Graphical Abstract: Chrono-pharmacology-based antiplatelet therapy. Mammalian Rev-erbα expression in platelets shows a circadian rhythm and reaches its highest expression at 9 a.m., a time corresponding to the occurrence of major adverse cardiovascular events. Increased Rev-erbα expression leads to an increased platelet aggregation, while both genetic deletion and pharmacological inhibition of Rev-erbα decreased multiple aspects of mouse and human platelet activation. Rev-erbα platelet-specific knockout mice (Plt.-specific Rev-erbα KO mice) have prolonged bleeding time and reduced extent of thrombosis in the ferric chloride-induced thrombosis mouse model and reduced infarct size after experimental acute myocardial infarction.
Ischaemic heart disease remains the leading cause of morbidity and mortality worldwide. Its most serious manifestation is acute myocardial infarction (AMI), an acute thrombotic occlusion of a large epicardial coronary artery leading to myocardial ischaemia, necrosis, and ultimately heart failure. The increased activation of circulating platelets is associated with myocardial infarct size, indicating that platelet activation is a major determinant of disease prognosis. Platelets are mainly known for their role in thrombosis; however, they also significantly contribute to atherosclerotic plaque development and progression before and after the atherothrombotic events. Inhibition of platelet activation and aggregation is an ultimate therapeutic goal in patients with AMI. However, while substantial breakthroughs in antiplatelet therapy have been achieved, the underlying mechanisms of (residual) platelet activation in patients with AMI remain poorly understood.
Epidemiological studies have previously reported a marked circadian periodicity in the onset time of AMI. Indeed, the occurrence of major adverse cardiovascular events has daily patterns peaking at 6–9 a.m. These events are linked to our chronobiology. The human body displays a 24 h temporal rhythmicity driven by central or peripheral circadian clocks. These clocks regulate ~10% of genes within each cell and govern a number of cardiovascular processes, including blood pressure, heart rate, endothelial function, platelet aggregation, and thrombolytic activity. For instance, platelet aggregability experiences a peak in the morning and has been associated with increased risk of AMI and sudden cardiac death. Altogether, the morning peak in adverse cardiovascular events may be potentially related to the endogenous circadian clock of platelet aggregation and coagulation.
The circadian clock is a molecular mechanism that consists of several loops. The core loop genes BMAL1 and CLOCK form a heterodimer to bind to the DNA E-box, a promoter element of clock-controlled genes, to activate their transcription and guarantee the rhythmic activation of a large number of genes. Mice lacking the master circadian gene BMAL1 display a prothrombotic activity, while the daily variation in platelet aggregation was lost in circadian rhythm-altered Clock mutant mice. These Clock mutant mice exhibited increased infarct size and increased immune cell infiltration after AMI. Additionally, polymorphisms in CLOCK and BMAL1 clock genes might be an extra risk factor for MI. These results suggest that disturbances of circadian rhythms enhance AMI risk and have a crucial function in the onset of the disease. Among the BMAL1- and CLOCK-targeted genes, REV-ERBα, a nuclear circadian receptor, controls the core loop's transcriptional activity by repressing BMAL1 rhythmic expression. Although REV-ERBα is a component of the circadian clock system, recent studies unveiled a broader role for REV-ERBα in pathological conditions, including local inflammatory diseases, heart failure, and cancer. Still, the expression profile and biological function of REV-ERBα in platelets have until recently been unappreciated.
The study Shi et al. published in this issue of the European Heart Journal uncovered the role of the platelet circadian nuclear receptor REV-ERBα in platelet activation and thrombus formation. The authors observed a daily oscillation of REV-ERBα expression in human platelets derived from healthy young volunteers independent of gender.REV-ERBα expression increased by two-fold at 9 a.m., reaching its highest peak, a time corresponding to the occurrence of major adverse cardiovascular events, compared with its lowest level at 5 p.m. Strikingly, the authors also report a four-fold increase of REV-ERBα expression at 9 a.m. in platelets derived from patients with acute ST-elevation AMI. This is in accordance with a previous study demonstrating increased REV-ERBα expression in human cardiac biopsies in the morning hours. Next, the authors confirmed that Rev-erbα is also present in murine platelets and displays a circadian rhythm that positively correlates with platelet aggregation. In their study, the authors showed that both the global and the platelet-specific Rev-erbα knockout mice exhibited significantly prolonged tail bleeding time without altering the platelet count or microstructures. It is important to note that global genetic disruption of either Clock or Bmal1 did affect the platelet count, suggesting that Rev-erbα could be a more exciting and safer therapeutic target in that aspect. Next, they challenged their Rev-erbα platelet-specific knockout mice with well-established experimental thrombosis models. Absence of platelet-specific Rev-erbα almost doubled the mean time to occlusion and reduced by two-thirds the size of the thrombus, underlining the importance of the prothrombotic role of Rev-erbα in murine platelets. Importantly, Shi et al. subjected their mice to AMI. Platelet-specific Rev-erbα deletion reduced the presence of microthrombi two-fold, decreased the infarct size by ~30%, and subsequently improved the cardiac function compared with control mice. Mechanistically, Shi et al. propose that Rev-Erbα controls platelet activation by binding to Oligophrenin1 (Ophn-1), a RhoA GTPase-activating protein known for its critical role in the reorganization of the cytoskeleton, which is a major process required for stable platelet adhesion and thrombus formation. Platelet-specific Rev-erbα knockout in mice significantly disrupted RhoA activity, a main downstream target of Ophn-1, and down-regulated the phosphorylation levels of RhoA effectors from the ERM (ezrin/radixin/moesin) family. Considering also that Rev-erbα modulates inflammatory responses in macrophages through the production and release of proinflammatory cytokines involved in the recruitment of classical monocytes in atherosclerotic plaques as well as in the infarcted heart, it emerges as a potential therapeutic target for the treatment of atherosclerotic and atherothrombotic cardiovascular disease.
Circadian genes are highly associated with diseases, and 56 of the top 100 best-selling drugs in the USA target a product of a circadian rhythm, suggesting that drugs are time-of-day administration sensitive. Nevertheless, new prospective chronotherapeutic studies are needed to investigate both the influence of time of administration of the available drugs and to develop new drugs that safely target circadian components. Indeed, aspirin intake is now considered for chronotherapy in randomized clinical trials of patients with stable cardiovascular disease. In theory, the occurrence of major adverse cardiovascular events could be reduced by targeting the circadian rhythm of prothrombotic or proinflammatory genes utilizing chrono-pharmacology-based therapeutic strategies. Chrono-pharmacology-based therapy targeting REV-ERBα has recently emerged as a promising cardioprotective therapeutic strategy. Perioperative myocardial injury is transcriptionally orchestrated by REV-ERBα in patients undergoing aortic valve replacement. Inhibition of Rev-Erbα signalling by its synthetic antagonist SR8278 conferred cardiomyocyte protection against myocardial injury in an ex vivo murine model of hypoxia–reoxygenation. The present study evaluated a chrono-pharmacology-based anti-platelet therapy by giving a peptide that selectively inhibits Rev-erbα (Graphical Abstract). Molecular docking experiments were performed to confirm the binding affinity and target sites between a known Rev-erbα antagonist compound SR8278 and the Rev-erbα receptor in mice and humans. The authors found that similar to the genetic deletion, pharmacological inhibition of Rev-erbα significantly inhibits human and mouse platelet aggregation and activation (Graphical Abstract). Treatment of human platelets with the inhibitory peptide SR8278 elicited reduced activity of RhoA and phosphorylation of ERM, similar to the defective events documented in Rev-Erbα-deficient murine platelets. Therefore, a platelet-targeted Rev-erbα inhibition could be the optimal choice to avoid off-target effects on other cell types. This is especially important because Rev-erbα agonists have been reported to improve adverse cardiac remodelling in a murine model of ischaemia/reperfusion through down-regulation of the cardiac inflammasome in fibroblasts. Although the molecular clocks are preserved among different tissues, these studies suggest the presence of tissue-specific effects of circadian rhythm-related genes. Further studies are called to investigate the role of circadian rhythm in various cardiovascular tissues and cells in health and disease.
REV-ERBα is a unique circadian gene belonging to both the molecular circadian clock and nuclear receptor families. As a result, REV-ERBα may be a pharmaceutical target that controls many downstream genes related to atherothrombosis. An alternative approach would be to identify REV-ERBα-targeted genes to generate chrono-pharmacology-based therapy approaches. Interestingly, the chrono-pharmacological treatment of Ccl2 signalling, a known target of Rev-erbα, has shown promising results in mouse models of both atherosclerosis and acute lung inflammation. The discovery of clock elements in platelets lays the foundation for a novel chrono-pharmacological-based antiplatelet therapy to inhibit or ameliorate acute thrombotic events including AMI. Future studies are needed to ascertain the relevance of the circadian platelet clock and its target genes, and explain the time-dependent onset of AMI. We believe that from now on future drug development should also consider the drug target's circadian rhythmicity and metabolism throughout the 24 h day in order to optimize its efficacy for the benefit of the patients.
Eur Heart J. 2022;43(24):2335-2337. © 2022 Oxford University Press
Copyright 2007 European Society of Cardiology. Published by Oxford University Press. All rights reserved.