Disrupting the key circadian regulator CLOCK leads to age-dependent cardiovascular disease

    Disrupting the Key Circadian Regulator CLOCK leads to Age-Dependent Cardiovascular Disease Faisal J. Alibhai, Jonathan LaMarre, Cristine J. Reitz, Elena V. Tsimakouridze, Jeffrey T. Kroetsch, Steffen-Sebastian Bolz, Alex Shulman, Samantha Steinberg, Thomas P. Burris, Gavin Y. Oudit, Tami A. Martino PII: DOI: Reference:

S0022-2828(17)30008-1 doi:10.1016/j.yjmcc.2017.01.008 YJMCC 8510

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

30 November 2016 12 January 2017 16 January 2017

Please cite this article as: Alibhai Faisal J., LaMarre Jonathan, Reitz Cristine J., Tsimakouridze Elena V., Kroetsch Jeffrey T., Bolz Steffen-Sebastian, Shulman Alex, Steinberg Samantha, Burris Thomas P., Oudit Gavin Y., Martino Tami A., Disrupting the Key Circadian Regulator CLOCK leads to Age-Dependent Cardiovascular Disease, Journal of Molecular and Cellular Cardiology (2017), doi:10.1016/j.yjmcc.2017.01.008

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Disrupting the Key Circadian Regulator CLOCK leads to Age-Dependent Cardiovascular

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Disease

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Faisal J. Alibhaia, Jonathan LaMarrea, Cristine J. Reitza, Elena V. Tsimakouridzea, Jeffrey T. Kroetschb, Steffen-Sebastian Bolzb, Alex Shulmana, Samantha Steinberga, Thomas P. Burrisc,

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Gavin Y. Ouditd, and Tami A. Martinoa*

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Centre for Cardiovascular Investigations, Department of Biomedical Sciences, University of

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Guelph, Canada; b

Department of Physiology, University of Toronto, Canada;

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Louis, Missouri, USA;

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Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St

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Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute,

University of Alberta, Edmonton.

Running Title: CLOCK regulates age-dependent cardiovascular disease

*Correspondence to: Tami A. Martino, Centre for Cardiovascular Investigations, Biomedical Sciences/OVC Room 1646B, University of Guelph, Canada, N1G2W1, (519)-824-4120 x54910; [email protected]

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease

Abstract

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The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm

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disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the

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circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac

heart

weight,

hypertrophy,

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physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in dilation,

impaired

contractility,

and

reduced

myogenic

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responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten

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gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts.

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Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3β, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates

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the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3β, S6K1 phosphorylation are increased, consistent with the development of agedependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in

growth

and

renewal;

disrupting

Clock

leads

to

age-dependent

cardiomyopathy.

Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

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Alibhai et al. CLOCK regulates age-dependent cardiovascular disease

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease

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Keywords:

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Circadian, Cardiovascular, Aging, Cardiac hypertrophy, MicroRNA

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Word Count: 6,130

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Number of Figures: 6

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Number of Tables: 2

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease 1. Introduction Circadian rhythms underlie daily variations in cardiovascular physiology including heart rate

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[1], blood pressure [2], and cardiac contractility [3]. Circadian rhythm disruption is a major risk factor for cardiovascular disease and is associated with adverse health consequences [4-6].

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Recently there have been major advances in our understanding of daily rhythmicity, and it’s

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relevance to the pathogenesis and treatment of cardiac hypertrophy and heart failure [3, 7-12]. However, the underlying mechanisms responsible are generally clinically unappreciated, and

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their role in cardiac hypertrophy with aging is not known.

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At the molecular level circadian clocks in all cells enable us to entrain to environmental cues and anticipate the differing physiologic demands of daily events, including those of the cardiovascular system. The circadian mechanism at its most basic level is a 24-hour

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transcription and translation loop present in virtually all cells including cardiomyocytes. The positive arm consists of a heterodimeric pairing of CLOCK and BMAL1, which bind to promoter

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E-boxes to induce expression of the repressors PERIOD (PER), CRYPTOCHROME (CRY), and REV-ERB. In the negative arm, PER and CRY are phosphorylated by CASEIN KINASE, inhibit their own transcription, and the 24-hour cycle begins again. There are many excellent reviews [13-16]. The circadian mechanism also regulates daily patterns of tissue-specific factors, comprising ~10% of the genes [17-19] and proteins [3] in the heart. CLOCK [20] is a core component of the circadian mechanism [21-23] and plays a role in regulating diurnal cardiac metabolism, contractile function, and gene and protein rhythms [1, 3, 4, 19]. However, whether CLOCK plays a role in cardiac growth and renewal, and conversely how disrupting CLOCK adversely affects heart structure and function, especially with aging is not known. The risk of cardiovascular disease increases with age and the majority of heart failure diagnoses and deaths occur in those older than 70 years of age [24, 25]. Aging is associated with structural changes in the heart including myocardial thickening [26], cardiomyocyte hypertrophy [27], and cardiac fibrosis [28]. Moreover there is a decline in cardiac function [29, 5

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease 30], which is most apparent when the cardiovascular system is challenged, for example by exercise [31]. The average lifespan of humans has increased over the last 2 centuries [32],

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Therefore understanding the mechanism that regulate age-dependent changes for healthy cardiovascular physiology are of clinical relevance, especially for maintaining high quality of life

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in the elderly population.

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In this study we investigate the role of CLOCK in the pathophysiology of cardiac aging. Here we use ClockΔ19/Δ19 mice which have a mutation in the Clock gene resulting in a protein

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incapable of transcriptional activation [22]. We demonstrate that ClockΔ19/Δ19 mice develop age-

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dependent cardiovascular disease. Moreover, we show that CLOCK is a key mediator of mRNAs and miRNAs in the PTEN-AKT signal pathways involved in cardiac hypertrophy. In vivo and in vitro experiments confirm that Clock regulates these signal pathways important for

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cellular hypertrophy. Moreover, pharmacological modulation of the circadian mechanism using the REV-ERB agonist SR9009 targets these pathways and attenuates cardiac hypertrophy in

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vivo. Identifying a direct role for CLOCK in cardiac remodeling could have therapeutic implications, especially for individuals subjected to circadian rhythm disruption such as shift works, individuals with sleep disorders, and in the aging population.

2. Materials and methods

2.1 Mice Male homozygote ClockΔ19/Δ19, heterozygote ClockΔ19/+ and wild type littermates on a congenic C57Bl/6J background [20] were obtained from our University of Guelph mouse breeding core, which sources its colony breeders from Jackson Laboratory [33]. Mice were housed under a 12hour light (L):12-hour dark (D) cycle with standard chow and water provided ad libitum, and aged in the Central Animal Facilities at the University of Guelph. For sample collection animals were euthanized with isoflurane and cervical dislocation. For histology, hearts were perfused 6

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease with 1M KCl, fixed in 10% neutral buffered formalin for 24hrs, and processed. Paraffin embedded hearts were sectioned at the level of the mid papillary and stained with Masson’s

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trichrome or picrosirius red. For molecular studies tissues were frozen in liquid nitrogen and stored at -800C until assayed. The effects of SR9009 were assessed in 21 month old

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ClockΔ19/Δ19 and WT mice. Animals were administered the drug (100 mg/kg, i.p., b.i.d) or vehicle

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(15% cremophor) at ZT0 and ZT12 for 28 days. TAC mice were treated for 2 weeks with SR9009 following induction of pressure overload. For studies in P0 mice, hearts were collected

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at ZT07, two hearts were pooled per collection and a total of 6 hearts per genotype (n=3 pooled

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samples) were used. All studies were approved by the University of Guelph Institutional Animal Care and Use Committee and in accordance with the guidelines of the Canadian Council on

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Animal Care.

2.2 Actigraphy

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Wheel-running experiments were performed as described [34]. Individually housed mice were entrained to a diurnal 12:12 L:D cycle for 2 weeks, followed by transfer to constant darkness (circadian cycle, DD). Data were analyzed using ClockLab (ActiMetrics).

2.3 Radiotelemetry

PA-C10 murine telemetry probes (Data Sciences) were used to monitor and collect blood pressure and heart rate data from conscious, freely moving animals [7]. Data were acquired for 30 seconds every 5 minutes over 72 hours, collated into 1-hour bins according to zeitgeber time, and averaged to yield a 24-hour plot.

2.4 Cardiac Pathophysiology Cardiac pathophysiology was performed using established techniques at 4, 8, 12, 18 and 21 months of age [33]. Serial echocardiography was done using a i13L 14MHz linear-array 7

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease transducer on a GE Vivid 7 Dimension ultrasound system. Hemodynamics were recorded in mice anesthetised under isoflurane, using a 1.2Fr pressure catheter (Transonic), and the ADI

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Power Lab system with Lab Chart 7.

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2.5 Pressure Myography

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Cremaster skeletal muscle resistance arteries were isolated at ZT07 from 21 month old mice, canulated on glass micropipettes, and used for functional experiments as described [35]. For

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myogenic responsiveness, vessels were subject to 20 mmHg stepwise increases in transmural

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pressure (from 20 to 100 mmHg). The maximal diameter was measured under Ca2+-free conditions using the same stepwise protocol. Depolarization-induced vasoconstriction was

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determined using i) L-phenylephrine (PE, 1 nmol/L-10 μmol/L), or ii) KCl (60 mmol/L).

2.6 MicroRNA Arrays and database

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Total RNA was isolated from 4 and 21 month old WT and ClockΔ19/Δ19 hearts collected at ZT06 using the miRNeasy Mini Kit (Qiagen). For miRNA microarray profiling, total RNA from 12 individual samples was used. Samples were digested (RNase Free DNase set, Qiagen), validated using RNA ScreenTape (RIN >7; Agilent) and Nanodrop (260/280 ≥ 2.0; Thermo Scientific), and analyzed using the Affymetrix GeneChip miRNA 4.0 array which interrogates all mature miRNA sequences in miRBase v20 (G.E.O. Accession: GSE84151). Bioinformatics analyses were performed using the extraction software (v10.7.1.1) and GeneSpring Gx v11.5 (Agilent). Genome locations were from miRBase [36]. Validated target mRNAs were from Tarbase v7.0 [37]. KEGG pathways were from miRPath v2.0 [38]. All miRNAs > 50 Raw fluorescence units (RFUs) are listed our novel database at www.circadianmicrorna.com.

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease 2.7 qRT-PCR To determine mRNA expression profiles we used the Power SYBR Green RNA-to-CT one Step

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Kit (Life Technologies) on a ViiA7 real time PCR system (Applied Biosystems) using primers listed in Supplementary Table 6. For studies with SR9009, the total RNA was isolated from

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hearts collected at ZT07 using the RNeasy kit (Qiagen). Relative expression was determined

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using the ΔΔCT method normalized to histone, as described [19]. To validation miRNA expression, qRT-PCR was performed using the qScript microRNA cDNA Synthesis Kit and the

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PerfeCTa SYBR Green Super Mix (Quanta Biosciences). All miRNA primers were purchased

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from Quanta Biosciences. Relative miRNA expression was determined using the ΔΔCT method, normalized to RNU6. qRT-PCR was performed using n=3 individual hearts/group, and n=2

2.8 Western Blots

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technical duplicates.

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Protein extracts were prepared with CelLytic MT Cell Lysis Reagent (Sigma) supplemented with cOmplete protease and PhosSTOP phosphatase inhibitor cocktails (Roche). Lysates were subjected to SDS-PAGE and transferred to PVDF membrane (Bio-Rad) for immunoblotting. Primary antibodies were from Cell Signaling, as follows: phospho-PTEN (Ser380/Thr382/383) (#9554, 1:1000), PTEN (#9559; 1:1000), phospho-AKT (Ser473) (#4060; 1:1000), AKT (#4691; 1:1000), phospho-GSK-3β (Ser9) (#9323; 1:1000), GSK-3β (#9315; 1:1000), phospho-PRAS40 (Thr246) (#2997, 1:1000), PRAS40 (#2691; 1:1000). Loading was normalized to total protein using the Mem-code PVDF kit (Thermo Scientific). Proteins were visualized using SignalFire Elite ECL reagent (12757, Cell Signalling).

2.9 Comprehensive Lab Animal Monitoring System (CLAMS) CLAMS (Columbus Instruments) was used to monitor the metabolic parameters of 21 month old WT and ClockΔ19/Δ19 mice when administered SR9009 or vehicle (15% cremophor). Animals 9

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease were acclimatized for 2 days in the CLAMS after which metabolic parameters were measured

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for 24 hours.

2.10 Transverse Aortic Constriction

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Young (8 weeks old), WT mice were subjected to transverse aortic constriction (TAC) to induce

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pressure overload cardiac hypertrophy, as described previously [7, 10, 39]. Briefly, mice were anesthetized with isoflurane and intubated. A thoracotomy was performed via an incision in the

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second intercostal space, and the transverse aorta distal to the left subclavian artery, was

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constricted using a 27-gauge needle. Sham mice underwent the same procedure but the aorta was not constricted.

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2.11 Isolation, culture and treatment for hypertrophy of mouse cardiac myocytes We isolated and cultured neonatal mouse cardiomyocytes using standard techniques [40]. Cells

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were plated at a density of 500 cells/mm2 in 4-well LabTek chamber slides coated with collagen type I (Sigma). The following day cells were transferred to serum free media containing 0.1 mM BrdU for 24 hours. On day 2 cells were treated with either IGF-1 (10 ng/ml, Sigma), or FBS (10%), or LiCl (20mM) for 3 days, after which they were stained for α-actinin (A7811; Sigma 1:500) using Alexa Fluor 488 (A11029; Life Technologies, 1:500). Cell size was determined using Cell Profiler as described previously [41].

2.12 Statistics All values are mean ± SEM. The data were assessed for normal distribution and similar variance between groups using SigmaPlot 12 (Systat Software Inc.). We used a two-sided Student’s t-test for comparisons between 2 groups. For the aging studies in mice, we used a two way ANOVA with Tukey post hoc, p<0.05 are considered significant.. Statistical analyses of 24 hour gene rhythms was done using JTK_CYCLE v2.0 in R v3.1.2 [42]. JTK_CYCLE is used 10

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease to identify rhythmic components and provide an estimate of period, phase and amplitude. The JTK p-value is the estimated probability of rejecting the null hypothesis. Here the null hypothesis

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is a dataset that is not rhythmic. A threshold of p<0.05 was used to identify rhythmicity The JTK q-value is the false discovery rate at which a gene is called cyclic. For further details see

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Hughes et al. (2010) [43]. We selected sample size for murine studies based on the numbers

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typically reported in the literature.

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3. Results

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3.1 ClockΔ19/Δ19 mice develop age-dependent cardiac hypertrophy and fibrosis The mutant ClockΔ19 allele extends the circadian period from ~23.4hr in the WT mice, to Δ19/Δ19

homozygote mice.

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24.3hr in the ClockΔ19/+ heterozygotes, and to 28.2hr in the Clock

Representative wheel running actigraphy under normal light:dark (LD) and circadian conditions

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(DD) are shown on the left , and quantification of period under DD are on the right (Fig. 1A). The amplitude of mean arterial pressure (MAP) in ClockΔ19/Δ19 mice is also blunted in the light period (murine sleep time) (Fig. 1B). Diurnal profile is shown on the left, and the light and dark phase quantification of MAP is shown on the right (Fig. 1B). These activity and MAP observations are consistent with those previously reported in ClockΔ19/Δ19 mice [2, 20]; however this affects the heart with aging has not been previously investigated. Thus we next looked at cardiovascular parameters. To investigate the role of Clock in cardiac structure and function we first examined agedependent changes in heart weight (HW). HW was significantly greater in the ClockΔ19/Δ19 mice by 18 months of age as compared to ClockΔ19/+ and WT mice (Fig. 1C). Moreover, HW:body weight and HW:tibia length were also significantly greater by 18 months of age compared to ClockΔ19/+ and WT mice (Fig. 1D). These observations are consistent with the notion that the ClockΔ19/Δ19 mice develop cardiovascular disease. Moreover, histopathology revealed that aged 11

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease ClockΔ19/Δ19 hearts had significantly increased cardiomyocyte hypertrophy and interstitial fibrosis, as compared to ClockΔ19/+ and WT mice (Figs. 1E, F). Collectively these findings show the

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3.2 ClockΔ19/Δ19 mice develop cardiovascular disease with age

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development of obvious cardiac hypertrophy and cardiac fibrosis in the aging ClockΔ19/Δ19 heart.

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Given that Clock underlies age-dependent changes in heart structure we next examined how these correlated with cardiac functional outcomes. First cardiac structure and function were

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examined using serial echocardiography from 4 to 21 months of age. Compared with the WT

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and ClockΔ19/+ mice, ClockΔ19/Δ19 mice had greater adverse remodelling with significantly increased left ventricular (LV) dimensions by 12 months of age, followed by decreased ejection fraction (%EF) and fractional shortening (%FS) (Figs. 2A-C, and Table 1).

Because the

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differences in the ClockΔ19/Δ19 mice could be correlated to cardiovascular hemodynamic

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responses, we next measured these in vivo. We found that aging ClockΔ19/Δ19 mice showed

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worse functional profiles in LV end-systolic and LV end-diastolic and arterial pressures (Fig. 2D, and Table 2). Concurrent with a drop in MAP, is reduced total peripheral resistance (TPR) and impaired myogenic tone (Fig. 2E, Table 2, and Supplementary Fig. 1). The disease phenotype is restricted to the aging ClockΔ19/Δ19 mice. The young animals do not develop pathology, nor impaired cardiac function and appear healthy, with all cardiovascular parameters similar to the WT and ClockΔ19/+ littermates. The heart disease that develops in the aging ClockΔ19/Δ19 animals does so only over an extended period time, and the cardiac and vascular systems decompensate with age.

3.3 Dysregulation of miRNA expression in ClockΔ19/Δ19 hearts Since Clock is a transcriptional regulator of the core circadian mechanism [22], we next evaluated the effects on gene expression in the heart. Clock drives Per2 expression, and ClockΔ19/Δ19 hearts haves significantly reduced Per2 amplitude by JTK_CYCLE (Fig. 3A). 12

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Second, Clock also drives Rev-Erbα, and ClockΔ19/Δ19 hearts have reduced Rev-Erbα expression in the heart (Fig. 3A). Lastly, since Rev-Erbα is a key repressor of Bmal1 and Clock, we

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anticipated and found that ClockΔ19/Δ19 hearts have increased expression of these genes (Fig. 3A) [44, 45]. The statistical outcomes for the JTK_CYCLE analyses are in Supplementary Table

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1. Thus these data show that Clock underlies the diurnal cycling of the circadian mechanism

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genes, which in turn are postulated to drive the output of genes and proteins important for the heart [1, 3, 17, 18].

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The core circadian mechanism genes drive the output of a wide variety of mRNAs. These

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mRNAs in turn have an important role in producing proteins in the heart. Up to ~13% of the mRNAs [18] and 8% of the proteins [3] in the heart exhibit diurnal rhythms. Many of these products are involved in key cellular processes in the heart; time of day of expression is

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important for heart structure and function [5, 15]. Moreover, it has been shown that miRNAs also influence cardiac growth pathways and renewal pathways in the heart [46, 47]. Thus we next

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looked at miRNA expression in ClockΔ19/Δ19 and WT hearts at 4 and 21 months of age. Microarray profiling revealed 17 miRNAs that were differentially expressed (p<0.05, foldchange>1.4) in young ClockΔ19/Δ19 versus WT hearts (Fig. 3B and Supplementary Table 2). All miRNAs expressed in the hearts of the WT and ClockΔ19/Δ19 mice are provided in our database at www.circadianmicrorna.com. Hierarchical cluster analysis (Fig. 3C) and functional mapping (Fig. 3C, and Supplementary Table 3) revealed that the greatest number of target genes of the altered miRNAs in ClockΔ19/Δ19 hearts were involved in the PTEN-Akt signalling pathway. For example, primary transcripts from the miR-17-92 family and their cognate miRNAs are negative regulators of PTEN [48, 49]; we found that this family of miRNAs are have reduced expression in young ClockΔ19/Δ19 hearts (Figs. 3D, E). Moreover, these miRNAs do not oscillate with respect to time-of-day in WT hearts. In addition, there are no oscillations in ClockΔ19/Δ19 hearts. However, all of the miRNAs were significantly reduced in ClockΔ19/Δ19 hearts at virtually all time points over the day night cycle (ZT, 3, 7, 11, 19, 23) as compared to WT hearts (Supplementary Fig. 2). 13

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Finally, these miRNAs exhibit age-dependent increased expression by 21 months of age (Figs. 3F, G). Thus Clock plays a role in expression of regulatory miRNAs that target the PTEN-AKT

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pathway in the heart.

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3.4 Disruption of AKT signalling in ClockΔ19/Δ19 hearts

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Given the significant relationship between miRNAs and the PTEN-AKT pathway, we next investigated whether Pten mRNA was rhythmically expressed in the heart. As rhythmic Pten

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expression has not yet been shown in the heart we next determined whether expression is

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rhythmic and driven by the endogenous circadian mechanism independent of the light:dark cycle. We found a significant circadian rhythm in Pten mRNA in WT hearts under constant darkness (JTK p-value = 4.2 x 10-3) (Fig. 4A). Furthermore, we determined whether expression

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is rhythmic under the normal light:dark conditions that animals and humans live under. We found a significant rhythm in Pten mRNA in WT hearts in the diurnal environment (JTK p-value

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= 0.02) (Fig. 4B), In contrast to the results in our WT animals, Pten mRNA expression exhibits a loss of rhythmic oscillation, as well as increased levels over 24 hours in ClockΔ19/Δ19 hearts (Figs. 4A, B). The statistical outcomes for JTK-CYCLE analyses are in Supplementary Table 4. Next, we investigated if PTEN protein exhibits a diurnal rhythm in the heart, by western blot (Fig. 4C). We found that PTEN protein abundance is rhythmic in WT hearts (JTK p-value = 0.038) (Fig. 4D). Given that PTEN is rhythmic in the WT heart, we next investigated the profiles in ClockΔ19/Δ19 hearts. PTEN protein abundance exhibits a loss of rhythmic oscillation (JTK pvalue=1.00), as well as increased levels over 24 hours in ClockΔ19/Δ19 hearts, as compared to WT hearts (Figs. 4C, D). In contrast, phospho-PTEN abundance did not oscillate in WT or ClockΔ19/Δ19 hearts over 24 hour periods (Fig. 4C and Supplementary Fig. 3). Taken together these results indicate that PTEN oscillations occur at the level of the protein. Given that PTEN is a negative regulator of AKT (S473) phosphorylation, we next investigated whether our findings correlated with changes in downstream phospho-AKT 14

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease expression (Fig. 4E). We found that in WT hearts, AKT exhibited rhythmic phosphorylation (JTK p-value =

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with increased levels during the dark

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In ClockΔ19/Δ19 hearts, there was a loss of rhythmic phosphorylation (JTK p-value=1.00), and blunted phospho-protein levels during the dark phase (Fig. 4F). Moreover, when we

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plot ClockΔ19/Δ19 PTEN values over the light (animals asleep) or dark (animals awake) period, we

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see a loss of diurnal rhythms with increased levels of PTEN in the dark (Fig. 4G). Conversely, there is a loss of diurnal rhythms for phospho-AKT, and reduced levels in the dark (Fig. 4H).

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Thus although the time-of-day changes are interesting, it is also worth noting that these occur

that develop in the ClockΔ19/Δ19 hearts.

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over many days and weeks, which could be a driving factor underlying the structural changes

In light of these observations, we next performed a series of experiments using young

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ClockΔ19/Δ19 mice and found a significant cascade effect. PTEN is the main negative regulator of

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downstream AKT signalling. Increased PTEN abundance in young ClockΔ19/Δ19 hearts was

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associated with reduced AKT activation and a loss of AKT (S473) phosphorylation rhythms inClockΔ19/Δ19 hearts (Fig. 4E-H). Two direct downstream targets of AKT namely GSK-3β (S9) and PRAS40 (T246), as well as the indirect target S6K1 (T389), also showed a loss of diurnal rhythms in phosphorylation in ClockΔ19/Δ19 hearts consistent with an altered signalling cascade (Fig. 4I). The rhythmic JTK_CYCLE values are in Supplementary Table 5.

3.5 ClockΔ19/Δ19 cardiomyocytes and hypertrophy We next studied the effects of the ClockΔ19/Δ19 mutation on AKT-signalling and cardiac growth using neonatal cardiomyocytes (Fig. 5A). FBS is a general activator of cell growth [40] and treatment stimulated both WT and ClockΔ19/Δ19 cardiomyocytes to increase to similar cell size (Fig. 5B).However, the ClockΔ19/Δ19 cells were smaller compared to WT cells and thus showed a greater fold-change from baseline (Fig. 5C). IGF-1, a potent activator of AKT-signalling [50], increased cell size in WT but not ClockΔ19/Δ19 cardiomyocytes, consistent with impaired AKT15

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease activation in these cells (Fig. 5D). Conversely LiCl stimulated the growth of ClockΔ19/Δ19 cardiomyocytes, consistent with LiCl-mediated inactivation of the elevated GSK-3β activity in

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these cells (Fig. 5E). These data provide further support that Clock is a key regulator of the AKT signalling axis regulating cardiomyocyte growth pathways.

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One key element of the studies in Fig. 5 was to examine how a known AKT activator (IGF-1)

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influences cardiac hypertrophy in ClockΔ19/Δ19 cardiomyocytes. However, since we are interested in expression of the miRNAs in the intact animal in vivo, we also assessed expression of the

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miR-17-92 family in ClockΔ19/Δ19 and WT hearts from P0 mice (the same day at which cells are collected for culture). We found increased expression of all miR-17, miR-18a, miR-19b, miR-

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20a, and miR-92a in ClockΔ19/Δ19 compared to WT hearts (Fig. 5F). Expression of these miRNAs more resembles aged ClockΔ19/Δ19 hearts consistent with the notion that during disease states

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the heart reactivated fetal like features.

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3.6 Increased AKT signalling in aged ClockΔ19/Δ19 hearts Chronic dysregulation of AKT signalling causes maladaptive cardiac remodeling [51]. Thus we next assessed AKT signalling in aged (21 month old; ZT07) ClockΔ19/Δ19 versus WT mice. We observed that phosphorylation of AKT (S473) and its downstream targets S6K1 (T389) and GSK-3β (S9) were increased in the old ClockΔ19/Δ19 hearts (Fig. 6A). In the aged ClockΔ19/Δ19 mice, chronic Clock disruption led to increased activation of AKT signalling pathways leading to inactivation of GSK-3β, consistent with the development of cardiac hypertrophy in these animals. Importantly, GSK-3β S9 phosphorylation also occurs in failing human hearts [52, 53], and this is consistent with the development of reduced cardiac function (Fig. 2) and increased gene expression of the heart failure (HF) biomarkers Nppa and Nppb in the old ClockΔ19/Δ19 hearts (Fig. 6B).

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease 3.7 Pharmacological modulation and hypertrophy We next sought to examine whether modulation of the circadian mechanism downstream of

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Clock, using the REV-ERB agonist SR9009 [54], is efficacious in our model. We found that SR9009 increased oxygen consumption in the aged WT mice (Fig. 6C), consistent with effects

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observed in young mice [54]. Interestingly, SR9009 also increased oxygen consumption in the

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old ClockΔ19/Δ19 mice (Fig. 6D). Although not as severe as the ClockΔ19/Δ19 mice, old WT mice also have increased heart size and decreased cardiac function by 21 months of age (Fig. 1 and

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2). Therefore we further tested whether pharmacological modulation in mice with a functional

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Clock gene could alter AKT activation consistent with our hypothesis. First we show that SR9009 is effective at altering circadian mechanism function in WT mice as Bmal1 and Reverbα mRNA expression were reduced in the hearts mice treated with SR9009 compared to

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vehicle treated mice (Fig. 6E). Moreover, we show that SR9009 reduced AKT (S473) activation

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in WT hearts (Fig.6F), while AKT activation was not reduced in ClockΔ19/Δ19 hearts (Fig. 6G).

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Furthermore, there is a concurrent up-regulation of Atrogin-1 in the hearts SR9009 treated WT mice (Fig. 6H (which inhibits AKT-dependent cardiac hypertrophy [55]) and a reduction in heart weight (Fig. 6I). To assess whether agonist treatment has a direct effect on cardiac size we used the TAC model. We found that SR9009 significantly reduced LVIDd (Fig. 6J), HW (Fig. 6K), and HW:BW ratio (Fig. 6L) compared to vehicle treated TAC mice. This was not associated with a reduction in blood pressure as measured by in-vivo hemodynamics (Supplementary Table 7). In sham mice SR9009 did not affect any morphometric, echocardiographic, or in-vivo hemodynamic parameters supporting that reduction of cardiac hypertrophy was specific to the TAC mice. Collectively these results suggest that SR9009 acts directly to reduce cardiac hypertrophy, consistent with our findings in aged WT mice.

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease 4. Discussion Here we investigated the role of Clock in cardiac aging. Using ClockΔ19/Δ19 mice we show for

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the first time that loss of Clock leads to the development of age-dependent cardiovascular disease. ClockΔ19/Δ19 mice develop increased heart weight, cardiomyocyte hypertrophy and

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interstitial fibrosis by 21 months of age, compared to WT littermates. Moreover, aging

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ClockΔ19/Δ19 mice exhibit LV remodeling characterized by increased LV dilation and reduced function by echocardiography and in-vivo hemodynamics analyses. In order to investigate the

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underlying molecular changes we first examined microRNA expression in the heart. We found

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that a loss of Clock reduces expression of the primary transcript and mature miRNAs of the miR-17-92 family, which plays an important role in PTEN-AKT signaling. Consistent with these, changes in miRNA expression, ClockΔ19/Δ19 mice have altered diurnal activation of PTEN-AKT

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pathway genes and proteins. To further establish circadian regulation of AKT signalling we used both in-vitro and in-vivo approaches. We show a direct functional consequence in neonatal

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cardiomyocytes, as hypertrophy responses were impaired in ClockΔ19/Δ19 cardiomyocytes treated with the AKT activator IGF-1. Moreover, we show pharmacological inhibition targets to this pathway in vivo as SR9009 reduced cardiac AKT activation in functional WT but not the impaired ClockΔ19/Δ19 mice. This is consistent with the notion that the Clock gene is required for SR9009 to modulate AKT activation. Collectively, these results demonstrate that Clock plays an important role in normal cardiovascular aging and that the Clock mutation leads to agedependent cardiovascular disease. Although it is well established that circadian rhythms play a role in the timing of onset of adverse cardiovascular events [4, 5, 56-58], their role in cardiac aging is poorly understood. Here we showed that disturbing Clock leads to the development of age-dependent heart disease. This is consistent with our earlier experimental paradigm showing that altering the circadian mechanism through genetic or environmental disturbances exacerbates existing heart disease. For example, disturbing rhythms exacerbates cardiac remodelling after myocardial 18

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease infarction in mice; maintaining rhythms benefits infarct healing and outcome [33]. In addition, circadian rhythm disruption reduces hypertrophic responses and impairs cardiac function in

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mice subjected to pressure-overload induced cardiac hypertrophy [10]. At the molecular level, disruption of the circadian clock alters cardiac gene rhythms [1, 3, 10], protein abundance [3],

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and cardiomyocyte function [19]. Thus the circadian mechanism is vital for normal

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cardiovascular function, and disturbing the mechanism factor Clock is consistent with altered gene and protein profiles leading to disturbed cardiomyocyte function and the development of

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cardiovascular disease.

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In our study, the aged ClockΔ19/Δ19 mice have cardiovascular disease at multiple levels, including cardiac hypertrophy, fibrosis, reduced % ejection fraction and % fraction shortening, decreased contractility, and reduced myogenic responsiveness. Previously Martin Young and

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colleagues reported that cardiomyocyte specific Clock mutant (CCM) mice develop agedependent cardiac hypertrophy and fibrosis but not contractile dysfunction [11]. This is

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consistent with our findings that the circadian mechanism regulates key components of cardiomyocyte growth pathways. In addition, our data suggest that the pathogenesis and pathophysiology of age-dependant cardiomyopathy depends disruption of the circadian mechanism at multiple levels including heart and vasculature. The circadian mechanism in the vasculature and plays a role in vascular structure and function [57, 59, 60]; future studies investigating the interaction between the heart and regulation of myogenic response and vascular tone are worthy of investigation. Our data further demonstrate that Clock plays a direct role in cardiac miRNA expression as loss of Clock changes the profiles in the heart, especially those related to cardiac growth and renewal pathways. Importantly, we found altered expression of the miR-17-92 family in ClockΔ19/Δ19 hearts. This miRNA family is important for cardiac structure and function, as experimentally transgenic overexpression induces cardiomyocyte proliferation and is associated with lethal cardiomyopathy and arrhythmogenesis in mice [48, 61]. 19

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease In terms of mechanism, we observed changes downstream of miR-17-92 in ClockΔ19/Δ19 mice including altered phosphorylation patterns and blunted activation of AKT, GSK3β, PRAS40 and

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S6K1; these are all components of the PTEN-AKT signalling pathway regulating cardiac hypertrophy. Intriguingly, loss of Clock specifically in cardiomyocytes alters phosphorylation

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patterns of AKT and GSK-3β [62], Moreover, altering activation of AKT and its downstream

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mediator mTOR [51], or inhibition of GSK-3β [63], directly targets cardiac hypertrophy in vivo. It’s worth noting that Clock disruption on this pathway occurs on a daily basis, and also occurs

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over many days, weeks, and years to lead to heart disease. This is consistent with the notion

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that increased AKT signalling is linked to maladaptive remodeling when activated on a long term basis [51, 53]. Thus, there could be an uncoupling of AKT regulation due to disease pathways being activated with age as part of the remodeling process. Collectively, these studies

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demonstrate a key role for Clock in regulating cardiac growth, and disruption is associated with exacerbation of hypertrophy pathways with aging.

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In the future it would be interesting to look at the relative contributions of the period misalignment caused by the Clock mutation vs. impaired CLOCK mediated transcription in the development of age-dependent cardiomyopathy. In addition, determining the role of other core circadian factors in cardiac aging, especially those that have been shown to play a role in disease pathophysiology warrants future investigation [9, 34, 64-67]. Notably, it will be important to determine whether these core circadian factors mediate cardiac aging through circadian or circadian-independent pathways. Identifying the role of circadian mechanism factors in aging could have therapeutic implications, especially for individuals subjected to circadian rhythm disturbance (e.g. shift work, sleep disorders) in the aging population. In terms of clinical implications, disturbed circadian rhythms in humans are associated with increased risk of heart disease and worse outcomes [68-70]. For patients with heart failure, a major concern is impaired oxygen consumption, in part because it’s a limiting factor for rehabilitory exercise [71]. Notably, we found that pharmacological targeting of the circadian 20

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease mechanism using the agonist SR9009 improved oxygen consumption in our old animals. This drug has also been shown to increase oxygen consumption and exercise capacity in healthy

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young mice [54, 72], and is aimed to improved skeletal muscle performance related to sports endurance [73]. We also found that SR9009 attenuates cardiac hypertrophy in TAC mice,

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consistent with our findings that the circadian mechanism plays a key role in regulating cardiac

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growth pathways. This could also open new doors for reducing cardiac hypertrophy in patients with heart disease. It is worth nothing that we gave the drug twice daily for effect, based on the

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original identification of SR9009 [54]. Recent studies have suggested that there could be an

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optimal time for dosing during the light period [74]; this might be worthy of consideration especially if drugs targeting the circadian mechanism move towards clinical translation. Future studies using pharmacological targeting of the circadian mechanism can open new avenues to

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5. Conclusions

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treat cardiovascular patients.

In summary, ClockΔ19/Δ19 mice develop cardiac hypertrophy. In ClockΔ19/Δ19 mice there is dysregulation of the core circadian gene mechanism, output genes and miRNA posttranscriptional regulators. Functional mapping identified the PTEN-AKT signalling pathway as targets of Clock-induced cardiac hypertrophy. Although other miRNA pathways may also be involved, our results indicate that Clock is a key regulator of AKT signalling for triggering cardiac hypertrophy in vivo. These experiments demonstrate that there is novel potential for targeting the circadian mechanism to reduce cardiac hypertrophy; Investigating strategies are a promising new area for treating patients with heart disease.

Funding Sources This work was supported by a grant from the Heart and Stroke Foundation of Canada to T.A. Martino. 21

ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Acknowledgements We thank Dr. Lynne O’Sullivan for her clinical cardiovascular advice, and Dr. David Wright for

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the use of the CLAMS.

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Disclosures

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None.

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Fig 1. Aging ClockΔ19/Δ19 mice develop cardiac hypertrophy and fibrosis. (A) Actigraphy of WT,

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ClockΔ19/+, ClockΔ19/Δ19 mice under diurnal (12hr light:12hr dark) and circadian (constant darkness; DD) conditions (left) and period quantification under DD (right), n=3/group, *p<0.05

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vs. all other groups by one way ANOVA followed by Tukey post hoc. (B) Diurnal rhythms in

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mean arterial pressure (MAP) in WT as compared to ClockΔ19/Δ19 mice (left) and photoperiod quantification (right), n=3/group, *p<0.05 light vs dark for WT. (C) Heart weight (HW). (D)

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HW:body weight (HW:BW) ratio (top) and HW:tibia length (HW:TL) (bottom) in ClockΔ19/Δ19, ClockΔ19/+, and WT mice at 4, 8, 18 and 21 months of age, n=4-6/group, *p<0.05 by two way

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ANOVA followed by Tukey post hoc. (E) Representative histopathology images stained with

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Masson’s trichrome or picrosirius red, and (F) quantification of myocyte cross sectional area (MSCA) and interstitial fibrosis in 4 and 21 month old ClockΔ19/Δ19, ClockΔ19/+, and WT hearts,

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Fig 2. Aging ClockΔ19/Δ19 mice exhibit increased LV remodelling and impaired vascular function.

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(A) Serial echocardiography at 4, 8, 12, 18 and 21 months of age showing increased LV internal dimensions at diastole (LVIDd) and systole (LVIDs) by 12 months, and (B) reduced ejection

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fraction (%EF) by 18 months, n=9/group. (C) Representative mid papillary M-mode images. (D)

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In vivo hemodynamics show decreased LV end systolic pressure (LVESP), and increased diastolic pressure (LVEDP) with aging, n=4-6/group (left). *p<0.05 as calculated by a two way

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ANOVA followed by Tukey post hoc. (E) Total peripheral resistance (TPR) decreases in

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ClockΔ19/Δ19 with age (left), n=4-6/group for each age, *p<0.05 ClockΔ19/Δ19 vs. all other groups at the same age as calculated by a two way ANOVA followed by Tukey post hoc. Myogenic responsiveness of cremaster skeletal muscle resistance arteries isolated from 21 month old

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mice, n=11-12 arteries from 4 mice/group, *p<0.05 by Student’s t test (right). All values are

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Fig. 3. Dysregulation of rhythmic gene and miRNA expression in ClockΔ19/Δ19 hearts. (A)

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Illustration of the circadian mechanism, and altered circadian rhythms in the core mechanism genes Per2, Rev-erbα, Bmal1, and Clock in ClockΔ19/Δ19 hearts, n=3 hearts/time point/group,

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CT=circadian time (constant darkness). (B) Heat map illustrating differentially expressed

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miRNAs in young ClockΔ19/Δ19 hearts (and Supplementary Table 2), n=3 hearts/group. (C) Hierarchal cluster analysis of pathways targeted by differentially expressed miRNAs using

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miRPath. KEGG identifications are in Supplementary Figure 3. Expression of (D) the primary

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miR-17-92 transcript, and (E) the mature miRNAs in 4 month old ClockΔ19/Δ19 hearts, and (F) upregulation of the primary miR-17-92 transcript and (G) and mature miRNAs, by 21 months of

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age, n=3 hearts/group, *p<0.05 by Students t test. Values are mean ± SEM.

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Fig. 4. Disrupted AKT signalling in young ClockΔ19/Δ19 hearts. (A) Circadian and (B) diurnal

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rhythms in Pten gene expression and (C) Phosphorylated and total PTEN protein abundance in WT and ClockΔ19/Δ19 hearts, n=3 hearts/time point/group, (D) Time-of-day quantification of total

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PTEN abundance in WT and ClockΔ19/Δ19 hearts . (E) Representative western blot and (F)

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quantification of AKT phosphorylation at S473. Average photoperiod quantification of (G) total PTEN and (H) pATK. (I) Representative western blots (left) and quantification (right) of GSK-3β

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phosphorylation at S9, PRAS40 phosphorylation at T246, and S6K1 phosphorylation at T389.

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Graphs represent phospho/total protein abundance, n=3 hearts/time point, *p<0.05 by Students t-test. JTK_CYCLE data are provided in Supplementary Tables 1, and 4. Values are mean ±

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Fig 5. Impaired cardiac hypertrophy in ClockΔ19/Δ19 cardiomyocytes following IGF-1 treatment.

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(A) Neonatal ClockΔ19/Δ19 and WT cardiomyocytes stained with anti-α-actinin (green) and dapi (blue) used for cell size quantification. (B) Cell size following vehicle and 10% FBS treatment.

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(C) Greater fold change in ClockΔ19/Δ19 cardiomyocytes from baseline with FBS treatment as

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compared to WTs, n=5-8/group. (D) Impaired cardiac hypertrophy in ClockΔ19/Δ19 cardiomyocytes following treatment with 10ng/mL insulin like growth factor-1 (IGF-1) with cell size (left) and fold

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change from vehicle (right), n=5-8/group. (E) Treatment of cells with 20mM lithium chloride

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(LiCl) (n=3-4/group). Cell size (left) and fold change from control (right), *p<0.05 by Students t test. Each n-value represents a separate isolation and culture of neonatal cardiomyocytes and each culture were run in duplicate. (F) Expression of the miR-17-92 family in P0 hearts collected

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Fig. 6. Clock, AKT signalling and cardiac remodeling. (A) Representative western blots (left)

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from 21 month old WT versus ClockΔ19/Δ19 hearts, and densitometry quantification (right) of phospho-AKT (S473), phospho-S6K1 (T389), and phospho-GSK-3β (S9), n=3 hearts/group

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collected at ZT07. (B) Nppa and Nppb gene expression in 21 month old WT and ClockΔ19/Δ19

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hearts, n=3 hearts/group. (C) Oxygen consumption (VO2) in old WT mice following SR9009 treatment (100mg/kg, i.p, b.i.d, 28 days) or vehicle (15% cremophor), and (D) in ClockΔ19/Δ19

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mice, n=4/group, *p<0.05 by Students t test. (E) SR9009 (SR9) treatment leads to reduced

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expression of Bmal1 and Rev-erbα, n=3/group collected at ZT07, *p<0.05 by Student’s t-test. (F) Representative western blots of AKT activation (left), and quantification of phospho/total AKT in WT hearts (right). (G) Quantification of phospho/total AKT in ClockΔ19/Δ19 hearts. (H)

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Atrogin-1 expression by qRT-PCR in WT hearts following SR9009 treatment, and (I) heart weight/tibia length. n=4/group, *p<0.05 by Students t-test. (J) LVIDd was reduced in TAC mice

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treated with SR9009 for 2 weeks. Moreover, (K) HW and (L) HW:BW was also reduced in TAC SR9009 mice vs. vehicle treated controls. Values are provided in Supplementary Table 7. n=8/TAC group and n=5/Sham group, *p<0.05 vs. all groups by two way ANOVA followed by Tukey post-hoc. Values are mean ± SEM.

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Table 1 Echocardiography at 4, 8, 12, 18 and 21 months of age ClockΔ19/+

Wild Type

9

9

9

LVIDd (mm)

4.86±0.06**

4.52±0.05*

4.37±0.03

LVIDs (mm)

3.45±0.06**

3.00±0.03

2.85±0.06

FS (%)

28.98±0.76**

33.61±0.64

34.86±0.99

EF (%)

61.72±1.30**

n

NU

SC

RI

21 Month

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ClockΔ19/Δ19

68.88±0.84

70.58±1.28

0.80±0.01

0.81±0.01

0.84±0.02

PWTd (mm)

0.81±0.01

0.78±0.01

0.79±0.01

LV Mass (mg)

170.88±7.23**

142.38±3.53

136.32±2.23

LV/BW (mg/g)

4.86±0.33**

3.36±0.18

3.33±0.15

421±9

428±4

436±10

LVIDd (mm)

4.63±0.06**

4.35±0.05

4.30±0.04

LVIDs (mm)

3.20±0.05**

2.85±0.06

2.80±0.05

FS (%)

30.84±0.60**

34.51±0.81

34.89±0.52

64.96±0.88**

70.01±1.06

70.77±0.70

IVSd (mm)

0.81±0.01

0.79±0.01

0.78±0.01

PWTd (mm)

0.79±0.01

0.77±0.01

0.77±0.01

LV Mass (mg)

150.58±4.68**

130.96±2.30

127.27±2.24

LV/BW (mg/g)

3.79±0.35*

2.88±0.12

3.07±0.13

431±5

454±11

466±10

4.42±0.04*

4.20±0.02

4.23±0.04

EF (%)

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HR (bpm)

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HR (bpm) 12 Month LVIDd (mm)

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2.59±0.03

2.66±0.04

FS (%)

35.85±0.46

38.13±0.52

37.12±0.50

EF (%)

72.00±0.61

74.75±0.62

73.54±0.61

IVSd (mm)

0.76±0.01

0.74±0.01

PWTd (mm)

0.74±0.01

0.71±0.01

LV Mass (mg)

128.32±4.22*

112.39±1.60

116.25±2.30

LV/BW (mg/g)

2.78±0.09

2.57±0.14 460±9

460±12

0.74±0.01

2.83±0.12

LVIDd (mm)

4.23±0.04

4.13±0.03

4.09±0.04

LVIDs (mm)

2.57±0.04

2.52±0.04

2.48±0.06

FS (%)

39.34±0.95

39.55±0.36

39.28±0.90

76.04±1.06

76.45±0.41

76.12±1.00

0.74±0.01

0.72±0.01

0.72±0.01

0.73±0.01

0.71±0.01

0.71±0.01

LV Mass (mg)

114.96±2.95

106.83±2.03

105.19±3.11

LV/BW (mg/g)

3.03±0.10

2.99±0.14

3.03±0.13

449±8

450±6

478±13

LVIDd (mm)

4.13±0.03

4.02±0.03

4.04±0.03

LVIDs (mm)

2.43±0.05

2.44±0.03

2.43±0.03

FS (%)

41.25±0.85

39.29±0.44

39.70±0.35

EF (%)

78.19±0.94

76.15±0.54

76.64±0.40

IVSd (mm)

0.73±0.01

0.72±0.01

0.71±0.01

PWTd (mm)

0.72±0.01

0.69±0.01

0.70±0.004

HR (bpm)

408±8

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HR (bpm)

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109.54±1.96

100.12±0.99

100.92±1.54

LV/BW (mg/g)

3.69±0.18

3.62±0.13

3.76±0.14

435±8

466±16

489±10

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LVIDd; left ventricle internal dimensions at diastole, LVIDs; LV internal dimensions at systole,

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FS; fractional shortening, EF; ejection fraction, IVSd; septal wall thickness at diastole, PWTd;

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posterior wall thickness at diastole, LV; left ventricle, BW; body weight, HR, heart rate. n=9/group, *p<0.05 vs. all other groups of same age; **p<0.01 vs. all other groups of same age

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ACCEPTED MANUSCRIPT Alibhai et al. CLOCK regulates age-dependent cardiovascular disease Table 2 In vivo hemodynamics at 4,8,18 and 21 months of age ClockΔ19/Δ19

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LVESP (mmHg)

83.01±4.15*

102.35±6.35

100.31±1.00

LVEDP (mmHg)

6.10±1.54*

2.06±0.75

1.78±0.33

dp/dt max (mmHg/s)

6465.71±676.82*

9781.44±932.97

8829.16±374.43

dp/dt min (mmHg/s)

-5669.03±604.70*

-8686.69±1013.86

-8276.03±122.42

SBP (mmHg)

92.13±2.01*

101.48±2.49

99.72±1.09

DBP (mmHg)

57.64±1.54*

65.38±1.49

65.76±0.94

MAP (mmHg)

69.14±1.42*

77.41±1.75

77.08±0.81

TPR (mmHg/mL/min)

2.25±0.09*

2.87±0.12

2.68±0.04

93.33±2.03*

102.02±1.29

105.23±0.93

3.20±1.68

2.31±0.73

1.86±0.67

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LVEDP (mmHg)

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dp/dt max (mmHg/s)

7507.83±658.23

9360.69±707.45

8390.26±377.84

dp/dt min (mmHg/s)

-7851.31±664.73

-9550.18±886.80

-8438.52±622.71

SBP (mmHg)

96.53±1.32

100.13±1.42

104.56±1.35

DBP (mmHg)

59.72±1.65

66.22±0.82

63.74±2.14

MAP (mmHg)

71.99±1.17*

77.52±0.1.01

77.35±1.84

2.77±0.12

3.19±0.11

2.99±0.17

LVESP (mmHg)

98.61±1.07

102.97±1.17

101.98±1.61

LVEDP (mmHg)

0.28±1.13

1.75±0.88

0.25±0.77

dp/dt max (mmHg/s)

8758.68±563.19

9038.40±525.96

9676.84±383.15

dp/dt min (mmHg/s)

-8963.90±570.06

-8858.86±540.43

-9135.13±572.25

TPR (mmHg/mL/min) 8 Month

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101.01±1.27

101.22±1.51

DBP (mmHg)

59.83±1.69

65.24±0.98

62.06±2.01

MAP (mmHg)

72.98±0.87

77.09±0.91

75.12±1.14

TPR (mmHg/mL/min)

2.76±0.11

3.19±0.09

LVESP (mmHg)

98.76±1.14

102.62±1.01

LVEDP (mmHg)

0.87±1.07

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dp/dt min (mmHg/s)

-8645.00±832.50

1.56±1.14

9565.88±416.16

9417.49±185.42

-9070.42±360.75

-8488.80±258.49

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102.21±1.35

0.83±0.36

98.02±1.13

99.22±0.88

97.98±0.74

DBP (mmHg)

62.22±0.57

63.66±1.02

63.45±0.52

MAP (mmHg)

74.15±0.34

75.51±0.52

74.96±0.43

3.01±0.09

3.17±0.18

3.11±0.09

500.02±15.20

536.35±22.39

504.02±20.15

TPR (mmHg/mL/min)

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SBP (mmHg)

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dp/dt max (mmHg/s)

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3.02±0.09

LVESP, left ventricle (LV) end systolic pressure; LVEDP, LV end diastolic pressure; dP/dtmax and dP/dtmin, maximum and minimum first derivative of LV pressure; SBP, systolic blood pressure (BP); DBP, diastolic BP; MAP, mean arterial pressure; TPR, total peripheral resistance; HR, heart rate. n=4-6/group, *p<0.05 ClockΔ19/Δ19 vs. all other groups of same age as calculated by two way ANOVA followed by Tukey post hoc. Values are mean ± SEM.

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Highlights ClockΔ19/Δ19 mice develop age-dependent cardiac hypertrophy

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ClockΔ19/Δ19 hearts express dysregulated miRNAs in PTEN-AKT pathway

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This pathway is crucial for regulating cardiac hypertrophy

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Targeting the circadian mechanism modulates hypertrophy pathways in vivo and in vitro

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Applying circadian biology leads to new approaches for understanding heart disease

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