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Am J Physiol Heart Circ Physiol 293: H2385-H2393, 2007. First published July 6, 2007; doi:10.1152/ajpheart.01361.2006
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Circadian rhythms in myocardial metabolism and contractile function: influence of workload and oleate

David J. Durgan,1 Michael W. S. Moore,1 Ngan P. Ha,1 Oluwaseun Egbejimi,1 Anna Fields,1 Uchenna Mbawuike,1 Anu Egbejimi,1 Chad A. Shaw,2 Molly S. Bray,1 Vijayalakshmi Nannegari,1 Diane L. Hickson-Bick,3 William C. Heird,1 Jason R. B. Dyck,4 Margaret P. Chandler,5 and Martin E. Young1

1United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics; 2Department of Molecular and Human Genetics, Baylor College of Medicine; 3Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, Texas; 4Cardiovascular Research Group, Department of Pediatrics and Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; and 5Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio

Submitted 13 December 2006 ; accepted in final form 27 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Multiple extracardiac stimuli, such as workload and circulating nutrients (e.g., fatty acids), known to influence myocardial metabolism and contractile function exhibit marked circadian rhythms. The aim of the present study was to investigate whether the rat heart exhibits circadian rhythms in its responsiveness to changes in workload and/or fatty acid (oleate) availability. Thus, hearts were isolated from male Wistar rats (housed during a 12:12-h light-dark cycle: lights on at 9 AM) at 9 AM, 3 PM, 9 PM, and 3 AM and perfused in the working mode ex vivo with 5 mM glucose plus either 0.4 or 0.8 mM oleate. Following 20-min perfusion at normal workload (i.e., 100 cm H2O afterload), hearts were challenged with increased workload (140 cm H2O afterload plus 1 µM epinephrine). In the presence of 0.4 mM oleate, myocardial metabolism exhibited a marked circadian rhythm, with decreased rates of glucose oxidation, increased rates of lactate release, decreased glycogenolysis capacity, and increased channeling of oleate into nonoxidative pathways during the light phase. Rat hearts also exhibited a modest circadian rhythm in responsiveness to the workload challenge when perfused in the presence of 0.4 mM oleate, with increased myocardial oxygen consumption at the dark-to-light phase transition. However, rat hearts perfused in the presence of 0.8 mM oleate exhibited a markedly blunted contractile function response to the workload challenge during the light phase. In conclusion, these studies expose marked circadian rhythmicities in myocardial oxidative and nonoxidative metabolism as well as responsiveness of the rat heart to changes in workload and fatty acid availability.

fatty acids; glucose; glycogen; triglyceride

CIRCADIAN RHYTHMS IN CARDIOVASCULAR PHYSIOLOGY and pathophysiology are well established. Heart rate, blood pressure, cardiac output, platelet aggregability, myocardial infarction, arrhythmias, and sudden cardiac death all exhibit marked circadian rhythmicities in both humans and animal models (3, 7, 12, 18, 23, 33). To date, these observations have been attributed primarily to diurnal variations in multiple extracellular stimuli (i.e., neurohumoral factors) such as autonomic and sympathetic activity (23, 24, 32). However, it is becoming increasingly clear that the intrinsic properties of cardiovascular components change over the course of the day, suggesting that oscillations in responsiveness to extracellular stimuli may contribute toward circadian rhythmicities in cardiovascular events (36, 37).

Myocardial metabolism and contractile function are inextricably interlinked. For example, increased energy demand during periods of elevated workload is balanced by increased oxidative and nonoxidative metabolism (17). An inability of the heart to maintain adequate ATP supply will in turn adversely affect contractile function. Furthermore, many nutrients, such as fatty acids, act as more than just a fuel. Thus, prolonged imbalances between fatty acid availability and rates of myocardial fatty acid oxidation result in accumulation of detrimental fatty acid derivatives within the cardiomyocyte. This so-called lipotoxicity is associated with myocardial contractile dysfunction, as observed during diabetes mellitus (38). One way in which the heart counteracts lipotoxicity is through promotion of fatty acid utilization via transcriptional, translational, and posttranslational events, thereby reducing accumulation of detrimental intramyocellular fatty acid derivatives (1, 11, 20, 25, 38). However, increased fatty acid utilization is also associated with reduced myocardial efficiency acutely through a number of potential mechanisms (5, 6, 19, 22, 27, 34). Given that fatty acids are a less efficient substrate than carbohydrate, in terms of ATP generated per oxygen molecule consumed, it has been proposed that substrate switching toward increased reliance on fatty acid oxidation (and decreased reliance on glucose oxidation) contributes toward fatty acid-induced depression of cardiac efficiency (6, 22, 28, 34). However, theoretical O2 requirement differences for fatty acid and carbohydrate metabolism do not account fully for the depression in myocardial efficiency in the face of increased fatty acid availability, suggesting that nonoxidative mechanisms also contribute to energy wastage (28).

Both workload and plasma fatty acids exhibit marked circadian rhythms in mammals (30, 36). Despite this, little information is available regarding circadian rhythms in responsiveness of the myocardium to these external factors. We (15, 30) have recently reported that the rodent heart exhibits a circadian rhythm in responsiveness to fatty acids in terms of induction of beta-oxidation promoting genes (i.e., transcriptional response). Therefore, we decided to investigate whether the heart exhibits circadian rhythmicities in its acute response to changes in workload and fatty acid availability. We hypothesized that the heart would exhibit greatest responsiveness to increases in workload during the dark phase (more active) and greatest responsiveness to fatty acids during the light phase (less active, i.e., times at which these extracardiac stimuli peak). The present study reports that the rat heart exhibits 1) marked circadian rhythms in oxidative and nonoxidative metabolism, 2) modest circadian rhythms in responsiveness to a workload challenge, and 3) a marked circadian rhythm in oleate-induced depression of contractile function and efficiency (at high workloads).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Wistar rats (400–500 g; Charles River) were housed at the Animal Care Center of the Children's Nutrition Research Center, Baylor College of Medicine. All animals were housed under controlled conditions (23 ± 1°C, 12:12-h light-dark cycle) and received standard laboratory chow and water ad libitum. Ten days prior to being killed, animals were housed in a separate environment-controlled room, within which a strict 12:12-h light-dark cycle regime was enforced [lights on at 9 AM; zeitgeber time (ZT)0]. On the day of the experiment, hearts were isolated from anesthetized rodents (chloral hydrate, 500 mg/kg ip) at 9 AM (ZT0/24), 3 PM (ZT6), 9 PM (ZT12), or 3 AM (ZT18). For isolated working rat heart studies, hearts were placed immediately in ice-cold Krebs-Henseleit bicarbonate (KHB) buffer containing 5 mM glucose prior to perfusion (see below). For myocardial glycogen and triglyceride level measurements in nonperfused hearts, isolated hearts were freeze-clamped immediately following isolation and stored at –80°C prior to measurements (see below). All animal experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

Isolated working rat heart perfusions. Through the use of the isolated working rat heart preparation, circadian rhythmicity of myocardial function and metabolism was determined ex vivo, as described previously (31). Briefly, isolated hearts were perfused initially in a retrograde fashion (i.e., Langendorff perfusion) with KHB buffer containing 5 mM glucose. Following a brief perfusion (~5 min), hearts were perfused in the working mode in a nonrecirculating manner with KHB buffer containing 5 mM glucose, 10 µU/ml insulin, and 0.4 or 0.8 mM oleate conjugated to 1% BSA (fraction V, fatty acid free; Serologicals). Oleate-BSA perfusates were dialyzed overnight, followed by filtration, prior to use. The perfusate also contained tracer amounts of [U-14C]glucose (40 µCi/l) and [9,10-3H]oleate (60 µCi/l). After 20 min (i.e., perfusion time 0–20) of normal workload (NWL) perfusion conditions (15 cm H2O preload, 100 cm H2O afterload), hearts were perfused for an additional 20 min (i.e., perfusion time 20–40) under high workload (HWL) conditions (15 cm H2O preload, 140 cm H2O afterload + 1 µM epinephrine). Both functional and metabolic measures were made at 5-min intervals throughout the perfusion protocol. Steady-state rates presented in this study are those obtained during the last 10 min of both the low- (i.e., average of perfusion times 15 and 20) and high-workload (i.e., average of perfusion times 35 and 40) conditions.

Cardiac power and efficiency, as well as rates of myocardial oxygen consumption (MVO2), exogenous oleate oxidation, exogenous glucose oxidation, and [14C]glucose incorporation into glycogen, were determined as described previously (8, 17). Rates of net lactate release (i.e., lactate derived from both extracellular glucose as well as intracellular glycogen) were measured spectrophotometrically (8). Rates of 14C-labeled lactate release were determined by separation of [14C]glucose and [14C]lactate through ion exchange chromatrography (8). Endogenously derived lactate was calculated as the difference between net lactate and [14C]lactate release rates for each individual heart. Reliance on exogenous glucose (REG), exogenous oleate (REO), and endogenous substrates (RES), which normalize for alterations in absolute rates of oxidative metabolism, was calculated for each individual heart as follows: REG = [glucose oxidation rate x 6 (O2 molecules for complete glucose oxidation) x 100%]/[MVO2], REO = [oleate oxidation rate x 25.5 (O2 molecules for complete oleate oxidation)] x 100%/[MVO2], RES = 100% – (REG + REO).

Myocardial glycogen and triglyceride content measurements. Myocardial glycogen and triglyceride contents were measured from homogenate extracts using enzymatic-based spectrophotometric methods, as described previously (2, 9, 35).

Lipid fractionation studies. Lipid extraction and fractionation was performed according to Bligh and Dyer (4). Following extraction, lipids were separated into distinct fractions by thin-layer chromatography using silica gel plates. Standards for diacylglycerol, triglyceride, phospholipids, and cholesterol esters were run in parallel for accurate identification of sample fractions.

Statistical analysis. Repeated-measures analysis of variance was conducted to investigate the effects of time across workload conditions. Stata version 8SE (Stata, College Station, TX) was used for this analysis. A full model including interactions was conducted to test for differences in response to workload across time. The null hypothesis of no-model effects was rejected at P < 0.05. In addition, Bonferroni-corrected pairwise comparisons were conducted to identify significant differences between groups.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Circadian rhythms in responsiveness of the heart to increased workload. To investigate whether the rat heart exhibits a circadian rhythm in its responsiveness to increased workload, myocardial metabolism and contractile function were measured in hearts perfused under both NWL and HWL (in the presence of a physiological oleate concentration, 0.4 mM). Data are shown for both the entire 40-min perfusion protocol as well as at steady state. During NWL, time of day had no significant influence on either initial (i.e., perfusion time 0) or steady-state cardiac power, MVO2, or myocardial efficiency as measured ex vivo (Fig. 1, A, B, and C). These parameters were all significantly altered during the HWL, with increased cardiac power and MVO2, and concomitant decreased cardiac efficiency relative to the NWL (Fig. 1). During the HWL, MVO2 exhibited a significant time-workload interaction, peaking at the dark-to-light phase transition (Fig. 1B), whereas responsiveness of cardiac power and efficiency were not significantly influenced by time of day (Fig. 1, A and C).


Figure 1
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  		Fig. 1. Circadian rhythms in cardiac power (A), myocardial oxygen consumption (MVO2; B), cardiac efficiency (C), exogenous glucose oxidation rate (D), exogenous oleate oxidation rate (E), reliance on exogenous glucose oxidation (F), reliance on exogenous oleate oxidation (G), and reliance on endogenous substrate oxidation (H) for rat hearts perfused under normal (NWL; 100 cm H2O) and high (HWL; 140 cm H2O plus 1 µM epinephrine) workloads. Hearts were isolated at zeitgeber time (ZT)0/24 (9 AM), ZT6 (3 PM), ZT12 (9 PM), and ZT18 (3 AM) and perfused in the presence of 0.4 mM oleate. Data are shown throughout the perfusion protocol (i) and at steady state (ii). Values are shown as the mean ± SE for between 8 and 10 observations at each time point. *P < 0.05, time effect; $P < 0.05, workload effect; #P < 0.05, time-workload interaction. See RESULTS for individual time point comparisons.

 
Increasing the workload significantly increased rates of both exogenous glucose and oleate oxidation, independent of time of day (Fig. 1, D and E). Unlike rates of exogenous oleate oxidation, steady-state exogenous glucose oxidation rates exhibited significant circadian rhythmicities (at both NWL and HWL), with increased rates observed in the middle of the dark phase. For example, exogenous glucose oxidation rates are 1.9-fold higher at ZT18 vs. ZT6 at NWL (0.430 ± 0.054 vs. 0.226 ± 0.022 µmol·min–1·g dry wt–1, respectively). In contrast, time of day had no influence on initial rates of exogenous glucose oxidation (Fig. 1D, i). When reliance on exogenous and endogenous substrates is calculated, consistent with previously published studies (17), we find that increasing the workload significantly increases reliance on exogenous glucose and decreases reliance on exogenous oleate, with no significant effect on endogenous substrate reliance, at all times of the day investigated (Fig. 1, F, G, and H). In the case of reliance on exogenous oleate oxidation a significant time-workload interaction was observed, with greatest repression at the dark-to-light phase transition (Fig. 1G).

We hypothesized that increased channeling of pyruvate toward oxidative metabolism during the dark phase might account for the marked circadian rhythms in exogenous glucose oxidation rates. As such, we next measured circadian rhythms in lactate release from rat hearts perfused in the presence of 0.4 mM oleate (Fig. 2). Rates of net and 14C-labeled lactate release were not significantly influenced by the time of day (Figs. 2, A and B). Rates of endogenously derived lactate release exhibited a marked circadian rhythm, peaking at ZT6 (2.0-fold higher rates at ZT6 vs. ZT0/24 during NWL; Fig. 2C). Rates of net, 14C-labeled, and endogenously derived lactate release were all increased during HWL (Fig. 2, A, B, and C). The 14C-labeled lactate release rate to exogenous glucose oxidation ratio (an indirect marker of the coupling of pyruvate oxidation with glycolysis) tended to be higher during the light phase, although this did not reach statistical significant (Fig. 2D).


Figure 2
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  		Fig. 2. Circadian rhythms in rates of net lactate release (A), 14C-labeled lactate release (B), endogenously derived lactate release (C), and 14C-labeled lactate release rate to exogenous glucose oxidation rate ratio (D) for rat hearts perfused under NWL (100 cm H2O) and HWL (140 cm H2O + 1 µM epinephrine). Hearts were isolated at ZT0/24 (9 AM), ZT6 (3 PM), ZT12 (9 PM), and ZT18 (3 AM) and perfused in the presence of 0.4 mM oleate. Data are shown throughout the perfusion protocol (i) and at steady state (ii). Values are shown as the mean ± SE for between 8 and 10 observations at each time point. *P < 0.05, time effect; $P < 0.05, workload effect. See RESULTS for individual time point comparisons.

 
Given the marked circadian rhythmicities in endogenously derived lactate release from rat hearts (likely derived from glycogen), we next sought to investigate whether myocardial glycogen levels exhibit circadian rhythmicities. Figure 3A shows significant oscillations in glycogen content of nonperfused rat hearts, with the highest levels at the dark-to-light phase transition (1.8-fold higher at ZT0/24 vs. ZT12). Glycogen content was next measured in hearts freeze-clamped at the end of the perfusion protocol. Somewhat surprisingly, Fig. 3B shows that hearts perfused at ZT6 have 2.0-fold higher glycogen content at the end of the perfusion compared with hearts perfused at ZT18. In contrast, rates of net [14C]glucose incorporation into glycogen over the entire perfusion period were identical for hearts perfused at ZT6 vs. ZT18 (Fig. 3C).


Figure 3
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  		Fig. 3. Circadian rhythms in glycogen content of nonperfused hearts (A), glycogen content of hearts at the end of the perfusion protocol (B), and net incorporation of [14C]glucose into glycogen of perfused hearts (C). Hearts were isolated at ZT0/24 (9 AM), ZT6 (3 PM), ZT12 (9 PM), and ZT18 (3 AM). Values are shown as the mean ± SE for between 6 and 10 observations at each time point. *P < 0.05, time effect; {dagger}P < 0.05, ZT6 vs. ZT18. See RESULTS for individual time point comparisons.

 
Like glucose, fatty acids can be channeled into either oxidative or nonoxidative pathways. Data presented in Fig. 4 show a circadian rhythm in triglyceride content of nonperfused rat hearts, suggesting a potential circadian rhythm in the channeling of fatty acids into nonoxidative pathways. As such, we sought to investigate whether rates of fatty acid channeling into di- or triacylglycerol, phospholipid, and/or cholesterol ester synthesis differed for rat hearts perfused ex vivo at ZT6 vs. ZT18 in the presence of 0.4 mM oleate. Table 1 shows that hearts perfused at ZT6 exhibit increased rates of di- and triacylglycerol, as well as phospholipid, synthesis compared with hearts perfused at ZT18. No significant differences were observed in rates of cholesterol ester synthesis between the two perfusion times investigated.


Figure 4
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  		Fig. 4. Circadian rhythms in triglyceride contents of nonperfused hearts. Hearts were isolated at ZT0/24 (9 AM), ZT6 (3 PM), ZT12 (9 PM), and ZT18 (3 AM). Values are shown as the mean ± SE for 6 observations at each time point. *P < 0.05, time effect. See RESULTS for individual time point comparisons.

 

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  		Table 1. Rates of [3H]oleate incorporation into cholesterol ester, phospholipid, and di- and triacylglycerol pools

 
Circadian rhythms in responsiveness of the heart to oleate. We (15) have recently shown that the heart exhibits a circadian rhythm in its responsiveness to fatty acids at a transcriptional level. Therefore, we tested the hypothesis that the heart exhibits circadian rhythms in its acute response to fatty acids in terms of myocardial metabolism and function. Hearts were perfused in the presence of an elevated oleate concentration (0.8 mM) under both NWL and HWL conditions. We find that perfusing rat hearts with 0.8 mM oleate conjugated to 1% BSA is similar to 1.2 mM oleate conjugated to 3% BSA in terms of contractile function and rates of substrate utilization (see Supplemental Table S1; supplemental data can be found on the AJP - Heart and Circulatory Physiology Web site). Furthermore, unbound oleate was undetectable with 0.8 mM oleate conjugated to 1% BSA when assayed with acrylodated intestinal fatty acid-binding protein, suggesting that saturation of BSA fatty acid-binding sites did not occur (Hickson-Bick DL and Young ME, unpublished observations). At normal workload, increasing the oleate concentration from 0.4 to 0.8 mM had no significant effects on cardiac power, MVO2, or efficiency regardless of the time of day investigated (Fig. 1, A, B, and C, vs. Fig. 5, A, B, and C). Increasing the oleate concentration from 0.4 to 0.8 mM significantly (P < 0.001) decreased exogenous glucose oxidation rates and concomitantly increased exogenous oleate oxidation rates, independent of the time of day (Fig. 1, D and E, vs. Fig. 5, D and E). Similarly, increasing the oleate concentration from 0.4 to 0.8 mM significantly (P < 0.001) increased reliance on exogenous oleate oxidation and concomitantly decreased reliance on exogenous glucose and endogenous substrate oxidation, independent of the time of day (Fig. 1, F, G, and H, vs. Fig. 5, F, G, and H).


Figure 5
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  		Fig. 5. Circadian rhythms in cardiac power (A), MVO2 (B), cardiac efficiency (C), exogenous glucose oxidation rate (D), exogenous oleate oxidation rate (E), reliance on exogenous glucose oxidation (F), reliance on exogenous oleate oxidation (G), and reliance on endogenous substrate oxidation (H) for rat hearts perfused under NWL (100 cm H2O) and HWL (140 cm H2O + 1 µM epinephrine). Hearts were isolated at ZT0/24 (9 AM), ZT6 (3 PM), ZT12 (9 PM), and ZT18 (3 AM) and perfused in the presence of 0.8 mM oleate. Data are shown throughout the perfusion protocol (i) and at steady state (ii). Values are shown as the mean ± SE for between 7 and 8 observations at each time point. Scales are identical to those for hearts perfused in the presence of 0.4 mM oleate (Fig. 1), allowing ease of comparisons. *P < 0.05, time effect; $P < 0.05, workload effect; #P < 0.05, time-workload interaction. See RESULTS for individual time point comparisons.

 
We next investigated whether myocardial metabolism and/or contractile function exhibited circadian rhythms during HWL in the presence of elevated oleate levels (0.8 mM). Cardiac power exhibited a significant time-workload interaction in the presence of 0.8 mM oleate, with the lowest cardiac power observed for hearts perfused at ZT6 (Fig. 5A). Similar observations were observed for cardiac efficiency, although the time-workload interaction did not reach statistical significance (P < 0.09; Fig. 5C). Indeed, HWL significantly (P < 0.05) reduced cardiac efficiency only for hearts perfused at ZT6 in the presence of 0.8 mM oleate (Fig. 5C). MVO2 increased during HWL, independent of the time of day (Fig. 5B). Increasing the workload increased rates of both exogenous glucose oxidation and exogenous oleate oxidation for hearts perfused in the presence of 0.8 mM oleate at HWL, independent of the time of day (Fig. 5, D and E). When reliance on exogenous and endogenous substrates was calculated, increasing the workload (in the presence of 0.8 mM oleate) was found to increase exogenous glucose oxidation reliance, decrease exogenous oleate oxidation reliance, and increase endogenous substrate oxidation reliance (Fig. 5, F, G, and H). Significant time-workload interactions were observed for reliance on exogenous oleate oxidation and endogenous substrate oxidation, with the greatest effect of increased workload observed at the light-to-dark phase transition (Fig. 5, G and H).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary goals of the present study were to investigate whether the rat heart exhibits circadian rhythmicities in its responsiveness to alterations in workload and/or fatty acid availability at the levels of myocardial metabolism and contractile function. Through the use of the isolated working rat heart preparation, we expose circadian rhythmicities in 1) myocardial metabolism, 2) responsiveness to an acute increase in workload, and 3) responsiveness to an acute increase in fatty acid availability. More specifically, the rat heart exhibits 1) increased reliance on exogenous glucose oxidation during the dark (more active) phase, 2) decreased glycogenolysis capacity during the light phase, 3) increased channeling of fatty acids into nonoxidative pathways during the light phase, and 4) greatest susceptibility to oleate-induced depression of cardiac power and efficiency in the middle of the light phase (i.e., ZT6) during high workload, an effect that cannot be explained by increased rates of exogenous fatty acid oxidation.

Circadian rhythmicity in myocardial metabolism. The intrinsic properties of numerous organs fluctuate over the course of the day both in anticipation and response to environmental stimuli (16, 36). Such plasticity involves a plethora of molecular events at transcriptional, translational, and posttranslational levels. Consistent with this concept, Reddy et al. (26) have recently shown that at least 20% of all soluble hepatic proteins undergo significant circadian rhythms, many of which are involved in metabolism. Martino et al. (21) report that at least 13% of all myocardial genes exhibit significant circadian oscillations in expression in the rodent heart. These genes are clustered within distinct groups according to phase of the oscillation and known biological function, including those known to influence transcription, protein turnover, cellular signaling, and metabolism. Through the use of a hypothesis-testing approach, we have reported circadian oscillations in numerous myocardial metabolic genes known to regulate both carbohydrate and fatty acid metabolism (13–15, 29, 30, 36, 37, 39–41). Taken together, these observations have led us to hypothesize that the heart exhibits circadian rhythmicities in metabolism (which, in turn, potentially influence contractile function) (36).

The present study characterizes fully circadian rhythmicities in myocardial metabolism for the rat. Consistent with our previous findings (39), we report higher rates of exogenous glucose oxidation during the middle of the dark phase (Fig. 1D). Decreased rates of exogenous glucose oxidation during the light phase are associated with higher rates of lactate release at this time (Figs. 1D and 2). Indeed, the 14C-labeled lactate release rate to exogenous glucose oxidation ratio tends to be higher for hearts perfused during the light phase, particularly at high workloads, suggesting that a poorer coupling of glycolysis with pyruvate oxidation occurs at this time (Fig. 2D). Of the lactate parameters measured in the present study, endogenously derived lactate release exhibited the most striking circadian rhythms, being elevated during the light phase (Fig. 2C). This observation suggested that glycogenolysis was potentially increased for rat hearts perfused at this time. However, Fig. 3 shows that, although myocardial glycogen contents are identical for nonperfused hearts at ZT6 and ZT18, at the end of our perfusion protocol glycogen content was 2.0-fold higher in hearts perfused at ZT6 vs. ZT18. Given that net incorporation of [14C]glucose into glycogen did not differ between hearts perfused at ZT6 vs. ZT18, these observations suggest decreased glycogenolysis for rat hearts perfused at ZT6. Elevated rates of endogenously derived lactate release observed at ZT6 are therefore likely secondary to decreased coupling of glycolysis and pyruvate oxidation at this time.

In addition to circadian rhythms in carbohydrate metabolism, the rat heart exhibits significant oscillations in nonoxidative fatty acid metabolism. We report that the rat heart channels fatty acids into di- and triacylglycerol, as well as phospholipid, synthesis to a greater extent during the light phase (Table 1). In contrast, little variation in myocardial oleate oxidation is observed over the course of the day, consistent with our previously published observations (39). Interestingly, increased capacity of the rat heart for nonoxidative fatty acid metabolism during the light phase is synchronized with increased circulating fatty acids in the in vivo setting at this time (30).

Circadian rhythmicity in responsiveness of the heart to changes in workload. Circadian rhythms in cardiovascular parameters are well established. For example, at the sleep-to-wake transition, increased sympathetic, autonomic, and adrenergic stimulation, as well as increased vascular resistance, are all associated with increased heart rate and cardiac output. Despite these observations, it is not known whether the myocardium exhibits a circadian rhythm in its responsiveness to these extracardiac stimuli. We hypothesized that the rat heart would exhibit a circadian rhythm in the responsiveness to simulated exercise (i.e., increased afterload plus adrenergic stimulation), such that a greater responsiveness would be observed during the dark phase (i.e., when this stimulus typically increases in the rodent). The isolated working rat heart was utilized to remove potentially confounding acute neurohumoral influences present in vivo. Additionally, hearts were allowed to reach a steady state at NWL before the increased workload challenge. We report that the rat heart exhibits a circadian rhythm in its responsiveness to increased workload, with the greatest responsiveness at the end of the dark phase (in terms of myocardial oxygen consumption and reliance on exogenous oleate oxidation; Fig. 1, B and G). Future studies are required to elucidate the mechanism(s) responsible for this observation, such as circadian rhythms in responsiveness to beta-adrenergic stimulation.

Circadian rhythmicity in fatty acid-mediated depression of myocardial function and efficiency. Circulating fatty acids exhibit a circadian rhythm, with increased levels during the light phase in the ad libitum-fed nocturnal rodent. This observation led us to hypothesize that the rodent heart would exhibit increased responsiveness to fatty acids during the light phase for the effective utilization of fatty acids when available. However, we have recently reported that the rodent heart exhibits the greatest responsiveness to fatty acids during the dark phase, at the transcriptional level, which is mediated by the circadian clock within the cardiomyocyte (15). These observations suggest that the rodent heart may have evolved to anticipate periods of food scarcity (as opposed to availability), such that fatty acids remain elevated in the circulation when the rodent in the wild is initially unsuccessful in its forage for food. The present study investigated the hypothesis that the rodent heart also exhibits circadian rhythms in its acute (metabolic) response to fatty acids. Counter to our hypothesis, we report that increasing fatty acid availability ex vivo increased oleate oxidation, and concomitantly decreased glucose oxidation, in a time-of-day-independent manner (Fig. 1, D and E, vs. Fig. 5, D and E).

Studies performed in multiple laboratories (5, 6, 19, 22, 27, 34) report that increased fatty acid availability depresses myocardial function and efficiency. These observations are often explained in terms of increased oxygen requirement for the full oxidation of fatty acids compared with carbohydrate. Given that the rat heart exhibits the lowest reliance on exogenous glucose oxidation in the middle of the light phase (i.e., ZT6), we hypothesized that hearts would exhibit increased susceptibility to fatty acid-induced myocardial inefficiency at this time. Consistent with this hypothesis, we found that hearts isolated at ZT6 exhibit the greatest susceptibility to oleate-induced depression of myocardial efficiency (and cardiac power) at high workloads (Fig. 5, A and C). Somewhat unexpectedly, our data suggest that the effects of oleate to reduce myocardial efficiency in the middle of the light phase are potentially due to mechanisms independent of altered exogenous oleate oxidation rates. This suggestion stems from the observations that rates of oleate oxidation increase in a time-of-day-independent manner when oleate availability is increased from 0.4 to 0.8mM (Figs. 1E and 5E), yet increased oleate availability decreases myocardial contractile function and efficiency during HWL only at ZT6 (Figs. 1, A and C, and 5, A and C). In contrast, nonoxidative oleate metabolism does exhibit a circadian rhythm, being increased during the light phase (i.e., ZT6; Table 1). These data have led us to speculate that channeling of fatty acids in nonoxidative pathways contribute toward fatty acid-induced depression of myocardial contractile function and efficiency.

Perspectives, study limitations, and future directions. The major limitation of this study relates to its descriptive nature. The specific mediators of circadian rhythms in myocardial metabolism, as well as responsiveness to workload and fatty acid challenges, remain unidentified. For example, one testable hypothesis is that circadian rhythms in pyruvate dehydrogenase (PDH) activity account for the reciprocal circadian rhythms in glucose oxidation and lactate release. Consistent with decreased PDH kinase 4 gene expression at ZT9, we (36) previously reported that the fraction of PDH in the active form (an inverse marker of phosphorylation status) reaches a peak at ZT12 in nonperfused rat hearts. However, the peak in PDH activity of nonperfused rat hearts is 6 h prior to the peak in glucose oxidation rates in perfused hearts, suggesting that PDH activity changes during the perfusion protocol (in a time-dependent manner) and/or mechanisms independent of PDH phosphorylation may play an important role in myocardial glucose oxidation circadian rhythms (e.g., malate/aspartate shuttle). It could be argued that increased rates of glucose oxidation during the dark phase are secondary to residual neurohumoral influences prior to heart isolation, such as higher circulating insulin levels at this time. However, careful inspection of the perfusion time course data reveals time-of-day-independent rates of glucose oxidation at the initiation of the perfusion. An additional possibility is that increased myocardial insulin sensitivity during the dark phase may account for the observed circadian rhythms in myocardial metabolism. Indeed, the current perfusions were performed in the presence of a basal level of insulin (10 µU/ml; a concentration similar to that observed in fasted rats). To address this possibility, we compared rat hearts perfused in either the absence or presence of 10 µU/ml insulin (at ZT18); this basal level of insulin had no effect on myocardial metabolism ex vivo (see Supplemental Table S2).

A second limitation of the present study is the use of only cardiac power as an indicator of cardiac function (as opposed to pressure-volume loops, for example). However, despite the use of this index of cardiac function, the data clearly expose a circadian rhythm in the susceptibility of the heart to oleate-induced depression of cardiac function and efficiency. An additional concern relates to the use of oleate as the sole fatty acid present in the perfusate as well as the use of glucose as the sole carbohydrate. In the latter case, previously published studies (10) have shown not only that the heart utilizes lactate as a significant source of energy but also that compartmentalization of lactate metabolism occurs. Through the use of stable isotopic labeling, future studies will likely improve our understanding of circadian rhythms in myocardial lactate metabolism.

Summary. The present study exposes marked circadian rhythms in 1) myocardial oxidative and nonoxidative metabolism, 2) responsiveness of the rat heart to a workload challenge, and 3) susceptibility of the rat heart to oleate-induced depression of cardiac function and efficiency. As summarized in Table 2, hearts perfused in the middle of the light phase are markedly different from hearts perfused during the dark phase.


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  		Table 2. Characteristics of the isolated working rat heart when perfused during the middle of the light phase (i.e. ZT6) compared with hearts perfused under identical conditions in the middle of the dark phase (i.e. ZT18)

 

   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Grants HL-074259 (M. E. Young) and HL-075747 (A. Fields and U. Mbawuike).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Young, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Dept. of Pediatrics, 1100 Bates St., Houston, TX 77030 (e-mail: meyoung{at}bcm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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