Although there are genetic risk factors for CVD, increased CVD prevalence has been linked to environmental factors including exposure to Cd and other toxic metals Most research in humans and animal models has focused on the significance of chronic Cd exposure during adulthood. Although Cd inefficiently crosses the placenta 12 , there is growing evidence that maternal exposure to Cd can impact child development and health 2 , 20 , 41 , However, molecular mechanisms of Cd toxicity in this context have been challenging to study in humans.
To better understand the impact and mechanisms of action of developmental Cd exposure on cardiovascular health, we established a mouse model of maternal Cd exposure. Two doses of Cd were chosen to reflect a low and a high dose relevant to human exposure levels. However, geographic clusters can contain higher levels of Cd as shown by a study in North Carolina identifying regions with up to 1.
The mean level of blood Cd in the US population has been reported to be 0. The mean level of Cd detected in the blood of F 0 50ppm mice was approximately Additionally, fetal growth restriction was observed primarily in the F 1 50ppm mice, a phenotype consistent with Cd exposure in humans 47 , providing further support that the high dose is relevant to the study of Cd-associated human health outcomes. Using this model, we have shown that maternal Cd exposure is associated with increased heart weight at birth and can program hypertension during adulthood.
Our data showing that maternal Cd-induced hypertension is observed only in females is consistent with sex-specific responses to Cd reported in other studies 48 , Our hybrid mouse system will provide a valuable model for dissecting the genetic basis of differences in susceptibility to Cd-induced hypertension.
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We did not detect any differences compared to F 1 0ppm controls at this age, suggesting that molecular changes in early life may be sufficient to program this phenotype. We therefore assayed trace element levels at birth and, strikingly, demonstrated perturbation of all elements measured. Many of these elements are important for cardiovascular development and function.
For example, Na was significantly increased in pup blood and livers due to maternal Cd exposure; excess dietary Na is a significant contributing factor to hypertension Additionally, Se levels were significantly decreased in pup tissues in response to maternal Cd exposure; Se is a vital component of antioxidant enzymes, and diseases due to Se deficiency present with enlarged hearts, congestive heart failure, and other altered cardiac features Fe deficiency may be one of the most significant contributing factors to the reduced birth weight, increased heart weight at birth, and hypertension observed in our study.
Fe deficiency is associated with increased oxidative stress that may damage cells in the heart and endothelial lining of vasculature 53 , possibly contributing to the susceptibility to hypertension. The heart responds to chronic stress by increasing in size to increase blood flow, oxygen, and nutrient delivery to tissues 54 , potentially explaining the increased heart weight at birth.
Not surprisingly, a severe Fe deficiency is associated with an increased risk of CVD in humans We propose that perturbed trace element homeostasis could represent one of the earliest events in the mechanisms leading to the phenotypes observed in this study. The direction of changes of many of the trace element levels in fetal tissues correlated with those seen in maternal tissues; this correlation highlights the importance of including a pre-conception exposure window in animal models of maternal exposure, as maternal exposures in reality often begin prior to pregnancy.
Additional trace elements in F 0 50ppm tissue appeared to correlate with fetal levels, but were not statistically significant, likely due to small sample sizes. Thus, many of the essential trace elements seen perturbed in the pups may be due to existing Cd-induced perturbations in the mother. It is also possible that the trace element levels are further exacerbated at the placental level, as Cd can interfere with nutrient transport across the placenta Zn and Fe are the most well-studied elements affected by maternal Cd exposure due to their similar chemical properties to Cd and shared mechanisms of absorption Here, we show a significant increase in blood Zn and Cu, yet a significant decrease in liver Zn and Cu, which could explain why no overall differences were observed in whole rat fetuses.
Our RNA-seq data from hearts at birth provide further support for early life molecular changes programming susceptibility to hypertension in adulthood. To our knowledge, this is the first time that a global transcriptomic analysis has been performed in the heart in the context of Cd exposure.
We show changes in expression in genes implicated in hypertension, abnormal heart and cardiovascular system development, and increased heart size. Our RNA-seq data are further enriched for multiple mechanistic pathways that contribute to these diseases such as hypoxia, altered cellular energy, ROS, nitric acid homeostasis, and altered metal homeostasis, consistent with our trace element analysis.
Mechanisms through which maternal and adult exposure to Cd affect health may differ as the placenta acts as a barrier to Cd, but no such barriers exist when exposed to Cd postnatally. Considering that little if any Cd enters fetal circulation, maternal Cd exposure may exert its effects indirectly, such as impacting placental development and function 57 , 58 , Blood pressure was measured using the non-invasive tail cuff method to limit stress associated with surgery and recovery.
The technique has been extensively used and is appropriate for measures of obtaining overall arterial pressure rather than instantaneous pressure in response to a stimulus.
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There are limitations to this technique; for example, tail cuff arterial pressure does not always mirror central arterial pressure Measurements are also susceptible to fluctuations in the environmental temperature, although mice were maintained on a heat pad and under a blanket during the procedure to control for this.
In summary, based on our assessment of trace element levels and transcriptomic analysis of newborn hearts, we propose that maternal Cd exposure leads to a nutrient-poor environment in the offspring during development, which leads to increased heart weight and susceptibility to hypertension during adulthood.
Further studies using our mouse model will be required to test this hypothesis empirically. Our observations provide novel insights into mechanisms underlying Cd-associated CVD. Multiple cohorts of females were exposed to Cd for 5 weeks. The study was not blinded, but females from all groups were treated simultaneously within their cohorts. After 5 weeks, females were mated with previously unexposed males of the opposite strain and of similar age. Cd exposure continued throughout mating and pregnancy, and was discontinued once a litter was observed Fig.
Cd exposure did not affect water consumption or female body weight during the 5-week preconception exposure window data not shown. Hybrid F 1 offspring were generated to enable analyses of allele-specific gene expression and DNA methylation for a separate study KH, MC, manuscript in preparation. Sex was determined through observation of the gonads. Experiments were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology. Blood pressures were measured on a random subset of 5. Animals were restrained on a heating pad and covered with a towel to maintain body temperature.
Blood pressure data used for analysis were taken on the fourth or fifth consecutive day of testing. Only readings in which a heart rate was detected were used. Trunk blood of 6-month-old F 1 mice was collected immediately after cervical dislocation and decapitation in 1.
Protein concentration was calculated according to kit instructions. Blood was weighed after collection as measuring volume was not technically practical. Multi-calibration standard solutions were purchased through Spex Certiprep or Inorganic Ventures, Inc. Male and female pups were equally represented in each treatment group. Maternal strain was determined to not significantly influence the levels of elements, so B and C F 0 data were pooled within tissue type and Cd dose. RNA was suspended in nuclease-free water and quantified using a Nanodrop Library enrichment was performed and indexes for each sample were added during the protocol-specified PCR amplification.
Amplified library fragments were purified and checked for quality and final concentration using an Agilent Tapestation D chip, Agilent Technologies combined with a Qubit fluorometer ThermoFisher. Libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina HiSeq. The software package Real Time Analysis RTA was used to generate raw bcl, or base call files, which were then de-multiplexed by sample into fastq files using bcl2fastq Conversion Software v2.
The Trimmomatic-programmable FastQC was performed again to verify the quality of trimmed reads. The Mouse RNASeq DeSEQ2 pipeline published by Cyverse 63 was used for indexing, aligning, read counts, and differential expression analysis; all analyses were performed using the recommended parameters.
Aligned reads were counted using the HTSeq-count Differential gene expression analysis was performed using the DeSEQ2 multifactorial pairwise comparisons app. Supplementary Fig. DNase I treatment was performed prior to reverse transcription as part of the Qiagen AllPrep kit protocol. Standards, the no template control NTC , and all other tested samples were each run in triplicate. Amplification efficiencies were calculated by determining the slope of the regression between the log values of the standard concentrations and the average Ct value of the standards.
Polr2a was not significantly differentially expressed between treatment groups data not shown and was used as a reference gene. Outliers, as defined by a point which falls more than 1.
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Wadhwa, P. Ronco, A. PLoS One , 9 6 Lee, H. Nutrients 7 11 , — Filipic, M. Mechanisms of Cadmium Induced Genomic Instability. Sigel, A. King, K. BMC Public Health 15 , 1—12 Nishijo, M. BMJ Open 7 e , 1—7 Piasek, M.
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