Disrupting the key circadian regulator CLOCK leads to age-dependent cardiovascular disease.

The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in heart weight, hypertrophy, dilation, impaired contractility, and reduced myogenic responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts. Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3ß, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3ß, S6K1 phosphorylation are increased, consistent with the development of age-dependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in growth and renewal; disrupting Clock leads to age-dependent cardiomyopathy. Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

Day-night dependence of gene expression and inflammatory responses in the remodeling murine heart post-myocardial infarction.

Diurnal or circadian rhythms are fundamentally important for healthy cardiovascular physiology and play a role in timing of onset and tolerance to myocardial infarction (MI) in patients. Whether time of day of MI triggers different molecular and cellular responses that can influence myocardial remodeling is not known. This study was designed to test whether time of day of MI triggers different gene expression, humoral, and innate inflammatory responses that contribute to cardiac repair after MI. Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 2-h time window either shortly after lights on or lights off, and the early remodeling responses at 8 h postinfarction were examined. We found that sleep-MI preferentially triggers early expression of genes associated with inflammatory responses, whereas wake-MI triggers more genes associated with metabolic pathways and transcription/translation, by microarray analyses. Homozygous clock mutant mice exhibit altered diurnal gene expression profiles, consistent with their cycling before onset of MI. In the first 8 h, crucial for innate immune responses to MI, there are also significant differences in sleep-MI and wake-MI serum cytokine responses and in neutrophil infiltration to infarcted myocardium. By 1-wk post-MI, there are differences in survivorship between the sleep and wake MI mice that could be explained by the different molecular and cellular responses. Our whole body physiology, tissues, and cells exhibit endogenous daily rhythms, and understanding their role in triggering effective responses after MI could lead to new strategies to benefit patients with cardiovascular disease.

Consequences of Circadian and Sleep Disturbances for the Cardiovascular System.

Circadian rhythms play a crucial role in our cardiovascular system. Importantly, there has been a recent flurry of clinical and experimental studies revealing the profound adverse consequences of disturbing these rhythms on the cardiovascular system. For example, circadian disturbance worsens outcome after myocardial infarction with implications for patients in acute care settings. Moreover, disturbing rhythms exacerbates cardiac remodelling in heart disease models. Also, circadian dyssynchrony is a causal factor in the pathogenesis of heart disease. These discoveries have profound implications for the cardiovascular health of shift workers, individuals with circadian and sleep disorders, or anyone subjected to the 24/7 demands of society. Moreover, these studies give rise to 2 new frontiers for translational research: (1) circadian rhythms and the cardiac sarcomere, which sheds new light on our understanding of myofilament structure, signalling, and electrophysiology; and (2) knowledge translation, which includes biomarker discovery (chronobiomarkers), timing of therapies (chronotherapy), and other new promising approaches to improve the management and treatment of cardiovascular disease. Reconsidering circadian rhythms in the clinical setting benefits repair mechanisms, and offers new promise for patients.

Therapeutic applications of circadian rhythms for the cardiovascular system.

The cardiovascular system exhibits dramatic time-of-day dependent rhythms, for example the diurnal variation of heart rate, blood pressure, and timing of onset of adverse cardiovascular events such as heart attack and sudden cardiac death. Over the past decade, the circadian clock mechanism has emerged as a crucial factor regulating these daily fluctuations. Most recently, these studies have led to a growing clinical appreciation that targeting circadian biology offers a novel therapeutic approach toward cardiovascular (and other) diseases. Here we describe leading-edge therapeutic applications of circadian biology including (1) timing of therapy to maximize efficacy in treating heart disease (chronotherapy); (2) novel biomarkers discovered by testing for genomic, proteomic, metabolomic, or other factors at different times of day and night (chronobiomarkers); and (3) novel pharmacologic compounds that target the circadian mechanism with potential clinical applications (new chronobiology drugs). Cardiovascular disease remains a leading cause of death worldwide and new approaches in the management and treatment of heart disease are clearly warranted and can benefit patients clinically.

Male-Specific Cardiac Dysfunction in CTP:Phosphoethanolamine Cytidylyltransferase (Pcyt2)-Deficient Mice.

Phosphatidylethanolamine (PE) is the most abundant inner membrane phospholipid. PE synthesis from ethanolamine and diacylglycerol is regulated primarily by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2(+/-) mice have reduced PE synthesis and, as a consequence, perturbed glucose and fatty acid metabolism, which gradually leads to the development of hyperlipidemia, obesity, and insulin resistance. Glucose and fatty acid uptake and the corresponding transporters Glut4 and Cd36 are similarly impaired in male and female Pcyt2(+/-) hearts. These mice also have similarly reduced phosphatidylinositol 3-kinase (PI3K)/Akt1 signaling and increased reactive oxygen species (ROS) production in the heart. However, only Pcyt2(+/-) males develop hypertension and cardiac hypertrophy. Pcyt2(+/-) males have upregulated heart AceI expression, heart phospholipids enriched in arachidonic acid and other n-6 polyunsaturated fatty acids, and dramatically increased ROS production in the aorta. In contrast, Pcyt2(+/-) females have unmodified heart phospholipids but have reduced heart triglyceride levels and altered expression of the structural genes Acta (low) and Myh7 (high). These changes together protect Pcyt2(+/-) females from cardiac dysfunction under conditions of reduced glucose and fatty acid uptake and heart insulin resistance. Our data identify Pcyt2 and membrane PE biogenesis as important determinants of gender-specific differences in cardiac lipids and heart function.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Short-term disruption of diurnal rhythms after murine myocardial infarction adversely affects long-term myocardial structure and function.

RATIONALE: Patients in intensive care units are disconnected from their natural environment. Synchrony between environmental diurnal rhythms and intracellular circadian rhythms is essential for normal organ biology; disruption causes pathology. Whether disturbing rhythms after myocardial infarction (MI) exacerbates long-term myocardial dysfunction is not known. OBJECTIVE: Short-term diurnal rhythm disruption immediately after MI impairs remodeling and adversely affects long-term cardiac structure and function in a murine model. METHODS AND RESULTS: Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 3-hour time window, randomized to either a normal diurnal or disrupted environment for 5 days, and then maintained under normal diurnal conditions. Initial infarct size was identical. Short-term diurnal disruption adversely affected body metabolism and altered early innate immune responses. In the first 5 days, crucial for scar formation, there were significant differences in cardiac myeloperoxidase, cytokines, neutrophil, and macrophage infiltration. Homozygous clock mutant mice exhibited altered infiltration after MI, consistent with circadian mechanisms underlying innate immune responses crucial for scar formation. In the proliferative phase, 1 week after MI, this led to significantly less blood vessel formation in the infarct region of disrupted mice; by day 14, echocardiography showed increased left ventricular dilation and infarct expansion. These differences continued to evolve with worse cardiac structure and function by 8 weeks after MI. CONCLUSIONS: Diurnal rhythm disruption immediately after MI impaired healing and exacerbated maladaptive cardiac remodeling. These preclinical findings suggest that disrupted diurnal rhythms such as found in modern intensive care unit environments may adversely affect long-term patient outcome.

Chronomics of pressure overload-induced cardiac hypertrophy in mice reveals altered day/night gene expression and biomarkers of heart disease.

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload-induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease.