Circadian Disruption and the Molecular Clock in Atherosclerosis and Hypertension

Abstract

Circadian rhythms are crucial for maintaining vascular function and disruption of these rhythms are associated with negative health outcomes including cardiovascular disease and hypertension. Circadian rhythms are regulated by the central clock within the suprachiasmatic nucleus of the hypothalamus and peripheral clocks located in nearly every cell type in the body, including cells within the heart and vasculature. In this review, we summarize the most recent preclinical and clinical research linking circadian disruption, with a focus on molecular circadian clock mechanisms, in atherosclerosis and hypertension. Furthermore, we provide insight into potential future chrono-therapeutics for hypertension and vascular disease. A better understanding of the influence of daily rhythms in behavior, such as sleep/wake cycles, feeding, and physical activity, as well as the endogenous circadian system on cardiovascular risk will help pave the way for targeted approaches in atherosclerosis and hypertension treatment/prevention.

Graphical Abstract.

INTRODUCTION

Hypertension remains the leading modifiable risk factor for cardiovascular disease (CVD) and all-cause mortality globally. This is partly due to high blood pressure (BP) being a major contributor to atherosclerosis, which itself is a dominant cause of most cardiovascular diseases^1–3. Many cardiovascular parameters display circadian rhythms, which naturally recur approximately every 24 hours, including BP, heart rate (HR), vascular endothelial function, coagulation factors, circulating catecholamines, and vasoconstrictors (e.g. endothelin-1 (ET-1)) and vasodilators (e.g. acetylcholine) release (reviewed in⁴). Disruption of circadian rhythms is associated with negative health outcomes including cardiovascular disease and hypertension⁵. Importantly, there are increased frequencies in adverse cardiovascular events during the morning hours compared with the rest of the day and at night^6–8. Furthermore, BP that does not dip at night (<10% decrease from daytime to nocturnal BP), as well as nocturnal hypertension, increases the risk for hypertension-induced organ damage^9,10 and cardiovascular mortality¹¹. Together, emphasizing the importance of understanding the influence of daily rhythms in behavior, such as sleep/wake cycles, feeding, and physical activity, as well as the endogenous circadian system on cardiovascular risk.

The circadian clock, a highly conserved system, has evolved to prepare our body for the anticipated 24-hour light/dark cycle and allow for predictive adaptations to meet the demands of the environment. The circadian system is composed of the central clock within the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clocks located in nearly every cell type in the body, including cells within the heart and vasculature. The central clock has long been referred to as the “master clock” that synchronizes the peripheral clocks, but peripheral clocks have been shown to be cell autonomous. Yoo et al. found peripheral clocks, such as in the liver and kidney, are capable of self-sustained oscillations independent of input from the central clock. Furthermore, rhythms differed between tissues suggesting the presence of tissue-specific zeitgebers (timing cues)¹². Circadian rhythms are driven by a transcription-translation feedback loop, referred to as the molecular clock, that occurs roughly every 24 hours, involving core clock proteins Brain and muscle ARNT-Like 1 (BMAL1), Circadian Locomoter Output Cycles Protein Kaput (CLOCK), Period (PER) and Cryptochrome (CRY). BMAL1 and CLOCK act as the positive arm of the circadian clock as BMAL1/CLOCK heterodimer bind to E-box response elements within promoter regions of target genes to induce transcription. PER and CRY are transcribed and once translated, translocate back into the nucleus to inhibit BMAL1/CLOCK, acting as negative feedback. The secondary regulatory loop involves transcription of REV-ERBα/β, also known as nuclear receptor subfamily 1 group D member 1/2, and RAR Related Orphan Receptor (ROR)α/β which mediate opposing actions to either inhibit or activate BMAL1 transcription via interaction with a ROR response element. A tertiary feedback loop (D-loop) also exists which involves D-site albumin-binding protein/Thyrotroph embryonic factor/Hepatic leukemia factor (DBP/TEF/HLF) and Nuclear factor, interleukin 3 regulated (NFIL3) binding to D-box response elements in promoter regions of RORα and RORβ (Figure 1). These regulatory loops mediate the rhythmic expression of nearly 50% of all genes expressed. For detailed review of what is currently known regarding the clock mechanism, see^13–15.

Figure 1.

Core components of the molecular clock. A transcription-translation feedback loop involving transcription factors BMAL1 and CLOCK that bind to E-box response elements within promoter regions of target genes, including PER1/2/3, CRY1/2, REV-ERBα/β, RORα/β, DBP, TEF, and HLF (positive arm). Once translated, PER and CRY dimerize and translocate back into the nucleus inhibiting BMAL1/CLOCK to repress their own transcription (negative arm). ROR activate BMAL1 and NFIL3 transcription via interaction with ROR response elements, whereas REV-ERB inhibit BMAL1 and NFIL3 (secondary loop). DBP, TEF, and HLF interact with D-box response elements to activate PER1/2/3, REV-ERBα/β, and RORα/β transcription, whereas NFIL3 represses this (tertiary loop). Created with Biorender.

In this review, we discuss the importance of circadian rhythms and the circadian clock in maintaining vascular function and BP, and the most recent advances from preclinical and clinical research in circadian disruption in atherosclerosis and hypertension. Furthermore, we consider future chrono-therapeutics in hypertension and vascular dysfunction prevention.

CIRCADIAN RHYTHMS IN THE VASCULATURE

Circadian rhythms in cardiovascular and endothelial function are well established. Blood flow¹⁶ and the sensitivity of blood vessels, such as the aorta, mesenteric, and renal arteries, to vasoconstrictors or vasodilators varies according to the time-of-day both ex vivo^17–20 and in vivo in rodents^21,22, and in humans^23,24, which can subsequently influence time-of-day variations in BP. Indeed, upon awakening, there is an elevation in BP, accompanied with increased levels of blood coagulation factors, increased catecholamines⁸, angiotensin II²⁵, and cortisol levels²⁶. BP then plateaus throughout the day before a nocturnal decline by 10–20%. This pattern also occurs in nocturnal animals, where BP peaks during the night which is the animal’s active period.

The circadian clock is present in all cardiovascular cell types²⁷. Oscillating clock gene expression has been confirmed in the human heart where they found rhythmic gene expression of BMAL1, CRY1, PER1, and PER2 from left papillary muscle of male and female patients undergoing orthotopic heart transplantation²⁸. This has also been shown in rodent hearts^27,29, as well as cultured cardiomyocytes³⁰. An important study by Davidson et al. found, using Per1::Luciferase rats to measure Per1 gene oscillation by bioluminescence, that Per1 was rhythmic in heart tissue explants and cultured arteries and veins. Moreover, the authors, as well as others, have found that circadian clock oscillations differed across the vascular tree, suggesting the clock mechanism is more complex than the current paradigm and may function differently across the vasculature tree^27,31. Mouse models of knocking out clock genes emphasize the importance of the clock in vascular function, with knockout (KO)s exhibiting BP changes^20,32, endothelial dysfunction³³, smooth muscle dysfunction²⁰, and remodeling of the vasculature³⁴. The role of specific clock genes in vascular function and BP regulation is discussed in greater detail below.

With the clock being present in cardiovascular tissues, it is reasonable to hypothesize that some genes are rhythmically expressed in these tissues. A previous problem in studies investigating this was lack of resolution with only 2 time points. Several informative studies including Rudic et al.³⁵ and Zhang et al.¹⁴ focused on addressing this gap in our knowledge. Rudic et al. performed a microarray on aorta every 4 hours for 48 hours from 4–6-week-old male C57BL/6 mice that were placed in conditions of constant darkness to assess free-running circadian rhythms (i.e. a rhythm that is not entrained by 24-hour light/dark cycle). Three hundred and thirty genes (5–10% of the transcriptome) were rhythmic and were relevant to the circadian clock, protein folding and degradation, vascular integrity, glucose and lipid metabolism, and response to injury³⁵. Zhang et al. performed RNA sequencing (RNAseq) on multiple tissues, including heart and aorta, from 6-week-old male C57BL/6 mice placed in constant darkness. RNAseq data showed that 6% and 4% of protein-coding genes are transcribed in a circadian manner in the heart and aorta, respectively. Interestingly, examples of rhythmic genes were Vegfa (vascular endothelial growth factor) and its two membrane-bound receptors Flt1 and Kdr, which are involved in angiogenesis¹⁴. With these studies focused on males, a key consideration for future studies should be whether there are sex-specific differences in rhythmically expressed genes in cardiovascular tissue and whether changes in the clock and/or the rhythmicity of these genes have implications in pathophysiology in males and females.

THE CIRCADIAN CLOCK IN VASCULAR DYSFUNCTION AND HYPERTENSION

Disturbance of the clock machinery has been associated with increased incidence of hypertension, coronary artery disease, atherosclerosis and other cardiovascular pathologies in both human and animals. In this section, we will discuss several genetic animal models that have been key to understanding the influence of the clock in cardiovascular function.

BMAL1

Studies from global and tissue-specific Bmal1 deficient rodents have increased our understanding of the role of clock protein BMAL1. Mice lacking BMAL1 are behaviorally arrhythmic, exhibit a wide range of organ damage, and have shortened lifespans. They also exhibit lowered BP, with a lack of diurnal variation in both BP and HR^32,36. A study by Lefta et al. found Bmal1-deficient mice have age-associated decreases in cardiac performance, thinning of the myocardial walls, and dilation of the left ventricle, all characteristics of dilated cardiomyopathy³⁷. Furthermore, these mice exhibit arrhythmias, hyperglycemia³⁶, and dyslipidemia³⁸. Interestingly, studies performed in global Bmal1 KO Sprague Dawley rats showed a similar BP lowering phenotype as seen in mice, but maintained the circadian rhythm of BP³⁹. An effort has been made using genetic models to understand the role of the clock in individual cells types. Selective deletion of BMAL1 from vascular smooth muscle cells (VSMCs) in mice compromised BP circadian rhythm with a decreased BP²⁰. Furthermore, BMAL1 in the perivascular tissue⁴⁰, liver⁴¹, adrenal gland⁴², and in segments of the kidney has been shown to play a role in BP control^43–46 (further details of these studies are reviewed in ^47,48). These studies highlight the vital role of BMAL1 in BP homeostasis.

BMAL1 gene expression and rhythm, as well as changes in PER1–3, CRY1/2, and CLOCK expression, were attenuated in human plaque-derived VSMCs, suggesting the clock may be involved in the process of atherosclerosis⁴⁹. Studies have shown that global or liver-specific deletion of BMAL1 in mice induces hyperlipidemia and enhances atherosclerosis⁵⁰. Anea et al assessed whether BMAL1 disruption altered the response of blood vessels to chronic reduction in blood flow, which simulate vascular remodeling during atherosclerosis and hypertension. They found global Bmal1 KO mice display increased pathological remodeling, with increased wall thickening, vascular injury, and endothelial dysfunction, which they suggested was due to an impairment of nitric oxide signaling which is critical to vascular function³⁴. VSMC-specific Bmal1 KO mice exhibit aggravated atherosclerosis lesions by promoting VSMC migration and monocyte transmigration by inhibiting Rac Family Small GTPase 1 (RAC1) activity⁵¹. Further evidence for the role of BMAL1 in atherosclerosis was highlighted in a study where wild-type mice developed robust arteriosclerotic lesion following transplantation of aortic graft from Bmal1-deficient mice⁵². So far, the absence of functional BMAL1 is detrimental, but it has been found to be beneficial for cardiovascular health. For example, Yang et al. found reduced atherosclerosis lesion burden following high fat diet in both male and female mice lacking BMAL1 in myeloid cells, delaying atherogenesis and limits the formation of abdominal aortic aneurysm⁵³. In addition to this, this deletion caused a reduced pro-inflammatory response in the aorta during atherogenesis⁵³. HR and BP remain unaltered in these mice. Together, these studies highlight that BMAL1 is important in physiological function, with an emphasis on inflammation, which when disturbed can contribute to progression of atherosclerosis. It should be noted that these studies were predominately performed in male animals so there is a need to investigate the role of BMAL1 in females.

CLOCK

Disruption and/ or mutation of CLOCK in mice causes reduced lifespan⁵⁴ and impairs circadian behavior⁵⁵. Curtis et al. demonstrated mutated Clock (Clock^mut) male mice exhibit disrupted circadian variation in BP and HR, which was also seen in global Bmal1 KO mice previously discussed³². Notably, Clock^mut mice showed disrupted BP during the light phase and HR during the dark phase³². Zuber et al. showed that Clock KO male mice on a C57BL/6 background resulted in lower BP compared to wild-type mice, with no changes in BP rhythm⁵⁶. Additionally, Clock^mut mice on a Jcl/ICR background showed dampened diurnal rhythm of BP and HR, interestingly with the effect abolished after adrenalectomy, suggesting a role for the adrenal gland in diurnal rhythms of BP and HR⁵⁷.

Carotid atherosclerotic plaques are associated with hypercholesterolemia and hyperglycemia. It has long been known that glucose and lipid homeostasis exhibit circadian variation^58,59, however the specific clock mechanisms are not completely understood. Rudic et al. demonstrated Clock^mut mice exhibited suppressed diurnal variation in glucose and triglycerides³⁸. Clock^Δ19/Δ19ApoE^−/− mice displayed both hypercholesterolemia and hyperglycemia, and enhancement of atherosclerosis⁶⁰, suggesting a protective role for CLOCK in atherosclerosis progression. This is supported with a study assessing carotid artery plaques from patients with carotid artery stenosis and found that carotid artery plaque was strongly associated with loss of CLOCK expression⁶¹. Together, highlighting a role for CLOCK in glucose and lipid homeostasis which could influence atherosclerosis development/progression.

PER1/2

Studies from our group show that PER1, part of a negative arm of the clock which inhibit BMAL1:CLOCK activity, is important for BP regulation in different strains of mice^62,63. Interestingly, this was only found in male mice, with global female Per1 KO mice being protected from the salt-sensitive (SS) non-dipping hypertension observed in male Per1 KO^62–64, suggesting a sex-specific role of PER1 in BP regulation. A role for PER1 in BP regulation was also supported by a recent study where global Per1 KO Dahl SS male rats displayed an exacerbated hypertensive phenotype following a high salt diet, which was associated with loss of circadian synchrony in mean arterial BP⁶⁵. The circadian rhythm of HR was altered in Dahl SS Per1 KO rats as well, with KO rats exhibiting a significantly lower amplitude of HR. Little is known about the role of PER1 in vascular function, requiring future investigation. PER2 has been shown to play a role in maintenance of cardiovascular function. Per2 mutant mice had aortic endothelial dysfunction involving diminished production of nitric oxide and prostaglandins, which was associated with increased release of vasoconstrictor cyclooxygenase-1³³. To examine the mechanism behind PER2 influencing endothelial function, endothelial cells were assessed in Per2 mutant mice. This study found that Per2 mutation led to Akt-dependent premature vascular senescence, which was associated with both impaired endothelial progenitor cell function and angiogenesis⁶⁶.

CRY1/2

CRY1/2 are also part of a negative feedback loop of the clock. Doi et al. demonstrated that male mice lacking both Cry1 and Cry2 (Cry-null mice) exhibited SS hypertension⁶⁷. This effect was associated with increased mRNA levels of Hsd3b6 (3β-hydroxyl-steroid dehydrogenase), a member of the steroid synthesis pathway, which lead to an enhanced enzymatic activity of 3β-HSD (3β-hydroxysteroid dehydrogenase-isomerase) resulting in an augmented production of aldosterone that contributes to hypertension⁶⁷. CRY1/2 are also important in generating the circadian rhythm of baroreflex sensitivity. Cry-null mice showed increased BP and HR in both light and dark phase. These mice also had amplified baroreflex sensitivity which was partially regulated by α1-adrenoceptor-mediated vasoconstriction²¹.

Atherosclerotic patients have been shown to have lower serum CRY1 mRNA levels⁶⁸. Whether CRY1 is involved in progression of atherosclerosis is not known but a study by Yang et al. showed overexpression of Cry1 in ApoE^−/− male mice significantly attenuated the expression of pro-inflammatory cytokines including Interleukin (IL)-6, IL-1, Tumor Necrosis Factor (TNF)α, and Nuclear Factor kappa B (NF-kB). Additionally, these mice also had reduced plasma levels of total cholesterol, triglyceride, and low-density lipoprotein (LDL) cholesterol, and they are protected from plaque development⁶⁸. Together these studies demonstrate a critical role for CRY1 and CRY2 in maintaining normal vascular function.

REV-ERBα/β and RORα/β

Limited work has investigated the role of REV-ERBs and RORs in development and/progression of atherosclerosis and hypertension. Work over 20 years ago found that, using the staggerer mouse (spontaneous mutation in Rorα gene), RORα is required for normal contractility in VSMCs in small resistance arteries⁶⁹, and contributes to plasma high-density lipoprotein (HDL) levels and the development of atherosclerosis after exposure to an atherogenic diet⁷⁰. Since then, there have been limited studies in this area. Song et al. have generated cardiomyocyte-specific Rev-erbα/β double KO mice and recently found these mice have heart failure with dilated cardiomyopathy⁷¹. Interestingly, studies have explored candidate drug molecules to target REV-ERBα/β in cardiovascular disease, which is discussed in more detail below.

DBP/TEF/HLF

Again, limited studies have examined the role of the tertiary feedback loop (D loop) in vascular function. Dbp/Tef/Hif triple KO mice develop cardiac hypertrophy and left ventricular dysfunction associated with low BP⁷². Catecholamines, potent vasoactive hormones, have been suggested as central clock signals to entrain peripheral clocks, including the vascular clocks. Norepinephrine and epinephrine, via activations of β2 and α1-adrenergic receptors, are able to alter expression of Dbp expression in both mouse and human aortic VSMCs in vitro. Although, this was not the case in vivo as in mice that cannot synthesize norepinephrine and epinephrine (dopamine β-hydroxylase (DBH) KO) or adrenergic receptor antagonism, peripheral oscillations were not altered⁷³. An effort has been made to examine how vascular Dbp is regulated, but additional work is needed to understand vascular Dbp function.

EVIDENCE OF CIRCADIAN DISRUPTION IN ATHEROSCLEROSIS AND HYPERTENSION RISK

Disruption in circadian rhythms is defined as misalignment between behavioral rhythms and endogenous circadian rhythms. Animal models have increased our understanding of the consequences of circadian misalignment. One in particular is the tau mutant hamster, which have a single point mutation in the gene coding casein kinase 1 ε. These hamsters have shortened circadian periods, with homozygotes having 20-hour period, heterozygotes 22-hour compared with 24 hours in wild-type⁷⁴. Interestingly, heterozygous tau mutant hamsters have decreased longevity, severe cardiac hypertrophy, myocardial fibrosis, and collagen deposition when maintained on their normal 14:10-hour light/dark cycle, which was not seen in homozygous mutants even though they are arrhythmic⁷⁵. Remarkably, when these hamsters were maintained on a 22-hour circadian period (12:10-hour light/dark), which aligns with their endogenous circadian rhythm, this prevented the cardiac phenotype and restored longevity. A similar result was also found when the central clock was removed by SCN ablation in tau mutants maintained on a 14:10-hour light/dark cycle, providing evidence for uncoupling of central and peripheral clocks driving this cardiac phenotype. These data emphasize that circadian misalignment has worse cardiac outcomes compared with lack of rhythms^74–76. Circadian misalignment often seen in shift workers has been associated with increased risk of cardiovascular disease and hypertension in humans⁵. In this section, we discuss the influence of circadian disruption, caused by shift work, artificial/abnormal light exposure, sleep disorders, and aging, on atherosclerosis and hypertension in both human and animal studies and explore potential mechanisms behind this.

Shift work

Shift work, referring to working outside the typical working hours of 7 AM and 6 PM, has been reported to be associated with increased risk of cardiovascular disease and hypertension⁷⁷. An estimated 12% of the working population in Canada reported exposure to shift work⁷⁸. Studies have explored the association between shift work and atherosclerosis. Haupt et al. recruited 2510 subjects >45 years old where 698 were previously shift workers and performed carotid ultrasound to evaluate carotid intima-media thickness. They found that shift work was associated with both atherosclerosis and myocardial infarction (MI) but was dependent on duration of exposure as shift work exposure of >20 years had the highest risk of MI⁷⁹. Furthermore, a study assessing the relationship between shift work and subclinical atherosclerosis was also conducted in young adults (24–39 years old) and found that shift work accelerated atherosclerotic processes in males but not females⁸⁰. Similarly, in a study of 3582 Chinese steelworkers assessing the association between rotating night shift work and carotid atherosclerosis, they found that male rotating night shift workers had higher odds of carotid plaque, which was not seen in females⁸¹. The mechanisms behind the sex differences seen in these studies merits further investigation.

Results from studies evaluating the risk of hypertension on shift workers remains inconclusive. A meta-analysis of 27 studies, with a total of 394,793 individuals, found an association between shift work and hypertension in male shift workers. When assessing specifically night shift workers, there was no association in shift work at night and higher risk of hypertension⁸². In contrast, a study assessing risk in 2151 shift/night workers found greatest risk for hypertension in individuals who works 95–100% night shift⁸³. A few reported no association between shift work and hypertension^84,85. This inconsistency could be due to differences in many factors such as working environments, type/duration of rotating schedules, controlling for variables such as smoking status and body mass index (BMI), and regularity of BP monitoring. Ensuring consistency and reporting of the timing of BP monitoring is important as BP is rhythmic, as previously described. Measuring ambulatory BP accounts for 24-hour BP variation. Interestingly, shift work has been reported to change BP dipping status on initial days of working but reverts to dipping pattern after 4 days⁸⁶. There have been limited studies investigating the impact of shift work on BP rhythm. Hypertensive patients can present with abnormal BP rhythm including non-dipping, reverse dipping (nocturnal risers), and extreme dipping (>20% between nocturnal BP and daytime BP), and alterations in rhythm as well as amplitude of BP worsens cardiovascular outcome^10,87. A recent meta-analysis of 50 studies concluded BP dipped during sleep in shift workers but studies again varied on duration and type of shift work⁸⁸. Further research to evaluate the effect of shift work on BP rhythm in individuals with and without hypertension is needed.

Studies in humans and rodents have explored mechanisms behind the association between circadian misalignment and cardiovascular risk using shift work/jet lag protocols. An example of this is a forced desynchrony protocol where 10 participants were subjected to a 28-hour behavioral cycle under dim light, causing an increase in wake BP by 3 mmHg with sleep BP not assessed⁸⁹. This was accompanied with decreased sleep efficiency. Another protocol relevant to shift workers is the circadian misalignment protocol, which involves a 12-hour inverted behavioral and environmental cycles for 3 days. Full details of this protocol are described in⁹⁰. Morris et al. found a similar magnitude of BP increase but was mainly driven by increases in sleep BP, and reduced systolic BP dip, which are both predictors of adverse cardiovascular events and all-cause mortality. HR was increased during sleep but decreased during wake periods. Furthermore, circadian misalignment increased systemic inflammatory marker C-reactive protein (CRP) and pro-inflammatory markers TNF-α and IL-6⁹⁰. Changes in inflammatory status was also found in hyperlipidemic heterozygous APOE 3-Leiden. Cholesteryl Ester Transfer Protein (CETP) female mice exposed to weekly alternating 12-hour light/dark cycles to model shift work. Researchers found that weekly light shifts increased atherosclerosis development with increased lesion macrophages, increased gene expression of pro-inflammatory markers, including Tnfα, and increased expression of the chemokine CCL2 in the aorta vessel wall⁹¹.

Artificial/abnormal light exposure

Artificial and abnormal light exposure is common in today’s society and can have negative impacts on our health, as discussed above. Even an advance of 1 hour during daylight savings transition sees increased reports of cardiovascular events^92–94. There is now proposed legislation for a shift to permanent daylight saving time in Canada. For a very informative perspective of the potential cardiovascular implications on this legislative change, see⁹⁵. The use of light-emitting electronic devices has drastically increased in recent years. A representative survey of 1508 adults from the United States revealed 90% used a light-emitting electronic device 1 hour before bed at least a few times a week⁹⁶. Exposure to light at night can impact the circadian clock, as the central clock is entrained by light and it causes a phase-delay as well as acute suppression of melatonin, longer sleep latency, and impaired morning alertness⁹⁷. Furthermore, exposure to light at night is associated with higher prevalence of cardiovascular risk factors like hypertension⁹⁸ and was found to be associated with increased nighttime BP in elderly individuals⁹⁹. Abnormal light exposure also occurs in the intensive care unit (ICU) where patients are critically ill. Light levels have been assessed in ICU, with daytime light levels between 30–165 lux compared with a sunny day between 32000–60000 lux. Night levels ranged from 2.4–145 lux and during procedures varying throughout the day can be a maximum of 10000 lux¹⁰⁰. This constant exposure to light can have negative impacts on health. This was explored in ApoE^−/− mice to test the impact of light exposure on atherosclerosis. Male, but not female, ApoE^−/− mice housed in constant light for 12 weeks had exacerbated atherosclerosis and increased total serum cholesterol compared with mice on a normal 12-hour light/dark cycle¹⁰¹. Interestingly, the same research group found the same finding with exacerbated atherosclerosis and increased total serum cholesterol concentration but only in female ApoE^−/− mice when exposed to a chronic light/dark shift, where the light/dark cycle was advanced by 6 hours every week for 12 weeks¹⁰². This suggests there are sex-specific differences in response to misaligned or arrhythmic circadian disruption. The mechanisms behind these differences merit investigation.

Preclinical studies have also looked at simulating the ICU. Alibhai et al. investigated the effect of short-term circadian disruption following MI in male mice. After MI, mice were exposed to 5 days of “ICU simulation” (10-hour light/dark cycle) before returning to a normal 12-hour light/dark cycle. This short-term circadian disruption caused impaired healing and adverse cardiac remodeling, likely to negatively affect prognosis¹⁰³. This supports a role for the circadian mechanism in inflammatory responses and highlights the potential risks of our society’s abnormal light exposure.

Poor sleep

Sleep health, including duration, timing, regularity, efficiency, satisfaction, and impact on daytime alertness, has been recently included as an important component for cardiovascular health in American Heart Association Life’s Essential 8¹⁰⁴. Poor sleep has been linked with cardiovascular risk factors and outcomes^105–108. There is also evidence suggesting irregular sleep increases the likelihood of subclinical atherosclerosis^109–112. This comes from the Multi-Ethnic Study of Atherosclerosis (MESA) study, which is a study of 6814 asymptomatic men and women aged 45–84 from racially and ethnically diverse backgrounds across the United States to investigate the characteristics of subclinical cardiovascular disease and risk factors that predict progression. Participants with greater sleep duration irregularities were more likely to have a higher burden of atherosclerosis, with higher coronary artery calcium (CAC) and abnormally low ankle brachial index (ABI)¹¹². Individuals with obstructive sleep apnea (OSA), characterized by repetitive episodes of cessation of inspiratory airflow lasting ≥10 seconds, in the MESA study was associated with prevalent CAC only¹⁰⁹, with short (<6 hours) and long (>8 hours sleep) associated with abnormal ABI¹¹⁰.

Individuals that suffer from short sleep and/or OSA have increased risk for hypertension as well, with the more severe the OSA, the higher the hypertensive risk (recently reviewed in¹¹³). Severe OSA has also been linked with greater odds of having non-dipping BP (moderate to severe OSA, 67% higher odds), suggesting OSA can influence BP variation¹¹⁴. Furthermore, patients with non-dipping BP have a higher risk of OSA, creating a disruptive circadian loop¹¹⁵.

Together, these findings highlight that sleep health is indeed an important component of cardiovascular health and consistent sleep durations, timing, and regularity may be an important lifestyle recommendation for prevention of cardiovascular disease and hypertension.

Aging

Aging promotes the development and progression of hypertension and atherosclerosis. The proposed mechanisms behind this phenomenon are complex and include, but are not limited to, chronic inflammation, oxidative stress, and cell senescence. For detailed reviews on potential mechanisms behind aging promoting atherosclerosis, see^116–119. Furthermore, the process of aging causes changes in circadian rhythms including activity and sleep rhythms, as well as hormone levels including melatonin^120–122. Aging reduces the number of rhythmically expressed genes in multiple tissues, including the heart, of C57BL/6 male mice¹²³. Interestingly, the heart showed the least change in rhythmic genes from young (6 months) to old mice (27 months) with a 34% decline compared with other tissues examined such as the kidney with a ~75% decline. The aged related changes in gene expression in the heart of old mice were enriched for stress responses. This study highlights the impact of aging on the circadian clock function and could subsequently influence the development and progression of hypertension and atherosclerosis. Further work is needed to explore the role of the molecular circadian clock in vascular aging in both males and females.

CHRONOTHERAPY

Modifiable risk factors such as diet, exercise, and BP are targeted to treat cardiovascular diseases¹²⁴. Here, we have highlighted that a disrupted circadian system is a potential novel mechanism contributing to atherosclerosis and hypertension. A promising therapeutic approach for targeting/preventing atherosclerosis and hypertension is circadian medicine, with the goal to leverage the power of circadian biology to optimize therapeutic strategies. Below, we discuss recent and ongoing clinical trials in potential circadian therapeutic approaches that could be beneficial to individuals with atherosclerosis and/or hypertension.

Chrono-medicine

Many pharmacological targets for atherosclerosis and hypertension display rhythmic gene expression, such as targets for angiotensin II receptor blockers, β-adrenoceptor blockers, and aldosterone receptor blockers, with some prescription drugs having a half-life of ≤ 6 hours¹⁴. Therefore, time-optimized treatment could be beneficial for these patients. Several clinical trials have examined the effects of bedtime dosing of antihypertensive drugs on nighttime BP dipping and cardiovascular outcomes. However, the effects have been conflicting. The HOPE (Heart Outcomes Prevention and Evaluation) Study, Syst-Eur (Systolic Hypertension in the Elderly), and Syst-China (Systolic Hypertension in China) found evening dosing, alongside morning dosing for management of BP in most patients, reduced the risk of cardiovascular outcomes^125–127. Bedtime compared with after awakening dosing was evaluated in 2156 hypertensive Spanish patient (the MAPEC study; Ambulatory BP Monitoring for Prediction of Cardiovascular Events). Bedtime dosing improved BP, as well as dipping profile, and reduced total cardiovascular events (MI, coronary revascularization, and heart failure) by 61%¹²⁸. Similar findings were also reported in the HYGIA trial, which evaluated 19084 hypertensive patients¹²⁹. These studies provide promise of chronotherapy however, limitations of the study design have been noted as there was a high risk of bias, as well as number and class of antihypertensive drugs prescribed varied^130–133. Several studies have found no such benefit of evening dosing of antihypertensive drugs^134–136. With the need for an independent, large, randomized trial to investigate whether evening dosing, compared with morning, of usual antihypertensive drugs improved cardiovascular outcomes, the Treatment in Morning versus Evening (TIME) study was designed with >20000 participants; results were recently published in The Lancet¹³⁷. The study found no difference in major cardiovascular outcomes between morning and evening dosing, with the authors suggesting dose time should not be a significant consideration in BP management. It is worth noting that the patients were not stratified by dipping status, so perhaps nighttime drug administration would still benefit non-dippers.

Recent studies have explored candidate drug molecules to target the circadian clock, such as REV-ERBα/β (an inhibitor of BMAL1), for treatment of various disease states. Previous studies have shown decreased REV-ERBα/β in the heart of cardiomyocyte-specific Bmal1 KO mice, and the dysfunction in the heart clock was associated with alterations in cardiac mitochondrial function, metabolism, signaling, and contractile function. SR-9009, a selective REV-ERBα/β agonist, normalized cardiac glycogen synthesis rates, cardiomyocyte size, interstitial fibrosis, and contractility in these mice¹³⁸. Interestingly, REV-ERBα antagonism with SR8278 or Reverbα gene KO in isolated mouse heart ameliorated MI when administered at the onset of the active period in rodents¹³⁹. These studies highlight a potential versatile, but complex, role of targeting REV-ERBα/β in cardiovascular disease. Several studies have explored different compounds targeting the circadian clock machinery (reviewed in¹⁴⁰), but there is a demand for future work to examine the effects of these drugs on preventing/treating atherosclerosis and hypertension. At present, there are several clinical trials ongoing investigating chronotherapeutic interventions for treatment of hypertension and cardiovascular disease which are summarized in Table 1.

Table 1.

Ongoing clinical trials investigating chronotherapeutic interventions for treatment of hypertension and cardiovascular disease

Clinical trial	Participants details	Duration	Study design	Expected study completion
Chronotherapy in Hypertension – a Study of Blood Pressure Levels Following Intake of Antihypertensive Medication in the Morning or at Bedtime (NCT05322967)	120 participants aged 40–75 years on stable anti-hypertensive treatment and blood pressure <150/95 mmHg	1 year	Changes in blood pressure, renal function, LDL and HDL-cholesterol after taking antihypertensive medication in the morning or at bedtime.	March 1, 2023
The SleepWell Study (NCT05299723)	20 patients who experienced acute coronary syndrome (ACS)	1 year	Combined chronotherapy intervention consisting of morning bright light therapy and evening blue light blocking administration.	April 15, 2023
Diurnal Blood Pressure in those at Increased Risk of Cardiovascular Disease (NCT04522765)	120 participants with increased risk of cardiovascular disease	3.5 years	Ambulatory blood pressure and arterial stiffness with day and night blood and urine sampling assessed in patients at increased risk of cardiovascular disease compared with health controls	August 1, 2023
Sleep, Hypertension, and Nocturia: a Multicomponent Approach for Comorbid Illnesses	30 community-dwelling older adults (>65 years old) who take ≥ 1 daily non-diuretic antihypertensive medication, with a mean systolic blood pressure >135 mmHg and awake ≥ 2 times nightly to void	1 year	Patients assigned behavioral sleep intervention to improve sleep and nocturia or switching time of antihypertensive administration	June 30, 2023
SHIFTPLAN (NCT05452096)	176 shift workers	9 months	Effect of shift work interventions, including ergonomic shift scheduling and an educational program, on fatigue, sleep and health	August, 2023
Effect of Antihypertensive Medication Timing on Morbidity and Mortality (BedMed; NCT02990663)	3440 hypertensive patients (estimated enrolment)	4 years	Patients assigned to ingest ≥ 1 hypertension medication in the morning or evening.	December 31, 2023
BedMed Frail (NCT04054648)	1200 hypertensive patients who are residents in a participating long-term care facility (estimated enrolment)	2 years (estimate)	Patients assigned to ingest ≥ 1 hypertension medication in the morning or evening.	January, 2024
Exercise and Vascular Function in Postmenopausal Females with Hypertension (NCT05597033)	47 postmenopausal females with hypertension	1 year	Exercise performed in the morning versus early evening on blood pressure and measures of blood vessel health in postmenopausal females with hypertension	February 15, 2024
Treatment of Hypertension During Sleep (THADEUS; NCT03457168)	5320 hypertensive individuals	>10 years	Effects of intensive control of asleep systolic blood pressure proposed by new ACC/AHA guidelines (<100 mmHg) versus conventional control (<120 mmHg) to reduce cardiovascular morbidity and mortality in hypertensive individuals	March, 2031

Sleep therapy

Sleep therapy by encouraging maintenance of regular sleep schedules and durations may be a potential intervention to reduce cardiovascular disease risk as the MESA study previously described showed that irregular sleep duration and timing were associated with measures of subclinical atherosclerosis¹¹². Clinical trials to investigate if improving sleep regularity as a lifestyle intervention can reduce cardiovascular risk are needed.

Light therapy

The consequences of abnormal light exposure, seen in shift work, has been previously discussed. Visible blue light exposure, at doses similar to sunlight, decreased BP and arterial stiffness and increased endothelial function by nitric oxide release in 14 healthy male individuals¹⁴¹. This could have benefits for shift workers. In fact, a study found that 12-week light therapy improved BP dipping status and glucose tolerance, with reduced plasma catecholamine levels, in 24 rotating night shift workers¹⁴². Future work into the potential short- and long-term benefits of light therapy for individuals who are shift workers or patients in the ICU is warranted.

Time-restricted feeding

Time-restricted feeding (TRF), where the eating window is confined to <12 hours, has recently emerged as a potential therapeutic for improving cardiometabolic health. In male mice, TRF to their rest period caused BP rhythm to be inverted, so peaked during the rest period instead of active with no change in 24-hour BP¹⁴³. Therefore, suggesting feeding entrained BP rhythm in mice. Five weeks of daytime TRF restricted to 6 hours lowered BP, but had no effect on arterial stiffness, LDL or HDL cholesterol in prediabetic men¹⁴⁴. Although, 12 weeks of daytime TRF for 10 hours did lower LDL cholesterol in men and women with metabolic syndrome, as well as lowered BP and increased restful sleep¹⁴⁵. BP rhythm was not assessed in these studies but would be of interest in future studies to explore the effect of TRF on BP rhythm. The data regarding the effectiveness of TRF in modulating cardiometabolic health is limited but studies so far are promising. Larger trials assessing long-term TRF effects, as well as exploring precise mechanisms, are necessary.

Chrono-exercise

Exercise has long been recognized as beneficial for cardiovascular health¹⁴⁶. Furthermore, being more active is included as an important component for cardiovascular health in American Heart Association Life’s Essential 8¹⁰⁴. Less is known about whether timing of exercise matters. Small-scale timed 12-week exercise interventions have been carried out on shift workers 2 hours prior to their night shift which improved physical exercise capacity and arterial stiffness in healthy individuals¹⁴⁷, but had no influence on 24-hour BP¹⁴⁸. A recent systematic review has recently examined the time-dependent effects on physical activity, both acute and chronic exercise interventions, on cardiovascular risk from 16 studies between January 2000 and June 2022 with sample sizes between 11–275 participants diagnosed with obesity, hypertension, diabetes, and coronary heart disease¹⁴⁹. The findings were that there was some evidence that cardiovascular risk factors in adults may be time-dependent to physical activity but with small sample sizes, a larger randomized controlled trial is needed to determine whether synchronizing time of exercise to the individual’s circadian rhythm improves the benefits of exercise on cardiovascular health.

CONCLUSION AND FUTURE PERSPECTIVES

The circadian system is a complex system that is not fully understood. This review emphasizes that the circadian system greatly influences cardiovascular function and disruption of circadian rhythms and the circadian clock have potential detrimental consequences on cardiovascular risk (Figure 2).

Figure 2.

Circadian misalignment and disruption in the molecular clock function have potential negative impacts on cardiovascular risk. Circadian rhythms are important for vascular function and subsequently, blood pressure control. Studies in humans and rodents suggest circadian misalignment, through shift work, abnormal light exposure, poor sleep, and aging, and disruption in the molecular clock function (e.g. clock gene mutation) can increase the likelihood of atherosclerosis and hypertension. There are several clinical trials ongoing investigating whether chronotherapeutic interventions, including light and sleep therapy, are beneficial for treatment of hypertension and cardiovascular disease. Created with Biorender.

A key area that should be a future focus is the role of the circadian clock in females, with important considerations for pregnancy and in aging. Throughout this review, the lack of female data, particularly in preclinical studies, across the lifespan is evident. Recent studies have highlighted the importance of the circadian clock in pregnancy. In preeclampsia, a pregnancy-related hypertensive condition which is an independent risk factor for cardiovascular disease, blunting of the circadian rhythm of maternal BP has been reported, with severe cases reporting reversed rhythm (reviewed in ¹⁵⁰). Furthermore, the placenta has a molecular clock, which interacts with both the maternal and embryo clock¹⁵¹. Interestingly, robust clock gene expression differences were found when comparing placentas¹⁵², as well as primary placental macrophages¹⁵³, from women with and without preeclampsia. Together, these studies highlight novel mechanisms of the clock in pregnancy that could be leveraged for the prevention and treatment of preeclampsia. Another area of interest is organ-organ clock communication. Clock crosstalk has been suggested between maternal, placental, and embryo clocks but this concept remains to be explored for other organs. This should be a focus for future studies as well as exploring whether aberrant clock crosstalk contributes to progression of disease.

In the modern world, it is impossible to avoid many of these circadian disruptions, but a better understanding of the impact of circadian disruption, such as shift work and aging, on cardiovascular risk is imperative for development of novel targeted preventive and therapeutic strategies. Several ongoing clinical trials investigating light and sleep therapy in shift workers and in the elderly, highlighted in Table 1, could be very informative. Furthermore, the use of diverse animal models of disturbed clock conditions and cell/tissue specific gene manipulations are warranted to better understand the heterogeneity of the clock system and the role of circadian clock in development and progression of atherosclerosis and hypertension.