Therapeutic Potential of REV-ERB Circadian Transcription Factors in Cardiac Function: A Comprehensive Review

From organisms to cells, time is crucial for biological functions. The heart adapts to daily changes in workload and hormone levels and is associated with sleep and eating patterns. Cardiovascular function also fluctuates in healthy individuals, with blood pressure and electrophysiology varying by approximately 8%–20% in 24 hours. The cortisol and renin-angiotensin system affect diastolic function, which varies day and night. There were also 24-h changes in cardiac aspects such as signaling pathways, metabolism, and contractility. Research has also shown that heart processes depend on the circadian clocks. There is a dependence of cardiovascular diseases on the time of day: ischemic events and arrhythmias peak in the early morning (such as heart attack and stroke). Specific times of day become associated with clotting and inflammatory indicators, making it more likely for the patient to have cardiovascular events, including possibly fatal arrhythmias. In order to treat it, it is important to understand the circadian rhythms in heart health. The disruption of clocks in mice causes heart disease and reduced lifespan. For optimum cardiovascular health and disease control, it is necessary to understand and utilize circadian rhythm. [1, 2] The molecular system of circadian clocks is comprehensively based on gene transcription with many feedback loops. In mammals, the components of the core are two transcription factors, Brain and Muscle Arnt-like protein-1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) (and sometimes NPAS2 for CLOCK). These substances activate numerous genes, such as those inducing negative feedback loops; in particular, period (PER1/2/3), cryptochrome (CRY1/2), and nuclear receptor subfamily 1 group D (REV-ERB (α and β)). [1] In 1989, REV-ERBα was identified as a transcriptional repressor that has a greater impact on circadian rhythms than REV-ERBβ. REV-ERBα is showing promise as a treatment target for sleep-related disorders, as well as metabolic conditions, including elevated lipid levels, sugar imbalances, and weight gain, owing to its role in regulating circadian and metabolic genes, and beyond, with implications in various diseases, including cardiovascular disorders and cancer. REV-ERBα/β's circadian drug metabolism role sparks chronopharmacology interest. [3] The secondary feedback loop is essential for the regulation of the circadian clock system by Bmal1 expression. CLOCK and BMAL1 secondarily induce REV-ERBα and REV-ERBβ that interact with retinoic acid-related orphan receptor (RORα and RORγ) to inhibit Bmal1 synthesis at the ROR response region of the Bmal1 promoter. Downstream genes that contribute to metabolism and inflammation, which are required for circadian clock homeostasis, are regulated by REV-ERBs. [4] An additional way the circadian clock influences heart health is through REV-ERB signaling—by changing the timing of expression of clock-controlled genes (CCGs) and through its impact on heart insulin signaling and energy use [1] (Figure 1).

Figure 1: The basic clock mechanism of the circadian system, which is a hierarchical system, uses transcriptional and translational feedback loops to drive endogenous biological timekeeping. As transcriptional repressors, REV-ERBs function in this system (top right). Clock-controlled genes (CGCs) are genes that, either alone or in combination, are modulated by core clock transcription factors, resulting in rhythmic expression. Integrated light/dark cycles and peripheral clock synchronization are functions of the suprachiasmatic nucleus (SCN) master clock, which lies at the top of this hierarchy. This synchronization takes place by controlling temporal elements like temperature, feeding patterns, mechanical stimulation, and hormonal and neural impulses, which collectively maintain the synchronization of cellular processes.

Circadian Rhythms and Their Role In Cardiovascular Health

Circadian rhythm is a natural, self-regulating cycle lasting 24 hours that governs the metabolism of organisms. [5, 6] This internal clock produces daily rhythms and manages synchronized fluctuations in numerous neuroendocrine signals and metabolism. [6, 7] A large number of recent studies have linked disturbances to these rhythms (produced by long-distance travel, shift work, or irregular eating habits) with disorders of the central nervous system, metabolic disorders, cardiovascular illness, cancer, and more. [6, 8, 9] The circadian clock gene functions at the cellular level through transcription-translation feedback loops. Here, two negative-feedback pathways are regulated by the Clock and brain and muscle Arnt-like protein-1 (Bmal1). Rev-Erbα and Rev-Erbβ are twin paralogues of the Period/Cryptochrome (Per/Cry) complex, which regulates these pathways. Negative feedback loops function by directly expressing Bmal1 (via Rev-Erbα/β) or lowering Bmal1/Clock transactivation activity (through Per/Cry) and inhibiting Bmal1 activation (through ROR). Moreover, clock-controlled genes (CCGs) like O-linked β-N-acetylglucosamine (O-GlcNAc) signaling, nicotinamide adenine dinucleotide+ (NAD+)-dependent sensors, nicotinamide phosphoribosyltransferase (NAMPT), silent information regulator sirtuin 1 (SIRT1), and AMP-activated protein kinase (AMPK) connect circadian rhythms to metabolic controls. Nuclear receptors (NRs) in metabolic tissues also exhibit circadian rhythms and directly influence Bmal1 transcription via ROR and Rev-erb. [6] Factors such as light, activity, diet, health conditions, and aging influence circadian rhythms. Potentially metabolism-related problems, such as type 2 diabetes and obesity, are linked to genetic differences in key clock genes, including Bmal1 and Clock. Around 50 percent of human genes exhibit circadian fluctuations in different tissues. Stem cell control, mitochondrial activity, and immune responses are affected by these rhythms. The interactions between Bmal1 and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) enable the relationship between mitochondria and the circadian clock. [6] Studies have revealed that morning shear stress and platelet aggregability can negatively impact unstable plaque patients. A biological clock similar to circadian rhythm controls daily cardiovascular fluctuations independently. Genes like CLOCK, BMAL1, PER, and others regulate cellular functions and gene expression. Cardiovascular circadian clocks are vital for functions such as vascular reactivity, wound healing, and inflammation regulation. Research using models has highlighted the significance of the cardiomyocyte circadian clock in maintaining cardiovascular health [2] (Figure 2).

Figure 2: Intrinsic factors, such as genetic elements (including age and mutations in clock genes), and extrinsic factors, such as environmental influences (sleep disturbances, irregular diets, nighttime light exposure, artificial lighting, electromagnetic waves, repeated jet lag, and shift work), can disrupt circadian rhythms. These disruptions in circadian clock oscillations are associated with the development of cardiometabolic diseases, central nervous system disorders, and cancer.

Molecular Mechanisms of The Circadian Clock

Rev-Erb: A Circadian Rhythm Repressor

Rev-Erbα (Nr1d1) and Rev-Erbβ (Nr1d2) are two components of the nuclear receptor (NR) family that critically regulate daily cycles, metabolism, and immune responses. [6, 10, 11] They are different from other NRs because they lack activation function-2 (AF2) in the ligand-binding area of their end part needed in other activator assemblies for teaming up with coactivator to turned on or off gene expression. Rev-Erbs primarily work by blocking gene activity related to our internal clock. They do this in two ways: either by simply getting in the way of another protein that activates genes, called ROR, at specific DNA sites (RORE), or by actively calling in a team of proteins (the Nuclear Receptor Co-repressor-Histone Deacetylase 3 (NCoR-HDAC3) complex) that shut down gene activity. [6] Additionally, Rev-Erbs are involved in managing how our bodies process sugars, fats, and energy, and they also influence the creation of fat tissue and the body's inflammatory response. [6, 8, 9]

Rev-Erb in the Molecular Clock Mechanism

REV-ERB is an essential component of the circadian clock mechanism in animals. The three interrelated feedback loops regulate the molecular clock. [3, 12] The main loop is driven by the BMAL1/CLOCK heterodimer-induced expression of the E-box-controlled genes PER and CRY. PER and CRY proteins translocate to the nucleus, whereupon they accumulate and then block BMAL1/CLOCK activity. When PER and CRY levels are reduced following degradation, the result is a new transcription cycle: PER and CRY separate from the BMAL1/CLOCK complex. Casein kinases (CKIδ and CKI?) and adenosine monophosphate-activated protein kinase (AMPK), when indicated for ubiquitination and proteasome degradation, control the breakdown of PER and CRY. [3, 13] F-box and leucine-rich repeat protein 3 (FBXL3) and F-box and leucine-rich repeat protein 21 (FBXL21) also participate in CRY ubiquitination; FBXL21 prevents CRY degradation. [3] REV-ERBs and RORs are expressed by BMAL1/CLOCK, which in turn suppresses and activates BMAL1 transcription and RORE/RevRE-controlled genes, respectively, in the second loop. Numerous physiological functions, such as immunological responses, metabolic equilibrium, and cardiovascular function, are influenced by these genes. E4 promoter-binding protein 4 (E4BP4) and albumin D-site binding protein (DBP), which govern D-box-controlled genes and PER2, are involved in the third loop. Although the profiles of their expression differ, all clock genes exhibit cyclic patterns [3] (Figure 3). REV-ERBα, which normally acts as a monomer, binds to specific half-site motifs on target gene promoters. It also acts occasionally as a dimer on neighboring motifs, recruiting a co-repressor to regulate transcription. It may repress and reduce gene expression via dynamic chromatin looping and enhancer-derived RNA suppression. In addition, E4bp4 is repressed and interacts with other transcription factors [3, 14] (Table 1).

Figure 3: REV-ERB in the Molecular Clock System: (A) Schematic Representation of the Molecular Clock Machinery: The mammalian molecular clock comprises three interconnected auto-regulatory feedback loops. These loops involve PERs/CRYs (depicted by light blue lines), REV-ERBs/RORs (gray lines), and DBP/E4BP4 (orange lines). (B) Circadian mRNA Expression Patterns: These depict the rhythmic expression of clock genes in mice. (C) Regulatory Patterns of REV-ERBα on Target Genes: REV?ERBα regulates transcription in multiple ways: directly via a single RORE (i), REVDR2 (ii), or two adjacent ROREs (iii), and indirectly through other transcription factors (iv). Source. [3] Adapted from Wang et al. According to the Creative Commons License.

Physiological Functions of Rev-Erbα/Β In the Heart

Cardiac disorders are one of the most prevalent diseases worldwide, with approximately 64.3 million sufferers affecting 11.8% of those populations older than 65 in developed countries. [6, 44, 45] Other effects of ischemic injuries include remodeling of the structure and function of the heart, with fibrosis defined as increasing deposition of extracellular matrix (ECM). Collagen type I (Col1) provides tensile strength to myocardial collagen, while collagen type III (Col3) endows flexibility. Fibrosis post-injury is due to growth factors and specific proteolytic enzymes released from the cardiac surface (extracellular matrix/ECM), which have shown circadian modulation in the hearts of humans and rodents. [6, 46–49] Mice that are genetically modified to lack the circadian gene Bmal1 have been observed to demonstrate signs of cardiomyopathy as well as a shorter lifespan, in addition to displaying other characteristics of explantation, which include hypertrophic aging hearts and interstitial degree of fibrosis. Likewise, cardiovascular diseases have been accompanied by shift work, for example, atrial fibrillation and coronary heart diseases. [6, 50–53] Circadian rhythms are vital in the conduct of activities within the body, such that there is a cyclic regulation of the expression of nuclear receptor proteins such as Rev-Erb proteins, which are associated with energy and metabolic processes, inflammation, and fibrotic processes. Rev-Erb suppression holds significant promise for heart and metabolic disease treatment, as well as organ fibrosis treatments for the liver, heart, and lungs. [6] The respective Rev-erb agonist SR9009 has been found to slow the rate of progression in heart failure in mouse models and cardiomyocyte hypertrophy, as well as fibrosis. [54] It enhances left ventricle function and post-MI survival. SR9009 revealed the downregulation of some cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP9); a lower level of immune cells; and also a decrease in myocardial injury. [55] In mice with pressure overload-induced cardiac hypertrophy due to transverse aortic constriction, it also diminished AKT expression and cardiac hypertrophy. [52] In addition, SR9009 also prevented atherosclerosis in low-density lipoprotein (LDL) receptor-deficient mice fed a Western diet through a decrease in M1 proinflammatory to M2 anti-inflammatory macrophages, further confirming the anti-inflammatory properties of SR9009 [6, 56] (Figure 4).

Figure 4: REV-ERBα/β are key mammalian circadian clock components that regulate the synthesis of cardiac glycogen, cardiomyocyte size, interstitial fibrosis in the heart's interstitial tissue, extracellular matrix components of the heart, cardiac hypertrophy, inflammatory cytokines in the heart, and contractile function of the heart.

Disruption Of Circadian Rhythms and Heart Disease

Effect of REV-ERBα Disruption in Heart Disease

The strategy of targeting REV-ERBα is emerging as a promising strategy to control inflammation. REV-ERBα has been shown to activate and improve heart failure and myocardial infarction [54, 55, 57] (Figure 5). REV-ERBα is also involved in NLRP3 inflammasome stimulation. REV-ERBα inhibits the function of the NLRP3 inflammasome, preventing heart failure, fulminant hepatitis, ulcerative colitis, and peritoneal inflammation in mice [29, 30, 57, 58] (Table 2).

Figure 5: How REV-ERBα Functions in Heart Tissue Clock-controlled genes (CCGs), which are important in heart diseases such as ischemia-induced reperfusion injury, are regulated by REV-ERBα. These include the NLRP3-mediated inflammasome, inflammatory cytokines, myeloid cell migration, macrophage infiltration, and particle-positive entity migration. Furthermore, in a tissue-specific way, REV-ERBα controls myocardial damage throughout the perioperative phase, which includes the release of cardiac troponin T. By directly controlling target genes or indirectly by modifying gene transcription through other transcription factors like E4BP4, REV ERBα influences gene expression.

REV-ERBα/β Agonism and Cardiac Function

The nuclear receptor superfamily has two subtypes of Rev-erb: Rev-erbα and Rev-erbβ. Rev-erbs are primarily present in the liver, skeletal muscle, adipose tissue, heart, and brain, and are specifically implicated in the development and regulation of daily rhythms. Structurally similar, Rev-erb β and α govern circadian rhythms, lipid and glucose metabolism, and other functions. The circadian cycles of cardiac muscle cells control energy processing, protein synthesis, ion levels, and gene activation. The control of this process requires Rev-Erbα/β and BMAL1. Cardiac muscle cells disrupted in BMAL1 impair the circadian rhythm, which in turn affects mitochondrial and metabolic processes. In the BMAL1-deficient murine heart, SR-9009 activates Rev-Erbα/β and enhances cardiac function by promoting glycogen production, contractility, and fibrosis. This treatment also provides cardioprotection against ischemia–reperfusion, atherosclerosis, and heart failure and protects against age-related decline in cardiac function. Rev-erb activators have the potential to prevent future cardiac events in individuals with metabolic syndrome or a recent history of myocardial infarction (MI). Despite little improvement in MI outcomes, early administration of Rev-erb agonists post-event may be harmful. These compounds could be novel treatments for heart failure, atherosclerosis, myocardial infarction, and metabolic syndrome complications, especially in patients with myocardial infarction. [1,55]

Rev-Erbα/Β In Cardiac Metabolism and Homeostasis

REV-ERBα/β Repression of E4bp4 and Activation of NAMPT in Cardiac Function

According to studies, the E4BP4 protein is overproduced in diseased human hearts, whereas its Drosophila counterpart, Vrille, causes cardiac hypertrophy by interfering with mitochondrial activity. Nicotinamide phosphoribosyltransferase (NAMPT), a crucial enzyme in the NAD+ salvage pathway, has both direct and indirect control over metabolism. REV-ERB nuclear receptors regulate E4BP4 expression to modulate Nampt expression and suppress NAD+ in the absence of REV-ERBs. Disrupted cardiac energy metabolism also leads to suppression, and such suppression is associated with metabolic disorders and dilated cardiomyopathy. Elevated levels of E4BP4 and decreased NAMPT and NAD+ levels are observed in unhealthy hearts, indicating a relationship between circadian rhythms and heart metabolism. REV-ERB-E4BP4-NAMPT axis targeting may potentiate the NAD+ metabolism to fight age-related and cardiovascular diseases well. [59]

The Obesity Paradox and REV-ERB’s Role in Cardiovascular Health

REV-ERB, a circadian core clock component, is critical for protecting the heart and is antifibrotic in multiple animal models. The survival and proliferation of cardiac fibroblasts (CFs) also depend on the maintenance of cardiac fibroblasts (CFs) in a healthy state, and pharmacological targeting of REV-ERB in cardiomyocytes and REV-ERBα/β Agonism and Cardiac Function . The nuclear receptor superfamily has two subtypes of Rev-erb: Rev-erbα and Rev-erbβ. Rev-erbs are primarily present in the liver, skeletal muscle, adipose tissue, heart, and brain, and are specifically implicated in the development and regulation of daily rhythms. Structurally similar, Rev-erb β and α govern circadian rhythms, lipid and glucose metabolism, and other functions. The circadian cycles of cardiac muscle cells control energy processing, protein synthesis, ion levels, and gene activation. The control of this process requires Rev-Erbα/β and BMAL1. Cardiac muscle cells disrupted in BMAL1 impair the circadian rhythm, which in turn affects mitochondrial and metabolic processes. In the BMAL1-deficient murine heart, SR-9009 activates Rev-Erbα/β and enhances cardiac function by promoting glycogen production, contractility, and fibrosis. This treatment also provides cardio protection against ischemia–reperfusion, atherosclerosis, and heart failure and protects against age-related decline in cardiac function. Rev-erb activators have the potential to prevent future cardiac events in individuals with metabolic syndrome or a recent history of myocardial infarction (MI). Despite little improvement in MI outcomes, early administration of Rev-erb agonists post-event may be harmful. These compounds could be novel treatments for heart failure, atherosclerosis, myocardial infarction, and metabolic syndrome complications, especially in patients with myocardial infarction. [1,55]

Rev-Erbα/Β In Cardiac Metabolism and Homeostasis

REV-ERBα/β Repression of E4bp4 and Activation of NAMPT in Cardiac Function

According to studies, the E4BP4 protein is overproduced in diseased human hearts, whereas its Drosophila counterpart, Vrille, causes cardiac hypertrophy by interfering with mitochondrial activity. Nicotinamide phosphoribosyltransferase (NAMPT), a crucial enzyme in the NAD+ salvage pathway, has both direct and indirect control over metabolism. REV-ERB nuclear receptors regulate E4BP4 expression to modulate Nampt expression and suppress NAD+ in the absence of REV-ERBs. Disrupted cardiac energy metabolism also leads to suppression, and such suppression is associated with metabolic disorders and dilated cardiomyopathy. Elevated levels of E4BP4 and decreased NAMPT and NAD+ levels are observed in unhealthy hearts, indicating a relationship between circadian rhythms and heart metabolism. REV-ERB-E4BP4-NAMPT axis targeting may potentiate the NAD+ metabolism to fight age-related and cardiovascular diseases well. [59]

The Obesity Paradox and REV-ERB’s Role in Cardiovascular Health

REV-ERB, a circadian core clock component, is critical for protecting the heart and is antifibrotic in multiple animal models. The survival and proliferation of cardiac fibroblasts (CFs) also depend on the maintenance of cardiac fibroblasts (CFs) in a healthy state, and pharmacological targeting of REV-ERB in cardiomyocytes and remodeling. While Nr1d1 and Nr1d2 encode REV-ERB isoforms, Nr1d1 is dominantly expressed in the heart. SR9009 might have systemic usage and can affect cardiomyocytes (30–50%) that make up healthy heart cells. REV-ERB is rhythmically expressed in all primary heart cell types. Although SR9009 decreased REV-ERB levels, cardioprotection still exists, suggesting that REV-ERB mechanisms may not be the only ones protecting the heart, and further exploration is needed. [63]

SR9009 in myocardial ischemia-reperfusion reduces heart failure via cardiac inflammasome

The aim of this study was to examine whether the REV-ERB agonist SR9009 might improve heart tissue after blood supply has been blocked in mice. The results showed that SR9009 decreased inflammation by preventing production of inflammatory compounds and inflammasome functions. Administering the drug one day after blood flow returned to the heart resulted in decreased immune cell infiltration, less tissue damage, and a lessened risk of heart failure. They found that mice lacking the REV-ERB protein did not benefit from SR9009. REV-ERB can potentially improve heart function following a heart attack in humans. [57]

Berberine: A Natural REV-ERB Modulator

Several findings identify berberine (extracted from Rhizoma Coptidis) as a REV-ERBα agonist. [58] It suppresses Bmal1–luciferase and Nlrp3–luciferase activity reporters. It also increases the repressive function of REV-ERBα in galactose-inducible transcription factor 4 (Gal4) co-transfection. Berberine also suppresses gene expression regulated by REV-ERBα in bone marrow-derived macrophages. [3] Berberine has been used for over 2000 years in Chinese and Ayurvedic medicine in order to treat metabolic and cardiovascular disorders. The medicinal uses of the plant are for conditions such as heart failure, stroke, diabetes, and non-alcoholic fatty liver disease. Pharmacological effects of berberine target adenosine monophosphate-activated protein kinase (AMPK), protein tyrosine phosphatase 1B (PTP1B), silent information regulator 1 (SIRT1), proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor (LDLR), peroxisome proliferator-activated receptor (PPAR), nuclear factor kappa-light-chain-enhancer of activated B (NF-κB), and modulation of gut bacteria to improve the effect of berberine on a number of health issues. [64]

Puerarin and Its Effects on Cardiac Function

On the other hand, puerarin extracted from Puerariae Radix, a traditional Chinese medicinal herb used for numerous diseases, including fever and liver problems, reversed REV-ERBα. [65] Chen et al.’s conclusion is also supported by luciferase reporter, Galactose-Inducible Transcription Factor 4 (Gal4) cotransfection, and gene expression studies. [66] Puerarin prevents cardiac fibrosis and inhibits the transforming growth factor beta (TGF-β) induced in endothelial-to-mesenchymal transition (End MT). [67] In cases of myocardial infarction (MI), puerarin provides protective benefits to the heart and protects endothelial cells from inflammation-related harm. [68] Puerarin decreases pulmonary vascular remodeling and improves adverse remodeling of the myocardium in ischemic rats. [69, 70]. Berberine and puerarin are very different structural entities from other synthetic REV-ERBα ligands, suggesting the identification of novel molecular scaffolds for such ligands. The selectivity of berberine and puerarin towards REV-ERBs is not validated [3] (Table 3).