Mechanisms for the adverse effects of late gestational increases in maternal cortisol on the heart revealed by transcriptomic analyses of the fetal septum

Abstract

We have previously shown in sheep that 10 days of modest chronic increase in maternal cortisol resulting from maternal infusion of cortisol (1 mg/kg/day) caused fetal heart enlargement and Purkinje cell apoptosis. In subsequent studies we extended the cortisol infusion to term, finding a dramatic incidence of stillbirth in the pregnancies with chronically increased cortisol. To investigate effects of maternal cortisol on the heart, we performed transcriptomic analyses on the septa using ovine microarrays and Webgestalt and Cytoscape programs for pathway inference. Analyses of the transcriptomic effects of maternal cortisol infusion for 10 days (130 day cortisol vs 130 day control), or ∼25 days (140 day cortisol vs 140 day control) and of normal maturation (140 day control vs 130 day control) were performed. Gene ontology terms related to immune function and cytokine actions were significantly overrepresented as genes altered by both cortisol and maturation in the septa. After 10 days of cortisol, growth factor and muscle cell apoptosis pathways were significantly overrepresented, consistent with our previous histologic findings. In the term fetuses (∼25 days of cortisol) nutrient pathways were significantly overrepresented, consistent with altered metabolism and reduced mitochondria. Analysis of mitochondrial number by mitochondrial DNA expression confirmed a significant decrease in mitochondria. The metabolic pathways modeled as altered by cortisol treatment to term were different from those modeled during maturation of the heart to term, and thus changes in gene expression in these metabolic pathways may be indicative of the fetal heart pathophysiologies seen in pregnancies complicated by stillbirth, including gestational diabetes, Cushing's disease and chronic stress.

Keywords: cortisol; fetal heart; late gestation; metabolism; mitochondria.

Fig. 1.

Volcano plots showing probes upregulated (red), downregulated (green), and unchanged (black) in fetal septa of ewes treated with cortisol from ∼120 to 130 days of gestation (top) or from ∼115 of gestation to term (bottom). The x-axis represents the difference in the expression between cortisol and control, changes in the positive direction being greater in cortisol than control. The y-axis is the negative log of the P value, the solid line represents P = 0.05, the level of significance accepted in the analyses; probes above this line were significantly differently regulated by cortisol in these septa. CON, control; CORT, cortisol; d, day.

Fig. 2.

Validation of the microarray and pathway analyses with quantitative real-time PCR. Microarray and pathway analyses suggested that TXNIP (top right), PDK4 (bottom right), and CYP26B1 (bottom left) gene expression were significantly increased in 140-day CORT (red) compared with 140-day CON (black) in the response to nutrient and to hypoxia pathways. Pathway analyses suggested that responses to nutrient were altered and PCR confirmed an increase in SOCS3, part of this pathway, but not found on the microarray. All of these genes were significantly differently expressed at P < 0.05 (shown by *); note the difference in y-axis scale between plots.

Fig. 3.

Volcano plot showing probes upregulated (red), downregulated (green), and unchanged (black) in fetal septa of 140 days gestation compared with 130 days gestation. The x-axis represents the difference between the expression at 140 and 130 days gestation, changes in the positive direction being greater at 140 days than at 130 days. Probes above the line indicating P = 0.05 were significantly differently regulated by maturation of these septa. Maturation significantly altered more genes than did cortisol at either age; the magnitude of change was also greater in many cases (Difference in scale of y-axes in Figs. 1 and 3 indicates a greater effect of gestational age than of cortisol on gene expression).

Fig. 4.

Volcano plots of the expression distribution of the probes on the array in the fetal septa, illustrating the maturational effects (difference between 140-day CON and 130-day CON, ○) with the probes altered in 130-day CORT (difference of 130-day CORT and 130-day CON) (top) or 140-day CORT (difference of 140-day CORT and 140-day CON) (bottom) shown in colors. Probes significantly differentially expressed with maturation are drawn above the solid line (P < 0.05). The red symbols are those probes whose expression was significantly increased by CORT (P < 0.05, at 130 day, top, or at term, 140 days, bottom) (i.e., 140 days vs 130 days in CON fetuses). The green symbols are the probes significantly decreased by CORT (P < 0.05; 130-day CORT, top, or 140-day CORT, bottom). Note that some genes are changed by CORT, but not by maturation (colored symbols below the significance line), and some were changed in the opposite direction by CORT compared with the effect of gestational age (e.g., red symbols on the negative x-axis side of the volcano plot, or green symbols on the positive x-axis side of the plot). In all cases y-axis values reflect the P values for gene expression due to the maturational effect.