In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation

Abstract

In utero exposure to diesel exhaust air pollution has been associated with increased adult susceptibility to heart failure in mice, but the mechanisms by which this exposure promotes susceptibility to heart failure are poorly understood. To identify the potential transcriptional effects that mediate this susceptibility, we have performed RNA sequencing analysis on adult hearts from mice that were exposed to diesel exhaust in utero and that have subsequently undergone transverse aortic constriction. We identified 3 target genes, Mir133a-2, Ptprf, and Pamr1, which demonstrate dysregulation after exposure and aortic constriction. Examination of expression patterns in human heart tissues indicates a correlation between expression and heart failure. We subsequently assessed DNA methylation modifications at these candidate loci in neonatal cultured cardiac myocytes after in utero exposure to diesel exhaust and found that the promoter for Mir133a-2 is differentially methylated. These target genes in the heart are the first genes to be identified that likely play an important role in mediating adult sensitivity to heart failure. We have also shown a change in DNA methylation within cardiomyocytes as a result of in utero exposure to diesel exhaust.-Goodson, J. M., Weldy, C. S., MacDonald, J. W., Liu, Y., Bammler, T. K., Chien, W.-M., Chin, M. T. In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation.

Keywords: PM2.5; Pamr1; Ptprf; heart failure; miR133a-2.

Figure 1.

Exposure and sample collection scheme.

Figure 2.

Genomic regions for bisulfite sequencing. Genomic regions highlighted in red boxes are areas that were targeted for bisulfite sequencing. Targets correspond to GM6307 promoter and first exon (A); Ptprf CpG islands 17, 24, and 79 (B); and Pamr1 promoter and first exon (C).

Figure 3.

Expression of miR133a-2, PTPRF, and Pamr1 after in utero DE exposure and TAC surgery, correlated with ventricular weight/tibia length measurements. A, C, E) ΔΔC_t values for miR133a-2 (A), Ptprf (C), and Pamr1 (E) in the 4 categories: FA sham-treated, DE sham-treated, FA TAC, and DE TAC. B, D, F) Corresponding linear regression values for miR311a-2 (B), Ptprf (D), and Pamr1 (F), plotting expression as a function of ventricle weight normalized to tibia length. *P < 0.05.

Figure 4.

Expression of Ptprf (A) and Pamr1 (B) in human heart failure samples. Significance was calculated by using 1-way ANOVA with post hoc Bonferroni. Data were derived from publicly available Affymetrix data set (NCBI GEO Accession No. GSE26887). **P < 0.005, ***P < 0.0005.

Figure 5.

Targeted bisulfite sequencing of GM6307 promoter and exon 1. The upper portion of the plot contains a karyogram of chromosome2, with a vertical red bar showing the region of interest; the horizontal bar just below the karyogram shows the zoomed-in region of the chromosome being inspected. The transcripts portion of the plot presents Universiy of California, Santa Cruz (UCSC) genome browser-style transcripts, where the orange bars represent exons and the gray lines represent introns. Arrows in the intronic region represent the direction of transcription. Below that, we present the observed (individual points) and smoothed (lines) percent methylation estimates for CpGs in this genomic region. Pink points and lines represent FA samples, whereas blue represent DE samples. In the lowest (coverage) portion of the plot we present the sequencing read depth (e.g., the number of sequencing reads that aligned over each CpG). The pink vertical bar indicates the genomic region that seems to be differentially methylated. This region spans 96 bp and contains 7 CpGs, all of which indicate a decrease in methylation in DE-exposed animals at a Wald value of P < 0.01.