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. Key Words: chronobiology Ⅲ ischemia/reperfusion Ⅲ myocardium N umerous aspects of cardiovascular physiology and pathophysiology demonstrate circadian rhythms. 1 In humans, heart rate, blood pressure, and cardiac output all increase in the early hours of the morning, as does the onset of adverse cardiac events, such as myocardial infarction. 2,3 These rhythms have been attributed primarily to time-of-day oscillations in neurohumoral influences, such as sympathetic or autonomic stimulation. 3,4 Although extracardiac factors undoubtedly play critical roles in modulation of cardiovascular function/dysfunction, increasing evidence suggests that intrinsic factors, such as cellautonomous circadian clocks, likely contribute. 1 Circadian clocks are transcriptionally based molecular mechanisms, composed of positive-and negative-feedback loops, with a free-running period of Ϸ24 hours. 5 This mechanism allows the cell to anticipate alterations in environmental stimuli, through time-of-day-dependent modulation of cellular responsiveness to extrinsic factors. 5 Circadian clocks have been identified/characterized in multiple cardiovascular-relevant cell types, including cardiomyocytes, vascular smooth muscle cells, and endothelial cells. 6 -8 Ubiquitous genetic ablation of circadian clock function markedly influences multiple cardiovascular parameters, including heart rate and blood pressure. 9 We have recently used a CCM (cardiomyocyte-specific circadian clock mutant) mouse to reveal regulation of myocardial gene expression, ␤-adrenergic responsiveness, metabolism, heart rate, and cardiac power by this mechanism. 10,11 Although circadian rhythms in myocardial infarction onset are well established, time-of-day oscillations in myocardial ischemia/reperfusion (I/R) tolerance have not been reported. Given that the cardiomyocyte circadian clock influences Original

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 ␤-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-ofday-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.Circadian clocks have emerged as critical regulators of energy metabolism (1, 2). Animal models wherein components of these cell autonomous mechanisms are genetically manipulated invariably exhibit altered energy balance, resulting in overt metabolic phenotypes (e.g. obesity or leanness). This concept is exemplified when either Clock or Bmal1 (two transcription factors at the core of the mammalian clock) are disrupted; Clock⌬19 mutant mice are obesity-prone, whereas Bmal1 null mice are lean (3, 4). Appreciation for links between circadian clocks and metabolism has grown further through demonstration that perturbations in metabolism (e.g. changes in nutrient availability, models of obesity, and diabetes mellitus, etc.) in turn influence the clock mechanism (5-7). Collectively, these observations have fueled identification of a number of posttranslational mediators that facilitate the interdependence of circadian clocks with metabolism. These include phosphorylation, ubiquitination, acetylation, and ribosylation of critical clock and/or metabolic components in time-of-day-dependent manners (1, 8 -14).Defining the role of a specific cell autonomous circadian clock in metabolic regulation through the use of animal models wherein clock components are genetically altered in a ubiquitous fashion is often hampered by the fact that time-of-day-dependent rhythms are altered at multiple levels (e.g. behavioral, neurohum...

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.Striking time-of-day-dependent oscillations are observed in multiple cardiometabolic parameters in both animal models and humans. These parameters range from levels of circulating nutrients and endocrine factors, neural activity, glucose tolerance, insulin sensitivity, feeding behavior, and energy metabolism (both at the individual tissue and whole body levels) to cardiovascular function (1-5). Significant alterations in many of these oscillations are observed in metabolic disease states (e.g. obesity, diabetes mellitus, and cardiovascular disease), suggesting that circadian misalignment may play an important role in the etiology of multiple pathologies (5, 6). Recent molecular/ genetic-based studies reinforce such a concept and suggest that intrinsic cellular circadian clocks play a pivotal role in mediating many, if not all, biological rhythms. Circadian clocks are transcriptionally based molecular mechanisms that generate self-sustained positive and negative feedback loops with a free running period of ϳ24 h (7); this molecular mechanism has been identified within essentially all mammalian cells (both central and peripheral). Circadian clocks confer the selective advantage of anticipation. In doing so molecular clocks enable the cell to prepare for an external stimulus before its onset, thereby maintaining optimal synchrony with the environment. Given marked time-of-day-dependent rhythms in energy supply (e.g. dietary nutrient intake) and de...