Number 44, 2009
Circadian rhythms and cardiovascular disease

Diurnal variations in myocardial metabolism

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Ju-Yun Tsai, Martin E. Young
USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Department of Pediatrics, Houston, Texas, USA
Correspondence: Dr Martin E. Young, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Department of Pediatrics, 1100 Bates Street, Houston, Texas 77030, USA.
Tel: +1 713 798 7567; fax: +1 713 798 7101; e-mail: meyoung@bcm.edu

Introduction
Diurnal variations (ie, fluctuations over the course of the day, in the presence of environmental cues, such as the light/dark cycle) are observed in myocardial biology [1]. Gene expression, cellular signaling, metabolic fluxes, and contractile function of the heart oscillate dramatically as a function of time [1–7]. For example, robust diurnal variations in heart rate and cardiac output are observed in both animal models and humans [5–7]. Similarly, the incidence of adverse cardiac events (eg, arrhythmias, sudden cardiac death, myocardial infarction) exhibits dramatic diurnal variations [8]. Myocardial contractile function and metabolism are inseparably interlinked [9]. Furthermore, significant evidence suggests that myocardial metabolism profoundly influences outcome after an adverse cardiovascular event (eg, myocardial infarction) [10]. It is therefore not surprising that myocardial metabolism also exhibits robust diurnal variations [11,12]. These oscillations in myocardial metabolism are attuned not only to sleep/wake cycles, but also to feeding/fasting cycles [11]. The purpose of this review is to provide a brief overview regarding diurnal variations in myocardial metabolism, with a specific emphasis on fatty acid metabolism. We will also highlight our current knowledge regarding the contribution of the cardiomyocyte circadian clock towards myocardial metabolism diurnal variations.

Diurnal variations in myocardial metabolism

General metabolism
Diurnal variations in metabolism have been observed across various species, at several levels. In mammals, oscillations in metabolism that are dependent on the time of day are observed at whole-body, organ, and cellular levels. To date, studies focusing on diurnal variations in myocardial metabolism have been carried out primarily in rodents. The rat heart exhibits profound diurnal variations in carbohydrate, fatty acid, amino acid, and coenzyme metabolism (summarized in Table I) [4,13–19]. For example, during the active phase, a propensity exists for glucose units (derived both extracellularly and from glycogen) to be fully oxidized [4,13]. This in turn would aid the myocardium in meeting the high energetic demand at this time. Evidence also exists suggesting that amino acid and protein turnover exhibit clear diurnal variations. For example, Rau and Meyer [17] have reported that net protein synthesis increases near the sleep-to-wake (light-to-dark) phase transition in the rat heart, which is independent of feeding/fasting cycles. Total free amino acid concentrations are increased in the rat heart during the sleep (light) phase, suggesting that myocardial concentrations do not simply increase postprandially [19]. High-performance liquid chromatography (HPLC) analysis reveals myocardial levels of arginine, serine, and tyrosine significantly elevated at this time (M. E. Young, unpublished observations). These observations lead to the hypothesis that myocardial accumulation of amino acids during the sleep phase allows anticipation of increased protein synthesis at the sleep-to-wake transition.

Table I. Diurnal variations in rat heart metabolism.

Fatty acid and triacylglycerol metabolism
Fatty acids are the primary energy source for the healthy myocardium, generating approximately 70% of the ATP utilized for contraction [20]. However, fatty acids are more than just a fuel and, when in excess, they exert cardiotoxic effects [21]. Significant sources of fatty acids for the heart include circulating non-esterified fatty acids, circulating lipoproteins, and endogenous myocardial TAG [22]. All these sources exhibit diurnal variations, as do both the acute and chronic responsiveness of the myocardium to fatty acids. Acutely, fatty acids depress myocardial contractile function and efficiency of the rat heart to the greatest extent during the sleep/resting phase [13]. In terms of metabolic flux, although fatty acid oxidation rates do not exhibit diurnal variations ex vivo, channeling of fatty acids into distinct non-oxidative pathways does, including phospholipid, diacylglycerol, and TAG synthesis [13]. Chronically, for the rat, transcriptional responsiveness of the heart to increased fatty acid concentrations peaks during the active phase [14].
Myocardial fatty acid utilization is regulated at several levels, including uptake, binding, activation, and channeling into oxidative or non-oxidative pathways, in addition to secretion (Figure 1). Uptake of long-chain fatty acids into the cardiomyocyte involves several proteins, including fatty acid translocase (FAT/CD36), fatty acid transport proteins (FATP), fatty acid binding proteins (FABP), and long-chain acyl coenzyme A (CoA) synthetases (ACSL) [23]. Uptake of fatty acids derived from lipoproteins also requires lipoprotein lipase (LpL) [22]. Many of these mediators of fatty acid uptake demonstrate diurnal variations in expression/activity. LpL activity oscillates in the rat heart, peaking during the sleep phase [24]. In contrast, FABP peaks in the middle of the active/awake phase in the rat heart [25]. More recently, we have reported significant diurnal variations in the expression of acsl1 and acsl3 in mouse and rat hearts, respectively [26].

Contribution of myocardial metabolism diurnal variations by the cardiomyocyte circadian clock
Diurnal variations in myocardial metabolism are regulated by an interplay of extrinsic neurohumoral factors (eg, sympathetic activity, circulated insulin, thyroid hormone, and corticosteroid levels, as well as circulating fuels such as glucose, fatty acids, triglycerides, and ketone bodies) and intrinsic cell autonomous circadian clocks. Circadian clocks are defined as a set of proteins that generate self-sustained transcriptional positive- and negative-feedback loops with a free running period of approximately 24 h [33]. This molecular mechanism provides the selective advantage of anticipation, enabling the cell to prepare for an environmental stimulus before its onset (eg, modulates responsiveness to neurohumoral stimuli), and ultimately regulating function and metabolism in a manner that is dependent on the time of day. After characterization of the cardiomyocyte circadian clock, we developed a mouse model of genetic ablation of this molecular mechanism [15,34]. Using this model, termed “cardiomyocyte-specific clock mutant” (CCM), we defined roles for the cardiomyocyte circadian clock as a mediator of diurnal variations in myocardial gene expression, signaling, metabolism, and contractile function [2].
Microarray studies comparing wildtype and CCM mice identified various metabolic genes as being regulated by the cardiomyocyte circadian clock [2]. These included genes influencing glucose (eg, pdk4), fatty acid (eg, acsl1), and general oxidative (eg, ndufa3/b3) metabolism [2]. Consistent with these alterations in gene expression, CCM hearts exhibit alterations in fatty acid oxidation (increased) and glycogenolysis (decreased) [2]. In addition, through use of CCM mice, we have shown that diurnal variations in the transcriptional responsiveness of the heart to fatty acids are mediated by the cardiomyocyte circadian clock [15]. Additional evidence for the latter includes persistence of diurnal variations in FA transcriptional responsiveness in cultured adult rat cardiomyocytes (under which conditions neurohumoral influences are ablated) [15].
Not surprisingly, myocardial TAG metabolism is markedly altered in CCM hearts. Microarray studies revealed that the cardiomyocyte circadian clock regulates several genes involved in myocardial fatty TAG metabolism (eg, agpat3, dgat2, adpn, ces3; Figure 1) [2]. Consistent with these observations, fasting-induced myocardial TAG synthesis is abolished in CCM hearts [15]. More recently, we have found that diurnal variations in TAG synthesis observed in wildtype hearts perfused ex vivo are abolished in CCM hearts [35]. Taken together, these observations show that the cardiomyocyte circadian clock regulates myocardial TAG metabolism.

Summary
The myocardium exhibits profound oscillations in metabolism that are dependent on the time of day. Many of these oscillations appear to precede (as opposed to proceed) feeding/fasting and sleep/wake cycles. This anticipation is probably conferred by the cardiomyocyte circadian clock. Impairment of the cardiomyocyte circadian clock or diurnal variations in neurohumoral factors (eg, fatty acids), or both, will result in uncoupling of the synchronization of the heart with the environment, potentially accelerating contractile dysfunction through inappropriate responses (eg, channeling fatty acids into lipotoxic pathways).

Acknowledgments
We wish to thank Douglas Burrin (Baylor College of Medicine) for his help with rat heart amino acid analysis by HPLC.

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