Diurnal rhythms in myocardial physiology (e.g., metabolism, contractile function) and pathophyiology (e.g., sudden cardiac death) are well establish and have classically been ascribed to time-of-day-dependent alterations in the neurohumoral milieu. Existence of an intramyocellular circadian clock has recently been exposed. Circadian clocks enable the cell to anticipate environmental stimuli, facilitating a timely and appropriate response. Generation of genetically modified mice with a targeted disruption of the cardiomyocyte circadian clock has provided an initial means for deciphering the functions of this transcriptionally based mechanism and allowed predictions regarding which environmental stimuli the heart anticipates (i.e., “anticipating anticipation”). Recent studies show that the cardiomyocyte circadian clock influences myocardial gene expression, β-adrenergic signaling, transcriptional responsiveness to fatty acids, triglyceride metabolism, heart rate, and cardiac output, as well as ischemia-reperfusion tolerance. In addition to reviewing current knowledge regarding the roles of the cardiomyocyte circadian clock, this article highlights putative frontiers in this field. The latter includes establishing molecular links between the cardiomyocyte circadian clock with identified functions, understanding the pathophysiological consequences of disruption of this mechanism, targeting resynchronization of the cardiomyocyte circadian clock for prevention/treatment of cardiovascular disease, linking the circadian clock with the cardiobeneficial effects of caloric restriction, and determining whether circadian clock genes are subject to epigenetic regulation. Information gained from studies investigating the cardiomyocyte circadian clock will likely translate to extracardiac tissues, such as skeletal muscle, liver, and adipose tissue.
- gene expression
cardiac form and function are far from static. In an unrelenting effort to meet the demands of the organism, the heart undergoes continuous adaptation to its environment, at molecular, structural, and functional levels. This is true over multiple time scales: beat to beat, daily, seasonally, as well as developmentally. Myocardial adaptation is critical, not only for development and physiology, but also in the etiology of cardiovascular disease. Central to the concept of adaptation is an ability of the heart to adequately respond to both intracellular and extracellular cues. Countless stimulus-response coupling mechanisms have been characterized, ranging from intracellular Ca2+ sensing during an individual heartbeat, to postprandial neurohumoral responses, and to hypertrophic adaptation to long-term elevations in sheer stress. Molecular characterization of cellular response mechanisms over the last several decades has uncovered a complex network of signaling cascades running both in parallel and series. Intramyocellular circadian clocks have emerged as a cornerstone in stimulus-response coupling. This mechanism is not only highly sensitive to intracellular and extracellular cues (some of which are integral clock components), but is capable of directly influencing numerous signaling components. Simply put, the circadian clock confers a selective advantage within a dimension of its own. That being, the circadian clock can anticipate a stimulus before it occurs.
The purpose of this review article is severalfold. First, historical evidence for the existence of a cardiomyocyte circadian clock will be presented. Next, the insights gained from recent studies designed to unravel the functional roles of the cardiomyocyte circadian clock will be outlined. Finally, putative future frontiers for this novel area of research will be highlighted. Using our current knowledge, attempts will be made to predict which environmental stimuli the cardiomyocyte circadian clock allows the heart to anticipate (i.e., “anticipating anticipation”).
CHARACTERIZATION OF THE CARDIOMYOCYTE CIRCADIAN CLOCK
As early as 1729, an appreciation for the existence of an internal timekeeping mechanism within terrestrial organisms has been documented (26). With the advent of molecular biology, appropriate tools became available for identification and characterization of the timekeeping mechanism components (so-called circadian clocks). Circadian clocks are defined as a set of proteins comprising a series of self-sustained transcriptional positive and negative feedback loops, with a free-running period of ∼24 h (26). The circadian clock confers the selective advantage of anticipation; by knowing the time of day, the cell/organ can prepare for a stimulus before its onset, enabling both a rapid and appropriate response.
Since the mammalian circadian clock has been reviewed previously (20, 26, 31, 68) and is detailed throughout the current Topic Review Series, only a brief overview is included here. Circadian clocks can be subdivided into three major components: input, the clock mechanism itself, and output. The mammalian clock mechanism itself is made up of more than 12 distinct core clock proteins. At the heart of this mechanism lies two transcription factors, Clock and Bmal1, which heterodimerize and bind to E-boxes within the promoters of target genes (27, 33). Target genes include several core circadian clock components, as well as numerous clock output genes (68). As such, output from the circadian clock initially manifests at the level of transcription, but ultimately results in altered cellular function in a time-of-day-dependent manner. Input factors (termed zeitgebers or timekeepers) entrain cell autonomous circadian clocks (32). Light is among the best characterized zeitgebers, which entrains the central “master” clock within the suprachiasmatic nucleus, via the retinohypothalamic tract (5). In contrast, peripheral clocks rely primarily on neurohumoral factors for entrainment (14).
A fully functional circadian clock has been identified within virtually all mammalian cells investigated to date, including cardiomyocytes. One of the first published studies reporting rhythmic expression of a circadian clock component within the heart was in 1998. Sakamoto et al. (62) observed diurnal (i.e., during normal light-dark cycles) oscillations in per2 gene expression in mouse hearts. Given well-established diurnal variations in cardiac function, we decided to characterize major circadian clock components and output genes in both normal rat hearts, as well as hearts isolated from rat models of human cardiovascular disease (pressure overload-induced hypertrophy and uncontrolled streptozotocin-induced diabetes mellitus). Consistent with predictions, all circadian clock genes investigated were expressed in the rat heart and did so in a diurnal manner, and these oscillations were altered in disease states (79, 80). Several laboratories have confirmed diurnal oscillations in expression of circadian clock genes in the rodent heart (15, 30, 36, 45, 49, 52, 57, 66). More recently, Leibetseder et al. (43) has reported rhythmic expression of circadian clock gene components in human hearts.
Oscillation of circadian clock gene expression for rodent (and human) hearts in vivo is not sufficient to conclude that a cell autonomous circadian clock resides within cardiomyocytes, for numerous reasons. First, oscillations in clock gene expression in vivo could be secondary to changes in the neurohumoral environment, as opposed to a self-sustained mechanism intrinsic to the cardiomyocyte. Second, the intact heart is composed of numerous cell types, including cardiomyocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts, any one of which could/will contribute to observed clock gene oscillations. These concerns prompted us to investigate whether cultured ventricular adult rat cardiomyocytes (ARCs) possessed a fully functional circadian clock. We found that expression of circadian clock genes oscillate in a circadian (i.e., independent of light-dark cycles) manner in cultured ARCs, with a temporal relationship essentially identical to that observed in vivo (21). These studies conclusively revealed the presence of a fully functional circadian clock within the cardiomyocyte. Following exposure of this molecular mechanism, the next step was to define functional roles.
PHYSIOLOGICAL ROLES FOR THE CARDIOMYOCYTE CIRCADIAN CLOCK
Identifying Cardiomyocyte Circadian Clock Functions
Definitively assigning roles for the circadian clock within a specific cell type is an arduous undertaking. Generally, when one addresses questions relating to the physiological function of a mechanism in vivo, genetically modified animal models are employed. Two primary questions arise: 1) which gene(s) should be targeted; and 2) in what manner (e.g., overexpressed or knocked out, cell-type specific or ubiquitous, inducible or constitutive). An enormous level of redundancy exists for the mammalian circadian clock. As such, genetic manipulation of a single circadian clock component does not always result in complete ablation of clock function. Currently, two strategies for single genetic manipulation have been successful in nullifying circadian clock function, both of which target the Clock/Bmal1 heterodimer. These are mutation of Clock (removal of exon 19, resulting in a dominant-negative mutant, termed ClockΔ19), and knockout of Bmal1 (termed Bmal1−/−) (12, 73). Impairment of circadian clock function has been observed for all cell types investigated in ClockΔ19 mutant and Bmal1−/− mice. Both models have impaired diurnal and circadian function at multiple levels, including behavior (e.g., feeding-fasting cycles, sleep-wake cycles), neurohumoral factors (e.g., glucose, insulin, norepinephrine), and tissue gene expression (12, 13, 61, 70, 73). Due to the profound global impact of whole body clock disruption, conclusions regarding the role of cell-type-specific circadian clocks are limited. Such a concept was exemplified recently by Lamia et al. (41). Comparisons were made for glucose homeostasis between Bmal1−/− mice and hepatocyte-specific BMAL1 null mice (41). Markedly different data were obtained between the two models. Bmal1−/− mice were shown to be glucose intolerant, while hepatocyte-specific BMAL1 null mice exhibit increased glucose tolerance. The reason for this discrepancy undoubtedly stems from the fact that glucose tolerance is the product of a complex interaction between multiple organs, including the liver, pancreas, adipose, skeletal muscle, and the central nervous system. Through generation of a tissue-specific model, Lamia et al. successfully assigned a role for the hepatocyte circadian clock in glucose homeostasis. This concept is of equal importance when interrogating potential roles of the cardiomyocyte circadian clock. Myocardial physiology is influenced by countless extracellular factors, many of which are altered in ClockΔ19 and Bmal1−/− mice. We, therefore, generated a model wherein the ClockΔ19 gene was specifically expressed in cardiomyocytes; these mice are termed CCM (cardiomyocyte-specific clock mutant). Extra cardiac circadian clocks are normal in CCM mice (8, 23).
Two primary criteria were established for considering a gene/process to be regulated by the cardiomyocyte circadian clock. These are that the process exhibits a significant diurnal/circadian oscillation in wild-type hearts, and that this oscillation is attenuated/abolished in CCM hearts (Fig. 1; an attenuated oscillation is defined as a decrease in the amplitude). Characterization of circadian clock components reveals that this molecular mechanism is temporally suspended at the initiation of the light phase in CCM hearts (8, 23). This observation has aided in identification of cardiomyocyte circadian clock-regulated genes. Genes that oscillate in wild-type hearts, but whose expression is temporally suspended at the initiation of the light phase in CCM hearts, are considered cardiomyocyte circadian clock-regulated genes. Two major classes of cardiomyocyte circadian clock genes were identified: those peaking at the light-to-dark phase transition in wild-type hearts (and chronically repressed in CCM hearts; Fig. 1A, scenario A) and those peaking at the dark-to-light phase transition in wild-type hearts (and chronically induced in CCM hearts; Fig. 1B, scenario B). Following an inevitable delay between transcription and translation of clock-controlled genes (6 h depicted in the figure, although this will vary between proteins), respective myocardial processes will be either chronically repressed (scenario A) or activated (scenario B) in CCM hearts. Currently, we have observed scenario A for diurnal variations in sensitivity to β-adrenergic stimulation, transcriptional responsiveness to fatty acids, triglyceride synthesis, heart rate, cardiac output, and ischemia-reperfusion tolerance (all peak in the middle of the dark phase in wild-type hearts and are chronically repressed in CCM hearts; see discussion below). Scenario B has not yet been identified for a cardiomyocyte circadian clock-regulated myocardial process.
The primary function of the circadian clock is to confer the selective advantage of anticipation. One way the clock accomplishes this is by time-dependent modulation of the sensitivity of the cell to extracellular stimuli. Therefore, an oscillation in wild-type hearts in vivo, and attenuation/ablation in CCM hearts, may be due to circadian clock-mediated diurnal variations in responsiveness of the heart to the in vivo milieu. To control for this distinct possibility, whenever possible, a candidate circadian clock process is investigated both in vivo and ex vivo.
Using the above-defined criteria, assignment of roles for the cardiomyocyte circadian clock has begun. These data provide evidence that the cardiomyocyte circadian clock influences myocardial gene expression, cellular signaling, metabolism, and contractile function. Each will be considered in turn.
Circadian clock output manifests initially at a transcriptional level. Identification of cardiomyocyte circadian clock-regulated genes is, therefore, an appropriate starting point for assignment of function. Consistent with such a concept, several microarray studies have been performed, defining time-of-day-dependent oscillations in gene expression for intact, wild-type hearts (45, 66). These important studies exposed dramatic diurnal/circadian oscillations in the transcriptome of the heart (expression of between 10 and 13% of myocardial genes oscillate in a time-of-day-dependent manner). We, therefore, decided to determine which of these gene expression oscillations were mediated by the cardiomyocyte circadian clock. Using the stringent criteria listed above, as well as a rigorous statistical analysis, microarray studies in wild-type and CCM mice identified 548 and 176 genes as being regulated by the cardiomyocyte circadian clock in atria and ventricles, respectively (8). Gene ontology analysis of the data revealed a concentration of cardiomyocyte circadian clock-regulated genes influencing transcription, cellular signaling cascades, and metabolism. For each of these categories, multiple genes were selected for validation by RT-PCR. One of these genes, nampt, has subsequently been confirmed as a direct circadian clock-regulated gene through independent promoter binding studies (53). Throughout this review, the identity and function of distinct cardiomyocyte circadian clock-regulated genes will be discussed. Importantly, ongoing studies within multiple laboratories are defining whether cardiomyocyte circadian clock-mediated oscillations in gene expression results in reciprocal changes in protein expression/activity and cellular function over the course of the day.
Two major cycles that occur on a daily basis are feeding/fasting and sleep/wake cycles. Humoral changes associated with these cycles, which are known to influence the myocardium, include oscillations in circulating insulin and epinephrine. The above described microarray studies have identified numerous kinases and phosphatases as being regulated by the cardiomyocyte circadian clock, several of which are known components of insulin (e.g., pik3r1) and β-adrenergic (e.g., prkar1a) signaling cascades (8). Regarding the latter, we have reported that epinephrine-induced augmentation of myocardial contractility and glycogenolysis both exhibit a diurnal variation in wild-type hearts, peaking in the middle of the active phase ex vivo (8). In contrast, responsiveness to epinephrine does not oscillate in CCM hearts, and responsiveness remains at trough levels observed for wild-type hearts. Collectively, these data show that the cardiomyocyte circadian clock modulates myocardial β-adrenergic signaling in a time-of-day-dependent manner. Beyond the aforementioned gene expression observations, little is known regarding cardiomyocyte circadian clock regulation of myocardial insulin signaling. Preliminary studies within our laboratory show that insulin-mediated Akt phosphorylation is attenuated in CCM hearts (unpublished observations). Clearly, additional studies are required.
Dey and colleagues have recently suggested that the activity of the protein phosphatase calcineurin exhibits a diurnal variation in the mouse heart (16). These studies report oscillations in multiple calcineurin-dependent activities, including rcan1.4 mRNA levels, nuclear localization of nuclear factor of activated T cells, and phosphorylation of both protein phosphatase 1 inhibitor 1 and phospholamban. In the case of rcan1.4 (also known as mcip1) mRNA, both microarray and RT-PCR studies confirm that this is a cardiomyocyte circadian clock-regulated gene (8). As such, it is possible that the cardiomyocyte circadian clock regulates calcineurin signaling in the heart.
What is becoming increasingly clear is that circadian clocks play an integral role in cellular metabolism (19, 29, 74). Myocardial metabolism and contractile function are also inseparably interlinked (63, 67). Considerable effort has, therefore, been placed on unraveling regulation of myocardial metabolism by the cardiomyocyte circadian clock (8, 10, 21–23, 25, 76, 78). The lessons learnt from CCM mice will likely be translated to other cell types, including skeletal myocytes, hepatocytes, and adipocytes.
Myocardial metabolism exhibits profound diurnal variations in both rat and mouse hearts, as reviewed previously (10, 25). Microarray analysis of wild-type vs. CCM hearts identified multiple cardiomyocyte circadian clock genes influencing glycogen (e.g., ppp1cc) and triglyceride (e.g., dgat2) metabolism (8, 25). Total glycogen and triglyceride levels oscillate in a diurnal manner, in multiple tissues, including the myocardium (22). Glycogen content oscillations persist in fasting rats, ruling out meditation by feeding/fasting cycles (35). Recent studies in ClockΔ19 mice support the concept that triglyceride metabolism is influenced by the circadian clock. For example, Turek and colleagues (70) report that ClockΔ19 mice exhibit augmented high-fat diet-induced obesity, associated with dyslipidemia, consistent with altered lipid homeostasis. Conversely, ClockΔ19 mice on the Jcl:ICR background are resistant to high-fat feeding-induced obesity and hepatic steotosis, likely due to abnormalities in lipid digestion/absorption, as well as differential hepatic gene expression (39, 54). Data concerning circadian clock-regulated lipid homeostasis in Bmal1−/− mice is also somewhat difficult to interpret mechanistically: young null mice initially exhibit increased adiposity, but subsequently develop accelerated lipoatrophy with age (11, 41). Two important points should be noted here. First, marked discrepancies regarding effects of circadian clock impairment on phenotype are background strain dependent. Second, few studies have formally assessed triglyceride metabolism in models of altered clock function. Instead, conclusions are typically drawn following measurement of circulating/cellular factors (e.g., triglyceride levels) and gene expression.
We have recently performed a series of studies designed to directly address the question of whether the cardiomyocyte circadian clock regulates oxidative and/or nonoxidative metabolism of fatty acids, including triglyceride turnover. Rationale for these studies includes identification of triglyceride metabolism genes as being clock regulated, as well as failure of CCM hearts to increase triglyceride synthesis during a 12-h fast (unlike wild-type hearts) (8, 23, 25). To directly measure triglyceride metabolism, wild-type and CCM mouse hearts were perfused ex vivo in the working mode at different times of the day, in the presence of radiolabeled tracers. Wild-type hearts exhibit a threefold diurnal variation in rates of triglyceride synthesis ex vivo, peaking in the middle of the active phase (69). Importantly, triglyceride synthesis diurnal variations are absent in CCM hearts, which exhibit rates comparable to trough rates in wild-type hearts. These data are the first to show that a peripheral circadian clock directly regulates triglyceride metabolism. At the same time, rates of fatty acid oxidation are chronically elevated in CCM hearts compared with wild-type littermate hearts (8, 69). However, fatty acid oxidation rates do not significantly oscillate in a diurnal manner in wild-type hearts. Elevated rates of fatty acid oxidation in CCM hearts are, therefore, potentially secondary to decreased triglyceride synthesis. Indeed, few genes known to directly influence fatty acid oxidation were identified as being directly regulated by the cardiomyocyte circadian clock.
We previously hypothesized that the cardiomyocyte circadian clock allows the heart to anticipate diurnal variations in fatty acid availability (23, 64, 76). The reasonings underlying this hypothesis were severalfold. Circulating fatty acids themselves exhibit a diurnal variation, which are dependent on feeding/fasting status (64). Fatty acids are the primary fuel utilized by the heart, providing ∼70% of the energy for myocardial contraction (1). However, when in excess, fatty acids depress contractile function of the heart, through channeling into so-called “lipotoxic” pathways (71, 77). The latter occurs in a diurnal manner, with the greatest fatty acid-induced depression of contractile function occurring during the sleep phase (22). During periods of increased fatty acid availability, the heart increases oxidative and nonoxidative (e.g., triglyceride) metabolism of fatty acids, through both transcriptional and posttranslational mechanisms (3, 77). The transcriptional responsiveness of the heart to fatty acids exhibits a diurnal variation, with increased responsiveness during the active phase (64). Diurnal variations in transcriptional responsiveness persist in ARCs and are abolished in CCM hearts, consistent with mediation by the cardiomyocyte circadian clock (23). Collectively, these observations are consistent with the model depicted in Fig. 2. Here, the cardiomyocyte circadian clock allows the heart to anticipate increased fatty acid availability during the active phase (due either to postprandial hyperlipidemia, or prolongation of the sleep phase fast), by promoting triglyceride synthesis and induction of genes involved in fatty acid oxidation/utilization. This, in turn, minimizes channeling of fatty acids into “lipotoxic” pathways. In contrast, exposure of the heart to increased fatty acids during the sleep phase (due to inappropriate feeding during the sleep phase and/or disease states associated with dyslipidemia) will result in channeling of excess fatty acids into lipotoxic pathways (due to decreased triglyceride synthesis and decreased transcriptional response). The detrimental effects of exposing the heart to an extracellular stress at an inappropriate time of the day is akin to “nondipping” hypertension, wherein increased cardiac injury is associated with increased stress during the sleep phase, a time of day at which blood pressure typically decreases in normotensive subjects (6, 56).
Myocardial Contractile Function
Diurnal variations in myocardial contractile function are well established, in both animal models and humans. Heart rate and cardiac output exhibit robust diurnal variations, peaking during the active phase (38, 55, 60, 72). Classically, time-of-day-dependent oscillations in these parameters have been attributed to diurnal variations in neurohumoral factors (60, 65, 76). However, recent studies suggest it is timely to reconsider this dogma. Note that circadian control of blood pressure will not be discussed here due to primary regulation via extracardiac influences, a lack of blood pressure differences in CCM mice, and a focus on this subject in other articles within this review series.
Initial studies in ClockΔ19 and Bmal1−/− mice reported somewhat predictable observations that heart rate diurnal variations are severely attenuated in these models of ubiquitous circadian clock dysfunction (13). Many of the neurohumoral factors known to influence heart rate are also dramatically altered in these models (e.g., autonomic stimulation, norepinephrine/epinephrine) (13). We, therefore, sought to determine the relative contribution of the cardiomyocyte circadian clock as a mediator of diurnal variations in heart rate. Telemetry studies revealed attenuated diurnal variations in heart rate in CCM mice in vivo, with a decrease in peak heart rate (during the dark phase); lower peak heart rate was also reported in ClockΔ19 and Bmal1−/− null mice (8, 13). Electrocardiogram telemetry subsequently confirmed sinus bradycardia in CCM hearts, in the absence of overt electrical abnormalities. Importantly, bradycardia persisted in CCM hearts ex vivo (8). Collectively, these data show that the cardiomyocyte circadian clock directly influences heart rate.
Similar to heart rate diurnal variations, we have observed persistence of cardiac output diurnal variations ex vivo, in both isolated working rat and mouse hearts (8, 22, 78). This promoted us to investigate cardiac output in CCM hearts. Consistent with mediation by the cardiomyocyte circadian clock, cardiac output did not oscillate in CCM hearts, which exhibit values similar to those observed for wild-type hearts during the sleep phase (i.e., the trough for this parameter) (8). Ongoing studies are underway to determine the mechanism(s) by which the cardiomyocyte circadian clock directly influences both heart rate and myocardial contractility. It is also noteworthy that contraction of the myocardium may, in turn, influence the timing of the circadian clock. Studies by Qi and Boateng (58) show a translocation of the Clock protein from the cytoskeleton to the nucleus during electrical stimulation.
PREDICTING POTENTIAL IMMINENT FRONTIERS
The concept that a circadian clock resides within the cardiomyocyte, which is capable of directly influencing myocardial physiology, is novel, exciting, and receiving increased interest from the scientific community. This field truly is in its infancy and, as such, is poised to venture down countless avenues. Here, I intend to briefly highlight just a few of the potentially imminent frontiers in this emerging field.
Identifying Direct Molecular Links Between the Cardiomyocyte Circadian Clock and Myocardial Physiology
Following identification of functional roles for the cardiomyocyte circadian clock, an entourage of questions arise regarding mechanisms. For example, which circadian clock transcription factors directly bind to the promoters of identified clock output genes, and which clock output proteins mediate changes in myocardial signaling, metabolism, and/or contractile function? The most straightforward mechanism would involve direct regulation of a gene by the Clock/Bmal1 heterodimer, and whose protein levels and activity correlate directly with the encoding mRNA, resulting in biological function oscillations (Fig. 1). However, regulation of a process by the cardiomyocyte circadian clock will potentially be more complex, including multiple clock output genes acting in distinct positive and negative manners. Regulation may be at the level of transcription, mRNA stability, translation, protein stability, phosphorylation, acetylation, compartmentalization, and/or cofactor availability (7, 18, 29, 31, 53, 68). Clearly, such an enormous level of complexity will hinder rapid assignment of molecular links between the cardiomyocyte circadian clock to specific myocardial processes.
An obvious question relates to whether the cardiomyocyte circadian clock is impaired in cardiovascular disease states, and, in turn, whether impairment of this molecular mechanism contributes toward the etiology of myocardial contractile dysfunction. Attempts have already been made to address both questions. Myocardial circadian clock gene expression oscillations are altered in animal models of pressure overload (e.g., aortic constriction, Dahl salt-sensitive rat), uncontrolled diabetes mellitus, obesity, simulated shift work (which increases the risk for cardiovascular disease), aging, and myocardial infarction (23, 28, 40, 49, 79, 80). In the latter case, circadian clock gene oscillations within the ischemic region of the myocardium became inactivated rapidly (<24 h), while clock gene oscillations remain unaltered in the nonischemic region (40). This suggests that an intraorgan dyssynchrony occurs following a myocardial infarction.
Whether the cardiomyocyte circadian clock plays a significant role in the etiology of cardiac dysfunction is less clear. Studies have been performed in animal models of global circadian clock dysfunction that indicate an increased susceptibility to cardiovascular disease progression (46). However, a distinct role for the cardiomyocyte circadian clock remains unknown. Given the rapid inactivation of the circadian clock following a myocardial infarction, and the established clinical observation that myocardial infarctions occur with a robust diurnal variation (increased incidence in the early hours of the morning), we recently hypothesized that the cardiomyocyte circadian clock influences myocardial ischemia-reperfusion tolerance (40, 51). Using the closed chest murine model of ischemia-reperfusion (thereby minimizing an acute induction of an immune response, which itself exhibits a diurnal variation), we found fourfold greater infarct sizes for wild-type hearts subjected to 45-min ischemia at the light-to-dark phase transition (24). Furthermore, this diurnal variation in ischemia-reperfusion tolerance is completely lost in CCM hearts, which have infarct sizes identical to trough values observed for wild-type hearts (24). These data expose a critical role for the cardiomyocyte circadian clock in modulating ischemia-reperfusion injury in a time-of-day-dependent manner.
Clearly, circadian clocks influence both cardiovascular physiology (e.g., responsiveness to fatty acids, heart rate) and pathophysiology (e.g., ischemia-reperfusion tolerance) and become altered/impaired in multiple animal models of cardiovascular disease. Elegant studies from Martino and colleagues (46) show that rodents with a genetic predisposition for dyssynchronization with the environment develop hypertrophic cardiomyopathy. Importantly, myocardial contractile dysfunction can be rescued by alterations in the light-dark cycle that resynchronize the rodent with the environment (46). Collectively, these studies suggest that targeting resynchronization of the cardiomyocyte circadian clock with the environment is a valid strategy for prevention/treatment of cardiovascular disease. Little is currently known regarding the identity of zeitgebers for the cardiomyocyte circadian clock. Initial in vitro studies suggest that norephinephrine and corticosterone are potential zeitgebers for the cardiomyocyte circadian clock (21, 81). However, genetic ablation of norepinephrine/epinephrine signaling or the glucocorticoid receptor has no reported effect on the timing of peripheral circadian clocks in vivo (2, 59). These observations can be interpreted, as neither norephinephrine nor corticosterone plays a significant role in the timing of peripheral circadian clocks during nonstressed conditions, or that a large number of redundant entrainment factors exist. Clearly, additional studies are required to define the zeitgebers for the cardiomyocyte circadian clock.
One of the strongest environmental factors influencing the timing of peripheral circadian clocks is food intake (14, 50). Given the amassing data in support of myocardial metabolism regulation by the cardiomyocyte circadian clock, we recently hypothesized that restricting ingestion of specific macronutrients (e.g., lipid) to distinct times of the day will synchronize the cardiomyocyte circadian clock with both circulating nutrients and myocardial metabolism (10). In doing so, myocardial function would be preserved by attenuating channeling of excess nutrients into “cardiotoxic” pathways. Consistent with this hypothesis, we found that restricting dietary lipid consumption to the beginning of the active phase preserves not only myocardial contractile function, but also multiple cardiometabolic syndrome parameters, including glucose tolerance and adiposity (9). In contrast, consumption of dietary fatty acids at the end of the active phase is associated with adiposity, glucose intolerance, and myocardial contractile dysfunction (9).
Aging, Caloric Restriction, and Sirtuins
Aging is associated with a host of physiological/pathophysiological alterations, including muscle wasting (sarcopenia), weight loss (advanced aging), mitochondrial DNA damage and dysfunction, oxidative stress, increased propensity for tumors, telomere shortening, reduced cellularity, hair graying and alopecia, reduced subcutaneous fat deposits, insulin resistance, decreased lifespan, as well as the development of cardiovascular disease (17, 28). Many of the pathological changes associated with aging can be prevented through caloric restriction (44). Pursuit of the molecular mechanisms mediating the benefits of caloric restriction strongly suggests a critical role for a family of NAD+-dependent deacetylases, known as sirtuins (SIRT) (48). Multiple SIRT isoforms have been identified in mammals. Of these, SIRT1 is strongly implicated as a mediator of the beneficial effects of caloric restriction (44, 48). Pharmacological activation of SIRT1 with resveratrol mimics many of the benefits of caloric restriction (34, 42). SIRT1 has recently been shown to be an integral component of the mammalian circadian clock, which is capable of directly associating with, and deacetylating, Bmal1 (53). Furthermore, circadian clock gene oscillations are attenuated with aging (28). Collectively, these observations lead to the hypothesis that one or more beneficial effects of caloric restriction are mediated through the circadian clock mechanism. Consistent with this hypothesis, Bmal1−/− mice exhibit many characteristics of accelerated aging, including muscle wasting (sarcopenia), weight loss, reduced subcutaneous fat deposits, glucose intolerance, and decreased lifespan (11, 37, 61).
Substantial evidence in both humans and animal models demonstrates that specific alterations in maternal nutrition (e.g., low-protein diet, caloric restriction, high-fat feeding) result in an increased susceptibility of the offspring to development of age-onset metabolic diseases, such as obesity, insulin resistance, and cardiovascular disease (47). Early studies examining birth records revealed a strong inverse correlation between birth weight and coronary artery disease in adulthood (4). Subsequent studies have confirmed these observations, as well as inverse correlations between birth weight with age-onset body mass index (BMI), insulin sensitivity, and blood pressure, in both humans and animal models (47). These observations lead to the hypothesis that nutritional signals at critical temporal windows within gestational and/or neonatal development result in a form of imprinting/reprogramming, which persists into adulthood. This hypothesis is often termed the Baker hypothesis.
The influence that early nutritional interventions have on cell autonomous circadian clocks is currently unknown. However, a select number of studies suggest that distinct interventions during gestational and neonatal development potentially influence this molecular mechanism in a manner that persists into adulthood. For example, Yoshihara et al. (75) have found that restricting access of pups to the dam during the first 3 wk of age results in altered locomotor diurnal variations in adult offspring. Given that diurnal variations in locomotor activity are driven primarily by the central circadian clock within the suprachiasmatic nucleus, these observations suggest that this early nutritional intervention permanently affects the timing of cell autonomous clocks. Additional studies are required to directly address the question of whether peripheral circadian clocks, such as the cardiomyocyte circadian clock, are permanently altered following early nutritional interventions, such as restriction of maternal protein, and/or whether this modulation of the circadian clock subsequently increases susceptibility to age-onset diseases, such as obesity, insulin resistance, and cardiovascular disease.
The goals of this review were to summarize our current knowledge regarding the functions of the cardiomyocyte circadian clock, and to highlight some of the imminent directions that this area of research may proceed. Continued research in this field will undoubtedly reveal regulation of multiple critical myocardial functions by the cardiomyocyte circadian clock.
This work was supported by the National Heart, Lung, and Blood Institute (HL-074259), the US Department of Agriculture/Agricultural Research Service (6250-51000-044), and Kraft Inc.
- Copyright © 2009 the American Physiological Society