Rev-erbα/β protein – a key component of the circadian clock – is involved in the development of heart disease

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Disrupting circadian rhythms, which change naturally on a 24-hour cycle, has been implicated in heart disease, but it is unclear how it leads to the condition.

A research team at Baylor College of Medicine and collaborating institutions investigated the function of the protein Rev-erbα/β, a key component of the circadian clock, on heart disease development in animal models and human patients.

The team reports in the journal Circulation that Rev-erbα/β in cardiomyocytes mediates a normal metabolic rhythm that enables the cells to prefer lipids as a source of energy during the animal’s resting time, daytime for mice.

Removing Rev-erbα/β disrupts this rhythm, reduces the cardiomyocytes’ ability to use lipids in the resting time and leads to progressive dilated cardiomyopathy and lethal heart failure.

“We studied how the Rev-erbα/β gene influenced the metabolism of the heart by knocking it out specifically in mouse cardiomyocytes,” said co-corresponding author Dr. Zheng Sun, associate professor of medicine, section of endocrinology, diabetes and metabolism and of molecular and cellular biology at Baylor. “Lacking the gene resulted in progressive heart damage that led to heart failure.”

To learn how Rev-erbα/β mediated its effects, the team analyzed gene and protein expression and a comprehensive panel of metabolites and lipids, during both the awake and sleep hours.

They found that the Rev-erbα/β gene is highly expressed only during the sleep hours, and its activity is associated with fat and sugar metabolisms.

“The heart responds differently to different sources of energy, depending on the time of the day,” explains co-corresponding author Dr. Lilei Zhang, assistant professor of molecular and human genetics and of molecular physiology and biophysics at Baylor. “In the resting phase, which for humans is at night and for mice in the day, the heart uses fatty acids that are released from fats as the main source of energy.

In the active phase, which is during the day for people and at night for mice, the heart has some resistance to dietary carbohydrates. We found that without Rev-erbα/β, hearts have metabolic defects that limit the use of fatty acids when resting, and there is overuse of sugar in the active phase.”

“We suspected that when Rev-erbα/β knockout hearts cannot burn fatty acids efficiently in the resting phase, then they don’t have enough energy to beat. That energy deficiency would probably lead to changes in the heart that resulted in progressive dilated cardiomyopathy,” said Sun, a member of Dan L Duncan Comprehensive Cancer Center.

To test this hypothesis, the researchers determined whether restoring the defect in fatty acid use would improve the condition.

“We know that fatty acid use can be controlled by lipid-sensing metabolic pathways. We hypothesized that if we fed the Rev-erbα/β knockout mice more lipids, maybe the lipid-sensing pathways would be activated, override the defect and consequently the heart would be able to derive energy from lipids,” Sun explained.

The researchers fed Rev-erbα/β knockout mice one of two high-fat diets. One diet was mostly high-fat. The other was a high-fat/high-sucrose diet, resembling human diets that promote obesity and insulin resistance. “The high-fat/high-sucrose diet partially alleviated the cardiac defects, but the high-fat diet did not,” Sun said.

“These findings support that the metabolic defect that prevents the heart cells from using fatty acids as fuel is causing the majority of the cardiac dysfunction we see in the Rev-erbα/β knockout mice. Importantly, we also show that correcting the metabolic defect can help improve the condition,” Zhang said.

Clinical implications in obesity paradox and chronotherapy

“There are three clinical implications from this work,” Sun said. “First, we analyzed the molecular clock function in heart tissues of patients with dilated cardiomyopathy who had received heart transplants to explore whether the clock function was associated with the severity of cardiac dilation in humans.

Tissue samples were taken at different times of the day and the ratio of the gene expression of the circadian genes Rev-erbα/β and Bmal1 was calculated providing a chronotype. We found that the heart chronotype correlates with the severity of cardiac dilation.”

“The second implication is that obesity and insulin resistance, long-known clinical risk factors for heart failure, can be paradoxically protective against heart failure, within a certain time window, probably by providing fatty acids in the resting phase,” Sun said.

Finally, the researchers explored the possibility of pharmacologically manipulating fatty acid and sugar metabolism to improve the condition. They found that while medications can help restore the altered metabolic pathways, it was important to give the drugs aligned with the internal circadian rhythm of the corresponding metabolic pathways. If the drugs were given out-of-sync with the pathway they were intended to restore, the treatment did not improve the cardiac condition.”

These findings highlight the importance of chronotherapy, the scheduling of medications according to the circadian rhythm, not just in this study, but for many other medications.

“Of the top 100 most prescribed drugs in the U.S., at least half of them have a target that is connected to a circadian rhythm,” Zhang said. “This indicates that for these drugs to be effective, they need to be taken in a time-specific way. Unfortunately, they are not. We want to emphasize the importance of taking the circadian rhythm into consideration when scheduling medications.”


Graphical Abstract
The role of circadian rhythms in cardiovascular physiology. (A) The circadian clock sustains a 24 h rhythm that regulates the cardiovascular system, including electrophysiological parameters, BP, cardiac contractility, coagulation, vascular function, and the cardiovascular involvement of the immune system. (B) The circadian clock is a molecular mechanism consisting of positive and negative feedback loops. (C) The circadian clock causes time-of-day variation of clock components as well as physiological parameters.

Circadian rhythms are endogenous (intrinsic) biorhythms that repeat approximately every 24 h. They allow the body to continuously anticipate day-to-night environmental variations consequent to the earth’s rotation. Circadian rhythms are present in all organisms. In humans, they play a central role in physiology and disease.

Many cardiovascular functions, such as blood pressure (BP),1,2 cardiac contractility, heart rate,3 and vascular resistance show 24 h, diurnal variations. These rhythms are the product of external (environmental and behavioural) factors and intrinsic (endogenous) circadian rhythms. Circadian rhythms are driven by circadian clocks. Humans possess two clock types:

(i) a central biological clock in the suprachiasmatic nucleus of the hypothalamus, that controls circadian rhythms via the autonomic nervous system and humoral mediators (e.g. cortisol, melatonin) and

(ii) peripheral clocks that locally enforce temporal governance in cells, such as the cardiomyocytes,4 vascular endothelial cells,5 smooth muscle cells,6 and cardiac progenitor-like cells.7 Both central and peripheral clocks are self-sustainable but can be altered and entrained by environmental factors (called Zeitgebers), such as light, physical activity, and food intake.

The circadian clock is a molecular mechanism that consists of clock proteins, such as CLOCK, BMAL1, PER1/2/3, and CRY1/2.8 In brief, CLOCK and BMAL form a heterodimer and induce transcription of PER and CRY proteins. The latter proteins subsequently form a complex and inhibit the transcription of CLOCK and BMAL1, thereby generating a negative feedback loop. This feedback loop is complemented by several other feedback loops, most notably REV-ERBα/β, a member of nuclear receptor family (and thus a pharmacological target of the circadian clock), and further regulated at different levels, including the post-translational and epigenetic level.

The circadian clock regulates transcription of ∼10–15% of all genes and proteins in the heart.9 Oscillation of these genes and proteins causes 24 h fluctuation in processes like cardiac cellular growth, cell adhesion, metabolism, apoptosis, fibrosis, electrophysiology,10 and contractile function.

An increasing number of studies support the idea that circadian rhythms affect almost all functions of the cardiovascular system and play central roles in cardiovascular disease and recovery. As a result, 24 h rhythms have evolved from a niche topic to one that is important in almost all preclinical and clinical research.

It is a factor that, like age or sex,11 may significantly impact the translation potential of cardiovascular research. In the current position paper of the European Society of Cardiology Working Group Cellular Biology of the Heart, we aim to provide key aspects on how circadian rhythms can be taken into account to improve clinical and preclinical studies in cardiovascular disease, with a focus on ischaemic heart disease.

Circadian rhythms and ischaemic heart disease

Circadian rhythms play a major role in cardiovascular disease at the level of incidence, pathophysiology, and outcome.41–43 Reviews have been written about this topic in specific cardiovascular diseases such as stroke44 and arrhythmias.38,45 Here, we focus on ischaemic heart disease in both its chronic and acute manifestations.

For decades, 24-h rhythms have been studied in the context of disease onset. Acute ischaemic heart disease (AMI) occurs more frequently in the early morning.46–49 This may be explained by a combination of factors, including the previously described morning increase in hemodynamic stress (surge in heart rate and BP), platelet aggregability, circadian leukocyte oscillations, and recruitment of inflammatory leukocytes from blood to plaque during this time-of-day.30,50–53

In 2010, Durgan et al.41 published a break-through study investigating circadian rhythms in tolerance of the heart to ischaemic insults. When myocardial ischaemia is induced at the sleep to wake transition (subjective morning) in an animal model, infarct size, fibrosis, and adverse remodelling were significantly worse compared to ischaemia at the wake-to-sleep transition (subjective evening). This illustrates that circadian rhythms are not only important in the incidence and development of ischaemic heart disease, but also play a major role in outcome of disease.41,42

Several clinical studies observed differences in plasma levels of creatine kinase after AMI that were similarly dependent on time-of-ischaemia onset.54–56 Some clinical data further suggest that morning onset of AMI is associated with increased risk of recurrent acute coronary syndromes and coronary atherosclerosis progression.57 However, other investigations failed to confirm an association between time-of-day at symptom onset and infarct size or long-term mortality in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI).58

The variable outcome between clinical studies has been discussed extensively.59,60 One of the limitations in humans is certainly the relatively high variability in patient characteristics [ethnic background, medication use, comorbidities (such as diabetes), chronotype, culprit artery, and time of ischaemia] as well as other study parameters (statistical methodology, study size, and outcome measure), which might render it difficult to validate an association between time-of-day of ischaemia onset and outcome.

To better assess direct causal relationships between circadian rhythmicity and infarct size, mouse models of AMI have been instrumental.61 Animal and in vitro models also help understand the processes involved in diurnal variation of AMI outcome. In support of a possible role for circadian clocks in ischaemic damage, genetic disruption of clock genes leads to an altered infarct size in mice.

Disruption of positive components of the molecular clockwork, such as Bmal and Clock caused an increased infarct size, whereas disruption of the negative component Per2 and Rev-Erbα reduced infarct size.41,42,62

This further supported the study of Durgan et al., 41 which used a cardiomyocyte-specific Clock-mutant mouse model to demonstrate that the diurnal variation in AMI outcome is orchestrated by the cardiomyocyte clock, possibly via a diurnal rhythm in ischaemia tolerance.28

Clock disruption in other cell types, such as the immune system and fibroblasts, may also contribute to the diurnal variation of AMI. Studies in wound healing, for example, demonstrate that 24 h variation in wound healing is caused by a rhythm in fibroblast activity, a process also important in post-AMI cardiovascular remodelling.63

Animal models also showed other important relations between outcome of ischaemic heart disease and circadian rhythms, for example, the effect of circadian disruption. In an experimental mouse model of permanent left coronary ligation, disruption of light/dark cycles promotes an unfavourable healing response after AMI.64 More specifically, infarcted mice were subjected to 10 h light/10h dark cycles over 5 days, resulting in cardiac dysfunction and poorer AMI tolerance.

Circadian disruption had significantly greater adverse remodelling with increased left ventricular internal systolic and diastolic dimensions, accompanied by decreased fractional shortening and ejection fraction. Other studies investigated time-of-day differences after myocardial infarction in depth, and found that in mice, AMI in the awake period triggers genes associated with metabolic pathways, whereas an AMI in the inactive period leads to up-regulation of genes associated with inflammation.65 This time-of-day effect of AMI on cardiac remodelling is regulated by the circadian clock.65 Vice versa, there is evidence that AMI may lead to circadian disruption, for example, in the beta-adrenergic receptor expression, thereby contributing to adverse cardiac remodelling.66,67

The first studies investigating time-of-day and ischaemic heart disease focused on melatonin, a hormone produced by the pineal gland, under the influence of light, and one of the input signals of the circadian clock.68 Both animal and patient studies suggest that endogenous melatonin levels correlate with lower ischaemia–reperfusion injury.69–72 Vice versa, AMI may lead to decreased melatonin levels.73,74

The relationship between melatonin and cardioprotection in ischaemic heart disease is complex and involves multiple processes including the regulation of the molecular circadian clock or direct effects of melatonin as a regulator of multiple prosurvival signalling cascades within the heart, an antioxidant and an anti-inflammatory molecule.75–77

In humans, disturbance of circadian rhythms is also associated with ischaemic heart disease.78 Circadian rhythm disturbance (e.g. by sleep deprivation or shift work) induces a misalignment between physical activity and intrinsic clocks, with adverse effects on cardiovascular parameters, healing responses, and remodelling.

Insufficient sleep, for example, affects the blood transcriptome and disrupts its circadian regulation.79 The identified genes, pathways and biological processes affected by insufficient sleep include circadian clock genes as well as inflammatory, immune, and stress response pathways.

The immune system appears to be a major contributor to the variation in AMI outcome. Humans have diurnal fluctuations in immune cell numbers.41,80 In particular, the innate immune system including the inflammasome, the first immune response following an AMI and involved in recruitment and activation of pro-inflammatory monocytes, is circadian regulated.81,82

Production and retention of neutrophils in the bone marrow is time-of-day dependent.83,84 Moreover, circulating neutrophils at the beginning of the active phase have higher capacity to migrate into the myocardium due to up-regulated C-X-C Motif Chemokine Receptor 2 expression.80 Other immune cells, such as classical monocytes are regulated by the circadian clock and involved in AMI outcome.85,86 Disruption of the molecular clock in these monocytes worsens inflammation.81,85 Recently, a study showed that the inflammatory role of the gut microbiome in AMI and heart failure is influenced by the circadian clock.87

The molecular circadian clock is not only important in AMI, but also plays a major role in chronic ischaemic heart disease. Disruption of the molecular clock can dampen BP rhythmicity, reduce the production of vasoactive hormones and cause endothelial dysfunction,88 thereby increasing the development of atherosclerosis.89

For example, the aortae of clock-mutant mice exhibit impaired cholesterol metabolism and enhanced atherosclerosis.90 Interestingly, the mechanism appears to be cell intrinsic as significant atherosclerosis develops when the aortae from clock-mutant mice are transplanted into wild-type mice.88 Pharmacological targeting of clock components decreased atherosclerosis in mouse models, likely secondary to effects on inflammation.53

Finally, to illustrate some relevant links beyond our focus on ischaemic heart disease, global and cardiomyocyte-specific clock-mutant mice develop dilated cardiomyopathy.16,91 Moreover, cardiomyocyte-specific down-regulation of BMAL1 results in reduced heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. The phenotype is linked with reduced circadian expression of the sodium and potassium channels, which may contribute to the sudden cardiac death observed in cardiomyocyte-specific Bmal knock-out mice.26,92

reference link : https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvab293/6368285


More information: Chronotype Myocardial Rev-erb-mediated diurnal metabolic rhythm and obesity paradox, Circulation (2022). DOI: 10.1161/CIRCULATIONAHA.121.056076

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