The purpose of the present study was to investigate whether chronic circadian dyssynchrony of the heart with its environment (through genetic temporal suspension of the cardiomyocyte circadian clock) would augment maladaptation to a circadian challenge (i.e., aging and/or simulated shift work). Counter to this hypothesis, CCM mice exhibit essentially identical responsiveness to aging and SSW relative to wild-type littermates, at whole body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels. However, CCM hearts appear to have a pro-hypertrophic phenotype, as observed through gravimetric, histological, transcriptional, and imaging analyses. Similarly, a second genetic model of selective disruption of the cardiomyocyte circadian clock (namely CBK mice) exhibits a pro-hypertrophic phenotype. To investigate further the potential role of the cardiomyocyte circadian clock in modulating responsiveness of the heart to hypertrophic stimuli, wild-type and CCM mice were treated with isoproterenol in a time-of-day-dependent manner. We report that isoproterenol induced cardiac hypertrophy and anf expression to the greatest extent when administered to wild-type mice at the awake-to-sleep transition (relative to administration at the sleep-to-wake transition). This diurnal variation in isoproterenol responsiveness is absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic stimuli.
The primary function of cell autonomous circadian clocks is to confer anticipation of extracellular/environmental stimuli, thereby promoting synchrony of the cell/organ/organism with its environment (Edery 2000
). As such, impairment or loss of this mechanism would likely promote maladaptive responses. With regards to the cardiovascular system, the recent exposure of circadian clocks as critical regulators of numerous aspects of cardiovascular function over the course of the day has led to speculation that impairment of these molecular timekeepers may precipitate cardiovascular disease (Young, et al. 2007
). Evidence in support of this hypothesis is several fold: 1) circadian clocks are altered/impaired in many animal models of increased cardiovascular disease risk (e.g., DIO, diabetes, hypertension, shift-work, etc.) (Durgan, et al. 2006
; Kohsaka, et al. 2007
; Kung, et al. 2007
; Kunieda, et al. 2006
; Mohri, et al. 2003
; Young, et al. 2001
; Young, et al. 2002
); 2) common environmental factors that are implicated as significant contributors towards cardiovascular disease in Western society (i.e., quality and quantity of calories ingested, physical activity, and sleep) are also known to be strong entrainment factors regulating circadian clocks (Burioka, et al. 2008
; Damiola, et al. 2000
; Kohsaka, et al. 2007
; Zambon, et al. 2003
); 3) genetic polymorphisms in human circadian clock genes have been shown to be associated with cardiovascular disease development, such as hypertension (Woon, et al. 2007
); and 4) multiple animal models of environment- and genetic- induced perturbations in molecular clocks generally exhibit increased susceptibility towards cardiovascular disease development (Sole, et al. 2009
). Several examples exist for the latter. This includes increased mortality in cardiomyopathic hamsters following light/dark cycle manipulation (Penev, et al. 1998
). Similarly, Martino et al
have reported that light/dark cycle manipulation augments myocardial dysfunction in mice subjected to pressure overload, an effect that could be reversed following re-entrainment to a normal light/dark cycle (Martino, et al. 2007
). More recently, the same group has shown that housing heterozygous tau
hamsters (that possess an altered circadian clock, with a periodicity of 22 hours) in a normal 24-hr light/dark cycle results in profound cardiorenal disease, which is absent when these rodents remain synchronized with their environment (by housing in a 22-hr light/dark cycle) (Martino, et al. 2008
). Targeted genetic ablation of circadian clock components has also been shown to influence susceptibility of mice to adverse cardiovascular stresses, such as vascular injury and high salt intake (Anea, et al. 2009
; Doi, et al. 2010
Although a number of studies have been performed investigating whether disruption/impairment of circadian clocks in a ubiquitous fashion influences the etiology of cardiovascular disease, few studies have similarly reported a role for distinct cell-type specific clocks. Through the use of CCM mice, we recently revealed that the cardiomyocyte circadian clock directly influences myocardial ischemia/reperfusion tolerance, in a time-of-day-dependent manner (Durgan, et al. 2010a
). However, the role of this clock in the etiology of cardiovascular disease in response to other clinically-relevant stressors remains unknown. To address whether the cardiomyocyte circadian clock plays a significant role in the development of cardiovascular disease in response to aging and/or light/dark cycle manipulations (i.e., simulated shift work), we utilized a genetic mouse model wherein the heart is dyssynchronized with the rest of the organism, through temporal suspension of the cardiomyocyte circadian clock (i.e., CCM mice) (Young 2009
). Contrary to expectation, wild-type and CCM littermates respond to aging and SSW in essentially identical manners (at whole body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels; – and –). These observations suggest that extra-cardiac factors likely play a dominant role in the adaptation/maladaptation of the myocardium to these distinct stresses (i.e., aging and light/dark cycle manipulation). Such factors will likely include paracrine and endocrine factors, many of which are themselves under direct control of non-cardiomyocyte circadian clocks. For this reason, ubiquitous disruption of circadian clocks likely augments cardiovascular disease progression to a greater extent then selective temporal suspension of the cardiomyocyte circadian clock.
One potential limitation of this aspect of the present study is the fact that neither aging nor simulated shift work precipitated cardiac dysfunction (in wild-type or CCM mice). It is conceivable that further aging and/or extending the light/dark cycle manipulation regime for greater than 16 weeks, might induce contractile dysfunction. However, approximately 20% of the aged mice died within 16 weeks of the SSW regime, thereby limiting justification for prolongation. Importantly, previous studies have shown that manipulation of the light/dark cycle for this duration is a significant stress, particularly for aged mice (Davidson, et al. 2006
During extensive characterization of wild-type and CCM littermates in the present study, we revealed a pro-hypertrophic phenotype in CCM mice, independent of age and SSW. More specifically, CCM mice exhibit a greater bi-ventricular weight-to-body weight ratio (), increased cardiomyocyte cross sectional area (), increased septal wall thickness (), and a transcriptional profile indicative of cardiac hypertrophy (i.e., induction of anf
, as well as a repression of mhcα
; ). Previous studies have shown that the genetic manipulation performed to disrupt the circadian clock can dramatically influence the resulting phenotype. For example, ubiquitous expression of the CLOCKΔ19 mutant protein has been reported to result in a pro-obesity phenotype, while knockout of BMAL1 in a ubiquitous manner results in a lean phenotype (Bunger, et al. 2005
; Turek, et al. 2005
). Genetic background of the mouse model also significantly influences the phenotype observed; CLOCKΔ19 mutant mice on the C57/Bl6J background are obesity-prone, while the same mice on either the ICR or CBA background are obesity-resistant (Kennaway, et al. 2007
; Oishi, et al. 2006
; Turek, et al. 2005
). To ensure the pro-hypertrophic phenotype observed in CCM mice was neither model nor background specific, we investigated a novel, second mouse model of temporal suspension of the cardiomyocyte circadian clock on a distinct genetic background (i.e., CBK mice on C57/Bl6J background). Similar to CCM hearts (Young 2009
), clock output from CBK hearts is essentially suspended at ZT0 (). Importantly, genetic disruption of the cardiomyocyte circadian clock in CBK mice was associated with a pro-hypertrophic phenotype ().
Given that CCM and CBK hearts both appear to be temporally suspended at the awake-to-sleep phase transition, we hypothesized that wild-type hearts may be more responsive to pro-hypertrophic stimuli at this time of the day. To test this hypothesis, mice were treated with the hypertrophic agonist isoproterenol at either the awake-to-sleep phase transition or the sleep-to-awake phase transition. Consistent with the hypothesis, wild-type mice exhibited greatest hypertrophic growth and anf induction when treated with isoproterenol at the awake-to-sleep phase transition (). In contrast, this diurnal variation in responsiveness to isoproterenol was absent in CCM mice (). Collectively, these data support the hypothesis that the cardiomyocyte circadian clock modulates responsiveness of the heart to pro-hypertrophic stimuli in a time-of-day-dependent manner.
An important question relates to the identity of potential mechanisms by which the cardiomyocyte circadian clock modulates responsiveness of the myocardium to hypertrophic stimuli. Similar to our data regarding hypertrophic responsiveness, Collins and Rodrigo reported that the inotropic response of adult cardiomyocytes to isoproterenol exhibits a time-of-day-dependent variation (Collins, et al.). Isoproterenol is a β-adrenergic agonist (Lefkowitz, et al. 2000
). Zhang et al
recently revealed a mechanistic link between β-adrenergic signaling and the circadian clock in hepatocytes (Zhang, et al. 2010
). Through a series of elegant studies, the investigators reported that cryptochromes (an integral clock component) can bind directly to Gsα, resulting in subsequent decreases in G-protein coupled receptor signaling (Zhang, et al. 2010
). Consistent with such a model, we report that CRY2 protein levels exhibit a diurnal variation in wild type, but not CCM or CBK hearts, with approximate 1.5-fold greater levels at ZT12 (the time of day at which isoproterenol-induced hypertrophy was lowest; ). These data are consistent with the hypothesis that the cardiomyocyte circadian clock influences myocardial β-adrenergic responsiveness, through time-of-day-dependent attenuation of Gsα by cryptochromes. However, it is important to note that additional potential molecular links may exist. For example, we have recently shown that the phosphorylation status of the growth signaling kinases Akt and GSK3β oscillates in wild-type hearts, in a cardiomyocyte circadian clock dependent manner (Durgan, et al. 2010a
); both Akt and GSK3β are known modulators of cardiac hypertrophic growth (Heineke, et al. 2006
). Whether CRY2, Akt and/or GSK3β serve as molecular links between the cardiomyocyte circadian clock and hypertrophic growth requires full elucidation.
The findings of the present study have significant clinical implications. Non-dipping hypertensive patients (whose blood pressure drops <10% during sleep, and therefore exhibit inappropriate stress on the heart during the sleep phase) exhibit increased left ventricular hypertrophy and are at increased risk of cardiovascular and renal disease (compared to dipping hypertensive subjects) (Bianchi, et al. 1994
; Hermida, et al. 2005
; Palatini, et al. 1992
). Similarly, obstructive sleep apnea (OSA) is associated with increased sympathetic stimulation on the heart during the sleep phase, adverse cardiac remodeling and increased risk of heart failure (Bradley, et al. 2009
; Kario 2009
). Recently, Sole and Martino hypothesized that the sleep phase corresponds to a time of increased myocardial renewal and growth, and as such pressure overload during this phase may accelerate the development of pathological hypertrophy (Sole, et al. 2009
). Our findings are consistent with this hypothesis, and also suggest an important mediatory role for the cardiomyocyte circadian clock.
In conclusion, we report that genetic, temporal suspension of the cardiomyocyte circadian clock has little impact on adaptation of the mouse to aging and/or simulated shift work. However, these studies highlight a novel potential role for the cardiomyocyte circadian clock in modulating responsiveness of the heart to pro-hypertrophic stimuli in a time-of-day-dependent manner.