Correction: Polyamine-associated changes in circadian gene expression and their relationship to GnRH-I expression.

Ventricular tachyarrhythmias and sudden cardiac death show a circadian pattern of occurrence in patients with heart failure. In the rodent ventricle, a significant portion of genes, including some ion channels, shows a circadian pattern of expression. However, genes that define electrophysiological properties in failing human heart ventricles have not been examined for a circadian expression pattern. Ventricular tissue samples were collected from patients at the time of cardiac transplantation. Two sets of samples (n=37 and 46, one set with a greater arrhythmic history) were selected to generate pseudo-time series according to their collection time. A third set (n=27) of samples was acquired from the nonfailing ventricles of brain-dead donors. The expression of 5 known circadian clock genes and 19 additional ion channel genes plausibly important to electrophysiological properties were analyzed by real-time polymerase chain reaction and then analyzed for the percentage of expression variation attributed to a 24-hour circadian pattern. The 5 known circadian clock gene transcripts showed a strong circadian expression pattern. Compared with rodent hearts, the human circadian clock gene transcripts showed a similar temporal order of acrophases but with a ≈7.6 hours phase shift. Five of the ion channel genes also showed strong circadian expression. Comparable studies of circadian clock gene expression in samples recovered from nonheart failure brain-dead donors showed acrophase shifts, or weak or complete loss of circadian rhythmicity, suggesting alterations in circadian gene expression. Ventricular tissue from failing human hearts display a circadian pattern of circadian clock gene expression but phase-shifted relative to rodent hearts. At least 5 ion channels show a circadian expression pattern in the ventricles of failing human hearts, which may underlie a circadian pattern of ventricular tachyarrhythmia/sudden cardiac death. Nonfailing hearts from brain-dead donors show marked differences in circadian clock gene expression patterns, suggesting fundamental deviations from circadian expression.

Evaluation of circadian gene expression changes in human peripheral blood cells as biomarkers of circadian disruption in shift workers: application to studies of breast and prostate cancer chemoprevention

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Retinoic acid induced 1 ( RAI1 ) encodes a dosage-sensitive gene that when haploinsufficient results in Smith-Magenis syndrome (SMS) and when overexpressed results in Potocki-Lupski syndrome (PTLS). Phenotypic and molecular evidence illustrates that haploinsufficiency of RAI1 disrupts circadian rhythm through the dysregulation of the master circadian regulator, circadian locomotor output cycles kaput ( CLOCK) , and other core circadian components, contributing to prominent sleep disturbances in SMS. However, the phenotypic and molecular characterization of sleep features in PTLS has not been elucidated. Using the Pittsburgh Sleep Quality Index (PSQI), caregivers of 15 school-aged children with PTLS reported difficulties in initiating sleep. Indeed, more than 70% of individuals manifested moderate to severe sleep latency, as defined by the PSQI. Moreover, these individuals manifested difficulties in sleep maintenance, with middle of the night and early morning awakenings. When assessing daytime sleepiness through the Epworth Sleepiness Scale, approximately 21% of the individuals manifested excessive daytime somnolence. This indicates that mild dyssomnia characterizes the majority of the sleep phenotype, with occasionally problematic daytime somnolence, a phenotype different than that expressed by individuals with SMS, where daytime sleepiness is a chronic problem. Gene expression analysis of the core circadian machinery in the hypothalamus of the PTLS mouse model ( Rai1 -Tg) found significant dysregulation of the transcriptional activators, Clock and Arntl , and the transcriptional repressors, Per1-3 and Cry1/2 , during both light and dark phases. These findings suggest a partial loss of circadian entrainment typically evoked by environmental photic cues. Examination of circadian clock gene expression in the Rai1- Tg mouse heart, liver, and kidney found unchanged expression of Clock and most of its downstream targets during both light and dark phases, suggesting an asynchronized circadian rhythm. Furthermore, examination of circadian gene expression in synchronized PTLS lymphoblasts revealed reduced transcripts of the Period ( PER1-3 ) family and normal expression of CRY1/2 . The finding that central circadian gene expression was altered while many peripheral circadian components were intact suggests a tissue-specific circadian uncoupling of the circadian machinery due to Rai1 overexpression. Overall, our results demonstrate that overexpression of RAI1 results in sleep deficiencies in individuals with PTLS due to a lack of properly regulated circadian machinery gene expression and highlight the importance of evaluating sleep concerns in individuals with PTLS.

Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as Bmal1 and Cry1, was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.

Circadian rhythms are twenty-four hour physiologic cycles present in all eukaryotes that control a variety of organismal processes, including metabolism, but the role of metabolism in control of circadian rhythms is still not well understood. Peripheral clocks such as those present in the liver control metabolic pathways such as glucose metabolism and respiration as well as amino acid metabolism. It has been recently demonstrated that the availability of the metabolite NAD (nicotinamide adenine dinucleotide) can feed back to control circadian rhythm. There is much interest in targeting glutamine metabolism in cancer, but it is still not fully understood how inhibition of glutamine metabolism affects normal cell physiology, including circadian rhythm. Here we show using the commonly-used circadian model U2OS osteosarcoma cells and other cell lines that glutamine withdrawal blocked proper circadian oscillation of gene expression. Glutamine withdrawal led to distinct and dramatic changes in circadian gene expression in several cell lines with highly different tissue origins, which could be rescued by addition of the cell permeable TCA-intermediate α-ketoglutarate. However, cells withdrawn from glutamine did not show signs of metabolic stress or impairment of the mTOR pathway. While alterations to histone modifications possibly stemming from impairment of αKG-dependent enzymes were observed, these did not explain the observed alterations in circadian rhythm. Rather, RNA-seq analysis of genetic changes after glutamine withdrawal and α-ketoglutarate rescue revealed strong induction of several genetic pathways associated with reactive-oxygen species (ROS) induction, particularly those resulting from chemotherapy or photodynamic therapy of cancer. Further supporting the importance of ROS in regulation of circadian rhythm, addition of cell permeable antioxidants rescued the disruption of circadian oscillation in the absence of glutamine. Finally, inhibiting expression of the key ROS-defense catalase phenocopied circadian rhythm disruption observed after glutamine withdrawal. Together, these data suggest that glutamine availability and metabolism are critical to support circadian rhythm and gene expression through modulation of intracellular ROS, and furthermore that cancer treatments that lead to induction of ROS could affect normal cellular circadian rhythm. We thank the following funding: NIH F32CA180370, NIH F32CA174148, NIH R01CA051497, LLS 610614 Citation Format: Brian J. Altman, Zachary E, Stine, Annie L. Hsieh, Ralph J. Deberardinis, Chi V. Dang. Mammalian glutamine metabolism controls circadian rhythm through regulation of reactive oxygen species. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2015-4708

Editor's evaluation: Comparative transcriptomic analysis reveals translationally relevant processes in mouse models of malaria

Introduction Altered circadian gene expression is hypothesized to underlie circadian dependent differences in airway resistance and risk for asthma exacerbation in pediatric asthma. Previously in ex vivo organotypic epithelial cell cultures we identified neutrophilic and IL-17 signaling genes as losing circadian rhythmicity in pediatric asthma. However, measuring circadian rhythms of gene expression in vivo is difficult due to the need for repeat sampling. Using bioinformatic approaches, circadian time can be inferred in single samples and rhythmic genes identified when sampling a larger number of individuals randomly over the course of the day. We analyzed circadian rhythmic gene expression using a publicly available data of gene expression in children with and without asthma at age 10 from the URECA study to identify genes with altered circadian rhythmicity in asthma. Methods Gene expression from nasal swabs obtained at age 10 in 352 children, 249 without asthma and 103 with asthma were analyzed using CYCLOPS to identify rhythmic genes. Circadian time was defined using a set of 100 rhythmic genes identified from ex vivo epithelial cell cultures. Differential rhythmicity was analyzed using CompareRhythms and phase set enrichment (PSEA) used to identify genes whose expression concentrated at specific circadian times. Results CYCLOPS analysis identified 3694 genes with rhythmic expression on nasal epithelial swabs. Genes for TGF-beta signaling, IL-6 signaling, and interferon-alpha responses had peak expression in the morning, whereas cell cycle and oxidative phosphorylation genes peaked in the evening. Differential rhythmicity identified 182 with altered rhythmicity in asthma, with genes for interferon and viral responses increasing rhythmicity in asthma and genes for granulocyte activation and neutrophil migration had reduced rhythmicity in asthma. Conclusion Circadian gene expression of neutrophil migration and granulocyte activation genes is decreased in pediatric asthma based on in vivo nasal swabs. Loss of circadian regulation may contribute to aberrant expression of airway inflammatory genes in pediatric asthma. Support (if any) SRS (WTP), Parker B Francis Fellowship (WTP), NIH K24AI150991(JSD)

Advanced sleep schedules affect circadian gene expression in young adults with delayed sleep schedules

Sleep is essential for maintaining brain myelin integrity. Emerging evidence suggests that poor sleep quality compromises the glymphatic system, a perivascular network crucial for brain waste clearance, leading to the accumulation of neuroinflammatory and toxic proteins, which may affect myelin integrity. Furthermore, poor sleep quality results in alterations in gene expression within the brain. We evaluated the associations among poor sleep quality, brain myelin integrity, and glymphatic clearance function as well as the impact of circadian clock gene expression on regional cortical myelin content. 50 poor sleepers (average age 71.08 ± 4.69 years; Pittsburgh Sleep Quality Index [PSQI] > 5) and 50 good sleepers (average age 73.04 ± 5.80 years; PSQI ≤ 5) were assessed. Myelin volume fraction (MVF) was quantified using magnetization transfer saturation imaging, and glymphatic function was noninvasively examined using diffusion tensor imaging along the perivascular space. Circadian gene expression was analyzed using postmortem brain tissue from the Allen Human Brain Atlas. Magnetic resonance imaging measures were correlated with cognitive and depression scores. Lower MVF was observed in the fronto-temporo-parietal and limbic regions as well as in major white matter tracts in poor sleepers compared with that in good sleepers. This reduction was linked to lower cognitive function scores and higher depressive scores. Poor sleepers also exhibited lower diffusivity along the perivascular spaces, mediating the relationship between poor sleep quality and demyelination. Regions with higher expression of CLOCK, CRY2, PER1, and PER2 exhibited greater MVF disparities between good and poor sleepers, whereas lower expression of CRY1 was associated with more pronounced differences. Poor sleep quality was associated with lower brain myelin integrity, correlating with reduced cognitive performance and increased depressive symptoms. These changes might be mediated by glymphatic clearance dysfunction and were associated with the differential expression of circadian clock genes.

Circadian Rhythms: Per2bations in the Liver Clock

Pancreatic cancer (PC), the fourth leading cause of cancer-related deaths, is characterized by high aggressiveness and resistance to chemotherapy. Pancreatic carcinogenesis is kept going by derangement of essential cell processes, such as proliferation, apoptosis, metabolism and autophagy, characterized by rhythmic variations with 24-h periodicity driven by the biological clock. We assessed the expression of the circadian genes ARNLT, ARNLT2, CLOCK, PER1, PER2, PER3, CRY1, CRY2 and the starvation-activated histone/protein deacetylase SIRT1 in 34 matched tumor and non-tumor tissue specimens of PC patients, and evaluated in PC derived cell lines if the modulation of SIRT1 expression through starvation could influence the temporal pattern of expression of the circadian genes. We found a significant down-regulation of ARNLT (p = 0.015), CRY1 (p = 0.013), CRY2 (p = 0.001), PER1 (p < 0.0001), PER2 (p < 0.001), PER3 (p = 0.001) and SIRT1 (p = 0.017) in PC specimens. PER3 and CRY2 expression levels were lower in patients with jaundice at diagnosis ( < 0.05). Having adjusted for age, adjuvant therapy and tumor stage, we evidenced that patients with higher PER2 and lower SIRT1 expression levels showed lower mortality (p = 0.028). Levels and temporal patterns of expression of many circadian genes and SIRT1 significantly changed upon serum starvation in vitro, with differences among four different PC cell lines examined (BXPC3, CFPAC, MIA-PaCa-2 and PANC-1). Serum deprivation induced changes of the overall mean level of the wave and amplitude, lengthened or shortened the cycle time and phase-advanced or phase-delayed the rhythmic oscillation depending on the gene and the PC cell line examined. In conclusion, a severe deregulation of expression of SIRT1 and circadian genes was evidenced in the cancer specimens of PC patients, and starvation influenced gene expression in PC cell lines, suggesting that the altered interplay between SIRT1 and the core circadian proteins could represent a crucial player in the process of pancreatic carcinogenesis.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock‐controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 h apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time‐dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day‐to‐night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night reduction in collagen I expression.Key pointsTendinopathy is a major clinical problem with unsolved pathogenesis.Previous work in mice has shown that a robust circadian rhythm is required for collagen homeostasis in tendons.The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue.Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues.We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy.

10 % of genome mRNA expression is rhythmic and these 24-hrs rhythms are under control of the circadian clock. Epidemiologic studies have revealed a clear link between the disruption of circadian rhythms and cancer development in humans. Growing evidence shows that circadian disruption is associated with development of malignant tumors, including breast cancer. Aim of this study was to investigate: the expression of circadian clock genes in human mammary epithelial cell line MCF10A and breast cancer cell lines MCF-7, ZR-75-1, BT-474 and if the multidrug resistance phenotype of cancer cells is associated with changes in circadian clock genes expression. We have found that Per1 expression significantly reduced in cancer cells. No correlation was detected between the expression of circadian clock genes and cancer breast cell lines drug resistance. Interestingly, the expression of Bmal1, Per1 and Cry1 were increased in multi-drug resistant MCF-7_D cells compare with the parent cells MCF-7 cells, however, if these changes in the expression contribute to the drug-resistance or not is not clear. These results argue for further study.

Effects of macronutrient manipulation on postprandial metabolic responses in overweight males with high fasting lipids during simulated shift work: A randomized crossover trial