood intake and reduced energy expenditure.30

3.5 Diurnal glucose metabolism in femaleClockD19/D19 miceDespite the altered metabolic profiles in the female ClockD19/D19 mice,glucose tolerance remains the same as their female WT littermates.Specifically, glucose tolerance testing at either ZT02 (rest phase) (Figure2F) or ZT14 (wake phase) (Figure 2G) revealed peak glucose levels at15 min post-glucose injection, followed by similar rates of glucose clear-ance to 2 h after injection in female ClockD19/D19 and WT mice.Consistent with normal glucose clearance, the area under the curve(AUC) is similar in female ClockD19/D19 and WT mice at ZT02 or ZT14(Figure 2H). Moreover, examination of blood glucose levels under ad lib-itum conditions demonstrated that both groups have normal diurnalblood glucose levels, as measured at ZT02 and ZT14 (Figure 2I). Thesefindings are significant because the female ClockD19/D19 mice maintainnormal blood glucose responses and are protected from heart dis-ease, which is in contrast, to previous work which showed that maleClockD19/D19 mice have abnormal glucose handling with aging.31

3.6 Substrate uptake in female ClockD19/D19

vs. male ClockD19/D19 miceSince the studies above indicated that the female ClockD19/D19 mice havedifferences in energy and glucose profiles as compared with the maleClockD19/D19 mice, and since this might help to explain why the femalesare protected from heart disease, we next investigated tissue substrateuptake by radiolabelled glucose and palmitate assays. In the heart,we found that female ClockD19/D19 mice maintain normal cardiac

3 H-deoxyglucose and 14C-palmitate uptake with aging, similar to femaleWT mice (Figure 3A). In contrast, male ClockD19/D19 mice have signifi-cantly increased cardiac glucose uptake compared with male WT mice(Figure 3A). In the liver, we found that female ClockD19/D19 mice also main-tain normal cardiac 3 H-deoxyglucose and 14C-palmitate uptake withaging, similar to female WT mice (Figure 3B). However, the maleClockD19/D19 mice have significantly increased liver substrate uptake com-pared with male WT mice (Figure 3B). Last, in skeletal muscle, we foundthat female ClockD19/D19 mice handle glucose the same, but have signifi-cantly reduced skeletal muscle fatty acid uptake, as compared with fe-male WT mice (Figure 3C). The males again are different; male ClockD19/D19

mice had no change in fatty acid skeletal muscle uptake comparedwith male WT mice (Figure 3C). Collectively, these data demonstratethat female ClockD19/D19 mice maintain normal substrate uptake withaging, which may help to protect them from heart disease, whereasthe male ClockD19/D19 mice have altered glucose and fatty acid uptakecompared with male WTs that could play a role in the pathogenesis ofage-dependent heart disease.

3.7 Cardiac lipidomicsPreviously we found a role for cardiac lipids in mediating sex-differencesin cardiac remodelling with aging in a different study which focused onlipid metabolism; however, we did not investigate the circadian system atthat time.32 The lipid composition of cells is influenced by ovarian hor-mones33,34 and ovarian hormones can protect against heart disease.35–37

In light of those observations, we next investigated the lipid compositionof the hearts of the female and male ClockD19/D19 mice and controls. Weperformed shotgun lipidomics (DI-MS/MS) to investigate the cardiac lipi-dome in aged mice. We were specifically interested in lipid profiles thatwere common to the female WTs, female ClockD19/D19 and male WTs,but altered in the male ClockD19/D19 animals with age-dependent cardio-myopathy. Total lipid content was extracted from the hearts, followed by

..............................................................................................................................................................................................................................

Table 2 Female ClockD19/D19 mice exhibit reduced LV contractile function with age

ClockD19/D19 ClockD19/1 WT

4 Month

LVESP (mmHg) 98.98 ± 1.87 100.98 ± 2.21 102.43 ± 0.89

LVEDP (mmHg) 1.29 ± 1.36 1.92 ± 1.25 1.31 ± 0.41

dp/dt max (mmHg/s) 9300.02 ± 643.12 8301.21 ± 555.65 9296.42 ± 863.03

dp/dt min (mmHg/s) –8569.22 ± 673.02 –8765.68 ± 562.74 –8693 ± 495.13

SBP (mmHg) 96.66 ± 1.76 97.31 ± 1.23 98.56 ± 1.47

DBP (mmHg) 62.45 ± 1.10 64.30 ± 1.26 65.36 ± 1.26

MAP (mmHg) 73.85 ± 1.26 75.30 ± 0.95 75.78 ± 0.55

HR (bpm) 532 ± 17 483 ± 20 541 ± 30

21 Month

LVESP (mmHg) 92.67 ± 1.34* 99.52 ± 1.02 100.29 ± 1.94

LVEDP (mmHg) 2.56 ± 0.83 2.89 ± 1.12 1.90 ± 0.77

dp/dt max (mmHg/s) 6374.51 ± 426.82** 8088.40 ± 437.04 8725.45 ± 596.38

dp/dt min (mmHg/s) –6662.27 ± 243.77** –7514.30 ± 693.18 –9521.94 ± 361.80

SBP (mmHg) 93.10 ± 2.34* 101.58 ± 1.28 101.36 ± 2.24

DBP (mmHg) 60.49 ± 1.94* 67.32 ± 2.34 66.50 ± 0.69

MAP (mmHg) 71.36 ± 1.95* 78.74 ± 1.76 78.12 ± 0.55

HR (bpm) 513 ± 19 548 ± 28 508 ± 31

LVESP, left ventricle end systolic pressure; LVEDP, LV end diastolic pressure; dP/dt max and min, maximum and minimum first derivative of left ventricular pressure; SBP, systolicblood pressure; DBP, diastolic BP; MAP, mean arterial pressure; HR, heart rate; n = 4–6/group for each age, *P < 0.05 ClockD19/D19 vs. all other groups of same age, and **P < 0.05ClockD19/D19 vs. WT by two way ANOVA followed by Tukey post hoc. All values are mean ± SEM.

Biological sex influences heart disease in Clock mice 5

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

Figure 1 Female ClockD19/D19 mice are protected from the development of age-dependent heart disease. (A) Wheel running actigraphy showing an increasein circadian period in homozygote ClockD19/D19 mice compared with all other genotypes under constant darkness, n = 3/group. White = Lights ON, Grey =Lights OFF. Serial echocardiography from 4 to 21 months; (B) LVIDd, (C) LVIDs, (D) EF, and (E) FS, n = 6 mice/group by two way ANOVA followed by Tukeypost hoc, *P < 0.05 ClockD19/D19 vs. all other groups. (F) HW, and (G) HW/BW at 4 and 21 months (n = 6 hearts/group) *P < 0.05 ClockD19/D19 vs. all other groupsby two way ANOVA. (H) Representative sections stained with Masson’s trichrome (left) and quantification (right) of MCSA. (I) Representative images stainedwith picrosirius red (left) and quantification (right) at 21 months of age (n = 4 hearts/group), *p < 0.05 by unpaired Student’s t-test. Values are mean ± SEM.

6 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

Figure 2 Behavioural and metabolic rhythms in female ClockD19/D19 and WT mice. Examination of whole body metabolism in 12 month old mice using theCLAMS: (A) % activity (left) and total activity (right), (B) % food intake, (C) RER, (D) oxygen consumption (VO2), and (E) energy expenditure (n = 6–8 mice/group), *P < 0.05 as indicated by two-way ANOVA followed by Tukey post hoc. Glucose tolerance test at 12 months of age measured at ZT 02 (F) and ZT14 (G). (H) AUC analysis of glucose tolerance tests at ZT 02 (left) and ZT 14 (right) (n = 6 mice/group). (I) Blood glucose levels at ZT 02 and ZT 14 underad libitum conditions (n = 9–10 mice/group). Values are mean ± SEM.

Biological sex influences heart disease in Clock mice 7

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.

mass spectrometry. Lipid View bioinformatics was used to compile theresults into the following groupings: phospholipids, triacylgylceride fattyacids, phospholipid fatty acids, and cardiolipins. We found general differ-ences in lipid content between male and female mice, as anticipated32

(see Supplementary material online, Tables S1 and S2). We also found dif-ferences in cardiolipin composition consistent with our hypothesis thatlipid content varies with cardiovascular remodelling in heart disease.Specifically, we found that the primary cardiolipin CL72:8 was lowest inmale ClockD19/D19 hearts (Figure 4A) compared with all other groups.Moreover, we found a shift in composition of the less abundant cardioli-pins. These changes include cardiolipin CL76:11 which were increased inmale ClockD19/D19 mice vs. all other groups (Figure 4B). In addition, cardioli-pin CL82:9 was increased and cardiolipin CL74:9 was decreased in maleClockD19/D19 mice with heart disease as compared with healthy male WTmice (Figures 4C and D). Collectively, these data show remodelling of lipidprofiles which correlate with increased cardiac remodelling in maleClockD19/D19 mice.

3.8 Activation of cardiac hypertrophypathwaysA mechanism by which disrupting CLOCK can promote cardiac aging isthrough the AKT and GSK-3 b signalling pathway; indeed we previously

reported this in the male ClockD19/D19 mice.19 Thus we next investigatedthese pathways in the female ClockD19/D19 mice as differential activationmight help to explain why the females are protected from age-dependent cardiac remodelling. Interestingly, we found that femaleClockD19/D19 mice exhibit a significant increase in AKT activation com-pared with female WT mice (Figure 4E), suggesting that CLOCK disrup-tion affects AKT activation regardless of sex. Moreover, GSK-3bphosphorylation was also significantly increased in female ClockD19/D19

hearts, consistent with increased AKT activation in these mice (Figure4E). Interestingly, despite differences in the AKT/GSK phosphorylationpathways, the female ClockD19/D19 mice maintained cytochrome c oxi-dase activity similar to female WT littermates (Figure 4F). This is in con-trast male ClockD19/D19 hearts, which have reduced cytochrome coxidase activity compared with male WT littermates (Figure 4F). Takentogether, these results show that female ClockD19/D19 mice have activa-tion of AKT/GSK pathways consistent with the presence of the Clockmutation, however this does not lead to changes in cytochrome c oxi-dase activity as was found in the development of age-dependent cardio-myopathy in male ClockD19/D19 mice (Figure 4G). Consistent with thisfinding, they also show no changes in cardiac glucose uptake and mito-chondrial function (Figure 4G), which may help to explain the cardio-protection despite the Clock mutation.

3.9 OVX female ClockD19/D19 mice exhibitage-dependent cardiac remodellingWe next hypothesized that ovarian hormones play a protective role andthat ovariectomy of female ClockD19/D19 mice should remove the pro-tective effect and lead to the development age-dependent cardiac re-modelling. To examine this, female ClockD19/D19 mice wereovariectomized (OVX) at 6 weeks of age, and their cardiac structure andfunction were evaluated as these animals aged and were compared withWT OVX mice. We found that OVX female ClockD19/D19 mice by8 months of age develop significantly increased left ventricular dilationevidenced by increased LVIDd (Figure 5A) and LVIDs (Figure 5B), as com-pared with OVX WT littermates. The hearts continued to dilate by12 months of age (Figures 5A and B), and HW is significantly increased(Figure 5C). In addition, the female OVX ClockD19/D19 mice no longermaintain normal blood glucose responses as intact females had, but in-stead show abnormal glucose handling. That is, at ZT02 (lights on, animalrest time), OVX ClockD19/D19 mice had increased blood glucose levelspost-injection, as compared with OVX WT mice (Figure 5D). Moreover,at ZT14 (lights off, animal wake time), OVX ClockD19/D19 mice also hadincreased blood glucose levels post-injection as compared with OVXWT mice (Figure 5E). Consistent with glucose intolerance in the OVXClockD19/D19 mice, AUC was significantly increased at ZT02 and ZT14,as compared with OVX WT mice (Figure 5F). Last, we examined cyto-chrome c oxidase activity and found that the OVX ClockD19/D19 micehave reduced cytochrome c oxidase activity compared with OVXWT mice (Figure 5G). Thus the loss of ovarian hormones in femaleClockD19/D19 mice recapitulates the heart disease phenotype, and meta-bolic dysregulation observed in male ClockD19/D19 mice, and is consistentwith a role of Clock in cardiac remodelling with age.

4. Discussion

In this study, we demonstrate that female ClockD19/D19 mice are pro-tected from the development of age-dependent heart disease, despitethe presence of the Clock mutation. Female ClockD19/D19 hearts appear

Figure 3 Glucose and fatty acid uptake in 12 month old femaleand male ClockD19/D19 and WT mice. Deoxyglucose and2-bromo[C14]palmitate uptake in the (A) heart (B) liver, and (C) skeletalmuscle (gastrocnemius) (n = 4–5/group), *P < 0.05 ClockD19/D19 vs. WTof same sex by two-way ANOVA followed by Tukey post hoc. All meas-urements were performed at ZT 06. Values are mean ± SEM.

8 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

Figure 4 Cardiolipin content and cardiac hypertrophy pathways. Cardiolipin (CL) composition of (A) CL72:8, (B) CL76:11, (C) CL82:9 and, (D) CL74:9(n = 6 hearts/group), *P < 0.05 as indicated and †P < 0.05 vs. all other groups by two way ANOVA followed by Tukey post hoc. (E) Western blots of phos-pho-AKT (S473), total AKT, phospho-GSK-3b (S9), total GSK-3b and total protein in female ClockD19/D19 and WT mice at 12 months of age (n = 7 hearts/group), *P < 0.05 by unpaired Student’s t-test. (F) Cytochrome c oxidase activity in mitochondria isolated from female and male ClockD19/D19 and WT hearts(n = 7/group), *P < 0.05 by two-way ANOVA followed by Tukey post hoc. (G) Schematic diagram of the physiological changes associated with cardiomyop-athy in male ClockD19/D19 and protection in female ClockD19/D19 mice. Values are mean ± SEM.

Biological sex influences heart disease in Clock mice 9

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

.healthy to 18 months of age, as shown by serial echocardiography andin vivo haemodynamics analysis. We also found no difference in HW, andmyocyte cross-sectional area as compared with female WT littermates.Investigation of diurnal metabolic parameters revealed that femaleClockD19/D19 mice have increased activity, food intake, carbohydrate

oxidation, and energy expenditure during the rest period. However, des-pite altered metabolic profiles the female ClockD19/D19 mice maintain nor-mal glucose tolerance and cardiac glucose uptake, similar to the femaleWT mice. Thus the female ClockD19/D19 mice maintain normal substrateprofiles, which may help protect against underlying genetic dysfunction.

Figure 5 OVX ClockD19/D19 mice develop LV dilation and metabolic dysfunction. (A) LVIDd, (B) LVIDs. (C) HW at 12 months of age (n = 7–9 mice/group),*P < 0.05 ClockD19/D19 vs. WT mice of same age by two way ANOVA followed by Tukey post hoc. Echocardiography was performed at 4, 8, and 12 monthsof age. (D) Glucose tolerance test at ZT 02 and (E) ZT 14. (F) AUC analysis for ZT 02 (left) and ZT 14 (right) (n = 6 mice/group), *P < 0.05 by two-wayANOVA followed by Tukey post hoc for GTT curves and *P < 0.05 by unpaired Student’s t-test for A.U.C analysis. (G) Cytochrome c oxidase activity in iso-lated mitochondria from OVX ClockD19/D19 and WT hearts (n = 6 mice/group), *P < 0.05 by unpaired Student’s t-test. Values are mean ± SEM.

10 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.In contrast to the male ClockD19/D19 mice which have altered metabolicprofiles and develop heart disease with age. AKT and GSK signallingwere increased in female ClockD19/D19 hearts, similar to the maleClockD19/D19 hearts. However, the female ClockD19/D19 hearts also hadpreserved cytochrome c oxidase activity, and differences in cardiolipincomposition, concurrent with the healthier cardiovascular profiles.These benefits were due in part to ovarian hormones, as ovariectomy ofthe female ClockD19/D19 mice recapitulated the heart disease phenotypein male mice. OVX female ClockD19/D19 mice developed increased LVdilation, increased HW, glucose intolerance and reduced cytochrome coxidase activity. Thus removal of ovarian hormones leads to cardiacremodelling in female ClockD19/D19 mice that was consistent with themale ClockD19/D19 mice, and supports a role of Clock in age-dependentcardiomyopathy.

One of the interesting findings of this study is that the adverse effectsof the circadian Clock mutation on the heart can be mitigated by femalebiological sex and ovarian hormones. Several recent circadian studiesusing genetic or environmental models provide further support for arole of biological sex in mediating the effects of circadian rhythm disrup-tion, consistent with our findings. For example, human females exhibitgreater impairment in cognitive performance following circadian dysyn-chrony, as compared with males in similar environments.38 In experi-mental animal models, survivorship after myocardial infarction is greaterin female mice as compared with male mice,39 and more specifically fe-males infarcted at sleep time have the greatest survivorship.40 It has alsobeen shown that female ClockD19/D19 mice exhibit lower survival follow-ing whole body irradiation compared with female WT mice while maleClockD19/D19 mice do not show changes in survival as compared withmale WT mice.41 Last, circadian disorganization caused by shifted lightdark cycles leads to reduced survival of male rats but not female rats fol-lowing middle cerebral artery occlusion.42 Thus Clock disruption mayinteract with factors associated with biological sex to modulate thepathologic effects of heart disease.

This is the first study to show a role for biological sex in mitigating theeffects of the Clock mutation on cardiac remodelling. However, the no-tion that females are protected from cardiovascular disease in general iswell established.23,43,44 For example females are protected from heartdisease as compared with males, with reduced cardiac remodelling inaging female rats45 and also less hypertrophy in pressure overload-induced cardiac hypertrophy in female mice.46 Metabolism also plays arole in male-female differences in heart disease.47 These can be driven inpart by differences in glucose handling,48,49 cardiac glucose uptake,50 andfatty acid oxidation profiles.51 In terms of our study, altered substrateutilization/cardiac lipid profiles and reduced cytochrome c oxidase activ-ity were associated with the development of age-dependent cardiomy-opathy in male ClockD19/D19 mice, whereas female ClockD19/D19 mice donot exhibit these metabolic changes and are protected from the devel-opment of heart disease with age. One key aspect we found is that therewere differences in the remodelling of cardiolipin profiles in a sex andgenotype manner.

Cardiolipins are a key component of the inner mitochondrial mem-brane important for mitochondrial bioenergetics,52 and composition isaltered in heart disease.53 Our female ClockD19/D19 mice maintain normalcardiolipin profiles and lack heart disease. In contrast, we observedchanges in cardiolipin profiles in the male ClockD19/D19 mice. Cardiolipinprofiles in the male ClockD19/D19 mice are consistent with those reportedin heart disease, as reduced 72:8 and increased 76:11 cardiolipin areassociated with heart failure in humans, and experimental heart failure inrats.53 Moreover, reduced 72:8 content is associated with reduced

cytochrome c oxidase activity.53 Collectively these studies suggest thatremodelling of cardiolipins leads to impaired mitochondrial function inmale ClockD19/D19 mice. Conversely, female ClockD19/D19 mice are pro-tected from age-dependent heart disease, mediated in part by preservedcardiolipin content and cytochrome c oxidase activity. We postulatethat the ovarian hormones protect against the decline in mitochondrialfunction, reducing age-dependent cardiac remodelling. In terms of futuredirections, it will be interesting to investigate serum lipid profiles as wellas the cardiac metabolome in female verses male ClockD19/D19 mice tofurther understand how female mice are metabolically protected fromthe Clock mutation.

It is interesting to note that there are differences in the ability of theskeletal muscle to utilize fatty acids compared with the heart in ClockD19/

D19 mice. Assuming that increase in whole body RER is reflecting a reduc-tion in skeletal muscle metabolism of fatty acids, there is no compensa-tory increase in glucose metabolism. One possible explanation for thisdifferential regulation is that these changes in substrate profiles are theresult of an interaction between tissue specific circadian regulation andcardiac specific changes associated with disease progression. In supportof this Zhang et al. have shown that rhythmic gene expression is regu-lated in a tissue specific manner, including genes critical for glucose andfatty acid metabolism.54 Moreover, the circadian clock has been shownto regulate genes crucial for substrate utilization in the heart55 and skel-etal muscle56 which likely contributes to tissue specific regulation. Last,there are changes in cardiac metabolism associated with heart diseaseincluding increased glucose utilization, as observed in male ClockD19/D19

mice.57,58 Collectively, these factors can help to explain why there is dif-ferential regulation of glucose and fatty acids between the heart and skel-etal muscle. Moreover, ovarian hormone regulation of circadian geneexpression and cardiac remodelling may explain why females are pro-tected against the metabolic derangement and cardiomyopathy withaging.

Although the female ClockD19/D19 mice are protected from heart dis-ease, OVX ClockD19/D19 mice develop age-dependent cardiac re-modelling, consistent with a role for Clock in this disease process. This isconsistent with the notion that ovarian hormones protect against heartdisease. For example, estrogen mitigates mechanisms of cardiomyocytehypertrophy, as demonstrated in rat cardiomyocytes in vitro.36

Moreover, estrogen replacement limits cardiomyocyte hypertrophy invitro, and preserves LV function and contractility in mice with pressureoverload-induced cardiac hypertrophy in vivo.37 Estrogens can interactwith the intrinsic circadian clock, and prevent abnormal metabolic pro-files caused by circadian disruption59 which helps to explain why our fe-male mice with a Clock mutation were protected. However, it’simportant to note that OVX leads to a loss of a variety of hormones.Although beyond the scope of this study, it would be interesting in fu-ture studies to examine how the different hormones interact withClock, and other components of the circadian mechanism to providecardio-protection with aging. For example, future studies using estro-gen supplementation as well as estrogen receptor specific agonists inOVX ClockD19/D19 mice will help delineate the pathways responsiblefor cardio-protection. Interestingly, we have recently shown that fe-male mice are less susceptible to adverse remodelling after myocardialinfarction (LAD ligation model) as compared with male mice.40 Giventhat increased AKT activation in the heart is associated with circadianclock disruption in male19,60 and female mice, it could be interesting todetermine sex-dependent differences in disease pathophysiology in thismodel as well in trans-aortic constriction, where AKT signalling is alsocrucial.

Biological sex influences heart disease in Clock mice 11

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.Although female biological sex confers some protection against the

circadian Clock mutation and heart disease in female mice, the males arenot protected. This has important clinical implications, particularly as cir-cadian rhythm disruption (e.g. in shift work, sleep disorders) is associatedwith increased risk of cardiovascular disease and worse outcomes, espe-cially in males.3,4,7,15,16 In addition, circadian timing of drug treatment(chronotherapy) benefits the efficacy of some cardiovascular therapiessuch as angiotensin converting enzyme inhibitors, and Aspirin.54,61–64

However, there are important sex differences that should be con-sidered, as the benefits of chronotherapy differ between male and femalepatients.65,66 Moreover, circadian mechanism modulators are currentlyunder development67,68 and hold therapeutic promise for reducing theprogression of heart disease; therapeutic benefits and potential side ef-fects should be investigated in both sexes. Therefore future investigationof how biological sex and the circadian mechanism interact to influencecardiac remodelling is critical for understanding the pathophysiology ofheart disease and for clinical benefit in males and females.

5. Conclusion

In summary, we show the first time that female ClockD19/D19 mice areprotected from the adverse effects of the Clock mutation and the devel-opment of age-dependent heart disease. Furthermore, we show thatovarian hormones play a key role in mediating the protective effects infemale ClockD19/D19 mice, as ovariectomy of ClockD19/D19 mice recapitu-lates the phenotype of metabolic dysfunction and adverse cardiac re-modelling with age. These data demonstrate a previously unrecognizedlink between biological sex and Clock in the development and protectionagainst heart disease with aging.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

FundingThis work is supported by grants from the Canadian Institute for HealthResearch (CIHR) and Heart and Stroke Foundation of Canada (HSFC) toT.A.M.

References1. Clarke JM, Hamer J, Shelton JR, Taylor S, Venning GR. The rhythm of the normal

human heart. Lancet 1976;308:508–512.2. Millar-Craig MW, Bishop CN, Raftery EB. Circadian variation of blood-pressure.

Lancet 1978;1:795–797.3. Alibhai FJ, Tsimakouridze EV, Reitz CJ, Pyle WG, Martino TA. Consequences of cir-

cadian and sleep disturbances for the cardiovascular system. Can J Cardiol 2015;31:860–872.

4. Martino TA, Young ME. Influence of the cardiomyocyte circadian clock on cardiacphysiology and pathophysiology. J Biol Rhythms 2015;30:183–205.

5. Paschos GK, FitzGerald GA. Circadian clocks and vascular function. Circ Res 2010;106:833–841.

6. Roenneberg T, Merrow M. Circadian clocks - the fall and rise of physiology. Nat RevMol Cell Biol 2005;6:965–971.

7. Reitz CJ, Martino TA. Disruption of circadian rhythms and sleep on critical illnessand the impact on cardiovascular events. Curr Pharm Des 2015;21:3505–3511.

8. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS,Weitz CJ. Role of the CLOCK protein in the mammalian circadian mechanism.Science 1998;280:1564–1569.

9. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, HastingsMH, Reppert SM. mCRY1 and mCRY2 are essential components of the negative limbof the circadian clock feedback loop. Cell 1999;98:193–205.

10. Griffin EA, Jr, Staknis D, Weitz CJ. Light-independent role of CRY1 and CRY2 in themammalian circadian clock. Science 1999;286:768–771.

11. Ko CH, Takahashi JS. Molecular components of the mammalian circadian clock. HumMol Genet 2006;15(Spec No 2):R271–R277.

12. Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature 2002;418:935–941.

13. Takahashi JS. Molecular components of the circadian clock in mammals. DiabetesObes Metab 2015;17(Suppl 1):6–11.

14. Morris CJ, Purvis TE, Hu K, Scheer FA. Circadian misalignment increases cardio-vascular disease risk factors in humans. Proc Natl Acad Sci U S A 2016;113:E1402–E1411.

15. Vyas MV, Garg AX, Iansavichus AV, Costella J, Donner A, Laugsand LE, Janszky I,Mrkobrada M, Parraga G, Hackam DG. Shift work and vascular events: systematic re-view and meta-analysis. Bmj 2012;345:e4800.

16. Knutsson A, Akerstedt T, Jonsson BG, Orth-Gomer K. Increased risk of ischaemicheart disease in shift workers. Lancet 1986;2:89–92.

17. Mistry P, Duong A, Kirshenbaum LA, Martino T. Cardiac clocks and preclinical trans-lation. Heart Fail Clin 2017;13:657–672.

18. Sole MJ, Martino TA. Diurnal physiology: core principles with application to thepathogenesis, diagnosis, prevention, and treatment of myocardial hypertrophy andfailure. J Appl Physiol (1985) 2009;107:1318–1327.

19. Alibhai FJ, LaMarre J, Reitz CJ, Tsimakouridze EV, Kroetsch JT, Bolz SS, Shulman A,Steinberg S, Burris TP, Oudit GY, Martino TA. Disrupting the key circadian regulatorCLOCK leads to age-dependent cardiovascular disease. J Mol Cell Cardiol 2017;105:24–37.

20. Alibhai FJ, Tsimakouridze EV, Chinnappareddy N, Wright DC, Billia F, O’sullivan ML,Pyle WG, Sole MJ, Martino TA. Short-term disruption of diurnal rhythms after mur-ine myocardial infarction adversely affects long-term myocardial structure and func-tion. Circ Res 2014;114:1713–1722.

21. Martino TA, Tata N, Belsham DD, Chalmers J, Straume M, Lee P, Pribiag H, KhaperN, Liu PP, Dawood F, Backx PH, Ralph MR, Sole MJ. Disturbed diurnal rhythm altersgene expression and exacerbates cardiovascular disease with rescue by resynchroni-zation. Hypertension 2007;49:1104–1113.

22. Martino TA, Oudit GY, Herzenberg AM, Tata N, Koletar MM, Kabir GM, BelshamDD, Backx PH, Ralph MR, Sole MJ. Circadian rhythm disorganization produces pro-found cardiovascular and renal disease in hamsters. Am J Physiol Regul Integr CompPhysiol 2008;294:R1675–R1683.

23. Mosca L, Barrett-Connor E, Wenger NK. Sex/gender differences in cardiovasculardisease prevention: what a difference a decade makes. Circulation 2011;124:2145–2154.

24. Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey PL, McDonald JD, DoveWF, Pinto LH, Turek FW, Takahashi JS. Mutagenesis and mapping of a mouse gene,Clock, essential for circadian behavior. Science 1994;264:719–725.

25. Podobed PS, Alibhai FJ, Chow C-W, Martino TA, Yamazaki S. Circadian regulation ofmyocardial sarcomeric titin-cap (Tcap, Telethonin): identification of cardiac clock-controlled genes using open access bioinformatics data. PLoS One 2014;9:e104907.

26. Podobed P, Pyle WG, Ackloo S, Alibhai FJ, Tsimakouridze EV, Ratcliffe WF, MackayA, Simpson J, Wright DC, Kirby GM, Young ME, Martino TA. The day/night prote-ome in the murine heart. Am J Physiol Regul Integr Comp Physiol 2014;307:R121–R137.

27. Clipperton-Allen AE, Lee AW, Reyes A, Devidze N, Phan A, Pfaff DW, Choleris E.Oxytocin, vasopressin and estrogen receptor gene expression in relation to socialrecognition in female mice. Physiol Behav 2012;105:915–924.

28. Tsimakouridze EV, Straume M, Podobed PS, Chin H, LaMarre J, Johnson R, AntenosM, Kirby GM, Mackay A, Huether P, Simpson JA, Sole M, Gadal G, Martino TA.Chronomics of pressure overload-induced cardiac hypertrophy in mice revealsaltered day/night gene expression and biomarkers of heart disease. Chronobiol Int2012;29:810–821.

29. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can JBiochem Physiol 1959;37:911–917.

30. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolicsyndrome in circadian Clock mutant mice. Science 2005;308:1043–1045.

31. Marcheva B, Ramsey KM, Buhr ED, Kobayashi Y, Su H, Ko CH, Ivanova G, Omura C,Mo S, Vitaterna MH, Lopez JP, Philipson LH, Bradfield CA, Crosby SD, JeBailey L,Wang X, Takahashi JS, Bass J. Disruption of the clock components CLOCK andBMAL1 leads to hypoinsulinaemia and diabetes. Nature 2010;466:627–631.

32. Basu P, Alibhai FJ, Tsimakouridze EV, Singh RK, Paglialunga S, Holloway GP, MartinoTA, Bakovic M. Male-specific cardiac dysfunction in CTP: phosphoethanolamine cyti-dylyltransferase (Pcyt2)-deficient mice. Mol Cell Biol 2015;35:2641–2657.

33. Leite RD, Prestes J, Bernardes CF, Shiguemoto GE, Pereira GB, Duarte JO,Domingos MM, Baldissera V, de Andrade Perez SE. Effects of ovariectomy and resist-ance training on lipid content in skeletal muscle, liver, and heart; fat depots; and lipidprofile. Appl Physiol Nutr Metab 2009;34:1079–1086.

34. Stark KD, Park EJ, Holub BJ. Fatty acid composition of serum phospholipid of preme-nopausal women and postmenopausal women receiving and not receiving hormonereplacement therapy. Menopause 2003;10:448–455.

12 F.J. Alibhai et al.

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..35. Xu Y, Arenas IA, Armstrong SJ, Davidge ST. Estrogen modulation of left ventricularremodeling in the aged heart. Cardiovasc Res 2003;57:388–394.

36. Pedram A, Razandi M, Aitkenhead M, Levin ER. Estrogen inhibits cardiomyocytehypertrophy in vitro. Antagonism of calcineurin-related hypertrophy through induc-tion of MCIP1. J Biol Chem 2005;280:26339–26348.

37. Donaldson C, Eder S, Baker C, Aronovitz MJ, Weiss AD, Hall-Porter M, Wang F,Ackerman A, Karas RH, Molkentin JD, Patten RD. Estrogen attenuates left ventricularand cardiomyocyte hypertrophy by an estrogen receptor-dependent pathway that in-creases calcineurin degradation. Circ Res 2009;104:265–275. 211p following 275.

38. Santhi N, Lazar AS, McCabe PJ, Lo JC, Groeger JA, Dijk DJ. Sex differences in the cir-cadian regulation of sleep and waking cognition in humans. Proc Natl Acad Sci U S A2016;113:E2730–E2739.

39. Wang F, Keimig T, He Q, Ding J, Zhang Z, Pourabdollah-Nejad S, Yang XP.Augmented healing process in female mice with acute myocardial infarction. GendMed 2007;4:230–247.

40. Bennardo M, Alibhai FJ, Tsimakouridze EV, Chinnappareddy N, Podobed P, Reitz CJ,Pyle WG, Simpson JA, Martino TA. Day-Night Dependence of Gene Expression andInflammatory Responses in the Remodeling Murine Heart post-Myocardial Infarction.Am J Physiol Regul Integr Comp Physiol 2016;311:R1243–R1254.

41. Antoch MP, Gorbacheva VY, Vykhovanets O, Toshkov IA, Kondratov RV, KondratovaAA, Lee C, Nikitin AY. Disruption of the circadian clock due to the Clock mutation hasdiscrete effects on aging and carcinogenesis. Cell Cycle 2008;7:1197–1204.

42. Earnest DJ, Neuendorff N, Coffman J, Selvamani A, Sohrabji F. Sex differences in theimpact of shift work schedules on pathological outcomes in an animal model of ische-mic stroke. Endocrinology 2016;157:2836–2843.

43. Berry JD, Dyer A, Cai X, Garside DB, Ning H, Thomas A, Greenland P, Van Horn L,Tracy RP, Lloyd-Jones DM. Lifetime risks of cardiovascular disease. N Engl J Med2012;366:321–329.

44. Piro M, Della Bona R, Abbate A, Biasucci LM, Crea F. Sex-related differences in myo-cardial remodeling. J Am Coll Cardiol 2010;55:1057–1065.

45. Forman DE, Cittadini A, Azhar G, Douglas PS, Wei JY. Cardiac morphology and func-tion in senescent rats: gender-related differences. J Am Coll Cardiol 1997;30:1872–1877.

46. Witt H, Schubert C, Jaekel J, Fliegner D, Penkalla A, Tiemann K, Stypmann J, RoepckeS, Brokat S, Mahmoodzadeh S, Brozova E, Davidson MM, Ruiz Noppinger P, GroheC, Regitz-Zagrosek V. Sex-specific pathways in early cardiac response to pressureoverload in mice. J Mol Med 2008;86:1013–1024.

47. Taegtmeyer H, Young ME, Lopaschuk GD, Abel ED, Brunengraber H, Darley-UsmarV, Des Rosiers C, Gerszten R, Glatz JF, Griffin JL, Gropler RJ, Holzhuetter HG, KizerJR, Lewandowski ED, Malloy CR, Neubauer S, Peterson LR, Portman MA, RecchiaFA, Van Eyk JE, Wang TJ. American Heart Association Council on BasicCardiovascular S. Assessing cardiac metabolism: a scientific statement from the amer-ican heart association. Circ Res 2016;118:1659–1701.

48. Balkau B, Shipley M, Jarrett RJ, Pyorala K, Pyorala M, Forhan A, Eschwege E. Highblood glucose concentration is a risk factor for mortality in middle-aged nondiabeticmen. 20-year follow-up in the Whitehall Study, the Paris Prospective Study, and theHelsinki Policemen Study. Diabetes Care 1998;21:360–367.

49. Skyler JS, Bergenstal R, Bonow RO, Buse J, Deedwania P, Gale EA, Howard BV,Kirkman MS, Kosiborod M, Reaven P, Sherwin RS. American Diabetes A, AmericanCollege of Cardiology F, American Heart A. Intensive glycemic control and the pre-vention of cardiovascular events: implications of the ACCORD, ADVANCE, and VADiabetes Trials: a position statement of the American Diabetes Association and aScientific Statement of the American College of Cardiology Foundation and theAmerican Heart Association. J Am Coll Cardiol 2009;53:298–304.

50. Zhang J, Duncker DJ, Ya X, Zhang Y, Pavek T, Wei H, Merkle H, U�gurbil K, FromAH, Bache RJ. Effect of left ventricular hypertrophy secondary to chronic pressureoverload on transmural myocardial 2-deoxyglucose uptake. A 31P NMR spectro-scopic study. Circulation 1995;92:1274–1283.

51. Doenst T, Pytel G, Schrepper A, Amorim P, Farber G, Shingu Y, Mohr FW,Schwarzer M. Decreased rates of substrate oxidation ex vivo predict the onset of

heart failure and contractile dysfunction in rats with pressure overload. CardiovascRes 2010;86:461–470.

52. Dolinsky VW, Cole LK, Sparagna GC, Hatch GM. Cardiac mitochondrial energy me-tabolism in heart failure: Role of cardiolipin and sirtuins. Biochim Biophys Acta 2016;1861:1544–1554.

53. Sparagna GC, Chicco AJ, Murphy RC, Bristow MR, Johnson CA, Rees ML, Maxey ML,McCune SA, Moore RL. Loss of cardiac tetralinoleoyl cardiolipin in human and ex-perimental heart failure. J Lipid Res 2007;48:1559–1570.

54. Zhang R, Lahens NF, Ballance HI, Hughes ME, Hogenesch JB. A circadian gene ex-pression atlas in mammals: implications for biology and medicine. Proc Natl Acad Sci US A 2014;111:16219–16224.

55. Bray MS, Shaw CA, Moore MW, Garcia RA, Zanquetta MM, Durgan DJ, Jeong WJ,Tsai JY, Bugger H, Zhang D, Rohrwasser A, Rennison JH, Dyck JR, Litwin SE, HardinPE, Chow CW, Chandler MP, Abel ED, Young ME. Disruption of the circadian clockwithin the cardiomyocyte influences myocardial contractile function, metabolism, andgene expression. Am J Physiol Heart Circ Physiol 2008;294:H1036–H1047.

56. Harfmann BD, Schroder EA, Kachman MT, Hodge BA, Zhang X, Esser KA. Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glu-cose homeostasis. Skelet Musc 2016;6:12.

57. Allard MF, Schonekess BO, Henning SL, English DR, Lopaschuk GD. Contribution ofoxidative metabolism and glycolysis to ATP production in hypertrophied hearts. Am JPhysiol 1994;267:H742–H750.

58. Leong HS, Grist M, Parsons H, Wambolt RB, Lopaschuk GD, Brownsey R, Allard MF.Accelerated rates of glycolysis in the hypertrophied heart: are they a methodologicalartifact? Am J Physiol Endocrinol Metab 2002;282:E1039–E1045.

59. Zhu L, Zou F, Yang Y, Xu P, Saito K, Othrell Hinton A, Jr., Yan X, Ding H, Wu Q,Fukuda M, Sun Z, Tong Q, Xu Y. Estrogens prevent metabolic dysfunctions inducedby circadian disruptions in female mice. Endocrinology 2015;156:2114–2123.

60. McGinnis GR, Tang Y, Brewer RA, Brahma MK, Stanley HL, Shanmugam G,Rajasekaran NS, Rowe GC, Frank SJ, Wende AR, Abel ED, Taegtmeyer H, LitovskyS, Darley-Usmar V, Zhang J, Chatham JC, Young ME. Genetic disruption of the cardi-omyocyte circadian clock differentially influences insulin-mediated processes in theheart. J Mol Cell Cardiol 2017;110:80–95.

61. Manfredini R, Fabbian F. A pill at bedtime, and your heart is fine? Bedtime hyperten-sion chronotherapy: an opportune and advantageous inexpensive treatment strategy.Sleep Med Rev 2016;33:1–3.

62. Martino TA, Tata N, Simpson JA, Vanderlaan R, Dawood F, Kabir MG, Khaper N,Cifelli C, Podobed P, Liu PP, Husain M, Heximer S, Backx PH, Sole MJ. The primarybenefits of angiotensin-converting enzyme inhibition on cardiac remodeling occurduring sleep time in murine pressure overload hypertrophy. J Am Coll Cardiol 2011;57:2020–2028.

63. Tsimakouridze EV, Alibhai FJ, Martino TA. Therapeutic applications of circadianrhythms for the cardiovascular system. Front Pharmacol 2015;6:77.

64. Bonten TN, Saris A, van Oostrom MJ, Snoep JD, Rosendaal FR, Zwaginga J,Eikenboom J, van der Meer PF, van der Bom JG. Effect of aspirin intake at bedtimeversus on awakening on circadian rhythm of platelet reactivity. A randomised cross-over trial. Thromb Haemost 2014;112:1209–1218.

65. Ayala DE, Hermida RC. Sex differences in the administration-time-dependent effectsof low-dose aspirin on ambulatory blood pressure in hypertensive subjects.Chronobiol Int 2010;27:345–362.

66. Giacchetti S, Dugue PA, Innominato PF, Bjarnason GA, Focan C, Garufi C, Tumolo S,Coudert B, Iacobelli S, Smaaland R, Tampellini M, Adam R, Moreau T, Levi F, GroupAIC. Sex moderates circadian chemotherapy effects on survival of patients withmetastatic colorectal cancer: a meta-analysis. Ann Oncol 2012;23:3110–3116.

67. Solt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, Lundasen T, Shin Y, Liu J,Cameron MD, Noel R, Yoo SH, Takahashi JS, Butler AA, Kamenecka TM, Burris TP.Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists.Nature 2012;485:62–68.

68. Chen Z, Yoo SH, Takahashi JS. Small molecule modifiers of circadian clocks. Cell MolLife Sci 2013;70:2985–2998.

Biological sex influences heart disease in Clock mice 13

Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvx185/4139791by University of Guelph useron 20 December 2017