Altered clock gene expression and vascular smooth muscle diurnal contractile variations in type 2 diabetic db/db mice

This study was designed to determine whether the 24-h rhythms of clock gene expression and vascular smooth muscle (VSM) contractile responses are altered in type 2 diabetic db/db mice. Control and db/db mice were euthanized at 6-h intervals throughout the day. The aorta, mesenteric arteries, heart, kidney, and brain were isolated. Clock and target gene mRNA levels were determined by either real-time PCR or in situ hybridization. Isometric contractions were measured in isolated aortic helical strips, and pressor responses to an intravenous injection of vasoconstrictors were determined in vivo using radiotelemetry. We found that the 24-h mRNA rhythms of the following genes were suppressed in db/db mice compared with control mice: the clock genes period homolog 1/2 (Per1/2) and cryptochrome 1/2 (Cry1/2) and their target genes D site albumin promoter-binding protein (Dbp) and peroxisome proliferator-activated receptor-γ (Pparg) in the aorta and mesenteric arteries; Dbp in the heart; Per1, nuclear receptor subfamily 1, group D, member 1 (Rev-erba), and Dbp in the kidney; and Per1 in the suprachiasmatic nucleus. The 24-h contractile variations in response to phenylephrine (α(1)-agonist), ANG II, and high K(+) were significantly altered in the aortas from db/db mice compared with control mice. The diurnal variations of the in vivo pressor responses to phenylephrine and ANG II were lost in db/db mice. Moreover, the 24-h mRNA rhythms of the contraction-related proteins Rho kinase 1/2, PKC-potentiated phosphatase inhibitory protein of 17 kDa, calponin-3, tropomyosin-1/2, and smooth muscle protein 22-α were suppressed in db/db mice compared with control mice. Together, our data demonstrated that the 24-h rhythms of clock gene mRNA, mRNA levels of several contraction-related proteins, and VSM contraction were disrupted in db/db mice, which may contribute to the disruption of their blood pressure circadian rhythm.     

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.     

Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator

The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.     

Early Chronotype and Tissue-Specific Alterations of Circadian Clock Function in Spontaneously Hypertensive Rats

Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.     

Development of arterial pressure rhythm and cardiac contraction rate and effects of beta-adrenomimetic agents]

Blood pressure (BP) and heart rate (HR) rhythms were studied in premature infants (299 profiles ranged 24-106 h at 20--min intervals) and 11-13-year-old children (19 profiles for 48 h at 15-min intervals) to explore ultradian-to-circannual rhythm characteristics in BP and HR in preterm human infant and to elucidate the influence of antenatal betamimetic (BM) exposure on adolescent BP and HR rhythms. A circannual modulation of the 3-h amplitude (A) or MESOR of systolic BP (SBP) and diastolic BP (DBP) was seen mainly in prematures with a positive family history of high BP on the father's (f+) side or with both a patro linous and matro-linous history (f+m+), the circannual modulation of HR ultradian A was statistically significant only in "f- m-" infants. In urine collected at 3 h intervals for 24-h spans from 21 premature infants Na+,K+ and 11-oxycorticosteroids had a significant circadian rhythm. 9 adolescents (BM+), which were exposed in utero to different BM doses, had a significantly higher SBP and DBP MESOR and numerically higher circadian A as compared to 10 controls (BM-); correlation (P less than 0.05) between BM dose and HR circadian A was found. DBP led SBP in 8 or 10 "BM-" but in 4 of 9 "BM+" (acrophase difference 17 min and 3 min correspondingly).(ABSTRACT TRUNCATED AT 250 WORDS)     

AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Background

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/Principal Finding

The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD⁺, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/Significance

This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Development of dilated cardiomyopathy in Bmal1-deficient mice

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1(-/-) mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1(-/-) mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure.     

Myocardial infarction and heart failure in the db/db diabetic mouse

Clinical studies have reported that the incidence and severity of myocardial infarction is significantly greater in diabetics compared with nondiabetics after correction for all other risk factors. The majority of studies investigating the pathophysiology of myocardial ischemia-reperfusion injury have focused on otherwise healthy animals. At present, there is a paucity of experimental investigations on the pathophysiology of heart failure in diabetic animals. We hypothesized that the severity of myocardial reperfusion injury and the development of congestive heart failure would be markedly enhanced in the db/db diabetic mouse. Accordingly, we studied the effects of varying durations of in vivo myocardial ischemia and reperfusion on the incidence of heart failure in db/db diabetic mice. Nondiabetic and db/db diabetic mice (10 wk of age) were subjected to 30, 45, or 60 min of left coronary artery occlusion and 28 days of reperfusion. Survival at 24 h of reperfusion was 100% in nondiabetic mice subjected to 30 min of myocardial ischemia and 88% in nondiabetic mice subjected to 45 min of myocardial ischemia. In contrast, survival was 53% in db/db diabetic mice subjected to 30 min of myocardial ischemia and 44% in db/db mice after 45 min of myocardial ischemia. Prolonged survival in nondiabetic mice was not significantly attenuated when compared during the 28-day follow-up period with all groups experiencing >90% survival. Prolonged survival was significantly decreased in db/db mice after both 30 and 45 min of myocardial ischemia compared with sham controls. Furthermore, we observed a significant degree or left ventricular dilatation, cardiac hypertrophy, and cardiac contractile dysfunction in db/db mice subjected to 45 min of myocardial ischemia and 28 days reperfusion. In nondiabetic mice subjected to 45 min of myocardial ischemia, we failed to observe any changes in left ventricular dimensions or fractional shortening. These studies provide a feasible experimental model system for the investigation of heart failure secondary to acute myocardial infarction in the db/db diabetic mouse.     

Effects of photoperiod reduction on rat circadian rhythms of BP, heart rate, and locomotor activity

The effects of a photoperiod reduction in the entrainment of circadian rhythms of systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and spontaneous locomotor activity (SLA) were determined in conscious Wistar rats by using radiotelemetry. Two groups of seven rats were maintained in a 12:12-h light-dark (12L/12D) photoperiod for 11 wk and then placed in a reduced photoperiod of 8:16-h light-dark (8L/16D) by advancing a 4-h darkness or by advancing and delaying a 2-h darkness for 6 wk. Finally, they were resynchronized to 12L/12D. Advancing a 4-h dark phase induced a 1-h advance of acrophase for SBP, DBP, and HR, but not for SLA. The percent rhythm, amplitude, and the 12-h mean values of all parameters were significantly decreased by the photoperiod reduction. When symmetrically advancing and delaying a 2-h dark phase, a 1 h 20 min delay of acrophases and a decrease in percent rhythms and amplitudes of SBP, DBP, HR, and SLA were observed. Only the 12-h mean values of HR and SLA were decreased. Our findings show that the cardiovascular parameters differ from SLA in phase-shift response to photoperiod reduction and that the adjustment of circadian rhythms to change from 12L/12D to 8L/16D photoperiod depends on the direction of the extension of the dark period.     

The Circadian Clock Maintains Cardiac Function by Regulating Mitochondrial Metabolism in Mice

Cardiac function is highly dependent on oxidative energy, which is produced by mitochondrial respiration. Defects in mitochondrial function are associated with both structural and functional abnormalities in the heart. Here, we show that heart-specific ablation of the circadian clock gene Bmal1 results in cardiac mitochondrial defects that include morphological changes and functional abnormalities, such as reduced enzymatic activities within the respiratory complex. Mice without cardiac Bmal1 function show a significant decrease in the expression of genes associated with the fatty acid oxidative pathway, the tricarboxylic acid cycle, and the mitochondrial respiratory chain in the heart and develop severe progressive heart failure with age. Importantly, similar changes in gene expression related to mitochondrial oxidative metabolism are also observed in C57BL/6J mice subjected to chronic reversal of the light-dark cycle; thus, they show disrupted circadian rhythmicity. These findings indicate that the circadian clock system plays an important role in regulating mitochondrial metabolism and thereby maintains cardiac function.     

Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ～4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.     

Disruption of Daily Rhythms by High-Fat Diet Is Reversible

In mammals a network of circadian clocks coordinates behavior and physiology with 24-h environmental cycles. Consumption of high-fat diet disrupts this temporal coordination by advancing the phase of the liver molecular clock and altering daily rhythms of eating behavior and locomotor activity. In this study we sought to determine whether these effects of high-fat diet on circadian rhythms were reversible. We chronically fed mice high-fat diet and then returned them to low-fat chow diet. We found that the phase of the liver PERIOD2::LUCIFERASE rhythm was advanced (by 4h) and the daily rhythms of eating behavior and locomotor activity were altered for the duration of chronic high-fat diet feeding. Upon diet reversal, the eating behavior rhythm was rapidly reversed (within 2 days) and the phase of the liver clock was restored by 7 days of diet reversal. In contrast, the daily pattern of locomotor activity was not restored even after 2 weeks of diet reversal. Thus, while the circadian system is sensitive to changes in the macronutrient composition of food, the eating behavior rhythm and liver circadian clock are specifically tuned to respond to changes in diet.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.