Long-term consequences of disrupting adenosine signaling during embryonic development

Abstract

There is growing evidence that disruption in the prenatal environment can have long-lasting effects on an individual's health in adulthood. Research on the fetal programming of adult diseases, including cardiovascular disease, focuses on epi-mutations, which alter the normal pattern of epigenetic factors such as DNA methylation, miRNA expression, or chromatin modification, rather than traditional genetic alteration. Thus, understanding how in utero chemical exposures alter epigenetics and lead to adult disease is of considerable public health concern. Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists including the methlyxanthines caffeine and theophylline are widely consumed during pregnancy. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). We examined the long-term effects of in utero disruption of adenosine signaling on cardiac gene expression, morphology, and function in adult offspring. One substance that fetuses are frequently exposed to is caffeine, which is a non-selective adenosine receptor antagonist. Over the past several years, we examined the role of adenosine signaling during embryogenesis and cardiac development. We discovered that in utero alteration in adenosine action leads to adverse effects on embryonic and adult murine hearts. We find that cardiac A1ARs protect the embryo from in utero hypoxic stress, a condition that causes an increase in adenosine levels. After birth in mice, we observed that in utero caffeine exposure leads to abnormal cardiac function and morphology in adults, including an impaired response to β-adrenergic stimulation. Recently, we observed that in utero caffeine exposure induces transgenerational effects on cardiac morphology, function, and gene expression. Our findings indicate that the effects of altered adenosine signaling are dependent on signaling through the A1ARs and timing of disruption. In addition, the long-term effects of altered adenosine signaling appear to be mediated by alterations in DNA methylation, an epigenetic process critical for normal development.

Keywords: Caffeine; Cardiac; Cardiomyopathy; Heart disease; Transgenerational.

Figure 1. Altered gene and miRNA expression in embryonic hearts exposed to in utero caffeine

Caffeine treatment of pregnant dams from E6.5–10.5 altered the expression of important cardiac genes in embryonic hearts at E10.5. In utero caffeine treatment altered expression of cardiac transcription factors, structural genes, DNA methylation enzymes, and miRNAs. Bars ± SEM represent fold changes normalized to β-actin or snRNA RNU6-2 and relative to individual controls (n=5–7, ** P<0.01, *** P<0.001 vs. vehicle).

Figure 2. Timing of in utero caffeine exposure leads to different cardiac phenotypes in adult offspring

The timing of in utero caffeine treatment leads to differences in adult cardiac function, gene expression, and phenotype. Exposure to caffeine from E6.5–9.5 leads the F1 generation to develop dilated cardiomyopathy with decrease % FS and increased Myh7 expression. In utero caffeine exposure from E10.5–13.5 leads to a hypertrophic cardiomyopathy in the F2 generation along with increased % FS and decreased Myh7 expression.