Circadian Rhythms in Cardiovascular Metabolism

Abstract

Energetic demand and nutrient supply fluctuate as a function of time-of-day, in alignment with sleep-wake and fasting-feeding cycles. These daily rhythms are mirrored by 24hr oscillations in numerous cardiovascular functional parameters, including blood pressure, heart rate, and myocardial contractility. It is therefore not surprising that metabolic processes also fluctuate over the course of the day, to ensure temporal needs for ATP, building blocks, and metabolism-based signaling molecules are met. What has become increasingly clear is that in addition to classic signal-response coupling (termed reactionary mechanisms), cardiovascular-relevant cells employ autonomous circadian clocks to temporally orchestrate metabolic pathways in preparation for predicted stimuli/stresses (termed anticipatory mechanisms). Here, we review current knowledge regarding circadian regulation of metabolism, how metabolic rhythms are synchronized with cardiovascular function, and whether circadian misalignment/disruption of metabolic processes contribute toward the pathogenesis of cardiovascular disease.

Introduction

In mammals, virtually all biological processes fluctuate over a normal 24hr period in association with daily changes in the environment and behaviors, such as light/dark, sleep/wake and fasting/feeding cycles.¹ Diurnal biological processes include both metabolism and cardiovascular function, which are considered inextricably interlinked.² Metabolism is defined broadly as all chemical reactions involved in the conversion or rearrangement of one molecule into another molecule. Accordingly, metabolism technically encompasses virtually all biological processes, ranging from the polymerization of nucleotides into RNA/DNA, the synthesis of proteins from precursor amino acids, and the generation of signal transduction secondary messengers, to intermediary metabolism. Intermediary metabolism is broadly defined as catabolic and anabolic reactions directly involved in the conversion of nutrient-derived compounds into cellular metabolites that are utilized for storage, structure, and/or work. When considering the influence of intermediary metabolism on cardiovascular function, the following often come to mind: 1) ATP synthesis through catabolic processes to meet energetic demands; 2) biosynthetic pathways for the generation of cellular constituent building blocks; 3) posttranslational modifications derived from intermediates within metabolic pathways; and 4) metabolism-derived signaling molecules. Underscoring the importance of metabolism, aberrant metabolic homeostasis is invariably associated with cardiometabolic disease states, including obesity, diabetes mellitus, hypertension, and heart failure. Although the term ‘cardiovascular’ theoretically refers to the heart and blood vessels, cardiovascular function is dependent upon an expansive system of organs and cells that extend beyond the heart and vessels. It is equally important to realize that the term circadian rhythm has a very specific definition. Originally derived from the Latin words ‘circa’ and ‘diem’ (meaning ‘about’ and ‘day’, respectively), the term circadian rhythm is casually linked with processes/factors that fluctuate over a 24hr period. However, within the chronobiology arena, designation as a circadian rhythm is limited to 24hr oscillations that occur independent of environmental factors. In other words, circadian rhythms are driven by endogenous mechanisms. Based on this reasoning, when discussing circadian rhythms in cardiovascular metabolism, we have elected to focus on current knowledge regarding endogenously driven 24hr rhythms in intermediary metabolism within cells/organs that impact cardiovascular function. For the sake of simplicity, we will refer to intermediary metabolism as metabolism henceforth. This article will also discuss how metabolic circadian rhythms are altered by and/or influence cardiovascular function, in response to physiologic perturbations and pathologic stresses. Finally, we speculate how these rhythms might be exploited for the prevention and/or treatment of cardiovascular disease.

Theoretical Evolutionary Advantage Conferred by Circadian Regulation of Metabolism

It is not surprising that 24hr fluctuations in metabolism have been reported for multiple organisms over the course of the day.³ Intuitively, one might imagine that these metabolic fluctuations are driven largely by daily changes in light intensity, temperature, humidity, and food availability, in parallel with changes in locomotion, feeding, and other behaviors. Indeed, whole-body metabolic rates are higher in mammals during the awake period, in association with increased physical activity and food intake at that time.⁴ Conversely, metabolic rates decrease during sleep.⁵ Under normal conditions, metabolism is exquisitely plastic in nature, exemplified by immediate changes in metabolic fluxes in response to physiologic perturbations. These reactionary adaptations are essential for maintaining metabolic homeostasis, and are often reported to be impaired during pathologic conditions.⁶ Consider a glucose tolerance test as an example: healthy individuals rapidly metabolize a sudden bolus of glucose as a homeostatic mechanism, whereas the (pre-)diabetic state is characterized by blunted glucose disposal.⁷ Intriguingly, the magnitude of metabolic plasticity varies over the course of the day, so much so that at specific times of the day an organism can approach levels of metabolic inflexibility mirroring that typically observed in pathologic states. Daily fluctuations in metabolic flexibility have important physiologic functions/consequences. Returning to glucose homeostasis as an example, prior to awakening, blood glucose levels rise secondarily to increased hepatic glucose output.^8–10 This event, termed the dawn phenomenon, appears to be a consequence of transient hepatic insulin resistance, to an extent that is somewhat similar to the (pre-)diabetic state.¹⁰ A hypothetical advantage of this phenomenon is to ensure that the organism has sufficient glucose for the energetic demands associated with increased cognitive function and physical activity upon awakening.

Temporal control of metabolic processes, leading to events such as the dawn phenomenon, raises an important concept that is worthy of further discussion. This concept is the ability of an organism to prepare for an anticipated event before it occurs. Typically, anticipation is considered an active cognitive function, requiring a state of consciousness.¹¹ It can, therefore, be difficult to comprehend how an individual cell or organ outside of the central nervous system is able to predict an event. However, persistent temporal cycling of selective pressures on Earth has resulted in the evolution of many forms of anticipation by organisms, which are observed at various periodicities. At the seasonal level, the thrifty gene hypothesis postulates that human metabolism has evolved to efficiently store excess calories as triglyceride in anticipation of limited nutrient availability during the winter months; the health consequence of this once evolutionary advantage is an increased risk of obesity in modern society.¹² In addition to cyclic seasonal variations, the periodicity of the day has remained relatively constant since the beginning of life on Earth. This selective pressure has led to the evolution of an approx. 24hr timekeeping mechanism in virtually all lifeforms. That timekeeping mechanism is the circadian clock. It is the circadian clock that confers the selective advantage of anticipation on a daily basis.¹³

Circadian clocks are cell-autonomous molecular mechanisms, comprised of a series of transcriptional-translational feedback loops, with a periodicity of approximately 24hrs.¹ Important conceptual points include that the: 1) clock mechanism is cell autonomous, such that an individual cell possesses all circadian clock components, which function even when a cell is in isolation; 2) clock is composed of transcriptional-translational modulators, whose activities feedback on one another in both positive and negative manners; and 3) activities of circadian clock components fit a cosine curve, with one peak and one trough over the course of the 24hr day.^1,13,14 Simultaneous to forming feedback loops, circadian clock components regulate a large number of cellular constituents, resulting in temporal governance of fundamental biological processes, such as transcription, translation, signaling, and metabolism.^15–17 Importantly, by temporally governing cellular constituents, circadian clocks induce daily fluctuations in the responsiveness of a cell/organ to extracellular stimuli.^1,18 In doing so, circadian clocks confer the selective advantage of anticipation, by allowing cells/organs to prepare for an event before it occurs.

Here, we have introduced two conceptual strategies underlying the regulation of biological processes: 1) reactionary, meaning response of a cell/organ to a stimulus; and 2) anticipatory, meaning preparation of a cell/organ prior to a predicted event. When considering mammals on a 24-hour timescale, reactionary stimuli will include a plethora of extrinsic factors that fluctuate in association with environmental and behavioral cycles.¹⁹ In contrast, the anticipatory mechanism is the circadian clock, which is intrinsic to the cell. In the in vivo setting, extrinsic and intrinsic factors interact, the product of which manifests as 24hr fluctuations in biological processes. This can be illustrated by reconsidering glucose tolerance. Cell autonomous circadian clocks are known to modulate insulin sensitivity over the course of the day.²⁰ If an organism consumes a carbohydrate-rich meal at a time of day during which insulin sensitivity is low, then glucose disposal will be delayed.²¹ Conversely, if the same carbohydrate-rich meal is consumed at a time of day during which insulin sensitivity is high, then glucose disposal rates are augmented. Similarly, by modulating the activity of enzymes involved in digestion/absorption and/or metabolism of a nutrient, circadian clocks influence the fate of that dietary nutrient depending on the time at which it is consumed.²² Such a phenomenon has been characterized in-depth for dietary lipids.²³ The relevance of these examples, in terms of cardiovascular function, stems from acknowledgement that impairment of either glucose or lipid metabolic homeostasis increases CVD risk.

The Mammalian Circadian Clock: Transcriptional, Translational, and Posttranslational Considerations

At the core of the mammalian circadian clock are a number of well-characterized transcriptional modulators, including CLOCK and BMAL1 (Figure 1).^24,25 Upon heterodimerization, these bHLH-PAS proteins bind to E-boxes within the promotor of target genes, resulting in induction of the respective transcript. These target genes include negative feedback loop components, such as Per1/2/3, Cry1/2, and Nr1d1/2; the latter encodes for REV-ERBα/β.^26–28 The transcriptional repressors REV-ERBα/β compete with ROR isoforms for binding at ROREs within promotors; this includes the Bmal1 gene promoter, resulting in attenuated expression.^28,29 Conversely, PER and CRY proteins heterodimerize, translocate back to the nucleus, and repress the transcriptional activity of CLOCK/BMAL1.²⁷ Consistent with the nature of these feedback loop mechanisms, core clock components exhibit 24hr oscillations at mRNA and protein levels, with distinct phases and amplitudes.¹⁵ For the mechanism to function with a periodicity of approximately 24hr, numerous PTMs are required, which modulate stability, cellular location, and/or activity of clock components.^30,31 Described clock component PTMs include phosphorylation, acetylation, ubiquitination, sumoylation, and O-GlcNAcylation.^30–32 The importance of PTMs was first appreciated for the PER proteins, whose phosphorylation enables an approximate 6hr phase delay between mRNA and protein oscillations.³³ Similarly, CLOCK/BMAL1 transcriptional activity is delayed by approximately 9hr relative to peak bmal1 and clock mRNA levels, secondary to PTMs.³⁴

Figure 1. Mechanisms by which the mammalian circadian clock influences cellular processes, such as metabolism.

The circadian clock (shown in blue) is composed of positive (e.g., BMAL1) and negative (e.g., REVERBα/β, PER1/2/3, CRY1/2) feedback loop components, as detailed within the main text. The circadian clock can influence cellular metabolism through transcriptional (i.e., canonical, shown in green), post-transcriptional (i.e., epigenetic, shown in red), and post-translational (i.e., non-canonical, shown in orange) mechanisms. The canonical mechanism involves direct binding of circadian clock components to upstream regulatory elements of clock-controlled genes (Ccg), resulting in oscillations in both mRNA and protein levels of target gene products; examples of metabolism-related targets are shown in green boxes. The epigenetic mechanism involves direct binding of circadian clock components to upstream regulatory elements of clock-controlled miRNA species, impacting both the circadian clock and metabolism through effects on mRNA stability and translation; examples are shown in red boxes. Non-canonical mechanisms involve direct binding of circadian clock components to pre-existing cellular constituents, leading to altered function; examples are shown in orange boxes. In addition, metabolism-related signals can feedback on the circadian clock mechanism. Illustration credit: Ben Smith.

In order to maintain their selective advantage, circadian clocks must remain sufficiently flexible, allowing for adjustment to fluctuations in the diurnal environment, which occur secondary to daily, seasonal, and geographic alterations.³⁵ The term zeitgeber is given to environmental and behavioral cues that ‘reset’ circadian clocks. Common zeitgebers for mammalian circadian clocks include light, food intake, and physical activity.³⁵ Typically, circadian clocks have been classified as either central or peripheral clocks; central clocks are located within specialized hypothalamic neurons of the SCN, whereas peripheral clocks are located in all other cells.³⁶ In mammals, light signals are transmitted to the SCN via the retino-hypothalamic tract, leading to entrainment of the central clock.^35,36 In turn, the SCN conveys entrainment to peripheral clocks via coordinated neurohumoral signals ranging from direct innervation to circulating endocrine factors.³⁷ SCN-induced entrainment of specific endocrine cells leads to secretion of additional zeitgebers, including cortisol, which aid in whole-body synchronization.^19,38 Entrainment can also be achieved through non-photic cues, including food intake; the power of this entrainment signal is underscored by observations that food consumption during the usual sleep period can phase shift peripheral clocks, while the SCN remains entrained to the light/dark cycle.³⁹ Consistent with the circadian clock mechanism, entrainment relies on transcriptional, translational, and posttranslational events. For example, activation of the glucocorticoid receptor leads to activation of the Per2 gene, while food intake promotes PKCγ-dependent phosphorylation of BMAL1.^40,41

Circadian clocks modulate cellular processes over a 24hr period, through numerous means. The canonical pathway involves temporal control of the transcriptome. More specifically, in addition to modulating transcription of one another, core clock components target promoters of output genes, leading to daily fluctuations in as much as 20% of a cell’s transcriptome.¹⁷ Clock output transcripts encode for proteins with diverse biological functions, ranging from cellular signaling, ion channels, cytoskeleton, and metabolism, to extracellular matrix and the secretome. Theoretically, if an oscillatory mRNA species results in rhythmic levels of the corresponding protein, then the biologic process would be predicted to fluctuate over a 24hr period.¹⁷ Somewhat surprisingly, although high amplitude oscillations have been reported for both the transcriptome and numerous biological processes, 24hr rhythms in the proteome are typically less impressive; the majority of proteins exhibit oscillations with <2-fold peak-to-trough differences.⁴² In addition, poor correlations exist between transcriptomic and proteomic circadian datasets. More specifically, <50% of proteins with 24hr rhythms can be readily explained by oscillations in their corresponding mRNA species.⁴³ Such observations suggest that circadian clocks employ a synchronized multi-modality approach when regulating a pathway/process and/or non-canonical mechanisms contribute (Figure 2). With regards to the synchronized multi-modality mechanism, it is important to recall a fundamental concept in metabolism: that being, everything is in flux. This is exemplified by the principle of substrate cycles, which is illustrated in Figure 3. Central to this concept is that under steady-state conditions, opposing reactions/pathways have baseline activity levels that are similar to one another; these substrate cycles contribute towards resting metabolic rates. In response to a stimulus, relatively small reciprocal alterations in opposing reaction rates result in comparably dramatic changes in net flux through a pathway (Figure 3). Non-canonical mechanisms include the direct binding of circadian clock components to preexisting cellular constituents, leading to rapid alterations in cellular location and/or activity. For example, when in the phosphorylated state, BMAL1 forms a complex with the translation initiation factors eIF3B and eIF4F, thereby promoting protein synthesis (Figure 1).⁴⁴ Similarly, several circadian clock components can directly alter cellular functions through mechanisms involving PTMs. Examples include: 1) PER2 inhibits protein synthesis and promotes autophagy via direct binding to the kinase complex mTORC1; 2) REV-ERBα binds to and activates OGT, leading to O-GlcNAcylation of cytosolic and nuclear proteins; and 3) CLOCK exhibits histone acetyltransferase activity, thereby affecting protein acetylation (Figure 1).^45–47 Given the direct links between mTORC1, OGT, and acetylation with metabolic processes, these examples will be revisited in subsequent subsections.

Figure 2. Evidence-based hypothetical mechanisms by which cell-autonomous circadian clocks orchestrate biological/cellular processes over a 24hr period.

A) A canonical pathway involves clock-induced high amplitude oscillations of a target gene, leading to high amplitude oscillations of the encoded protein (and its function); this simplistic mechanism requires short-half lives of both clock-controlled mRNAs and proteins. B) Synchronized control of regulators, such that 24hr oscillations of positive and negative regulators are antiphase, allowing the corresponding biological process to exhibit robust 24hr rhythms, even in the presence of low amplitude oscillations in protein levels. C) A non-canonical mechanism, wherein circadian clock components bind directly to cellular targets, affecting target activity/function over a 24hr period. Illustration credit: Ben Smith.

Figure 3. Substrate/Futile cycling promotes high amplitude fluctuations in biologic processes.

Illustration depicts a hypothetic scenario, wherein metabolite A (Met_A) can be reversibly converted into metabolite B (Met_B), through the actions of enzyme F (Enz_F; forward reaction) and enzyme R (Enz_R; reverse reaction). When the activities of Enz_F and Enz_R are similar, a substrate/futile cycle occurs, such that relatively small changes in the activities of the enzymes can lead to high amplitude fluctuations in net flux. For example, the illustration shows that 10% changes in enzyme activities, secondary to the actions of activators (Act) and inhibitors (Inh), led to a 2.9-fold change in flux. Such cycling is common for various aspects of biology, including metabolism and signaling (e.g., PTMs). Illustration credit: Ben Smith.

Impact of Cell Autonomous Circadian Clocks on Cardiovascular Metabolism

As highlighted in preceding sections, the interconnective nature of metabolism means that it impacts an array of fundamental biological processes that are critical for normal cellular function. This is true not only for cells classically considered to be within the cardiovascular system, such as cardiomyocytes, endothelial cells, VSMCs, fibroblasts, pericytes, and renal cells, but also cells outside of the system that are known to modulate cardiovascular function. Broadly speaking, cells outside of the cardiovascular system have the potential to impact cardiovascular metabolism through modulation of substrate availability and/or neurohumoral factor generation. These cells include intestinal epithelial cells, adipocytes, hepatocytes, skeletal myocytes, pancreatic cells, immune cells, and neurons. Unfortunately, it is not feasible to include a comprehensive discussion focused on circadian regulation of metabolism for all these cell types within an individual review article. For this reason, this subsection will focus only on the following select cell types: 1) cardiomyocytes; 2) endothelial cells and VSMCs; 3) fibroblasts; 4) intestinal epithelial cells; and 5) adipocytes. Whenever possible, circadian regulation of glucose and fatty acid metabolism, as well as protein/cellular consistent turnover, have been considered for the following reasons: 1) All cells/organs must contend with daily fluctuations in carbohydrate, lipid, and protein, as imbalances between macronutrient supply and utilization result in cellular/organ dysfunction, through gluco-, lipo-, and proteo- toxicity mechanisms; and 2) it is essential that metabolism-related quality control processes are employed to ensure normal functioning of organelles, such as mitochondria. Table 1 summarizes key observations reported for circadian regulation of metabolic parameters within the five focus cell types.

Table 1. Circadian regulation of metabolism in select cardiovascular-relevant organs/tissues/cells.

The table summarizes published studies supporting the concept that cell autonomous circadian clocks influence metabolism in the heart/cardiomyocytes, vessels/VSMC/endothelial cells, fibroblasts, intestine/IEC, and adipose tissue/adipocytes. Evidence is divided into categories that range from circumstantial/indirect evidence, such as altered transcript levels, to direct measures of metabolic flux, in alignment with a recent American Heart Association Scientific Statement.²¹⁸ Care has been taken to include studies that provide evidence of direct circadian regulation, using the following criteria: 1) 24hr rhythms in wildtype tissues in vivo; 2) 24hr rhythms in wildtype tissues ex vivo; 3) 24hr rhythms in wildtype cells in vitro; 4) perturbations following cell type specific genetic manipulation of a circadian clock component in vivo; and/or 5) perturbations following cell type specific genetic manipulation of a circadian clock component in vitro. To avoid secondary influences that germline genetic and/or environmental manipulations have on behaviors and neurohumoral factors, which in turn hinder understanding of the role of cell autonomous circadian clocks, these studies are not included in the table. Due to space constraints, each sub-category within a specific organ/tissue/cell has been limited to a maximum of 5 referenced studies.

	Indirect Evidence				Direct Evidence
Organ/Tissue/Cell Type	Transcript	Protein/PTM	Enzyme/Organelle Activity	Metabolites	Metabolic Flux
Heart/Cardiomyocyte	Omics: Transcriptomics in cardiomyocyte clock-disrupted hearts identified candidate metabolism-related transcripts49,61,63,164,209 Targeted: RT-PCR in cardiomyocyte clock-disrupted hearts or cultured cardiomyocytes identified clock-controlled metabolism-related transcripts, including: pik3r1, dgat2, nampt, bdh1, pdk4, ogt, glut4, ces1d, idh249,61,63,164,209	Omics: Proteomics in cardiomyocyte clock-disrupted hearts identified candidate metabolism-related proteins209,210 Targeted: Western blot in cardiomyocyte clock-disrupted hearts identified clock-controlled metabolism-related proteins, including: GLUT4, BDH1, NAMPT, HSL, OGT, p-AMPK, p-Akt, p-mTOR50,53,55,61,63	Enzyme Activities: Enzyme assays in cardiomyocyte clock-disrupted hearts identified clock-controlled metabolism-related enzymes, including: BDH, PDH, AMPK50,61,67 Organelle Activities: Organelle assays in cardiomyocyte clock-disrupted hearts or cultured cardiomyocytes identified clock-controlled metabolism-related organelles, including: Mitochondria, Lysosomes54,58,63,72,74	Omics: Metabolomics and lipidomics in wild-type and cardiomyocyte clock-disrupted hearts identified candidate metabolic pathways164,188,209 Targeted: Metabolite assays in cardiomyocyte clock-disrupted hearts identified clock-controlled metabolites, including: Glycogen, Triglyceride, NAD53,54,63,73	Tracer studies in cardiomyocyte clock-disrupted hearts identified clock-control of cardiac glucose uptake, glycolysis, glycogen turnover, glucose oxidation, oleate oxidation, triglyceride turnover, protein turnover, β-hydroxybutyrate oxidation49,50,53,55,61
Vessels/Endothelial Cell/VSMC	Omics: Transcriptomics in wild-type murine or primate aortas identified candidate metabolism-related transcripts17,90,211 Targeted: RT-PCR in Per2 knockdown endothelial cells identified clock-controlled metabolism-related transcripts, including: sirt391	Omics: None Targeted: Western blot in wild-type mouse aorta or Per2 knockdown endothelial cells identified clock-controlled metabolism-related proteins, including: p-NOS, SIRT391,94	Enzyme Activities: Enzyme assays in wild-type mouse aorta or Per2 knockdown endothelial cells identified clock-controlled metabolism-related enzymes, including: IDH, SUCLG, ACO, NOS91,94 Organelle Activities: None identified	Omics: None identified Targeted: None identified	Tracer and oxygen consumption studies in Per2 knockdown endothelial cells identified clock-control of endothelial oxidative metabolism, TCA cycle, and pentose phosphate pathway91
Fibroblast	Omics: Transcriptomics in synchronized fibroblasts in culture identified candidate metabolism-related transcripts212 Targeted: None identified	Omics: Proteomics in synchronized fibroblasts in culture identified candidate metabolism-related transcripts102 Targeted: None identified	Enzyme Activities: None identified Organelle Activities: None identified	Omics: None identified Targeted: None identified	None identified
Intestine/Epithelial Cell	Omics: Transcriptomics in wild-type primate intestines identified candidate metabolism-related transcripts211 Targeted: RT-PCR in wild-type or IEC clock-disrupted intestinal tissue or cultured IEC identified clock-controlled metabolism-related transcripts, including: sglt1, glut2, mtp, pept1, nhe3125,127,132,136,137	Omics: None identified Targeted: Western blot in wild-type and IEC clock-disrupted jejunum identified clock-controlled metabolism-related proteins, including: SGLT1, MTP125,131	Enzyme Activities: Enzyme assays in wild-type intestine and cultured Huh-7 cells identified clock-controlled metabolism-related enzymes, including: MTP131,132 Organelle Activities: None identified	Omics: None identified Targeted: None identified	Tracer studies in wild-type and IEC clock-disrupted mice identified clock-control of intestinal glucose and lipid absorption125,131,132
Adipose Tissue/Adipocytes	Omics: Transcriptomics in wild-type murine, primate and human adipose tissue, as well as adipocyte clock-disrupted adipose tissue identified candidate metabolism-related transcripts17,211,213–215 Targeted: RT-PCR in adipocyte clock-disrupted adipose tissue identified candidate metabolism-related transcripts, including: elovl6, scd1, ces1d144	Omics: None identified Targeted: None identified	Enzyme Activities: None identified Organelle Activities: None identified	Omics: Metabolomics and lipidomics in wild-type and adipocyte clock-disrupted adipose tissue identified candidate metabolic pathways144,216 Targeted: None identified	Glycerol release assay in synchronized adipocytes in vitro identified clock control of lipolysis217

i). Cardiomyocytes.

Cardiomyocytes are electrically responsive contractile muscle cells of the heart, which play an integral role in force generation during each heartbeat. Consistent with their need to contract continuously throughout life, cardiomyocytes are metabolic omnivores, being capable of metabolizing virtually any available substrate. Of the cardiovascular-relevant circadian clocks investigated to date, the role of the cardiomyocyte circadian clock in the temporal control of metabolism is understood to the greatest extent. Using multiple alternative experimental models of in vitro, ex vivo, and in vivo systems, coupled with genetic manipulation strategies, candidate and unbiased methodological approaches, it has been demonstrated that the cardiomyocyte circadian clock temporally governs an array of metabolic processes essential for normal contractile function.⁴⁸ Early studies employing cardiomyocyte-specific CLOCK mutant (CCM) mice revealed that the cardiomyocyte circadian clock augments cardiac glucose utilization during the active period, independent of cardiac work.^49,50 This includes temporal control of glucose uptake, glycolysis, pyruvate oxidation, and protein O-GlcNAcylation (Figure 4). Of these, cardiac glucose oxidation exhibits 2.5-fold higher rates during the active period, relative to the sleep period.⁵⁰ Given that the heart typically increases reliance on glucose to meet the energetic demands associated with acute increases in workload⁵¹, these observations led to the hypothesis that increased glucose utilization during the active period is in anticipation of greater physical activity at this time. Unlike glucose oxidation, cardiac fatty acid oxidation rates do not exhibit notable time-of-day fluctuations, consistent with a need for this process to meet the basal energetic needs of the heart.^49,52,53 However, cardiac triglyceride turnover fluctuates markedly over the course of the day, resulting in approximately 2-fold higher triglyceride levels in the heart towards the end of the active period.⁵³ This is observed at biochemical, histologic, and electron microscopy levels; in the latter case, both lipid droplet size and number fluctuate in cardiomyocytes over a 24hr period.^53,54 Using CCM mice, the cardiomyocyte circadian clock was shown to mediate rhythms in cardiac triglyceride turnover, potentially providing an intracellular fuel source for the anticipated upcoming sleep period.⁵³ Cardiomyocyte-specific BMAL1 knockout (CBK) mice have similarly been employed to study circadian regulation of cardiac protein synthesis, revealing that the cardiomyocyte circadian clock enhances myocardial protein synthesis at the beginning of the sleep period.^55,56 Phospholipid synthesis is also enhanced in the heart at this time, suggesting that the beginning of the sleep phase is important for the replacement of cellular constituents.⁵³ Consistent with this concept, autophagy peaks in the heart during the first 4 hours of the sleep phase, which is predicted to augment the removal of damaged cellular constituents.^57,58 Collectively, it appears that both oxidative and non-oxidative metabolism are temporally partitioned in the heart, due to governance by the cardiomyocyte circadian clock.

Figure 4. Regulation of cardiac metabolism by the cardiomyocyte circadian clock.

The cardiomyocyte circadian clock (depicted as a ‘clock’) has been shown to influence various aspects of cardiac glucose, fatty acid, amino acid, and ketone body metabolism. A ‘?’ indicates that direct evidence for cardiomyocyte circadian clock control of specific metabolic processes, such as the uptake of fatty acids, amino acids, and ketone bodies, does not currently exist. Illustration credit: Ben Smith.

A key question relates to how the cardiomyocyte circadian clock orchestrates cardiac metabolism. As highlighted in Figures 1 & 2, circadian clocks have the potential to act through numerous mechanisms. Conceptually, the simplest of these is the canonical pathway, which centers around the control of a gene whose protein product is ‘rate-limiting’ for the process. Numerous candidate genes have been explored, one of which includes Nampt. The encoded NAMPT protein is a key enzyme within the NAD salvage pathway, that maintains cellular levels of this critical cofactor.⁵⁹ The Nampt gene promoter/enhancer is regulated by the cardiomyocyte circadian clock through at least 3 distinct ways: 1) the CLOCK/BMAL1 heterodimer directly binds to E boxes, leading to transcriptional activation; 2) the CLOCK/BMAL1 target gene product KLF15 binds to two consensus sequences, activating transcription; and 3) the REV-ERBα/β target gene product E4BP4 binds to D boxes, repressing transcription.^60–64 Temporal coordination of CLOCK/BMAL1, KLF15, and E4BP4 results in 24hr oscillations in cardiac nampt mRNA levels, which are associated with NAMPT protein and NAD rhythms, all of which peak in the middle of the active period.^61,63,64 Increased cardiac NAD levels during the active period has been hypothesized to promote metabolism and signaling at this time.⁶⁵ However, it is noteworthy that 24hr rhythms in NAD levels persist in cardiomyocyte-specific REVERBα/β double knockout (csRevDKO) hearts, despite loss of cardiomyocyte circadian clock function and control of the Nampt gene.⁶³ This observation suggests that daily fluctuation in cardiac NAD levels may be mediated in part by extracardiac factors.

Similar to NAMPT, additional candidate mediators have been investigated in an attempt to define the mechanisms by which the cardiomyocyte circadian clock regulates cardiac metabolism. These include: 1) PIK3R1, a subunit of the insulin signaling cascade component PI3K, whose gene is activated by the CLOCK/BMAL1 heterodimer; 2) CES1D, a lipase, whose gene is repressed by E4BP4); and 3) DGAT2, an acyltransferase involved in TAG synthesis, whose gene is activated by the CLOCK/BMAL1 heterodimer, repressed by E4BP4, and repressed by the REV-ERBα/β-regulated miRNA let-7c-1–3p.^61,63,66 Transcript levels for pik3r1, ces1d, and dgat2 all peak in the heart during the active period.⁶¹ With regards to cardiac TAG turnover, Tsai et al. investigated the possibility that the cardiomyocyte circadian clock coordinately regulates multiple factors with established functions in lipolysis and lipogenesis.⁵³ Consistent with increased rates of net synthesis and accumulation of myocardial TAG during the active period, not only are dgat2 mRNA levels increased at this time, but factors promoting lipolysis were concomitantly repressed. The latter include hsl mRNA, phosphorylated HSL, and ATGL protein levels.⁵³ The opposite pattern was observed during the sleep phase, a time at which net lipolysis is augmented.⁵³ Importantly, 24hr oscillations in all these parameters were absent following genetic disruption of the cardiomyocyte circadian clock, consistent with the concept that this timekeeping mechanism orchestrates diurnal variations in TAG turnover through multi-level reciprocal regulation of opposing pathways. Evidence also exists supporting the concept that the cardiomyocyte circadian clock modulates myocardial glucose utilization through coordinated regulation of pik3r1 mRNA, GLUT4 and OGT protein), as well as pyruvate dehydrogenase activity; all of these peak during the active period, thereby synchronizing oxidative and non-oxidative glucose utilization.^50,52,61,67

As highlighted above, cardiac protein synthesis peaks at the beginning of the sleep phase, a time at which amino acid and ribosomal RNA levels, as well as phosphorylation/activity status of mTORC1, peak in the murine heart.⁵⁶ In the case of mTORC1 phosphorylation, the approx. 2-fold oscillation observed in the heart persists during fasting and is lost in CBK mice, indicating mediation by the intrinsic cardiomyocyte circadian clock.^56,57 Synchronization of these pro-translational factors is likely mediated by both canonical and non-canonical mechanisms. Consider mTORC1 as an example. mTORC1 activity is modulated by multiple binding proteins Mg²⁺ ions, and PTMs; all of these factors are circadian regulated in the heart. For example, RAGA and RAGD, the Mg²⁺ transporter SLC41A3, and the mTORC1 kinase Akt are all regulated by the cardiomyocyte circadian clock.^55,56,61,68 Conversely, the circadian clock component PER2 peaks in the heart during the active period, when cardiac protein synthesis is attenuated. Direct binding of PER2 to mTORC1 leads to inhibition, thus serving as a non-canonical mechanism.^45,50 It should be noted that in addition to regulating translation, mTORC1 influences a diverse array of biological processes, such as glucose and fatty acid metabolism, autophagy, macromolecular crowding, and ion homeostasis. This leads to the possibility that mTORC1 may serve as a critical mechanistic link between the cardiomyocyte circadian clock and numerous cardiac functions.⁶⁹ Consistent with this idea, administration of mice with rapamycin, a pharmacologic inhibition of mTOR, abolished time-of-day variations in spontaneous beating rate in ex vivo perfused hearts.⁷⁰

For non-cardiomyocyte cell types, various studies have reported circadian regulation of mitochondrial number, size, and oxidative metabolism through clock control of mitochondrial dynamics, mitochondrial structural proteins and enzymes, as well as substrate utilization.⁷¹ Such studies have led to the possibility that time-of-day oscillations in cardiac metabolism may be partially driven by the cardiomyocyte circadian clock influence over mitochondrial function. Initial studies investigated mitochondrial parameters in hearts of mouse models of cardiomyocyte circadian clock disruption at a single time of the day. More specifically, mitochondrial state 3 respiration is decreased in CCM hearts.⁴⁹ Similarly, transfection of neonatal rat cardiomyocytes with a dominant negative CLOCK mutant protein attenuates mitochondrial membrane potential in vitro.⁷² Deletion of BMAL1 in cardiomyocytes in vivo also attenuates complex I activity (whereas complex IV activity appears to be increased slightly).⁷³ Moreover, expression of mitochondrial fusion-related genes, Mfn1 and Opa1, is decreased in CBK hearts⁷⁴. Recently 24hr fluctuations in cardiac mitochondrial parameters have been investigated, revealing increased complex III activity in the heart during the active period, as well as a biphasic oscillation in total mitochondrial number; mitophagy exhibited a similar biphasic pattern, and was decreased in CBK hearts.⁵⁴ Additional studies are required to elucidate the extent to which the cardiomyocyte circadian clock modulates cardiac metabolism through perturbations in mitochondrial quality or quantity.

ii). Endothelial and Vascular Smooth Muscle Cells.

The vasculature serves critical functions, including delivery of oxygen, nutrients and hormones to target tissues, serving as a physical barrier to distinct circulating molecules and cells, as well as acting as a source of paracrine factors.⁷⁵ Blood vessels are composed of multiple cell types, including endothelial cells and VSMC. In both cases, metabolism is critical for maintenance of normal cellular function. For example, VSMC have a high reliance on glucose utilization, such that attenuation of glucose uptake impairs normal VSMC function.⁷⁶ Similar to VSMC, endothelial cell metabolism is highly glycolytic in nature.⁷⁷ It has been proposed that the low reliance of endothelial cells and VSMC on oxidative metabolism will spare oxygen for the tissues that the vascular bed serves.^78,79 Consistent with this ‘selfless’ concept, elegant studies by Son et al. have revealed that the vasculature plays an active role in the delivery of lipids to perivascular cells, in a CD36-dependent manner.⁸⁰ More specifically, endothelial-specific CD36 deletion attenuates the uptake of fatty acids in organs such as the heart, indicating that fatty acids potentially move through endothelial cells via active transport mechanisms. Although oxidative metabolism is perceived as relatively low in vascular cells, mitochondrial function is important for biosynthetic processes. Maintenance of mitochondrial function appears critical for endothelial cell processes such as proliferative and angiogenesis.^81,82 Amino acid metabolism also plays a pivotal role in vascular function, none more so than arginine metabolism; arginine serves as the substrate for NO generation, via the catalytic activity of NOS.⁸³ For additional information regarding the importance of metabolism for endothelial cell and VSMC function, the reader is referred to recently published reviews.^78,79

Circadian clocks are functional within the vasculature. Early evidence included studies by Davidson et al., revealing persistent 24hr rhythms of a bioluminescence reporter for the circadian clock in cultured vessel explants.⁸⁴ As with many mammalian cells, circadian clocks have been characterized within both endothelial cell and VSMC. For example, following serum-induced synchronization of endothelial cells in vitro, 24hr oscillations in bmal1 and per2 mRNA are observed.⁸⁵ Similar findings have been reported for VSMC.⁸⁶ Moreover, Nonaka et al. found that angiotensin II was sufficient to initiate bmal1, per2, and dbp mRNA 24hr oscillations in cultured VSMC.⁸⁷ Functional roles for circadian clocks within the vasculature have also begun to emerge. These include regulation of angiogenesis, coagulation, and injury response.^88,89 With regards to circadian control of vascular metabolism, some limited data is available. Indirect evidence includes transcriptomic analyses of murine vessels, revealing that a large portion of mRNA species exhibiting oscillations with a periodicity of 24hr encode for metabolism-related proteins.⁹⁰ Interestingly, Oyama et al. reported that knockdown of PER2 in cultured endothelial cells reduces oxidative metabolism, and that PER2 directly associates with several TCA cycle enzymes.⁹¹ With regards to arginine metabolism, NOS activity is regulated by the circadian clock via PTM and tetrabiopterin, as opposed to transcriptional control of the Nos gene.^92–94 Collectively, these observations suggest that circadian clocks within vascular cells likely regulate metabolism through canonical and non-canonical mechanisms. Future studies are required to define the extent to which circadian regulation of metabolism occurs in cells such as these, and the biological significance for vascular function.

iii). Fibroblasts.

Consistent with being the most abundant non-cardiomyocyte cell type in the heart, fibroblasts play vital roles in both physiologic and pathologic settings.⁹⁵ Secondary to their production of extracellular matrix components, fibroblasts are important for the maintenance of cardiac structural integrity.⁹⁶ Beyond this structural role, fibroblasts influence electrical conductance and mechanistic force distribution, as well as serving in an intercellular signaling capacity.^97–99 Following injury, activation of fibroblast-to-myofibroblast transition is not only an integral component of the normal repair/adaptation response (e.g., important for scar formation, to prevent cardiac rupture following an ischemic event), but also contributes towards disease progression. For example, fibroblast activation following an ischemic event is critical for scar formation, whereas myocardial stiffening secondary to excessive fibrosis impairs cardiac function.^100,101 As with virtually all cells, stimulus-response coupling results in metabolism-dependent perturbations in fibroblast function. Here, we will briefly review current knowledge regarding the fibroblast circadian clock, how metabolism influences fibroblast function, and will discuss hypothetical consequences of circadian regulation of fibroblast metabolism.

Fibroblasts were the first cell type for which an autonomous non-SCN circadian clock was described in mammals.¹⁴ More specifically, 24hr rhythms in circadian clock component transcripts persist in cultured fibroblasts, in the absence of environmental/extracellular stimuli.¹⁴ Employing a similar in vitro system, Hoyle et al. profiled the circadian proteome of fibroblasts, leading to the discovery that the fibroblast circadian clock modulates the efficiency of actin-dependent processes, including cell migration and wound healing.¹⁰² Evidence has emerged indicating that this clock also regulates epithelial-to-mesenchymal transition, fibroblast-to-myofibroblast differentiation, extracellular matrix remodeling, and fibrosis.¹⁰³ This is exemplified by observations that fibroblast-specific REV-ERBα knockout mice demonstrate worse fibrosis in response to lung injury.¹⁰⁴ Conversely, REV-ERBα agonists suppress TGF-β1-induced fibroblast-to-myofibroblast transition and pro-fibrotic phenotype.^105,106 Consistent with the knowledge that the CLOCK/BMAL1 heterodimer is immediately upstream of REV-ERBα, mice deficient for BMAL1 in the proximal tubule exhibit increased renal fibrosis in response to stress.¹⁰⁷ Collectively, these observations highlight that critical fibroblast functions are under circadian control.

Metabolic perturbations play fundamental roles in both fibroblast/myofibroblast function, as well as fibroblast-to-myofibroblast transition.¹⁰⁸ Upon activation, fibroblasts rapidly accelerate glycolytic flux, a metabolic switch known as the Warburg effect.¹⁰⁹ In doing so, this metabolic shift increases: 1) ATP availability for energetically demanding processes; 2) carbon availability for cellular constituent synthesis; and 3) generation of metabolic signals, including lactate and protein acetylation.¹⁰⁸ Both pharmacologic and genetic approaches have been employed to establish causation between increased glycolytic flux and myofibroblast activation. For example, attenuation of glucose uptake and/or glycolysis through use of 2-deoxyglucose, 3-bromopyruvate, or deletion of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 attenuates myofibroblast activation and/or collagen synthesis.^110,111 Conversely, collagen synthesis is augmented by culturing fibroblasts in the presence of high glucose levels.¹¹² Increased carbon flux along the glycolytic pathway promotes synthesis of lactate, acetyl-CoA, and numerous building blocks (e.g., amino acids, such as serine and proline).¹⁰⁸ Interestingly, evidence exists suggesting that lactate can function as a signaling molecule, and is sufficient to activate myofibroblasts.¹⁰⁹ Acetyl-CoA is utilized as a substrate for protein acetylation; indirect evidence suggests that this PTM may play a role in fibroblast differentiation.¹¹³ In addition to their use for cellular growth, amino acids are critical for the synthesis of collagen.¹¹⁴ It is also noteworthy that glutamine has emerged an important amino acid in myofibroblast activation, potentially by serving as a source of α-ketoglutarate.^114,115

Despite appreciation for clock control of metabolic processes, that the fibroblast circadian clock influences the fibroblast-to-myofibroblast transition, and that myofibroblast activation is dependent on cellular metabolism, we are unaware of studies directly investigating whether clock control of myofibroblast activation and/or collagen synthesis is through metabolic regulation. However, indirect evidence from other cell types exists. Key enzymes involved in both proline and glutamine catabolism are directly regulated by the cardiomyocyte circadian clock, including proline dehydrogenase and glutamine synthetase; 24hr oscillations in both of these enzymes are abolished in CCM and CBK hearts.^49,50,61 In the case of proline dehydrogenase, decreased levels observed in CBK hearts are potentially secondary to augmented miR-23b, and is associated with increased proline levels and cardiac fibrosis.^56,66,116 Although BMAL1 has been deleted specifically in cardiomyocytes in CBK hearts, the possibility exists that diminished cardiomyocyte proline catabolism increases proline availability for neighboring fibroblasts. In addition to the aforementioned clock control of glucose uptake in the cardiomyocyte subsection, lactate dehydrogenase has been reported to be directly regulated by the CLOCK/BMAL1 heterodimer in neurons, leading to 24hr oscillations in lactate synthesis; whether rhythms in lactate exposure impact myofibroblast activation is an intriguing possibility.¹¹⁷

iv). Intestinal Epithelial Cells.

When considering factors that impact cardiovascular metabolism, the contribution of substrate availability cannot be understated. All enzyme-catalyzed reactions are substrate dependent, making substrate availability a critical control modality. Secondary to physical barriers present in biology, numerous mechanisms regulating extracellular, intracellular, and subcellular substrate levels have evolved. In simplistic terms, circulating nutrient levels are dependent on feeding status, digestion/absorption, and clearance via cell/organ uptake. All of these processes are circadian regulated. Consistent with the location of the SCN within the hypothalamus, a brain region that is paramount in the control of food intake, circadian clocks reciprocally regulate various orexigenic and anorexic factors in a rhythmic manner, contributing to daily fluctuations in appetite and satiety (as reviewed elsewhere).¹¹⁸ The concept of circadian control of substrate uptake was introduced in the cardiomyocyte subsection, and holds true for various peripheral tissues. Interestingly, gastrointestinal function fluctuates dramatically over the course of the day at numerous levels, including gut motility, digestive enzyme secretion, microbiome composition, and nutrient absorption.¹¹⁹ IEC, also known as enterocytes, not only serve as a physical/biochemical barrier, separating host tissue from commensal bacteria within the intestinal lumen, but also play a pivotal role in nutrient absorption.¹²⁰ Circadian clocks significantly impact IEC functions, including proliferation, nutrient absorption, barrier function, gut microbiota interaction, and response to environmental challenges.^120,121 Given that substrate availability influences cardiovascular metabolism, this subsection will focus on how the IEC clock impacts the absorption of glucose, lipids, and peptides.

Multiple transporters involved in monosaccharide uptake exhibit 24hr oscillations in the gut; these include apical Na⁺/glucose cotransporter 1 (SGLT1), fructose transporter 5 (GLUT5), and hexose transporter GLUT2.^121–124 In the murine jejunum, transcript levels for these transporters peak at the beginning of the active period, likely secondary to direct induction by the CLOCK/BMAL1 heterodimer.^125,126 Indeed, targeted BMAL1 deletion in IEC severely attenuates rhythmic expression of sglt1 and glut2 mRNA.¹²⁵ A second mechanistic link between the IEC clock and SGLT1 may be afforded by PER1. More specifically, Balakrishnan et al. have demonstrated that PER1 downregulates sglt1 expression.¹²⁷ Unlike monosaccharides, which are initially transported from the gut to the liver via the hepatic portal vein, dietary lipids are packaged within chylomicrons by IEC, and subsequently exported into the lymphatic system. Anatomically, once released into the circulation, the heart is the first metabolically active organ to receive chylomicrons.¹²⁸ Consistent with an appreciable reliance on lipoproteins as a fuel, the heart has high lipoprotein lipase activity.¹²⁹ Accordingly, chylomicron availability dramatically impacts cardiac metabolism. Chylomicron assembly and release are facilitated by MTP within IEC.¹³⁰ Both MTP and lipid absorption display diurnal patterns in the intestine, with peak levels observed during the active period.¹³¹ It has been proposed that the CLOCK/BMAL1 heterodimer induces the transcriptional repressor SHP in IECs, which in turn directly binds to the MTP promoter in a rhythmic manner.^131,132 The clock output gene nocturnin also regulates mtp mRNA levels; nocturnin knockout mice exhibit low levels of MTP in the intestine, resulting in lipid retention within the IECs.¹³³ Regarding protein digestion/absorption, PEPT1 mediates the absorption of small peptides across the apical membrane.¹³⁴ Moreover, NHE3, located in the apical membrane of IEC, maintains the proton gradient required for peptide uptake.¹³⁵ Notably, both PEPT1 and NHE3 exhibit diurnal variations, which are likely secondary to both behaviors and the IEC clock.^22,136 In the latter case, PEPT1 has been reported to be regulated by the established clock output gene DBP, while NHE3 is regulated by the CLOCK/BMAL1 heterodimer.^137,138

v). Adipocytes.

Similar to the gut, adipose tissue plays an important role in metabolic homeostasis. Adipocytes not only influence circulating lipid availability through lipogenesis and lipolysis, but they also secrete a diverse array of adipokines, including adiponectin, leptin, and resistin.^139–141 The latter adipokines impact energy balance by regulating processes such as food intake, insulin sensitivity, and thermogenesis. Importantly, many adipocyte functions appear to be circadian regulated, leading to the possibility that adipose tissue contributes to 24hr oscillations in cardiovascular metabolism.¹⁴² When considering adipocyte metabolism, TAG turnover is at the forefront. In the fed state, increased circulating lipoproteins, glucose, and insulin promote adipose tissue TAG synthesis and storage.¹⁴³ Conversely, in the fasted state, stored TAG is hydrolyzed in response to lipolytic hormones, thereby increasing non-esterified fatty acid and glycerol levels in the blood.¹⁴³ In addition to the above-described effects of extracellular stimuli in TAG turnover, the adipocyte circadian clock appears to contribute towards diurnal rhythms. Genetic deletion of BMAL1 in adipocytes impairs 24hr rhythms in multiple transcripts encoding for enzymes involved in TAG turnover, associated with abolishment of plasma TAG daily rhythms.¹⁴⁴ At a mechanistic level, the lipogenesis/lipolysis genes elovl6, ces1d, and Scd1 are regulated by circadian clock components either directly or indirectly; indirect regulation may be via PPARγ and/or SREBP-1c.¹⁴⁴ In addition to lipid metabolism, the adipocyte circadian clock may influence glucose metabolism by regulating genes involved in glucose uptake, such as GLUT4.^145,146 Thus, the adipocyte circadian clock has the potential to impact metabolism of the cardiovascular system through its influence over nutrient availability.

Beyond its role as a storage organ, adipose tissue influences homeostatic processes through the synthesis of multiple paracrine and endocrine factors. These include proteins, lipid species, and exosomes.^147,148 Many of these factors appear to be circadian regulated.^19,149 For example, leptin levels fluctuate over the course of the day, peaking during the active period; in addition to fluctuating in response to increased food intake at this time, the adipocyte circadian clock contributes at the level of leptin secretion.¹⁴⁴ Moreover, 24hr rhythms in circulating leptin levels observed in humans appear to be independent of feeding/fasting cycles.¹⁵⁰ Evidence exists in support of adipocyte circadian clock control of the adiponectin gene, via CLOCK/BMAL1 regulation of PPARγ, which may contribute towards the diurnal rhythm observed in humans.¹⁵¹ Interestingly, BMAL1 deletion specifically in brown adipose tissue affected blood pressure in mice through alterations in perivascular adipose tissue angiotensin release.¹⁵² In addition to secreted proteins, adipocytes release numerous lipid species into the circulation in a circadian fashion. Some of these species, including distinct polyunsaturated fatty acids, act in a signaling manner.¹⁴⁴ It is noteworthy that secretion of exosomes has emerged as a critical cell-cell communication mechanism, whereby adipocytes can impact the function of other cells/tissues. Secreted exosomes are packaged with not only protein and lipid species, but also nucleic acids, such as RNA.¹⁵³ Recently, exosome composition has been reported to be circadian regulated, leading to the possibility that the adipocyte circadian clock may influence cardiovascular metabolism via this form of cellular communication.¹⁴⁹ Consistent with this concept, adipose-derived exosomes have recently emerged as a critical regulator of cardiac function.¹⁵⁴

Sex Differences in Circadian Regulation of Cardiovascular Metabolism

Sex differences in cardiovascular function and dysfunction are well-established.¹⁵⁵ Moreover, metabolic processes are also sex-dependent.¹⁵⁶ Appreciation of these observations raises questions regarding whether circadian control of cardiovascular metabolism differs between males and females. Unfortunately, very few studies have directly addressed this fundamentally important topic. With regards to the circadian clock mechanism itself, there appears to be little-to-no sex differences in cardiovascular tissues, such as the heart.¹⁵⁷ However, a recent study by Talamanca et al. revealed that only ~50% of the circadian transcriptome was similar between male versus female hearts.¹⁵⁸ Given that a significant proportion of oscillating genes within human hearts encode for proteins involved in glucose, lipid, and amino acid metabolism, these striking observations lead to the possibility that sexual dimorphism exists in circadian control of cardiovascular metabolism. This may reflect sex-dependent differences in the interaction of cell autonomous clocks with the neurohumoral milieu. Consistent with this concept, Alibhai et al. reported that although male germline CLOCK mutant mice develop metabolic perturbations and cardiac dysfunction with age, female mice do not.¹⁵⁹ More recently, Kane et al. reported sex-dependent 24hr oscillations in both mitochondrial parameters and TAG levels in the murine heart.⁵⁴ Future studies are required to comprehensively investigate the extent to which circadian regulation of cardiovascular metabolism is sex-dependent.

Metabolism as an Integral Circadian Clock Feedback Loop

As highlighted in prior sections, circadian clocks modulate metabolic processes through transcriptional, posttranscriptional, epigenetic, translational, and posttranslational mechanisms.^{15,44,45,160,161} Given the diverse nature by which circadian clocks regulate cellular metabolism over a 24hr period, it is not surprising that a number of metabolic signals feedback onto the clock mechanism. An underlying concept that cannot be understated is that metabolism extends beyond ATP synthesis, fuel store turnover and/or cellular constituent biosynthesis. Beyond these roles, metabolic pathways are critical for controlling the levels of signaling molecules and PTM precursors. Recent evidence has emerged indicating that these metabolic signals influence the levels and/or activity of circadian clock components, thereby forming integral feedback loops. They also afford a means by which extracellular stimuli/stresses act as zeitgebers, to reset the clock mechanism via changes in metabolism. Multiple metabolism-based feedback loops have been identified, some of which will be highlighted here briefly.

Consistent with daily fluctuations in energetic demand, both the phosphorylation status and activity of AMPK oscillates in the heart, peaking during the active period.⁵³ AMPK has been reported to influence the clock in two ways: 1) direct phosphorylation of CRY1, leading to degradation; and 2) phosphorylation of CK1ε, which in turn phosphorylates PER2, leading to degradation.^162,163 Consistent with the latter, PER2 protein levels steadily decrease in the heart during the latter half of the active period, reaching lowest levels approximately 6 hours after the peak activity of AMPK.^50,53 In addition to phosphorylation, various clock components undergo lysine acetylation, including PER1, PER2, and BMAL1.³¹ Acetylation is dependent on the nodal metabolite acetyl-CoA, which has recently been reported to be slightly increased in the heart during the active period.¹⁶⁴ Removal of this PTM is mediated by deacetylases, including NAD-dependent sirtuins; as highlighted in prior sections, the cardiomyocyte circadian clock regulates cardiac NAD levels, in part through NAMPT. Such observations have led to the hypothesis that rhythmic acetylation-deacetylation of core circadian clock components is critical for the normal function of this timekeeping mechanism.³¹ Another metabolism-related PTM includes protein O-GlcNAcylation, which has been reported to be circadian regulated in various tissues, including the heart.^50,165 Evidence suggests that BMAL1, CLOCK, PER1/2, and CRY1/2 are all O-GlcNAc modified, affecting their stability, cellular locality, and/or activity.¹⁶⁵ In the case of BMAL1, 24hr rhythms in protein O-GlcNAcylation peak in the heart during the active period.⁵⁰ Moreover, pharmacological perturbation of cardiac O-GlcNAcylation results in a rapid phase shift of the heart clock, consistent with a functional consequence of the PTM on the clock mechanism.⁵⁰ It is likely that additional metabolism-related PTMs, such as methylation, palmitoylation, succinylation, and/or hydroxylation, may play important roles in regulating circadian clock components in cardiovascular tissues.

Functional Consequences of Circadian Rhythms in Cardiovascular Metabolism

i). Physiologic Perturbations.

Several analogies can be drawn to help describe the potential impact of metabolic rhythms on cardiovascular function, and how they may operate to exert an evolutionary advantage in either the basal state or in response to physiologic stimuli. One example includes a ‘factory assembly line’ analogy. If production along an assembly line was to increase at a specific time of the day, all stations along the assembly line must temporally adjust their work rates in synchrony. Should synchrony not be achieved, leading to misalignment of work stations along the assembly line, then overall productivity would be adversely affected, and may even result in detrimental adverse events. Another analogy includes consideration of the body as a machine, who’s inner workings must operate in temporal alignment, for maintenance of function. Such an analogy may conjure images of cogs turning in synchrony, and consequentially jamming when misaligned. At a biological level, both analogies could be used when reflecting on circadian regulation of cardiovascular metabolism. Consider the aforementioned dawn phenomenon, wherein hepatic glucose output increases immediately prior to awakening, to ensure that the body’s energetic demands upon awakening are met with adequate fuel supply. If the model is simplified, by considering only the liver and heart, it could be reasoned that the hepatic clock promotes gluconeogenesis at a time when the cardiomyocyte clock promotes cardiac glucose catabolism; the net effect of this synchrony is that the energetic demands associated with increased cardiac contractility upon awakening are met. A key concept here is inter- organ/cell temporal alignment (Figure 5), wherein the hepatic clock anticipates the heart’s fuel needs at the same moment when the cardiomyocyte clock anticipates energetic demands associated with physical activity upon awakening. Such a concept leads to many questions, including: 1) how is inter- organ/cell temporal alignment achieved; 2) what happens if/when misalignment occurs; and 3) how do intrinsic synchrony mechanisms interface with acute environmental/behavioral perturbations. Here, we provide a brief overview regarding current knowledge of temporal alignment, with examples pertinent to metabolism and cardiovascular function.

Figure 5. Circadian control of inter-organ/cellular communication as a means of influencing cardiovascular metabolism.

A) Clock control of endocrine factor and nutrient release from the liver, intestine, and/or adipose tissue will impact cardiovascular metabolism over the course of the day. B) Diurnal variations in cellular communication within cardiovascular tissues is considered an important, and highly understudied area, which could impact metabolism during both health and disease. Illustration credit: Ben Smith.

Reductionism has been employed successfully in multiple scientific disciplines, as a strategy to address discrete, testable hypotheses. Over the past several decades, efforts have increased to integrate information gleaned from reductionist approaches, using systems biology approaches. Multiple levels of systems integration exist, including integration of transcriptional, translational, signaling, and metabolic processes within a cell, paracrine interactions between distinct cell types within an organ, and communication between two or more organs. Appreciation that cell and organ functions fluctuate over the course of the day has added an additional layer of complexity to systems biology. Conceptually, preservation of optimal biological functions requires temporal alignment at all levels, through the synchronized actions of distinct zeitgebers, that together fine-tune the timing of circadian clocks. The stereotypical zeitgeber is light, which resets the SCN. However, numerous non-photic environmental/behavioral-derived factors exist, such as temperature, pollutants, food intake, and physical activity.¹⁶⁶ Organs/cells are responsive to multiple zietgebers, albeit with some level of selectivity. For example, the SCN is highly sensitive to light, the liver to food intake, and skeletal muscle to physical activity.^35,166 The multi-factorial plasticity exhibited by circadian clocks confers both benefit and potential detriment. With regard to benefit, when optimal temporal synchrony of zeitgebers is achieved, high amplitude circadian clock output occurs, with alignment between multiple systems. In contrast, when zeitgebers are out of temporal order, cell/organ-specific influences result in circadian misalignment. Consider manipulation of food intake timing as an example. Mice are nocturnal, and consume approximately two-thirds of their daily food during the awake/dark period.¹⁶⁷ When light/dark, sleep/wake, and fasting/feeding cycles are reinforced by restricting food availability only to the awake/dark period, high amplitude circadian clock gene oscillations are observed in multiple tissues; in the case of peripheral tissues (e.g., liver, adipose tissue, skeletal muscle, and heart), circadian clock genes are in close alignment.^39,167 In contrast, when light/dark and sleep/wake cycles are dissociated from fasting/feeding cycles, by restricting food availability only to the sleep/light period, interorgan misalignment results. More specifically, the timing of SCN clock gene and physical activity rhythms remain aligned with the light/dark cycle, whereas liver, adipose tissue, heart, and skeletal muscle clock gene oscillations phase shift by approx. 8.4, 6.9, 3.9, and 3.5 hours respectively.^39,167 Moreover, clock gene oscillation amplitudes are diminished in several of these peripheral tissues.¹⁶⁷ In doing so, sleep/light phase restricted feeding in mice induces interorgan circadian misalignment. When considering the various metabolic interconnections highlighted in previous subsections within this review, it is unsurprising that this misalignment predisposes cardiometabolic disease.¹⁶⁸

It is noteworthy that in some instances, inter-organ misalignment of circadian-regulated metabolic processes may be advantageous. This can be illustrated by several well-established concepts in the field of metabolism. Consider the Cori cycle, which describes the metabolic inter-organ relationship between the liver and muscle during exercise.¹⁶⁹ More specifically, during exercise, hepatic glucose production is increased concomitant with augmented rates of muscle glucose utilization. In other words, at the same moment in time, two organs have similarly opposing metabolic profiles. The same is observed on the circadian timescale, such that at specific times of the day, metabolic processes appear to be opposing between distinct tissues, despite the alignment of circadian clocks. This is achieved in part through cell type-specific regulation of metabolic processes by circadian clocks. In the case of glucose metabolism, hepatic glucose output and cardiac glucose utilization are both augmented at the sleep-to-awake transition, in part due to differential clock control of these processes within the two organs. Another example involves lipid metabolism. More specifically, LPL activity exhibits time-of-day-dependent rhythms in multiple tissues, that are driven by cell autonomous circadian clocks.¹⁷⁰ However, these rhythms appear to be misaligned between the heart and adipose tissue; cardiac LPL activity peaks towards the middle of the sleep period, whereas adipose tissue LPL activity peaks during the active period.¹⁷⁰ These observations have led to the hypothesis that decreased LPL activity in the heart during the active period avoids over use of this substrate during the awake period, thereby preventing excessive lipid accumulation within the myocardium, and ensuring that excess dietary lipid is stored in adipose tissue. Conversely, during the sleep period, when both circulating and cardiac TAG levels decrease, LPL activity increases in the heart to help extract sufficient TAG-derived fatty acids from the plasma. A detrimental consequence of this circadian regulation is that the ‘inappropriate’ consumption of dietary lipid closer to the awake-to-sleep transition promotes cardiac steatosis and contractile dysfunction.^53,171,172

Circadian clocks confer the selective advantage of anticipation. However, cells/organs must maintain sufficient flexibility to ensure responsiveness to multiple alternative scenarios. For example, multiple events may occur upon awakening. These might include: 1) a need for bursts of strenuous physical activity during fight or flight encounters; 2) continuation of the sleep phase fast if the forage for food is not successful after awakening; and/or 3) dealing with increased nutrient availability if the forage for food is successful. Is it possible to anticipate all these scenarios simultaneously? In terms of cardiac glucose and fatty acid metabolism, these three behavioral scenarios result in two distinct metabolic profiles. Physical activity and consumption of a carbohydrate-enriched meal both result in increased myocardial glucose utilization, whereas prolongation of a fast or consumption of a lipid-enriched meal both result in increased myocardial fatty acid utilization.^51,57,173 Evidence exists that the cardiomyocyte circadian clock anticipates and prepares the heart for both metabolic profiles. The first metabolic profile was described within the cardiomyocyte subsection, wherein the cardiomyocyte circadian clock promotes glucose utilization upon awakening through multiple mechanisms, including augmentation of glucose uptake, glycolysis, and pyruvate oxidation (Figure 4). At the same time, the heart increases transcriptional responsiveness to fatty acids, through a coordinated increase in PPARα and PGC1α, in addition to a concomitant reciprocal decrease in REVERBα/β.^174,175 In doing so, if the heart is challenged with fatty acids at the being of the active period due to continuation of fasting or consumption of a lipid-enriched meal, then genes promoting fatty acid utilization are induced rapidly, resulting in increased fatty acid oxidation.^53,57,175 This ability of the heart to anticipate multiple scenarios is possible through orchestrated temporal regulation of metabolic processes by the cardiomyocyte circadian clock.

ii). Pathologic Stresses.

Disruption of circadian rhythms, secondary to behavioral, environmental and/or genetic perturbations, is often associated with an increased risk of cardiovascular disease in humans.^176–181 Similarly, various common cardiovascular disease risk factors are associated with impairment of circadian biology, manifesting at levels of altered rhythmicity in circadian clock genes, neurohumoral factors, and cellular/organ functions.^182–187 This includes altered 24hr rhythms in various metabolism-related parameters.^188,189 With regards to cardiovascular relevant tissues, high amplitude oscillations in circadian clock genes are attenuated in the heart during hypertension, hypertrophy, and following ischemia/reperfusion injury.^182,185,190 Moreover, during diabetes, the heart clock is phase-shifted.^183,191 Somewhat surprisingly, obesity has no significant effects on cardiac circadian clock component oscillations at the mRNA level, despite dramatic alterations in time-of-day-dependent fluctuations in the cardiac transcriptome, lipidome, and metabolism; this may indicate perturbed circadian output during obesity and/or altered temporal interaction of the heart with extra-cardiac factors.¹⁸⁸ Consistent with the latter possibility, inter-organ misalignment is observed during several of these disease states, which likely contributes towards pathologic outcomes.^192–194

It should be noted that there is a paucity of data concerning 24hr rhythms in cardiovascular metabolism during disease states. During hypertension, ischemic heart disease, and even diabetes mellitus, little is known regarding the extent to which metabolism is altered in cardiovascular-relevant tissues/organs. However, interrogation of circumstantial transcript data supports the idea that metabolic rhythms may be perturbed in many of these disease states. For example, during pressure overload-induced hypertrophy, 24hr oscillations in transcripts encoding for proteins involved in glucose metabolism, such as glut4 and pdk4 mRNA, are markedly attenuated in the heart.^52,182 Similar observations have been reported during ischemic heart disease and diabetes mellitus, although the extent to which these perturbed transcript rhythms affect metabolism remains to be established.^183,185 In contrast, the impact of obesity on day-night differences in cardiac metabolism have been investigated. More specifically, TAG synthesis diurnal variations were abolished in hearts of obese mice, despite a relative preservation in glucose metabolism rhythms.¹⁸⁸ This was associated with both aberrant day-night differences in the cardiac lipidome, as well as cardiac steatosis.¹⁸⁸ Importantly, reestablishment of circadian alignment through restriction of food intake to the active period normalized diurnal variations in cardiac lipid metabolism, reduced cardiac steatosis, and attenuated adverse cardiac remodeling in obese mice.¹⁸⁸ These observations highlight that during metabolic disease states, strategies targeting circadian alignment can be employed successfully for the treatment of cardiovascular disease. Translationally, time-of-day-restricted feeding has been investigated in overweight and shiftwork humans, leading to improvements in circulating lipid parameters and/or blood pressure.^195,196

In addition to the aforementioned studies, the impact of time-of-day-restricted feeding and cardiac function has been studied in animal models, for either augmentation or prevention of cardiovascular disease. For example, Tsai et al. first reported that the cardiac contractile dysfunction observed in mice following chronic high fat diet ad libitum feeding was prevented when food access was restricted to the active period.¹⁷¹ Similar findings have been reported in an invertebrate model.¹⁹⁷ Conversely, Peliciari-Garcia et al. observed that high fat feeding specifically at the end of the active period attenuated cardiac function relative to mice fed the same high fat meal at the beginning of the active period.¹⁷² Such observations are consistent with the American Heart Association recommendations, based primarily on epidemiologic observations, to eat “a greater share of the total calorie intake earlier in the day to have positive effects on risk factors for heart disease and diabetes mellitus”.¹⁹⁸

Despite a lack of knowledge concerning diurnal rhythms in cardiac metabolism during ischemic and non-ischemic heart disease, evidence exists suggesting that the manner with which the myocardium responses to acute stresses is time-of-day-dependent, likely due to metabolic fluctuations. Consider 24hr rhythms in cellular consistent turnover, as an example. The beginning of the sleep period is a time at which protein synthesis and autophagy are increased.¹⁹⁹ These processes are critical for the remodeling of the myocardium in response to both physiologic stimuli and pathologic stresses.²⁰⁰ Interestingly, acutely challenging the myocardium with pro-hypertrophic stimuli at this time leads to greater cardiac growth compared to the same challenge at the beginning of the active period.^56,201 Conversely, induction of an ischemic event at the beginning of the sleep period results in less damage to the myocardium, which may be secondary to enhanced cellular repair at this time.^65,202 Similarly, metabolism-related parameters such as glycogen levels, coupling of glycolysis-and-glucose oxidation, protein acetylation, ketone body metabolism, and autophagy generation have been linked to ischemic tolerance; all of these parameters are regulated by the cardiomyocyte circadian clock (Figure 4).^203–206 Many of these processes are also dependent on mitochondrial function, leading to the possibility that circadian regulation of mitochondrial quality control contributes towards 24hr rhythms in ischemic tolerance. Indeed, Rabinovich-Niktin et al. recently investigated clock control of mitophagy in the setting of simulated hypoxia in vitro.⁷² More specifically, overexpression of CLOCK attenuated hypoxia-induced mitochondrial damage and cell death, an effect that was dependent on the key autophagy factor ATG7.⁷² Studies such as these suggest that circadian disruption/misalignment may precipitate/augment cardiovascular disease through impairment of mitochondrial function.

Summary and Future Directions

Cell autonomous circadian clocks have emerged as a critical orchestrator of essentially all fundamental biological processes. None more so than metabolism. The depth of this relationship is underscored by the appreciation that metabolism is integrated within the inner workings of the clock machinery. In doing so, circadian clocks ensure that distinct metabolic processes occur at an appropriate time of the day, not only for the efficient functioning of that cell, but also with consideration of the temporal needs of neighboring cells/organs within the host (Figure 5). The latter requires constant checks and adjustments, to ensure temporal alignment within the organism, as well as alignment of the organism with the environment. Consistent with a need for metabolic plasticity, high amplitude 24hr oscillations are observed within the cardiovascular system at multiple levels (Table 1). The functional significance of these daily fluctuations is evident when they become aberrant, invariably resulting in precipitation of cardiometabolic and cardiovascular diseases. Despite this appreciation, several knowledge gaps remain, some of which are highlighted below within the following four main categories.

i). Molecular Link Identification.

The complexities of the mammalian circadian clock transcription-translation feedback loops, coupled with a divergence of ancillary interactions, has hindered the ability to define the precise mechanisms by which circadian clocks regulate metabolic processes. Similarly, metabolism homeostasis is achieved through a series of interconnecting pathways that are regulated with a high degree of flexibility and redundancy. Moreover, both circadian clocks and metabolism are, by definition, continually in flux. These attributes make defining the molecular links between circadian clocks and metabolism particularly challenging. Systems biology approaches (including machine learning) are needed to mine multi-omics information obtained from high temporal resolution samples/data, in order to define critical nodal points for clock control of metabolism.

ii). Physiologic Significance.

Within this review article, numerous examples of circadian control of metabolism are highlighted, and their likely significance is described. However, the majority of these studies have focused on specific metabolic pathways involved in ATP generation and/or nutrient storage, representing only a small fraction of cellular metabolism. In-depth flux analyses are required to illuminate the extent to which metabolic processes fluctuate over the course of the day in cardiovascular-relevant tissues; in doing so, novel roles for circadian control of metabolism are likely to be unveiled. Moreover, with the advent of single-cell metabolomics, insights regarding circadian regulation of metabolic pathways spanning two or more cell types may be gained. Additional studies are also required to reveal the extent to which metabolic relationships between distinct organs are under circadian control. Moreover, fundamental questions remaining to be answered include the extent to which circadian regulation of cardiovascular metabolism is impacted by distinct physiologic states, such as sex, pregnancy, and/or healthy aging.

iii). Pathologic Consequence.

Given that metabolic perturbations during disease states play causal roles in pathogenesis, and that maintenance of normal metabolic circadian rhythms is critical for cellular/organ function, a significant knowledge gap centers around understanding the extent to which circadian control of metabolism is impaired during CVD states. Similarly, the contribution of circadian misalignment in the pathogenesis of cardiovascular disease remains in its infancy, particularly with regards to inter-organ metabolic relationships.

iv). Manipulation for CVD Treatment/Prevention.

The translational potential of circadian control of metabolism for cardiometabolic diseases has begun to emerge. Lifestyle interventions designed to improve the circadian alignment of metabolism, such as time-of-day restricted feeding, have already yielded promising outcomes in humans. Whether similar interventions are beneficial in heart disease patients has yet to be reported. Similarly, little is known whether the time-of-day at which exercise is performed influences CVD outcomes, despite appreciation that this is an intervention that greatly affects metabolism and circadian clocks. Finally, it is worth noting that multiple pharmacological agents have been developed, which target distinct circadian clock components; many of these agents have yielded beneficial metabolic effects in animal models of cardiometabolic diseases.^207,208 Whether directly targeting the circadian clock in this manner will improve cardiovascular function in patients through improvements in metabolic processes remains unknown.

Abbreviations

ACO

acyl-CoA oxidase

AKT

protein kinase B

AMP

adenosine monophosphate

AMPK

adenosine monophosphate-activated protein kinase

ATG7

autophagy related 7

ATGL

adipose triglyceride lipase

ATP

adenosine triphosphate

BDH

β-hydroxybutyrate dehydrogenase

bHLH-PAS

basic helix-loop-helix PER-ARNT-SIM

BMAL1

brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like 1

CBK

cardiomyocyte-specific BMAL1 knockout

CCM

cardiomyocyte specific CLOCK mutant

CD36

cell differentiation 36

CES

carboxyesterase

CK1ε

casein kinase 1ε

CLOCK

circadian locomotor output cycles kaput

CoA

coenzyme A

CRY

cryptochrome

csRevDKO

cardiomyocyte-specific REVERBα/β double knockout

CVD

cardiovascular disease

DBP

D-box binding protein

DGAT

diacylglycerol acyltransferase

E4BP4

E4 binding protein 4

eIF

eukaryotic initiation factor

ELOVL6

elongation of very long chain fatty acids 6

GLUT

glucose transporter

HSL

hormone sensitive lipase

IDH

isocitrate dehydrogenase

IEC

intestinal epithelial cell

KLF15

kruppel like factor 15

LPL

lipoprotein lipase

MFN1

mitofusin 1

miRNAs

micro ribonucleic acid

mRNA

messenger ribonucleic acid

mTORC

mammalian target of rapamycin complex

MTP

microsomal triglyceride transfer protein

NAD

nicotinamide adenine dinucleotide

NAMPT

nicotinamide phosphoribosyltransferase

NHE3

sodium hydrogen exchanger 3

NO

nitric oxide

NOS

nitric oxide synthase

NR

nuclear receptor

OGT

O-GlcNAc transferase

OPA1

optic atrophy 1

PDH

pyruvate dehydrogenase

PDK4

pyruvate dehydrogenase kinase 4

PEPT1

peptide transporter 1

PER

period circadian protein

PGC1

peroxisome proliferator-activated receptor γ co-activator 1

PI3K

phosphoinositide 3-kinase

PIK3R1

phosphoinositide 3-kinase regulatory subunit 1

PKC

protein kinase C

PLIN1

perilipin 1

PPAR

peroxisome proliferator-activated receptor

PTM

posttranslational modification

RAG

RAS-related GTP binding

REV-ERB

nuclear receptor reverse-ERB

ROR

RAR-related orphan receptor

RORE

ROR-binding element

SCD1

stearoyl-CoA desaturase

SCN

suprachiasmatic nucleus

SGLT1

sodium/glucose cotransporter 1

SIRT

sirtuin

SLC

solute carrier

SREBP

sterol regulatory element binding transcription factor

SUCLG

succinyl-CoA ligase

TAG

triglyceride

TCA

tricarboxylic acid

TGF-β1

transforming growth factor β1