Accepted Manuscript Consequences of Circadian and Sleep Disturbances for the Cardiovascular System Faisal J. Alibhai, BSc, Elena V. Tsimakouridze, BSc, Cristine J. Reitz, BSc, W. Glen Pyle, PhD, Tami A. Martino, PhD PII:

S0828-282X(15)00050-1

DOI:

10.1016/j.cjca.2015.01.015

Reference:

CJCA 1548

To appear in:

Canadian Journal of Cardiology

Received Date: 31 October 2014 Revised Date:

25 December 2014

Accepted Date: 8 January 2015

Please cite this article as: Alibhai FJ, Tsimakouridze EV, Reitz CJ, Pyle WG, Martino TA, Consequences of Circadian and Sleep Disturbances for the Cardiovascular System, Canadian Journal of Cardiology (2015), doi: 10.1016/j.cjca.2015.01.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Consequences of Circadian and Sleep Disturbances for the Cardiovascular

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System

Faisal J. Alibhai, BSc, Elena V. Tsimakouridze, BSc, Cristine J. Reitz, BSc, W. Glen

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Pyle, PhD, Tami A. Martino, PhD

Guelph, Ontario, Canada.

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Cardiovascular Research Group, Department of Biomedical Sciences, University of

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Running Title: Consequences of Circadian and Sleep Disturbances on the Heart.

Correspondence: Tami A. Martino, Cardiovascular Research Group, Biomedical

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Sciences/OVC Room 1646B, University of Guelph, Canada, N1G2W1, (519)-824-4120

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x54910; [email protected]

Funding: Canadian Institutes of Health Research grant to T.A. Martino.

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Brief Summary That the circadian system plays a major role in regulating neural and endocrine pathways has long been known, and thus it is implicated in healthy cardiovascular

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physiology. Importantly, in recent years there is an increasing clinical appreciation that disturbing these rhythms significantly and adversely impacts the cardiovascular system, with profound health consequences. This review highlights the clinical and experimental

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consequences of circadian disturbance on heart disease.

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Abstract Circadian rhythms play a crucial role in our cardiovascular system. Importantly, there has been a recent flurry of clinical and experimental studies revealing the profound

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adverse consequences of disturbing these rhythms on the cardiovascular system. For example, circadian disturbance worsens outcome post-myocardial infarction with implications for patients in acute care settings. Moreover, disturbing rhythms

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exacerbates cardiac remodeling in heart disease models. Also, circadian dyssynchrony is a causal factor in the pathogenesis of heart disease. These discoveries have

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profound implications for the cardiovascular health of shift workers, individuals with circadian and sleep disorders, or anyone subjected to the 24/7 demands of society. Moreover, these studies give rise to two new frontiers for translational research. 1) Circadian rhythms and the cardiac sarcomere, which sheds new light on our

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understanding of myofilament structure, signalling and electrophysiology. 2) Knowledge translation which includes biomarker discovery (chronobiomarkers), timing of therapies (chronotherapy), and other new promising approaches to improve the management and

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treatment of cardiovascular disease. Reconsidering circadian rhythms in the clinical

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setting benefits repair mechanisms, and offers new promise for patients.

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Historical Introduction Jürgen Aschoff was a physician, scientist, and co-founder of the field of circadian biology. He introduced the notion that day and night (diurnal) rhythms in physiology are

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a fundamental feature in most living organisms including humans.1 We now know that rhythms are regulated by the circadian system, by orchestration in the hypothalamic suprachiasmatic nucleus, and through neural and hormonal outputs to all organs

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including the heart (Figure 1A, reviews2-7). Mechanistically, an exquisite underlying cellular clock mechanism has been identified, which controls molecular rhythms in

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virtually all cells including cardiomyocytes (Figure 1B, reviews8,9). Over the past five decades numerous clinical and experimental studies have revealed a crucial role for the circadian system in regulating healthy physiology. Most recently, studies by our group and others have shed light on the profound adverse health consequences of circadian

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disturbances, underlying cardiovascular, cancer, metabolic, respiratory, psychiatric and other disorders (e.g.6,10-15). This review focusses on the consequences of circadian

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disturbances on the pathogenesis and pathophysiology of heart disease.

Circadian Biology and Normal Cardiovascular Physiology

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i) Circadian Rhythms in Heart Rate (HR) and Blood Pressure (BP) Circadian rhythms are important for daily cyclic variation in mammalian physiology, for example our heart rate (HR) is highest during wake and lowest during sleep.16 Blood pressure (BP) also displays a daily rhythm, indeed Floras and colleagues documented for the first time the remarkable blood pressure surge that occurs at the time of waking and morning activity.17 Blood pressure is highest in the morning and falls progressively

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(~10%) to reach a nadir during sleep.18 These rhythms are consistent with the diurnal biases of our autonomic nervous system, which oscillate over 24 hour periods.19 Thus our cardiovascular physiology exhibits striking time-of-day dependent oscillations.

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Remarkably, diurnal variations in heart rate and cardiac contractility observed in vivo still persist ex vivo, for example when rodent hearts are perfused on a Langendorff apparatus.13,20 Thus it is not just the neurohormonal axes that contribute to time of day

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cardiac physiology, but also the intrinsic circadian properties of the heart. Over the last decade this has given rise to numerous experimental studies that show a direct role for

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the circadian mechanism on our daily cardiovascular physiology, as is demonstrated experimentally in rats21-24 and mice.13,25,26

ii) Daily Timing of Onset of Adverse Cardiovascular Events

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Adverse cardiovascular events are a leading cause of death worldwide and do not occur at random times throughout the day. An early morning peak of acute myocardial infarction (MI) was first reported in 1985,27 and in the decades since, despite changes in

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lifestyle and advances in medicine, the pattern of morning excess of MI persists.28-33 A diurnal pattern also occurs for infarct size,34-36 stroke,37 angina,38 ventricular

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tachyarrhythmia,39-41 defibrillation energy requirements,42 ventricular refractoriness,43 and sudden cardiac death.44-46 Physiologically, sympathovagal balance is thought to play a causal role, as patients with autonomic nervous system dysfunction do not exhibit the early morning peak in timing of onset of MI.47-49 Several studies have highlighted additional factors, many under circadian control, which increase the risk of adverse cardiovascular events, including diurnal rhythms in platelet activity50-53, thrombosis or

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thrombolysis54-56 and endothelial function.57 Mechanistically, a direct role for the circadian mechanism underlying time-of-day dependence has been demonstrated in

iii) Diurnal Variations in Cardiomyocyte Metabolism

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humans52,58 and animal models.34,59-61

As described above, cardiac function displays a time-of-day effect, driven in part by

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the sympathetic environment and the neurohormonal milieu. However, at the myocyte level, there is considerable new evidence that intrinsic cellular properties also occur in a

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circadian manner, as has been exquisitely detailed in studies by Young and colleagues. To summarize, this is especially evident from the ex vivo perfused rat or mouse heart models, which demonstrate persistence of time-of-day variations in cardiac metabolism, even in the absence of neural and hormonal cues.20,62,63 Day/night variations in cardiac

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metabolism allow the heart to switch between different substrates (mainly glucose and fatty acids) for energy sources depending on availability over the course of 24 hour periods (e.g. reviews64,65). These intrinsic cardiomyocyte processes are facilitated in

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part by the cardiomyocyte-specific circadian clock mechanism (e.g.13,66). This circadian mechanism regulates mRNA and protein expression of key metabolic factors in

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cardiomyocytes as described below, thus controlling cellular processes in a time-of-day dependent manner.

iv) Circadian Genomics

The heart is genetically a different organ in the day versus the night. The first large scale microarray study to demonstrate this revealed that ~8% of genes are rhythmic in

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murine hearts under circadian (constant dark) conditions.67 However, as humans (and medicine) exist in a diurnal and not circadian environment, we showed that ~13% of genes in murine hearts are rhythmic under normal day and night conditions.68 These

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genomic experiments recapitulate earlier findings of individual clock gene cycling on PCR analyses,69 and moreover, identify many new rhythmic genes important for cardiac growth, renewal, transcription, translation, signaling pathways, and metabolism.67,68 Our

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circadian heart microarray data (and that of other researchers) are available through an open access website created by the Hogenesch lab (Circadian Expression Profiles Data

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Base, Circa DB, http://bioinf.itmat.upenn.edu/circa/).70-73 Rhythmic cycling of the core circadian mechanism genes has also been recently demonstrated in human heart tissue, by PCR analyses.74 Consistent with these observations is the persistent 24 hour rhythmic oscillations in bioluminescence in heart and vascular tissue explants from

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transgenic rats where luciferase expression is driven by the circadian mechanism factor Per1.75 Moreover, rhythmicity has also been shown to occur at the cellular level, in cardiomyocytes, vascular smooth muscle cells, endothelial cells and fibroblasts in

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culture.76-80 It is worth noting that researchers generally perform rodent studies during the light period, during rodent sleep time, and results are frequently compared to human

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wake time. These molecular studies clearly demonstrate that timing of functional assessments is highly critical in interpreting experimental findings. In summary, the cell types in the heart possess circadian clock mechanisms which regulate a variety of clock controlled output genes, which in turn influence the time-of-day processes essential for heart structure and function.

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v) Circadian Proteomics Since it is the proteins and not directly the mRNA that underlies fundamental

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biochemical processes in cells, studies have recently begun to explore circadian variation in the cellular proteome. We found that ~8% of the soluble cardiac proteome exhibits diurnal variation in murine heart, by using a large-scale 2-dimensional

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difference in gel electrophoresis and mass spectrometry approach.81 The circadian mechanism plays a role in protein abundance in the heart, as cardiomyocyte-specific

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Clock mutant mice have altered diurnal abundance of proteins, including many rate limiting enzymes crucial for vital metabolic pathways in the heart.81 In terms of how time-of-day protein abundance is controlled, we reported that about half of the diurnal variation in the cardiac proteome can be accounted for by underlying rhythmic changes

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at the mRNA level.81 Our findings are consistent with a previous report by Reddy et al who similarly observed about 50% of mRNA fluctuations underlying the hepatic proteome.82 Most recently, Luck et al developed a statistical model showing that

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rhythmic production as well as degradation (half-life) at both the mRNA and protein levels underlies daily protein abundance.83 Additional mechanisms regulating diurnal

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molecular rhythms are glucocorticoid control of transcription,84,85 and time-of-day variations in chromatin architecture.86 Thus there is a dynamic nature to the cardiac proteome, which underlies diurnal variations in heart function.

Experimental Models of Circadian Disruption and Consequences for the Cardiovascular System

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As highlighted in the preceding section, circadian rhythms are fundamentally important for cardiovascular health. The heart displays clear differences in time-of-day organ, cellular, and molecular physiology. Questions arise, therefore, as to what

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happens when these rhythms are disturbed. Circadian rhythm disturbances can have multiple deleterious effects on organ physiology, with profound clinical implications for cardiovascular and other diseases (e.g. reviews6,10,87-89). This section will focus on

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disturbance on the cardiovascular system.

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experimental studies investigating the profound adverse effects of circadian rhythm

i) Diurnal Disturbance Worsens Outcome Post-MI

Myocardial infarction (MI) triggers a temporally orchestrated inflammatory response designed to clear the infarct of dead cells and debris, while at the same time activating

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reparative pathways for fibrosis and scar formation.90-92 We postulated that the circadian mechanism plays a key role in immune cell recruitment and infarct healing post-MI. That there is daily cycling of peripheral blood cells,93-96 diurnal reactivity of immunocytes,95,97

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and a circadian mechanism in immune cells98-102 provides indirect support for this hypothesis. Our recent study directly recognized for the first time that the circadian

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mechanism plays a causal role in immunocyte recruitment to infarcted myocardium, and that disturbing this mechanism worsens outcome post-MI.103 Mice were infarcted by left anterior descending coronary artery ligation (MI model), randomized to either a normal diurnal or disrupted light:dark environment for 5 days, and then maintained under normal diurnal conditions. Short term diurnal disruption altered innate immune responses crucial for scar formation, leading to maladaptive cardiac remodeling (Figure

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2). One of the most intriguing findings is a role for the circadian mechanism factor CLOCK in recruiting innate immune cells post-MI. This study has clinical relevance, as circadian and sleep disruptions can impair recovery in modern intensive care units.104Reconsidering how we practice medicine, by maintaining the diurnal environment

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and patients biological rhythms, provides a promising non-pharmaceutical approach to

ii) Diurnal Disturbance Worsens Cardiac Remodeling

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improve patient outcomes in acute clinical care settings.108

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The adverse consequences of diurnal disturbance on pre-existing heart disease was first demonstrated by Turek and colleagues, who revealed the deleterious effects on survival in cardiomyopathic hamsters subjected to chronic shifts in the light and dark cycle.109 We showed that diurnal rhythm disturbance exacerbates cardiac remodeling in

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mice with pressure-overload induced cardiac hypertrophy; and importantly, that the adverse remodeling only ceased when the animals were returned to their normal diurnal cycle.110 Thus, altering the photoperiod to one in which animals cannot entrain alters the

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relationship between the light and dark cycle, endogenous physiology, and cardiac gene and protein levels, such that individuals are “out of sync” with their environment. This

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has direct consequences on cardiac physiology and pathophysiology, exacerbating underlying heart disease.

iii) Circadian Dyssynchrony Causes Heart Disease The important consequences of circadian dyssynchrony on the heart are perhaps best exemplified by the +/tau hamster studies. These animals carry a mutation in the

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circadian clock mechanism gene casein kinase 1 epsilon (CK1e) such that they exhibit an internal circadian period of ~22 hours, even though they live in a 24 hour world.111 The +/tau heterozygotes exhibit abnormal entrainment to the 24 hour diurnal

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environment (earlier onset and a more fragmented activity profile),112 and reduced longevity, as compared to their wild type littermates.113 In terms of cardiovascular consequences, the Sole and Ralph labs demonstrated that circadian disorganization in

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the +/tau hamsters is a causal factor in the pathogenesis of dilated cardiomyopathy.114 Conversely, if the animals are housed in diurnal cycles appropriate for their genotype

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(22 hours), then rhythms normalize and the heart disease is prevented.114 Notably, the tau/tau homozygotes have a 20 hour period and do not entrain to a 24 hour light and dark cycle and thus they do not develop heart disease.114 Thus these studies demonstrate the importance of synchrony between the intrinsic circadian period and the

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external light dark environment, highlighting that abnormal entrainment by circadian dyssynchrony plays an etiologic role in the pathogenesis of heart disease.

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iv) Circadian Gene Mutations and Heart Disease Additional lines of evidence further support the notion circadian mechanism gene

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factors underlie cardiovascular pathophysiology; several recent studies are summarized below. 1) Whole body Bmal1-/- knockout mice exhibit a loss of diurnal variation in HR and BP,26 and develop dilated cardiomyopathy.115 2) Cardiomyocyte-specific Bmal1 knockout (CBK) mice also develop dilated cardiomyopathy even though the disturbance is only in the heart.66,116 3) In gene mapping studies it was found that Bmal1 associates with hypertension loci in spontaneously hypertensive rats.117 4) In a similar light, Bmal1

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(and also Npas2) polymorphisms are associated with hypertension in humans.117,118 5) Mice with simultaneous loss of the circadian output genes Dbp, Hlf, and Tef develop cardiac hypertrophy and left ventricular dysfunction.119 6) Cardiomyocyte-specific

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circadian mutant mice exhibit blunted diurnal HR variation, reduced responsiveness of the heart to workload changes, and altered cardiac metabolism.13,65,120 7) Per2 mice have attenuated injury in the coronary artery ligation model.121 8) Adenosine receptor

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A2b mediated stabilization of Per2 is protective against ischemic heart disease in mice.122 Collectively, these studies are crucial for demonstrating relevant relationships

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between the circadian mechanism and cardiovascular disease genesis and pathology.

Translational Consequences of Circadian and Sleep Disruption on the

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Cardiovascular System in Humans Many of the mechanistic studies described above that investigated the effects of circadian and sleep disturbances on the cardiovascular system, are performed in rodent

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models and cell culture. However, these have important translational applications for

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human health and for clinical cardiology, as described below.

i) Shift work and Heart Disease Circadian rhythm disturbances are relevant to humans. For example, the World Health Organization describes shift work as an occupational disturbance of circadian rhythms,123 and clinical studies support an association between shift work and heart disease. Mechanistically, shift work alters sympathetic and vagal autonomic activity,124

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and diurnal BP profiles,125 which may contribute to cardiovascular disease. Further supporting this notion, Sheer et al. demonstrated experimentally that the adverse effects

autonomic predictors of cardiovascular (and other) risk.126

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of circadian misalignment in healthy humans can affect endocrine, metabolic, and

Epidemiologic studies further support a link between shift work and heart disease. For example, an increased risk of ischemic heart disease was demonstrated in a

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longitudinal study of 504 paper-mill shift workers in Sweden.127 Moreover, an increased risk of coronary heart disease was reported for female U.S shiftwork nurses.128 Also, an

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increased risk for MI was shown by meta-analysis of 34 studies pertaining to shift disease.129

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atherosclerosis was demonstrated by cross sectional examination of 2,510 shift workers in Germany.130 Lastly, severe hypertension was demonstrated in a longitudinal study of

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6,495 Japanese male shift workers.131 Given that millions of North Americans and others engage in shift work or irregular work schedules at some point in their careers (e.g.132), it is

important to determine how

atypical work schedules impact on

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consequences.

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performance efficiency (e.g.133), while minimizing the potential for adverse health

ii) Circadian Rhythms and Insufficient Sleep Circadian rhythm disturbances are also relevant through their intersection with sleep because sleep is under both circadian and homeostatic control. Indeed, a role for circadian mechanism genetic factors on sleep has been recently discovered in humans. For example familial advanced phase sleep syndrome is associated with a missense

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mutation in the clock component hPER2, which alters the circadian period.134 These individuals go to sleep very early in the night time, and routinely wake up in the extreme early morning hours. Although a direct correlation between circadian factors influencing

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sleep and heart disease in humans has not yet been demonstrated, factors affecting sleep duration clearly have a direct effect on cardiovascular health. For example, in today’s society, 40% of North Americans do not achieve the recommended 7 hours of

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sleep,135 and short (9 hours) sleep durations are associated with an increased risk of heart disease.136 These findings are consistent with earlier reports of

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increased all-cause mortality risk (including ischemic heart disease) in individuals with sleeping patterns other than 7-8 hours per night.137,138 In addition to circadian mutations causing sleep changes, circadian-coupled factors disturbed by inadequate sleep may mechanistically contribute to heart disease pathophysiology (e.g.139-141). Intriguingly,

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there are molecular consequences on diurnal gene expression in the heart with as little as one night of sleep deprivation. This was demonstrated in mice following 3, 6, 9, and 12 hours of sleep deprivation, which altered expression of 2470 genes in the heart on

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microarray analyses.142

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iii) Chronotypes, Social Clocks, and Light at Night Circadian rhythms in some physiologic functions vary between humans (e.g. core body temperature, melatonin phase) leading to classifications of early/morning or late/evening chronotypes.143 Intriguingly, chronotype may influence cardiovascular disease, as evening types appear to be more at risk for cardiovascular changes (hypertension, HR, BP) as compared to morning types.144. A later chronotype is also

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associated with social jet lag - the misalignment of biological and social clocks145 - and prevalence of social jet lag in apparently healthy individuals may contribute to cardiovascular risk.146 Light at night including that from light-emitting diodes (LEDs) in

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television and computer screens has broad consequences for our health, as is the subject several interesting recent papers.147,148 These studies are relevant to contemporary society and shed new light on how our circadian phenotypes, lifestyle

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choices and the diurnal environment can impact cardiovascular health and disease.

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New Frontiers: Circadian Rhythms and the Cardiac Sarcomere In addition to the above experimental and clinical discoveries, there are new frontiers for circadian rhythms research important to our understanding of the cardiovascular system. The first involves circadian rhythms and the cardiac sarcomere, which is the

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main contractile apparatus in the heart. These studies give rise to new understanding of myofilament structure, signaling and electrophysiology, as detailed below.

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i) Circadian Rhythms and Myofilament Structure Cardiac myofilaments mediate heart performance (Figure 3, reviews149-152), and

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recent studies indicate a role for the circadian mechanism in affecting myofilament and thus cardiac function. For example, ex vivo rat hearts exhibit greatest contractile performance during the animal wake versus sleep time, on Langendorff analysis.20 Conversely, this variation in performance is ablated in hearts of diurnal disrupted mice.81 At a molecular level, day/night rhythms in cardiac myofilament activity are lost in cardiomyocyte-specific Clock mutant mice as compared to wild types, thus further

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supporting the notion cardiac contractile performance links to the circadian mechanism.81 That there are significant disruptions in sarcomere architecture in Bmal1-/mice provides further support for this notion.115 Our group also demonstrated a

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functional correlation between myofilament activity and protein phosphorylation as one mechanism for regulating diurnal myofilament function in the murine heart.81 Most recently we demonstrated that the expression of murine cardiac Titin-cap (Tcap,

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telethonin), a key regulator of sarcomere structure and function, is regulated by the circadian mechanism transcription factors CLOCK and BMAL1.153 In a similar light,

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Andrews and colleagues have shown links between skeletal muscles and the circadian clock mechanism.154 Thus collectively, these studies indicate that the circadian mechanism plays an important and previously unrecognized role in contractile muscle

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composition and performance.

ii) Circadian Rhythms and Myofilament Signaling cAMP-dependent protein kinase (PKA) targets the myofilament complex (troponin I,

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myosin binding protein C, titin, others) and thus has a powerful ability to affect myofilament function. PKA gene expression is regulated by the cardiomyocyte clock,13

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which may help explain diurnal variations in myocardial contractility. Additionally, there is diurnal variation in β-adrenergic receptor activation, a transmembrane receptor coupled to PKA activation in cardiac myocytes.155,156 Diurnal disruption decreases myofilament protein phosphorylation by PKA following MI in mice.103 Together, these findings show the importance of circadian variations in the β-adrenergic-PKA cascade, for myofilament regulation partly under control of the cardiomyocyte clock mechanism,

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and a pathway by which changes in myofilament function can influence the ability of the heart to respond to changes in driving factors. iii) Circadian Mechanism and Cardiac Electrophysiology

time of day fluctuations in contractile proteins.

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As alluded to above, the circadian mechanism plays a role in cardiac contractility via Mechanistically, this may also be

regulated intrinsically by ion channels in the heart that influence myofilament

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activation156,157 some of which exhibit diurnal variation. Time-of day dependent oscillations have been reported in the heart for the voltage dependent potassium

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channels Kv1.5 and Kv4.2158, and the L-type voltage-gated calcium channel subunit VGCCα1D.159 Also, L-type calcium channel current densities increase during the active period which is consistent with the nocturnal elevation in murine heart rate.155,157 Moreover, circadian variations in potassium channel interacting protein-2 (KChIP2)

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affect potassium channel opening in a manner that is consistent with changes in action potential duration and circadian variations in heart rate. In vivo, the kruppel-like factor 15 (circadian output gene) knockout mice display increased susceptibility to ventricular

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arrhythmias, and reduced expression of potassium channel interacting protein 2.160 Finally, there is loss of the rhythmic sodium channel voltage-gated type V alpha subunit,

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and increased susceptibility to arrhythmias, in inducible cardiomyocyte specific Bmal1-/mice.161 These studies provide new insights for understanding the diurnal nature of cardiac electrophysiology, heart rate regulation and arrhythmias.

New Frontiers: Translational Chronocardiology

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Another exciting new frontier is knowledge translation. This is important for rhythms research to have a beneficial impact on the quality of life for people with heart disease. One area of application is chronobiomarkers, where temporal gene (microarray) and

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protein (proteomic) meta-data are used to identify new biomarkers of heart disease. A second area of application is chronotherapy, which involves timing therapies to improve the management of cardiovascular (and other) diseases. These approaches and

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relevance to clinical cardiology are described in more detail below.

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i) Chronobiomarkers

Applying circadian concepts to clinical cardiology offers new opportunities for discovering biomarkers, by a variety of different technical approaches. For example, Ueda et al. estimate tissue time-of-day in mice by measuring expression of ~100 genes

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across a molecular time table on microarray analyses162. Body-time in mice163 and humans164 can also be by estimated using metabolomics studies. We discovered timeof-day biomarkers in murine cardiac hypertrophy by microarray profiling and a novel

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time series algorithm termed DeltaGene,165 and de novo chronobiomarkers in murine plasma samples over 24 hour periods.166 Collectively, these preclinical proof-of-concept

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studies are important for designing new approaches for biomarker discovery, by taking advantage of the body’s natural 24 hour molecular rhythms. Moreover, they have practical application for chronotherapy (described below), which requires easily accessible markers of body time to optimize the timing of drug treatments.

ii) Chronotherapy Benefits (Reverses) Cardiac Remodeling

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Chronotherapy improves therapeutic efficacy (and possibly reduces toxicity) by timing treatments with our daily physiology. For example, in a preclinical study on heart disease, we show that sleep-time administration of the short acting angiotensin

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converting enzyme inhibitor (ACEi) captopril markedly reduces cardiac remodeling in mice with pressure-overload induced cardiac hypertrophy; conversely, administration at wake-time has no measureable benefit on heart damage.167 Mechanistically, the sleep-

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time benefit is associated with diurnal rhythms in RAAS and cardiac genes and proteins, which peak during the rodent sleep-time. Indeed, there is significant potential for

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chronotherapy, as it has been recently reported that many best-selling drugs and the World Health Organization essential medicines have short half-lives for directly targeting gene pathways at specific times of day or night.168

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iii) Hypertension Chronotherapy

Chronotherapy can also be effective in the treatment of hypertension. Most people have a diurnal BP profile that exhibits a dip of ~10% at night.18 Conversely,

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hypertensive non-dippers169 or normotensives with attenuated nocturnal dips in BP170 have an increased risk of heart disease. Ambulatory BP monitoring, and especially

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covering the night time, is a useful prognostic indicator for heart disease,171-173 and is recommended by many international societies.174 Chronotherapy to treat hypertension at night helps to restore the day/night BP rhythms and reduces the risk of cardiovascular disease (reviews175-178). For example, both day and night drug administration manage daytime hypertension, however, only nighttime administration of ramipril179, or telmisartan180, or valsartan and amlodipine181 restores the nocturnal BP

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dip. Furthermore, the MAPEC study demonstrated prospectively that bedtime chronotherapy but not morning administration of anti-hypertensive medication improves

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BP control and reduces cardiovascular disease risk.182

iv) Chronotherapy and Other Cardiovascular Conditions: OSA, Hemodialysis, Aspirin Chronotherapy also holds significant clinical applicability in other conditions

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associated with heart disease. For example, obstructive sleep apnea (OSA) is a common respiratory disorder of sleep, with underlying circadian-coupled mechanisms

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that if left untreated can lead to the development and exacerbation of heart disease and heart failure (reviews183-188). Conversely, nocturnal therapy by continuous positive airway pressure (CPAP) benefits patients with OSA and heart failure, reducing sympathetic activity,189,190 decreasing BP during sleep, and improving left ventricular

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function.191-194 At a molecular circadian level OSA appears to cause circadian clock gene dysfunction, and nocturnal CPAP therapy helps to improve it. This was demonstrated by comparing hPer1 mRNA expression in peripheral blood cells of

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healthy subjects, OSA patients, and OSA patients treated with CPAP.195 Several additional lines of evidence further support the notion that timing of therapy

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benefits the cardiovascular system. For example, nocturnal hemodialysis of patients with cardio-renal disease reduces left ventricular hypertrophy196,197 and improves myocardial function,198 as compared to patients given conventional day time hemodialysis. In another study, low-dose aspirin taken at night reduces the circadian rhythm of platelet reactivity in healthy individuals; it is postulated that this may also reduce the risk of acute cardiovascular events during the peak morning hours.199 Thus

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these time-dependent effects of therapies have significant potential for benefitting many patients in clinical settings.

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Conclusions and Final Directions

In conclusion, clinical and experimental studies demonstrate the important role of circadian rhythms for healthy cardiovascular physiology. Disturbing rhythms has direct

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adverse consequences on cardiac physiology and pathophysiology. Future studies will undoubtedly provide new insights into the mechanisms and molecular pathways that

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drive these pathologies. In terms of translation, significant opportunities exist to increase our understanding of circadian rhythms on cardiovascular disease. For example, experiments investigating circadian regulation of cerebrovascular conditions and stroke are virtually unknown despite this being the third leading cause of death in Canada with

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an estimated 50,000 strokes per year.200 Also, metabolic disorders are considered major risk factors for cardiovascular disease, and thus circadian rhythms research applied to prevalent clinical conditions such as diabetes and obesity could be fruitful

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areas of investigation. Determining the relationship between the circadian mechanism and cardio-pulmonary conditions (eg. OSA, others) provides new opportunities to

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improve the diagnosis and treatment of heart and lung disease. Upcoming areas for knowledge translation include development of databases for patients with heart disease concurrent with circadian and/or sleep disorders, collecting circadian biometric data (e.g. 24-hour HR and BP profiles, gene and protein biomarkers) in chronobiology and sleep clinics, and chronotherapeutic strategies to improve the effectiveness of existing treatments. Thus circadian rhythms research advances our understanding of

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cardiovascular health and disease, and leads to novel strategies for management and treatment of heart disease in clinical settings.

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Heart

and

Stroke

Foundation

of

Canada.

Statistics.

Available

at:

http://www.heartandstroke.com/site/c.ikIQLcMWJtE/b.3483991/k.34A8/Statistics.

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htm#stroke. Accessed on August, 2014.

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Figure Legends Figure 1. Circadian Rhythms in Man. A) Schematic representation of the circadian hierarchical oscillator model and the outputs that influence cardiovascular processes.

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The suprachiasmatic nucleus (SCN) in the hypothalamus synchronizes peripheral organ clocks, including the heart, to entrain to the 24h light-dark environment. B) The molecular clock mechanism keeps 24h time via transcription-translation feedback loops.

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The primary negative feedback loop consists of BMAL1:CLOCK driven PER:CRY expression, PER:CRY phosphorylation by casein kinase 1 delta/epsilon (CKd/e), and

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PER:CRY self-repression. BMAL1:CLOCK also drives REV-ERBα expression, which represses BMAL1 transcription. This mechanism regulates expression of clock controlled genes (ccg) which regulate rhythmic biological processes.

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Figure 2. Short term diurnal rhythm disturbance in the first few days post-MI significantly worsens remodeling and outcome. Mice (C57Bl/6) were subjected to left anterior descending coronary artery ligation (MI model) then randomized to either a

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normal 12:12 light:dark (L:D) environment or a disturbed L:D environment for 5 days. After only 5 days, all animals were placed in normal 12:12 L:D for the duration of the

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experiment (8 weeks). Histologic analyses reveal that the disrupted-MI mice had greater left ventricular dilation and infarct expansion by 8 weeks post-MI compared to the normal-MI mice (Masson’s Trichrome). Overall, we showed that short term disturbance of circadian rhythms following myocardial infarction (MI) impairs alters immunocyte recruitment to infarcted myocardium and impairs healing; conversely, maintaining rhythms is crucial for benefiting wound healing and outcome.103

These preclinical

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findings offer promise for clinical translation, as maintaining diurnal rhythms in intensive and acute care units in the first few days post-injury can benefit healing and outcome. = normal diurnal environment.

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= disturbed L:D environment.

Figure 3. The Diurnal Cardiac Sarcomere. A) The cardiac sarcomere, delineated by two Z-discs, sits at the functional centre of myocytes. B). Cardiomyocyte contraction is a

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calcium-dependent interaction, with steric modulation by the troponin-tropomyosin complex, leading to myosin and actin cross-bridges. ATP cleavage by myosin heads

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provides energy for the power stroke that pulls actin towards the centre of the cell and produces cell shortening. We have shown that cross-bridge cycling exhibits a circadian rhythm and that disruption of CLOCK abolishes the diurnal cycling of cardiac

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myofilament activation.81 C). Key elements of the cardiac sarcomere.

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Figure 1. Circadian Rhythms in Man. Alibhai et al. B.

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A.

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Alibhai et al – Figure 2

Murine MI model, animals are randomized to either normal or short term diurnal rhythm disturbance post-MI

Short term diurnal rhythm and sleep disturbance (5 days)

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Normal diurnal environment

Normal diurnal environment

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8w post-MI (progression to heart failure)

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normal remodeling

worse remodeling and outcome

Short term diurnal rhythm and sleep disturbance in the first few days post-MI significantly worsens remodeling and outcome.

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Figure 3. The Diurnal Cardiac Sarcomere. Alibhai et al.