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Try out PMC Labs and tell us what you think. Learn More. All other data can be found in the Supplementary Excel file. Environmental factors play an important role in the etiology of cardiovascular diseases. Cardiovascular diseases exhibit marked sexual dimorphism; however, the sex-specific effects of environmental exposures on cardiac health are incompletely understood.
Perinatal and adult exposures to the metal lead Pb Columbus-MI adult sex linked to several adverse cardiovascular outcomes, but the sex-specific effects of this toxicant on the heart have received little attention. Perinatal environmental exposures can lead to disease through disruption of the normal epigenetic programming that occurs during early development. Two weeks prior to mating, dams were ased to control or Pb acetate 32 ppm water, and exposure continued until offspring were weaned at three weeks of age.
Enhanced reduced-representation bisulfite sequencing was used to measure DNA methylation in the hearts of offspring at five months of age. Although Pb exposure stopped at three weeks of age, we discovered hundreds of differentially methylated cytosines DMCs and regions DMRs in males and females at five months of age. Pathway analysis revealed altered methylation of genes important for cardiac and other tissue development in males, and histone demethylation in females.
Together, these data demonstrate that perinatal exposure to Pb induces sex-specific changes in cardiac DNA methylation that are present long after cessation of exposure, and highlight the importance of considering sex in environmental epigenetics and mechanistic toxicology studies. Environmental exposures that occur very early in life can have long-lasting influences on cardiovascular disease CVD risk [ 1 ], consistent with the Developmental Origins of Health and Disease DOHaD hypothesis.
In spite of this, the underlying Columbus-MI adult sex by which they do so are incompletely understood.
Developmental environments may influence CVD risk by altering the structure of the Columbus-MI adult sex, including the final of cells in the heart or density of blood vessels [ 2 ]. Exposures may also alter the expression of genes and their protein products critical for normal cardiac function, such as those of the mitochondrial electron transport chain [ 2 ]. There are considerable sex differences in the incidence Columbus-MI adult sex pathogenesis of CVDs [ 3 ].
For example, although men and women are both prone to ischemic heart disease, the pathogenesis differs between sexes, with males more frequently exhibiting obstructive coronary artery disease [ 3 ]. In contrast, ischemia in women is more often due to non-obstructive coronary artery disease or microvascular dysfunction. Likewise, women are more likely than men to experience lethal arrhythmias as a result of pharmacologic interventions antibiotics, antidepressants, antihistamines or genetic syndromes [ 34 ].
The molecular basis for these differences is unclear but likely involves genetic, epigenetic, and hormonal factors [ 3 ]. How these important sex differences interact with environmental exposures to influence cardiac health has not been investigated. One potential mechanism by which toxicant exposures early in life can lead to CVD is by interfering with the widespread epigenetic and transcriptional programming that drive normal cardiac development [ 1 ]. DNA methylation, or the addition of a methyl group to cytosine bases on DNA 5-methylcytosine Columbus-MI adult sex, plays a critical role in normal development, and disruptions in DNA methylation have been linked to environment-induced disease.
The development of the heart is characterized by dynamic changes in DNA methylation [ 5 ], and alterations in this epigenetic mark have been identified in a variety of cardiovascular disease states [ 156 ], as well as with toxicant exposures [ 678 ]. Notably, sex differences in epigenetic modifications and transcriptional profiles are present in the embryonic stem cell stage and persist throughout cardiac differentiation and into adulthood [ 910 ].
Thus, sex-specific alterations in DNA methylation by environmental exposures may have important implications for sexually dimorphic cardiovascular health. Recent incidents of Pb contamination in municipal water systems underscore the ongoing threat that Pb poses to human health in the US and worldwide. In addition to drinking water, common sources of Pb exposure in the US include household dust from Pb-based paint, imported consumer products, and industrial exposures [ 11 ].
Worldwide, e-waste recycling, traditional medicines, industrial emissions, and glazed ceramics comprise additional sources of exposure [ 11 ]. In addition to the established effects on the nervous system, the contribution of Pb to CVD mortality in the US is far greater than ly thought [ 13 ]. Pb exposure is linked to high blood pressure, myocardial infarction, stroke, and cardiac arrhythmias in humans and animals [ 131415161718 ]. In a rodent model, neonatal Pb exposure Columbus-MI adult sex to enhanced sensitivity to the arrhythmogenic effects of norepinephrine in adulthood, suggesting that early development is a critical window of susceptibility to the effects of Pb on cardiac health [ 1920 ].
In spite of established links between Pb and CVD, sex differences in these outcomes, and the underlying molecular mechanisms, are unclear. Early life exposure to Pb plays a clear role in reprogramming of DNA methylation in several non-cardiac tissues, and sex-specific effects of perinatal Pb exposure on DNA methylation have been reported [ 2122 ]. However, the sex-specific effects of developmental Pb exposure on the epigenome of the heart, and the implications this may have for disease, are unknown.
In this work, we hypothesized that Pb exposure during this critical window of development would also affect cardiac DNA methylation. To Columbus-MI adult sex this hypothesis, we utilized a mouse model of perinatal environmental exposure to investigate the effects of an environmentally relevant dose of Pb on DNA methylation in the hearts of adult male and female mice.
Mice utilized for this study have recently been described [ 8 ].
Procedures for Pb preparation and exposure were conducted exactly as outlined ly [ 24 ]. Pb-supplemented water was made by dissolving Pb II acetate trihydrate Sigma-Aldrich in a single batch of distilled water, and Pb concentrations were verified using inductively coupled plasma Columbus-MI adult sex spectrometry with a limit of detection of 1.
Dams were exposed to either control or Pb-supplemented drinking water for two weeks prior to mating, and exposure continued during gestation and lactation. After Columbus-MI adult sex on postnatal day 21, the resulting pups were weighed and switched to Pb-free drinking water Figure 1. Schematic of experimental de and sample collection. Dams were exposed to Pb 2 weeks prior to mating via drinking water. Maternal and, in turn, offspring exposure continued until weaning, when offspring reached 3 weeks of age. Health checks consisted of a general assessment of appearance fur coat intact and well-groomed, eyes clear, no s of fight wounds and behavior mobility, nest building, etc.
In addition to checks by lab personnel, a deated animal handler from ULAM checked these cues on a daily basis, and a veterinarian assessed the health status of the mice at least once a week. Euthanasia and tissue collection procedures were recently described [ 8 ]. DNA extraction 1—2 male and 1—2 female mice per litter and sequencing were performed as outlined ly [ 8 ].
Enhanced reduced representation bisulfite ERRBS was performed at the University of Michigan Epigenomics and Advanced Genomics Cores as described ly [ 2425 ], and each sample met the quality control criteria for next generation sequencing.
Bisulfite conversion efficiencies for all samples exceeded The average mapping efficiency was Libraries were multiplexed and sequenced over 2 lanes. On average, this method captured 4. DNA methylation analysis, including quality control, trimming, alignment, and methylation calling were conducted exactly as outlined ly [ 8 ].
Opposite strand CpGs at the same position were combined via destranding. Sex chromosomes were included in this analysis. We performed differential methylation testing on individual CpG sites DMCsand differentially methylated regions DMRs were identified in bp tiles using the same process. In order to be included in the analysis, sufficient sequencing coverage for a minimum of 4 samples from the Pb group and 4 samples from the control group was required.
Differentially methylated CpGs and regions were identified exactly as recently outlined [ 8 ]. Run was included as a covariate in the model to adjust for batch effects. After obtaining p -values, we adjusted for Columbus-MI adult sex testing using the FDR approach. To determine the distribution of differentially methylated sites across the genome, we used the annotatR R Bioconductor package v1. To determine whether the proportion of differentially methylated cytosines DMCs falling into each annotation was ificantly different from the total regions tested, we conducted a Chi Square test Columbus-MI adult sex 8 ].
Poly-enrich analysis was conducted using all DMRs with a p -value of at least 0. Sequenced re were trimmed via Trim Galore [ 28 ], and quality control was assessed with FastQC [ 29 ]. STAR was used for the alignment step [ 30 ]. Trimming, QC and alignment were all carried out with default parameters. Normalized read counts for Pb-exposed vs. Statistical analysis was conducted as noted in the next section. For heart weights and gene expression analysis lead vs. Litter-specific random effects were included to for within-litter correlation. Statistical analysis of overlapping genes or sites between groups was conducted using a hypergeometric test [ 33 ].
Exposure to Pb during gestation and lactation did not ificantly alter litter size, pup mortality, or the percentage of females in each litter, and animal weights at 5 months of age were not ificantly different between control and Pb exposed animals [ 24 ]. Pb exposure had no ificant effect on relative heart weights in either males or females at 5 months of age Figure 2. This finding may have been due to a relatively small sample size compared to our studies in which we observed phenotypic effects of Pb exposure [ 2234 ].
Heart weights, expressed as a percentage of body weight, were assessed for males A and females Columbus-MI adult sex at 5 months of age. Data were analyzed using linear mixed-effects regression with litter-specific random effects to for within-litter correlation. Black Columbus-MI adult sex represent control animals and red squares depict Pb-treated animals.
There were no statistically ificant differences in hearts from Pb-exposed animals compared to controls. In order Columbus-MI adult sex investigate the effects of gestational and lactational Pb exposure on DNA methylation, we utilized ERRBS to measure DNA methylation in isolated whole heart tissue from male and female offspring at 5 months of age. The total of cytosines and regions tested was similar between sexes Table 1 and Table 2. Volcano plots depicting differentially methylated cytosines DMCs for Pb exposed compared to control in males A and females B. Annotation summary plots depicting the total of CpGs tested in pink, hypermethylated differentially methylated cytosines DMCs in green, and hypomethylated DMCs in blue for each genomic annotation using the R annotatr package for males A and females B.
Top 10 differentially hypo- and hyper-methylated cytosines DMCs in each sex, ranked by methylation change. Top 10 differentially hypo- and hyper-methylated regions DMRs in each sex, ranked by Columbus-MI adult sex change. In order to determine the pathways enriched among the DMRs, we first performed analysis using Poly-Enrich [ 36 ], which has been shown to have a more accurate false positive rate than other pathway enrichment tests. We stratified our analysis by sex and direction of methylation change.
In both males and females, we observed enrichment for pathways relevant to cardiac function, including ion channel activity, but the were not statistically ificant Supplementary Tables S6—S9. Among males, DMRs were enriched for several pathways important for normal heart development and function, including the Notch and hedgehog aling pathways smoothened is a Columbus-MI adult sex of the hedgehog pathway [ 3738 ], as well as regulation of cardiac muscle hypertrophy Figure 5 A. Among females, DMRs were enriched for pathways associated with histone demethylation, arginine hydroxylation, and body morphogenesis Figure 5 B.
Pathways are arranged from most ificant top to least ificant bottom. We stratified the data by sex and direction of methylation and conducted separate analyses for each. In both males and females, there were fewer than differentially hypomethylated genes, so we included all of them in the analysis 35 hypo DMRs in females and 67 hypo DMRs in males. For differentially hypermethylated regions in both sexes, we included the top genes with the largest changes in methylation.
In females, the interactions among DMR-associated genes were not statistically ificant for hyper or hypomethylated genes. Many of the genes in these networks are important for normal heart development and function, or are associated with disease. Among hypermethylated regions, they included Afap1PrkceAtg5and Tmod1. Among hypomethylated regions, they Columbus-MI adult sex Bcas3Cux1, and Hnrnpu. DMR-associated genes in females showed no ificant network interactions and are not shown in the figure.
Panel A depicts genes associated with the top most hypermethylated DMRs based on percentage methylation change. Panel B depicts all hypomethylated DMRs, since there were fewer thanColumbus-MI adult sex
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