Authors: Brian P. Delisle (1Department of Physiology, University of Kentucky, Lexington, KY, USA), Abhilash Prabhat (1Department of Physiology, University of Kentucky, Lexington, KY, USA), Don E. Burgess (1Department of Physiology, University of Kentucky, Lexington, KY, USA), Isabel G. Stumpf (1Department of Physiology, University of Kentucky, Lexington, KY, USA), John J. McCarthy (1Department of Physiology, University of Kentucky, Lexington, KY, USA), Spencer B Procopio (2University of Florida, Gainesville, FL, USA), Xiping Zhang (2University of Florida, Gainesville, FL, USA), Karyn A. Esser (2University of Florida, Gainesville, FL, USA), Elizabeth A. Schroder (1Department of Physiology, University of Kentucky, Lexington, KY, USA; 3Department of Internal Medicine, University of Kentucky, Lexington, KY, USA)
Categories: Article, circadian clock, ion channels, arrhythmias, triggers, myocardial substrate, sudden cardiac death
Source: Journal of molecular and cellular cardiology
Authors: Brian P. Delisle, Abhilash Prabhat, Don E. Burgess, Isabel G. Stumpf, John J. McCarthy, Spencer B Procopio, Xiping Zhang, Karyn A. Esser, Elizabeth A. Schroder
Cardiologists have analyzed daily patterns in the incidence of sudden cardiac death to identify environmental, behavioral, and physiological factors that trigger fatal arrhythmias. Recent studies have indicated an overall increase in sudden cardiac arrest during daytime hours when the frequency of arrhythmogenic triggers is highest. The risk of fatal arrhythmias arises from the interaction between these triggers—such as elevated sympathetic signaling, catecholamine levels, heart rate, afterload, and platelet aggregation—and the heart’s susceptibility (myocardial substrate) to them. A healthy myocardial substrate has structural and functional properties that protect against arrhythmias. However, individuals with cardiovascular disease often exhibit structural and electrophysiological alterations in the myocardial substrate that predispose them to sustained lethal arrhythmias. This review focuses on how day-night and circadian rhythms, both extrinsic and intrinsic, influence the protective properties of the myocardial substrate. Specifically, it explores recent advances in the temporal regulation of ion channel gene transcription, drawing on data from comprehensive bioinformatics resources (CircaDB, CircaAge, and CircaMET) and recent RNA sequencing studies. We also examine potential mechanisms underlying the temporal regulation of mRNA expression and the challenges in linking rhythmic mRNA expression to corresponding changes in protein levels. As chronobiological research in cardiology progresses, we anticipate the development of novel therapeutic strategies to enhance the protective properties of the myocardial substrate to reduce the risk of fatal arrhythmias and sudden cardiac arrest.
Sudden cardiac death (SCD) is an unexpected death resulting from cardiovascular disease [1]. It is most often triggered by ischemic heart disease (e.g., coronary artery disease), congenital heart disease, or ion channelopathies [2, 3]. Estimates suggest that 30-80% of SCD cases stem from sudden cardiac arrest (SCA) due to ventricular arrhythmias in both men and women [4-6]. For over six decades, clinician-scientists have studied the timing of SCD and SCA events across the 24-hour cycle [7-9]. Cardiologists have examined daily patterns in SCD incidence to identify environmental, behavioral, and physiological factors that trigger arrhythmias in at-risk individuals, with the goal of modifying these factors to prevent SCA. Historically, these studies have identified time-of-day variations in the incidence of SCD, showing a morning peak among those with ischemic and non-ischemic heart disease [8]. Recent studies have failed to confirm a morning peak in SCA [10, 11]. Several factors may contribute to the loss of the morning peak in contemporary populations. Changes in medical practice, particularly the widespread use of beta-blockers and other cardiovascular medications that blunt morning surges in sympathetic activity, may have altered the temporal distribution of SCA. Modern lifestyle changes, including irregular sleep-wake cycles, shift work, and altered feeding patterns, may have disrupted the once-predictable morning increase in arrhythmogenic triggers. Additionally, improved management of cardiovascular risk factors and broader use of preventive therapies may have modified the substrate vulnerability that historically contributed to morning arrhythmia susceptibility. Recent analyses of SCA incidence suggest [10, 12-16] that societal and medical changes may have fundamentally altered the morning peak in the incidence of SCA. Still, the overall higher SCA incidence persists during daytime hours regardless of age, gender, and the presence or absence of coronary artery disease [17]. The increase in SCA during the daytime coincides with daily increases in several known arrhythmogenic triggers (e.g., higher sympathetic nervous system signaling, circulating catecholamine levels, heart rates, afterload, and platelet aggregation) [7, 17-21].
The interplay between arrhythmogenic triggers and the responsiveness of the myocardial substrate (structural and functional properties of the heart) influences the risk of arrhythmias that cause SCA [22]. The hearts of healthy individuals have myocardial properties that protect against triggered arrhythmias. These properties include efficient electrical coupling between cells, conduction velocity, and refractory period that protects against ectopic activity, afterdepolarizations, ventricular action potential dispersion, and re-entry. Those with ischemic heart disease, cardiomyopathies, or channelopathies have structural and electrophysiological alterations that lower the protective properties of the myocardial substrate and predispose these individuals to sustained deadly arrhythmias [23]. A simple model for explaining the day-night rhythm in SCA is that the increased frequency of arrhythmogenic triggers during the day leads to a higher incidence of sustained arrhythmias in people with a compromised myocardial substrate (Figure 1).
An important question is whether day-night rhythms in extrinsic and intrinsic circadian signaling contribute to a day-night rhythm in the protective properties of the myocardial substrate and the overall level of protection across the 24-hour cycle. Acute changes in protective properties could reflect changes in autonomic receptor sensitivity, second messenger-mediated signaling, and cross-talk between second messenger signaling pathways that alter ion channel function and Ca^2+^ cycling. Sustained alterations in myocardial protection could reflect daily transcriptional and posttranscriptional regulation that impacts the functional protein levels. This review discusses recent insights into extrinsic and intrinsic signaling mechanisms that regulate day-night rhythms in cardiac electrophysiology, recent advances and resources for understanding circadian regulation of ion channel gene transcription in the heart, and potential implications that transcriptional and posttranscriptional regulation have on the protective properties of the myocardial substrate.
Numerous studies report day-night rhythms in cardiac electrophysiology as being circadian. However, in chronobiology, circadian refers to intrinsic physiological rhythms that cycle with an approximate 24-hour periodicity, independent of external environmental or behavioral cues [22]. In this context, circadian rhythms are evolutionarily conserved feed-forward homeostatic mechanisms that anticipate predictable environmental changes across the 24-hour cycle [24]. Thus, although clinical studies may describe the 24-hour rhythms in cardiac electrophysiology as circadian, they are not measured in constant conditions. To avoid confusion, we refer to rhythms not measured in constant conditions as day-night rhythms because they reflect a combination of daily environmental, behavioral, and intrinsic circadian influences.
Day-night rhythms in cardiac electrophysiology can be non-invasively monitored using electrocardiography. Clinical studies indicate that RR and QT intervals follow day-night rhythms that peak in the early morning and reach their lowest point midafternoon [25]. In healthy individuals, autonomic nervous system signaling appears to primarily regulate the amplitude of the day-night rhythm of the RR interval. Studies on heart transplant recipients who have a "denervated" heart reveal a very low amplitude day-night heart rate rhythm (5-10 beats per minute) compared to the amplitude in transplant patients whose hearts have undergone reinnervation (20-25 beats per minute) [26]. The residual amplitude in the day-night rhythm observed in recipients with denervated hearts likely reflects additional physiological factors following a ~24-hour cycle, including day-night rhythms in core body temperature, circulating catecholamines, and extracellular K^+^ [27-30]. However, the status of the intrinsic circadian clock in the transplanted heart also requires consideration. Studies show that cardiac clock entrainment depends heavily on sympathetic stimulation [31-33], and circulating factors alone cannot restore cardiac rhythms in SCN-lesioned animals [34]. This suggests that transplanted hearts may experience disruption of both extrinsic autonomic regulation and intrinsic clock function, potentially contributing to the marked reduction in day-night cardiovascular rhythms. The relative contribution of these mechanisms to decreased rhythmicity in transplanted hearts remains an important area for investigation.
The QT interval is a key biomarker for arrhythmogenicity [35]. Since it varies with heart rate, the QT interval is corrected using specific formulas to produce the heart rate-corrected QT (QTc) interval. Individuals with abnormally short or long QTc intervals are at increased risk for life-threatening ventricular arrhythmias [36]. The day-night rhythm of the uncorrected QT interval closely aligns with the day-night rhythm of heart rate [25]. While some studies suggest that the QTc interval exhibits a day-night rhythm, this may depend on the correction formula used [37]. Like heart rate, the QTc interval is influenced by core body temperature, and fluctuations in body temperature throughout the day may contribute to a day-night rhythm in the QTc interval [38-43]. Earlier studies identified a morning spike in QTc interval duration shortly after waking [25]. This increase may have played a role in the higher incidence of arrhythmogenic SCD reported in earlier studies. However, as noted above, studies on more recent patient populations no longer identify a distinct morning peak in the incidence of SCA. Further investigation is required to determine whether the morning spike in the QTc still occurs in contemporary populations.
Electrocardiographic telemetry studies in mice and rats demonstrate that, like humans, animals exhibit day-night rhythms in cardiac electrophysiology. Animal models are valuable for mechanistically investigating how modifying environmental, behavioral, and circadian factors influence rhythms. Although mice and rats are nocturnal, their daily rhythms in cardiac electrophysiology, autonomic regulation of the heart, and core body temperature align with activity and feeding rhythms (similar to people) [30, 44-46], This observation suggests that the circadian mechanisms regulating the alignment of day-night rhythms in cardiac electrophysiology to daily rhythms in behavior are conserved.
The suprachiasmatic nucleus (SCN) of the hypothalamus plays a crucial role in entraining circadian rhythms. SCN neurons contain circadian clocks, which are molecular transcriptional-translational feedback loops that are entrained to the light-dark cycle via photic input through the retinohypothalamic tract [47, 48]. Light-entrained SCN neurons then communicate with other SCN neurons and brain nuclei to entrain the phase of circadian rhythms in neurohumoral signaling (e.g., autonomic, cortisol, catecholamines, melatonin, etc.) and behaviors (e.g., sleep, feeding) with the light cycle.
To explore the SCN's role in circadian rhythms, researchers can lesion the SCN in mice and rats. SCN lesions disrupt day-night rhythms in activity, feeding, body temperature, heart rate (RR interval), and QT interval rhythms [49-51]. Similar to SCN ablation, pharmacological inhibition of cardiac autonomic receptors also disrupts day-night rhythms in heart rate and QT intervals in mice [50]. These findings indicate that the SCN is central in regulating autonomically driven day-night rhythms in cardiac electrophysiology.
In addition to the extrinsic regulation of day-night rhythms in cardiac electrophysiology by the SCN, studies in mice have identified time-of-day changes in the intrinsic electrophysiological properties of the heart [52]. These studies were performed in isolated heart preparations at constant temperature and after one hour of ex vivo perfusion to mitigate the residual influence of autonomic signaling. The isolated hearts showed a significant time-of-day difference in cardiac electrophysiology, Ca^2+^ handling, and adrenergic responsiveness of the action potential duration. At the start of the dark cycle, the cardiac action potential duration, effective refractory period, and Ca^2+^ transient durations were shorter, the hearts were less responsive to adrenergic stimulation, and they were more resistant to arrhythmias compared to the other times of day tested. Hearts isolated from aged mice (18-20 month-old mice) did not show the same time-of-day difference in cardiac electrophysiological properties early during the dark cycle. The data suggests that aged mice may lose the time-of-day increase in myocardial protection.
Recent work suggests that some ex vivo changes in the electrophysiological properties of the myocardial substrate can be traced to day-night rhythms in transcriptional and translational mechanisms operating within individual cardiomyocytes [33, 50, 52-59]. The circadian clock mechanism entrains SCN neurons to the light-dark cycle and is also present in nearly all the cells in the body [60, 61], including cardiomyocytes [32]. Circadian clocks in peripheral tissues not only function as cellular timekeepers, but they also function to orchestrate the expression of genes important for tissue- and cell-specific functions. This includes several key cardiac ion channels that shape cardiac action potentials in the autorhythmic and working myocardium [53, 54, 56]. The core circadian clock mechanism involves the basic helix-loop-helix transcription factors BMAL1 and CLOCK (positive limb), which enhance transcription of Period (Per) and Cryptochrome (Cry) genes. This core loop is stabilized by an auxiliary feedback loop where REV-ERBα and ROR compete to regulate Bmal1 transcription through RORE elements. As REV-ERBα is also a direct target of BMAL1/CLOCK, this creates an interconnected regulatory network that generates robust ~24-hour rhythms. Transgenic studies show that the loss of Bmal1 in cardiomyocytes disrupts the cardiac circadian clock mechanism and prolongs the RR and QT intervals[56]. In addition, Bmal1 deficiency in cardiomyocytes decreases both circadian and steady-state levels of cardiac ion channel mRNA transcripts [62]. These changes correspond with reduced amplitudes in key depolarizing and repolarizing currents in cardiomyocytes, altered pacemaking activity, and delayed cellular repolarization properties in ventricular cardiomyocytes [46, 53, 57, 63]. In addition, studies using forced desynchrony protocols in people, where subjects live in a 20-hour day environment, demonstrate that 24-hour rhythms in heart rate persist even when disconnected from daily environmental and behavioral cycles [64] providing compelling evidence for intrinsic circadian control of cardiac function in humans. Another study in middle aged women found no significant day-night rhytgn in heart rate [65]. This differs from previous studies and suggests that aging may impact circadian regulation of cardiovascular function.
While these studies have provided valuable insight into intrinsic and circadian regulation of cardiac electrophysiology, they represent the tip of the iceberg for understanding the temporal regulation in the protective properties of the myocardial substrate. Researchers are now complementing these studies with high-throughput molecular-based discovery assays, like RNAseq, scRNA-seq, snRNA-seq, and ATAC-seq, to understand better how cardiac gene expression changes across the 24-hour cycle. This methodology identifies new avenues for exploring the temporal relationships of cardiac function on a broader scale, allowing for a more comprehensive examination of the complex interplay between environmental, behavioral, intrinsic, and circadian influences on the myocardial substrate.
Circadian signaling is vital in regulating the properties of the myocardial substrate. To better understand these complex relationships, we can utilize comprehensive circadian gene/protein expression databases developed to study circadian clock mechanisms [66-69]. By combining high-throughput gene expression data, functional annotations, and advanced visualization techniques, these databases offer a wealth of information examining the oscillating patterns of genes across varying conditions (darkness; light-dark; daytime-restricted-feeding; nighttime-restricted-feeding; age; sex). These platforms are available to identify rhythmically expressed gene (REGs) transcripts, analyze tissue-specific circadian patterns, and investigate the impact of environmental factors and behavior on rhythmic gene expression. Despite variances in collection protocols, it is remarkable how consistent much of the data is across the differing platforms. As our understanding of circadian cardiology grows, these databases will continue to serve as essential resources for unraveling the complex interplay between circadian rhythms, cardiac ion channel regulation, and arrhythmogenesis.
We queried four bioinformatic databases to generate Table 1, which examines the rhythmic expression of cardiac ion channel genes, regulatory subunit genes, and associated regulatory genes CircaDB (http://circadb.org) [66], CircaAge [67], CircaMET [69], and the GEO dataset GSE262714[68]). Table 1 allows for the quick identification of patterns that might require extensive cross-referencing of multiple databases to discover. For example, it is immediately evident whether a gene maintains rhythmicity across different conditions, exhibits sex-specific regulation, or changes with age. Within each section of Table 1, genes are listed in order of circadian consistency across databases as well as the robustness of the rhythms (p values). Table 1 also reveals coordinated regulation among functionally related genes that might not be apparent when examining individual genes in isolation. These cross-database, cross-condition patterns provide insights into regulatory networks and potential therapeutic timing that would be difficult to discern from individual database queries. Vertical comparisons show relative importance based on rhythm robustness and functional impact, while horizontal comparisons reveal how environmental and biological factors affect gene expression patterns. These integrated insights make the table a valuable resource for both focused investigation of specific proteins and broader understanding of temporal regulation in cardiac electrophysiology.
CircaDB [66] contains combined gene expression data from multiple experiments, tissues, and organisms and employs three separate algorithms to assess circadian gene expression with a resolution time scale of 2-4 hours. It is interactive because it allows users to restrict results by dataset and probability threshold. Column 2 of Table 1 shows CircaDB data from genes encoding cardiac ion channels or regulators of ion channel function. This data was collected from the male mouse heart in the absence of circadian time cues (darkness (D:D)) every two hours or from the male mouse heart collected every 4 hours under 12-hour light and 12-hour dark conditions (L:D). Constant darkness allows the internal circadian clock to "free run" with its intrinsic ~24-hour period. Gene expression may oscillate under light-dark conditions, but to be considered circadian, gene expression must oscillate in the absence of the light-dark cycle (i.e., constant conditions). Light-dark cycles and constant darkness have differing effects on animal behavior and physiology, mainly through a phenomenon termed masking. Masking refers to immediate behavioral and physiological responses to environmental signals that can override or obscure underlying circadian rhythms. Unlike true circadian rhythms that persist in constant conditions, masked responses occur as direct reactions to environmental cues. For example, light exposure can acutely suppress melatonin production regardless of circadian time[70-72], while feeding can immediately alter metabolic and cardiovascular parameters independent of the circadian clock[73-75]. Understanding masking is crucial when interpreting day-night rhythms in cardiac function, as observed changes may reflect either intrinsic circadian regulation or acute responses to environmental factors. Data presented in column 2 of Table 1 highlights the complex interactions between circadian rhythms and adaptations to light and darkness in mice. For instance, the K^+^ channel genes Kcna5, Kcnab1, Kcnb1, Kcnk2, and Kctd17 are rhythmically expressed under L:D conditions but lose rhythmicity in constant darkness, suggesting that the endogenous circadian clock does not drive these rhythms.
CircaAge (https://circaage.shinyapps.io/circaage/) [67], similar to CircaDB, contains gene expression data from multiple tissues harvested from ad libitum-fed male mice collected every 4 hours for 48 hours in constant darkness. Gene expression data for cardiac ion channel/regulatory genes from the CircaAge database is presented in column 3 of Table 1 and is designed to examine age-related changes in circadian gene expression. CircaAge enables researchers to perform several key analyses. It allows for investigating how rhythmic expression patterns of genes evolve with age, providing insights into the changing dynamics of circadian regulation in the aging tissues. CircaAge facilitates the identification of age-related shifts in both the timing and amplitude of oscillations in gene expression. This capability is important for understanding how the strength and precision of circadian control over cardiac function may alter as organisms age. Lastly, it offers a platform for directly comparing circadian rhythms between young, aged, and old tissues. By enabling these comparative analyses, CircaAge provides a comprehensive view of the circadian regulation of cardiac ion channel/ion channel regulatory gene expression throughout lifespan, potentially uncovering critical insights into age-related cardiac phenomena and disease risks. Several genes in Table 1 exhibit notable age-dependent alterations in their rhythmic expression patterns. For instance, Kcnh2 shows strong rhythmicity in Young (6 months old; p=1.5e-3) and Aged (18 months old; p=1.0e-4) mouse hearts but weaker rhythmicity in the Old group (27 months old; p=0.056), suggesting a decline in the relative circadian regulation of Kcnh2 with advanced age. Conversely, Kcnj15 develops a significant rhythm only in the Old age group (p=0.029), indicating the emergence of increased circadian regulation in later life. Diverse patterns of age-related changes in circadian gene expression could have important implications for understanding how the myocardial substrate and susceptibility to arrhythmias may change across the lifespan.
CircaMET (http://www.circametdb.org.cn/Index/index.html) [69] is a comprehensive resource that integrates circadian and metabolic data across multiple tissues. By leveraging CircaMET, scientists can identify potential metabolic regulators that influence the circadian rhythms of cardiac ion channels. Furthermore, it facilitates investigations into how disruptions in metabolic pathways might impact the timing and patterns of ion channel expression. This capability is important for understanding the potential cardiac consequences of metabolic disorders or circadian rhythm disruptions. CircaMet data in column 4 of Table 1 shows daytime restricted feeding (DRF) and night-time restricted feeding (NRF) data from female mouse hearts collected every 4 hours for 24 hours under L:D conditions. This column provides insight into how feeding time influences the expression patterns of cardiac ion channels and cardiac ion channel regulatory genes. For instance, Kcnh2 shows distinct phase relationships under different feeding schedules, with peak expression at ZT6 during daytime restricted feeding (DRF p=5.58e-4) shifting to ZT11 during nighttime restricted feeding (NRF p=3.86e-5). Through its integrative approach, CircaMET offers a unique platform for unraveling the complex relationships between metabolism and day-night expression patterns in mRNA transcript levels, potentially leading to testing for new strategies for managing heart health in the context of metabolic and circadian disturbances.
Column 5 in Table 1 (GEO dataset GSE262714) [67] provides recent data from our collaborative studies [68]. The data provides insights into sex differences in the circadian expression of cardiac ion channel genes in the ventricle control mice and mice with cardiomyocyte-specific knockout of Bmal1 collected under constant darkness. There are notable differences in rhythmicity and expression patterns between male and female mice for many of this column's ion channel/ion channel regulatory genes. These sex differences are observed in vehicle-injected control (iCSBmal1^+/+^) and the inducible cardiomyocyte-specific Bmal1 knockout (iCSBmal1^−/−^) in adult animals, suggesting clock-dependent and -independent sex differences. For instance, Kcnh2 mRNA transcripts show significant rhythmicity in both sexes but with different oscillation strengths (male wild-type p=1.81e-3; female wild-type p=1.4e-8). Some genes, like Kcna4, have mRNA transcripts that exhibit rhythmicity only in females (p=5.84e-3), while others, such as Kcnj3, show rhythmicity in mRNA transcripts only in males (p=0.027). Inducing the cardiomyocyte-specific Bmal1 knockout often alters these patterns, sometimes eliminating sex differences (e.g., Kcna4) and revealing new ones (e.g., Kcnd3). These sex-specific patterns in the circadian regulation of cardiac ion channel transcripts could have important implications for understanding sex differences in the responsivity of the myocardial substrate to arrhythmia triggers, arrhythmia risk, and response to cardiovascular therapies.
The heart is a complex organ composed of multiple cell types, with cardiomyocytes comprising 70-80 percent of the total volume of the mammalian heart but only 25-35 percent of its cellular makeup [76-78]. This heterogeneity presents challenges and opportunities for researchers seeking to understand cardiac function and regulation. Single-cell and single-nucleus analyses have emerged as powerful tools to address these complexities, offering significant potential for distinguishing between non-cardiomyocyte and cardiomyocyte mRNAs. While single-cell RNA sequencing (scRNA-seq) provides high-resolution transcriptomic data from individual cells, single-nucleus RNA sequencing (snRNA-seq) offers complementary advantages, particularly for tissues like the heart, where cell isolation can be challenging. snRNA-seq allows for profiling nuclear transcripts from intact tissue, potentially capturing information from cell types that may be underrepresented or lost during single-cell dissociation protocols. Together, these techniques provide unprecedented resolution for investigating circadian physiology within the heart, allowing researchers to map the circadian transcriptome of individual cardiac cell types. Single-cell and single-nucleus approaches can resolve cell-type specific rhythms, revealing distinct circadian patterns among cardiac cell populations that may be obscured in bulk tissue analyses. They also offer the potential to examine the circadian properties of cell-cell communication. By enabling this detailed examination of circadian rhythms at the cellular and nuclear levels, these technologies promise to reveal a novel understanding of the rhythmic expression of genes and their role in maintaining cardiac health, potentially uncovering differences in transcriptional regulation between cytoplasmic and nuclear RNA pools as well as identifying important novel ion channel regulatory mechanisms. Limited single-cell RNA seq data from the human heart is included in Table 1 (column 1). The superscript next to the gene name represents cell type-specific expression (^CM^Expressed in Cardiomyocytes; ^F^Expressed in Fibroblasts; ^E^Expressed in Endothelial Cells; ^SM^Expressed in Smooth Muscle; ^M^Expressed in Macrophages; ^P^Expressed in Plasma Cells; ^N^Expressed in Neutrophils; ^T^Expressed in T-cells; ^MC^Expressed in Mitotic Cells (Heart) [77].
The RNA sequencing techniques and datasets (Table 1) presented in this section offer complementary insights into cardiac ion channel gene expression patterns. While bulk RNA-seq examines broad rhythmic patterns, scRNA-seq and snRNA-seq provide high-resolution temporal and cellular insights. These methods, when combined, enable a comprehensive view of circadian gene expression across different cardiac cell types and subcellular compartments, advancing our understanding of the circadian physiology of the myocardial substrate.
These databases are particularly valuable in studying the complex temporal regulation of cardiac ion channels. They could potentially lead to a better understanding of arrhythmias, optimization of drug timing, and improved management of cardiovascular diseases in males and females across the lifespan. While specific findings and methodologies may vary, the overall conclusion that circadian rhythms play an important role in regulating cardiac electrophysiology and ion channel gene expression remains consistent between laboratories and across datasets. Future studies utilizing these combined methods will further elucidate the intricate relationship between circadian rhythms and the regulation of the myocardial substrate.
Our understanding of circadian rhythms in cardiac ion channel expression has evolved significantly in recent years. While the databases discussed previously provide a foundation for understanding the complex temporal patterns in ion channel expression, they also pave the way for more focused investigations into specific regulatory mechanisms. It has become clear that the progression from identifying circadian patterns to unraveling their intricate control mechanisms is still a work in progress. The following section will discuss potential pathways for regulating the circadian rhythms of the myocardial substrate.
Mammalian clock genes were first cloned in the late 1990s, [79-82] and the identification of circadian clock components in peripheral tissues soon followed [83, 84]. While earlier studies examined circadian gene expression (rhythmically expressed genes, (REGs)) in heart tissue [85, 86], Yamashita and colleagues were the first to specifically demonstrate and characterize day-night expression patterns in the cardiac K^+^ channel genes important for cardiac repolarization, Kcna5 (Kv1.5) and Kcnd2 (Kv4.2), in the rat heart. [55]. We now know that the regulation of rhythmically expressed ion channels is complex. Studies have demonstrated direct regulation by the core circadian clock mechanism and indirect mechanisms regulating rhythmic ion channel gene expression. This section will examine the current understanding of ion channel REG regulation.
Since initial studies demonstrated the circadian expression of Kcna5 and Kcnd2 mRNA transcripts, researchers have begun to examine the mechanisms behind these oscillatory expression patterns. A direct role for the circadian clock mechanism in regulating Kcnh2 [57], Scn5a [56], and Hcn4 [53] has been suggested. Kcnh2 encodes the pore-forming subunit of the voltage-gated K^+^ channel, Kv11.1, which is important for cardiac repolarization. Scn5a encodes the pore-forming subunit of the voltage-gated Na^+^ channel, Nav1.5, responsible for the rapid upstroke of the cardiac action potential. Hcn4 encodes the hyperpolarization-activated cyclic nucleotide-gated K^+^ channel, which is important for pacemaker activity (If). The mRNA expression pattern for each of these channels was shown to be circadian in mouse cardiac tissue (ventricle Kcnh2 and Scn5a; SA node Hcn4) and was disrupted with genetic disruption of the circadian clock mechanism in the heart. In addition, cardiomyocytes isolated from these mice resulted in a smaller Kv11.1 current (IKr) [57], Nav1.5 current (INa) [56], and HCN4 (If) [53] current. Transactivation by the circadian clock was demonstrated for all three channels utilizing promoter reporter luciferase assays [57].
Additionally, the circadian clock has been shown to exert indirect control over ion channel expression through intermediary transcription factors. This discovery adds a new dimension to our understanding of how daily rhythms influence cardiac electrophysiology. A prime example of this indirect regulation is the circadian control of the transcription factor Krüppel-like factor 15 (KLF15), as demonstrated by Jeyaraj et al [54]. Klf15 exhibits circadian oscillations under the direct control of the core clock proteins CLOCK and BMAL1. In turn, KLF15 protein regulates the transcription of Kcnip2, which encodes KChIP2, a voltage-gated K^+^ channel interacting protein. KChIP2 plays a role in modulating Kv4.2 channel proteins, thereby influencing cardiac repolarization. In addition, recent work has revealed that the clock-controlled transcription factor E4BP4 provides an additional regulatory layer for Kcnip2 expression, demonstrating how the circadian clock mechanism can modify cardiac electrophysiology through multiple transcriptional mechanisms [87].
Other important cardiac transcription factor mRNA transcripts, such as Tbx5 and Gata4, have also been observed to follow circadian expression patterns in the heart [58, 88, 89]. These factors are known to be critical for normal cardiac ion channel expression, suggesting that their rhythmic regulation could have far-reaching effects on regulating the protective properties of the myocardial substrate.
Other potential pathways contributing to the temporal regulation of the myocardial substrate include microRNAs (miRNA) and steroid or membrane receptor-mediated regulation. Similar to circadian clock transcription factors, miRNAs regulate ion channel gene expression, contributing to the post-transcriptional regulation of mRNA levels. In part, miRNAs influence cardiac function by targeting several key ion channel gene transcripts. For example, miRNAs regulate cardiac conduction by modulating the expression of Gja1 (which encodes connexin 43) and Kcnj2 (encoding the Kir2.1 inward rectifier K^+^ channel) [90, 91]. Connexin 43 is a component of gap junctions, facilitating intercellular communication and coordinated contraction in the heart. The Kir2.1 channel, on the other hand, is important for maintaining resting membrane potential and shaping the final phase of action potential repolarization. By modulating channel expression, miRNAs can significantly impact the duration and morphology of the cardiac action potential, thus affecting the overall cardiac substrate.
Recent studies have demonstrated that cardiomyocytes' core circadian clock machinery directly influences miRNA expression patterns. For example, cardiomyocyte-specific knockout of Bmal1 in mice, a central component of the circadian clock, led to upregulation of about 70% of the differentially expressed miRNAs (out of 47 examined) in the hearts of mice. Similarly, double knockout of REV-ERBα and REV-ERBβ, important circadian clock regulators, increased cardiomyocyte miRNA levels [92]. These data suggest that the circadian clock gene regulatory network in the heart affects miRNA expression, and changes in the miRNA expression may contribute to changes in the properties of the myocardial substrate.
Steroid hormones play a significant role in regulating the myocardial substrate in the heart through both genomic and non-genomic pathways. In the genomic pathway, steroid hormones bind to intracellular receptors, forming complexes that translocate to the nucleus and modulate the transcription of genes encoding ion channels and their regulatory proteins. For instance, sex hormones like estrogen and testosterone regulate the expression of K^+^ channels vital for cardiac repolarization [93-95]. and glucocorticoids, primarily cortisol in humans, act through the glucocorticoid receptor (GR) to influence the expression of various ion channels [59, 96, 97]. Steroid hormones can rapidly modulate ion channel function through non-genomic mechanisms [98, 99] as well. These effects occur within seconds to minutes and involve direct interactions between hormones and ion channels, altering their gating properties. For example, estrogen and glucocorticoids have been observed to rapidly affect L-type Ca^2+^ channels, potentially influencing contractility and arrhythmia susceptibility [98, 99]. Aldosterone has also been shown to exert rapid, non-genomic effects on Na^+^ and K^+^ currents in cardiac cells, which may contribute to its pro-arrhythmic potential in pathological conditions [100]. The expression of steroid hormone receptors themselves may be subject to circadian regulation, adding another layer of temporal control to the steroid-mediated regulation of ion channels. Sex hormones, glucocorticoids, and mineralocorticoids all contribute to this regulation, significantly affecting the properties of the myocardial substrate [59].
Lastly, indirect regulation of the day-night rhythms in the protective properties of the myocardial substrate includes daily rhythms in homeostatically regulated variables. For example, recent studies in mice show that day-night rhythms in core body temperature impact heart rate and ventricular repolarization, and circadian clock-mediated changes in intracellular soluble protein abundance can alter ionic gradients across the sarcolemma in cardiomyocytes to impact electrophysiology [30, 101] .
Currently, there is limited understanding of how cyclical changes in mRNA transcript availability translate into corresponding protein changes in cardiomyocytes. Shown in Figure 2A is the mRNA expression patterns from Bmal1 and Kcnh2 taken from the Circa Age database. Utilizing a simple model of translation [83] the phase delay and relative amplitude for the oscillating mRNA (dotted line) and protein levels (solid line) for BMAL1 (red) and Kv11.1 protein (blue) accumulation were calculated (Figure 2B). Studies in other tissues (e.g., liver) indicate that many REGs do not exhibit a direct correlation between mRNA and protein levels. Additionally, the amplitude of cycling proteins is generally much smaller than their mRNA counterparts [58, 102]. Consequently, mRNAs with small amplitude fluctuations are unlikely to result in significant rhythmic protein levels. Another important factor that determines whether REGs generate rhythmic expression in protein levels is protein half-life (Figure 2C); proteins with longer half-lives tend to accumulate over time. Continuous rhythmic mRNA expression is needed to maintain steady-state protein levels across the 24-hour cycle. Decreases in the amplitude of the rhythmic expression of mRNA in the heart are expected to reduce steady-state levels of protein, diminishing the protective properties of the myocardial substrate. This can be seen in Figure 2C utilizing a model from O’Hara et al. demonstrating increases in APD as Kv11.1 channel protein half-life is reduced [103, 104]. The model predicts that as the half-life of the Kv11.1 channel protein decreases, the impact on the time-of-day difference in the ventricular action potential duration increases. The half-life of wild-type Kv11.1 channel proteins has been estimated to be about 12 hours, but nonsense and missense KCNH2 mutations linked to the pro-arrhythmic long QT syndrome can decrease the Kv11.1 channel protein half-life to < 6 hours [105-107]. Although speculative, this raises the possibility that mutations that shorten the half-life of proteins encoded by circadian-regulated genes may unmask daily rhythms in cellular function and physiology. Research in the liver indicates that the rhythmic proteome is primarily regulated at the translational and posttranslational levels [83]. Although transcriptional studies have identified many REGs, only a subset will likely result in proteins with rhythmic expression patterns.
This review examines the complex interplay between circadian rhythms, cardiac electrophysiology, sudden cardiac arrest (SCA), and sudden cardiac death (SCD) with a specific focus on the role of the myocardial substrate. Recent data suggests the previously observed morning peak in SCA incidence is no longer evident in modern-day society. We propose that time-of-day disruption or more sustained changes that diminish the protective properties of the myocardial substrate may lead to a higher incidence of sustained arrhythmias during the day when the frequency of arrhythmogenic triggers is highest. We explored current data from various bioinformatic databases to illustrate rhythmic expression patterns of cardiac ion channel genes and their regulators and discuss the direct and indirect regulation of these genes by the circadian clock mechanism, as well as other regulatory pathways (microRNAs and steroid hormones) that would impact the properties of the myocardial substrate. In addition, we examine the importance of rhythmic gene expression in maintaining steady-state levels of ion channels in the heart. As chronobiological approaches continue to be integrated into cardiac electrophysiology research, it is anticipated that novel time-based therapeutic strategies will emerge to enhance protective properties of the myocardial substrate to reduce SCD risk.