Peripubertal serum dioxin concentrations and subsequent sperm methylome profiles of young Russian adults.

BACKGROUND: The association of exposure to endocrine disrupting chemicals in the peripubertal period with subsequent sperm DNA methylation is unknown. OBJECTIVE: We examined the association of peripubertal serum 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentrations with whole-genome bisulfite sequencing (WGBS) of sperm collected in young adulthood. METHODS: The Russian Children's Study is a prospective cohort of 516 boys who were enrolled at 8-9 years of age and provided semen samples at 18-19 years of age. WGBS of sperm was conducted to identify differentially methylated regions (DMR) between highest (n = 4) and lowest (n = 4) peripubertal TCDD groups. RESULTS: We found 52 DMRs that distinguished lowest and highest peripubertal serum TCDD concentrations. One of the top scoring networks, "Cellular Assembly and Organization, Cellular Function and Maintenance, Carbohydrate Metabolism", identified estrogen receptor alpha as its central regulator. CONCLUSION: Findings from our limited sample size suggest that peripubertal environmental exposures are associated with sperm DNA methylation in young adults.

Day-night dependence of gene expression and inflammatory responses in the remodeling murine heart post-myocardial infarction.

Diurnal or circadian rhythms are fundamentally important for healthy cardiovascular physiology and play a role in timing of onset and tolerance to myocardial infarction (MI) in patients. Whether time of day of MI triggers different molecular and cellular responses that can influence myocardial remodeling is not known. This study was designed to test whether time of day of MI triggers different gene expression, humoral, and innate inflammatory responses that contribute to cardiac repair after MI. Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 2-h time window either shortly after lights on or lights off, and the early remodeling responses at 8 h postinfarction were examined. We found that sleep-MI preferentially triggers early expression of genes associated with inflammatory responses, whereas wake-MI triggers more genes associated with metabolic pathways and transcription/translation, by microarray analyses. Homozygous clock mutant mice exhibit altered diurnal gene expression profiles, consistent with their cycling before onset of MI. In the first 8 h, crucial for innate immune responses to MI, there are also significant differences in sleep-MI and wake-MI serum cytokine responses and in neutrophil infiltration to infarcted myocardium. By 1-wk post-MI, there are differences in survivorship between the sleep and wake MI mice that could be explained by the different molecular and cellular responses. Our whole body physiology, tissues, and cells exhibit endogenous daily rhythms, and understanding their role in triggering effective responses after MI could lead to new strategies to benefit patients with cardiovascular disease.

Circadian regulation of myocardial sarcomeric Titin-cap (Tcap, telethonin): identification of cardiac clock-controlled genes using open access bioinformatics data.

Circadian rhythms are important for healthy cardiovascular physiology and are regulated at the molecular level by a circadian clock mechanism. We and others previously demonstrated that 9-13% of the cardiac transcriptome is rhythmic over 24 h daily cycles; the heart is genetically a different organ day versus night. However, which rhythmic mRNAs are regulated by the circadian mechanism is not known. Here, we used open access bioinformatics databases to identify 94 transcripts with expression profiles characteristic of CLOCK and BMAL1 targeted genes, using the CircaDB website and JTK_Cycle. Moreover, 22 were highly expressed in the heart as determined by the BioGPS website. Furthermore, 5 heart-enriched genes had human/mouse conserved CLOCK:BMAL1 promoter binding sites (E-boxes), as determined by UCSC table browser, circadian mammalian promoter/enhancer database PEDB, and the European Bioinformatics Institute alignment tool (EMBOSS). Lastly, we validated findings by demonstrating that Titin cap (Tcap, telethonin) was targeted by transcriptional activators CLOCK and BMAL1 by showing 1) Tcap mRNA and TCAP protein had a diurnal rhythm in murine heart; 2) cardiac Tcap mRNA was rhythmic in animals kept in constant darkness; 3) Tcap and control Per2 mRNA expression and cyclic amplitude were blunted in Clock(Δ19/Δ19) hearts; 4) BMAL1 bound to the Tcap promoter by ChIP assay; 5) BMAL1 bound to Tcap promoter E-boxes by biotinylated oligonucleotide assay; and 6) CLOCK and BMAL1 induced tcap expression by luciferase reporter assay. Thus this study identifies circadian regulated genes in silico, with validation of Tcap, a critical regulator of cardiac Z-disc sarcomeric structure and function.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Chronomics of pressure overload-induced cardiac hypertrophy in mice reveals altered day/night gene expression and biomarkers of heart disease.

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload-induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease.

The primary benefits of angiotensin-converting enzyme inhibition on cardiac remodeling occur during sleep time in murine pressure overload hypertrophy.

OBJECTIVES: Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. BACKGROUND: Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. METHODS: We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wake time for 8 weeks starting 1 week after surgery. RESULTS: TAC mice given captopril at sleep time had improved cardiac function and significantly decreased heart: body weight ratios, myocyte cross-sectional areas, intramyocardial vascular medial wall thickness, and perivascular collagen versus TAC mice given captopril or placebo during wake time. Captopril induced similar drops in blood pressure at sleep or wake time, suggesting that time-of-day differences were not attributable to blood pressure changes. These beneficial effects of captopril were correlated with diurnal changes in ACE mRNA expression in the heart. CONCLUSIONS: The ACE inhibitor captopril benefited cardiovascular remodeling only when administered during sleep; wake-time captopril ACE inhibition was identical to that of placebo. These studies support the hypothesis that the heart (and vessels) remodel during sleep time and also illustrate the importance of diurnal timing for some cardiovascular therapies.