David J. Durgan

The Cardiomyocyte Circadian Clock: Emerging Roles in Health and Disease

Circadian misalignment has been implicated in the development of obesity, diabetes mellitus, and cardiovascular disease. Time-of-day-dependent synchronization of organisms with their environment is mediated by circadian clocks. This cell autonomous mechanism has been identified within all cardiovascular-relevant cell types, including cardiomyocytes. Recent molecular- and genetic-based studies suggest that the cardiomyocyte circadian clock influences multiple myocardial processes, including transcription, signaling, growth, metabolism, and contractile function. Following an appreciation of its physiological roles, the cardiomyocyte circadian clock has recently been linked to the pathogenesis of heart disease in response to adverse stresses, such as ischemia/reperfusion, in animal models. The purpose of this review is therefore to highlight recent advances regarding the roles of the cardiomyocyte circadian clock in both myocardial physiology and pathophysiology (ie, health and disease).

The Circadian Clock within the Cardiomyocyte Is Essential for Responsiveness of the Heart to Fatty Acids

Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreaticβ-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: 1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and 2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. We report that both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle was associated with a re-entrainment of the circadian clock within the rat heart, which required 5–8 days for completion. Fasting rats resulted in the induction of fatty acid-responsive genes, an effect that was dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative CLOCK mutant, severely attenuated induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism.

Short Communication: Ischemia/Reperfusion Tolerance Is Time-of-Day–Dependent Mediation by the Cardiomyocyte Circadian Clock

Rationale: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown. Objective: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice. Methods and Results: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following 1 day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (zeitgeber time [ZT]12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following 1 month of reperfusion, prior ischemic event at ZT12 versus ZT0 resulted in significantly greater infarct volume, fibrosis, and adverse remodeling, as well as greater depression of contractile function. Genetic ablation of the cardiomyocyte circadian clock (termed cardiomyocyte-specific circadian clock mutant [CCM] mice) attenuated/abolished time-of-day variations in I/R outcomes observed in wild-type hearts. Investigation of Akt and glycogen synthase kinase-3&bgr; in wild-type and CCM hearts identified these kinases as potential mechanistic ties between the cardiomyocyte circadian clock and I/R tolerance. Conclusions: We expose a profound time-of-day dependence for I/R tolerance, which is mediated by the cardiomyocyte circadian clock. Further understanding of I/R tolerance rhythms will potentially provide novel insight regarding the etiology and treatment of ischemia-induced cardiac dysfunction.

Direct Regulation of Myocardial Triglyceride Metabolism by the Cardiomyocyte Circadian Clock

Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.

O-GlcNAcylation, Novel Post-Translational Modification Linking Myocardial Metabolism and Cardiomyocyte Circadian Clock

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.

Role of the Gut Microbiome in Obstructive Sleep Apnea-Induced Hypertension.

Individuals suffering from obstructive sleep apnea (OSA) are at increased risk for systemic hypertension. The importance of a healthy gut microbiota, and detriment of a dysbiotic microbiota, on host physiology is becoming increasingly evident. We tested the hypothesis that gut dysbiosis contributes to hypertension observed with OSA. OSA was modeled in rats by inflating a tracheal balloon during the sleep cycle (10-s inflations, 60 per hour). On normal chow diet, OSA had no effect on blood pressure; however, in rats fed a high-fat diet, blood pressure increased 24 and 29 mm Hg after 7 and 14 days of OSA, respectively (P<0.05 each). Bacterial community characterization was performed on fecal pellets isolated before and after 14 days of OSA in chow and high-fat fed rats. High-fat diet and OSA led to significant alterations of the gut microbiota, including decreases in bacterial taxa known to produce the short chain fatty acid butyrate (P<0.05). Finally, transplant of dysbiotic cecal contents from hypertensive OSA rats on high-fat diet into OSA recipient rats on normal chow diet (shown to be normotensive) resulted in hypertension similar to that of the donor (increased 14 and 32 mm Hg after 7 and 14 days of OSA, respectively; P<0.05). These studies demonstrate a causal relationship between gut dysbiosis and hypertension, and suggest that manipulation of the microbiota may be a viable treatment for OSA-induced, and possibly other forms of, hypertension.

Evidence Suggesting that the Cardiomyocyte Circadian Clock Modulates Responsiveness of the Heart to Hypertrophic Stimuli in Mice

Circadian dyssynchrony of an organism (at the whole-body level) with its environment, either through light-dark (LD) cycle or genetic manipulation of clock genes, augments various cardiometabolic diseases. The cardiomyocyte circadian clock has recently been shown to influence multiple myocardial processes, ranging from transcriptional regulation and energy metabolism to contractile function. The authors, therefore, reasoned that chronic dyssychrony of the cardiomyocyte circadian clock with its environment would precipitate myocardial maladaptation to a circadian challenge (simulated shiftwork; SSW). To test this hypothesis, 2- and 20-month-old wild-type and CCM (Cardiomyocyte Clock Mutant; a model with genetic temporal suspension of the cardiomyocyte circadian clock at the active-to-sleep phase transition) mice were subjected to chronic (16-wks) biweekly 12-h phase shifts in the LD cycle (i.e., SSW). Assessment of adaptation/maladaptation at whole-body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels revealed essentially identical responses between wild-type and CCM littermates. However, CCM hearts exhibited increased biventricular weight, cardiomyocyte size, and molecular markers of hypertrophy (anf, mcip1), independent of aging and/or SSW. Similarly, a second genetic model of selective temporal suspension of the cardiomyocyte circadian clock (Cardiomyocyte-specific BMAL1 Knockout [CBK] mice) exhibits increased biventricular weight and mcip1 expression. Wild-type mice exhibit 5-fold greater cardiac hypertrophic growth (and 6-fold greater anf mRNA induction) when challenged with the hypertrophic agonist isoproterenol at the active-to-sleep phase transition, relative to isoproterenol administration at the sleep-to-active phase transition. This diurnal variation was absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic stimuli. (Author correspondence: meyoung@uab.edu)

Alterations in the gut microbiota can elicit hypertension in rats

Gut dysbiosis has been linked to cardiovascular diseases including hypertension. We tested the hypothesis that hypertension could be induced in a normotensive strain of rats or attenuated in a hypertensive strain of rats by exchanging the gut microbiota between the two strains. Cecal contents from spontaneously hypertensive stroke prone rats (SHRSP) were pooled. Similarly, cecal contents from normotensive WKY rats were pooled. Four-week-old recipient WKY and SHR rats, previously treated with antibiotics to reduce the native microbiota, were gavaged with WKY or SHRSP microbiota, resulting in four groups; WKY with WKY microbiota (WKY g-WKY), WKY with SHRSP microbiota (WKY g-SHRSP), SHR with SHRSP microbiota (SHR g-SHRSP), and SHR with WKY microbiota (SHR g-WKY). Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. At 11.5 wk of age systolic blood pressure increased 26 mmHg in WKY g-SHRSP compared with that in WKY g-WKY (182 ± 8 vs. 156 ± 8 mmHg, P = 0.02). Although the SBP in SHR g-WKY tended to decrease compared with SHR g-SHRSP, the differences were not statistically significant. Fecal pellets were collected at 11.5 wk of age for identification of the microbiota by sequencing the 16S ribosomal RNA gene. We observed a significant increase in the Firmicutes:Bacteroidetes ratio in the hypertensive WKY g-SHRSP, as compared with the normotensive WKY g-WKY (P = 0.042). Relative abundance of multiple taxa correlated with SBP. We conclude that gut dysbiosis can directly affect SBP. Manipulation of the gut microbiota may represent an innovative treatment for hypertension.

A new rodent model for obstructive sleep apnea: effects on ATP-mediated dilations in cerebral arteries

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2 receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ~30% in MCAs from rats undergoing apneas compared with those from a sham control group (P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

Linking the Cardiomyocyte Circadian Clock to Myocardial Metabolism

IntroductionThe energetic demands imposed upon the heart vary dramatically over the course of the day. In the face of equally commanding oscillations in the neurohumoral mileu, the heart must respond both rapidly and appropriately to its diurnal environment, for the survival of the organism. A major response of the heart to alterations in workload, nutrients, and various neurohumoral stimuli is at the level of metabolism. Failure of the heart to achieve adequate metabolic adaptation results in contractile dysfunction.DiscussionSubstantial evidence is accumulating which suggests that a transcriptionally based timekeeping mechanism known as the circadian clock plays a role in mediating myocardial metabolic rhythms. Here, we provide an overview of our current knowledge regarding the interplay between the circadian clock within the cardiomyocyte and myocardial metabolism. This includes a particular focus on circadian clock mediated regulation of endogenous energy stores, as well as those mechanisms orchestrating circadian rhythms in metabolic gene expression.ConclusionAn essential need to elucidate fully the functions of this molecular mechanism in the heart remains.