Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

Abstract

The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.

Fig 1. In utero disruption of AHR signaling affects the adult cardiovascular function.

Naïve Ahr ^+/+ and Ahr ^-/- and high and low dose TCDD-exposed mice were assessed for cardiovascular function on postnatal days (PND) 60, 90, 150, and 270, corresponding to 2, 3, 5, and 9 months of age, respectively. Absolute left ventricle mass (mg) (A), normalized left ventricle mass (B), left ventricle volume (μL) (C), and ejection fraction (%) (D) were derived from echocardiographic examination of heart function. Conscious heart rate (F) and systolic blood pressure (G) were determined by the tail-cuff method on PND 270. Exercise endurance (minutes) was assessed by forced treadmill exercise on 2, 3, 5, and 9 months of age. All data is expressed as mean ± SEM where *p< 0.05.

Fig 2. In utero disruption of AHR signaling affects adult heart structure and transcriptome.

(A) Heart weights (normalized to body weight) at necropsy (10 months of age, PND 300)) from naïve Ahr ^+/+ and Ahr ^-/- and high and low dose AHR-ligand exposed mice. Wheat germ agglutinin (B) staining of 5-μm sections were used to highlight the cross-sectional myofibers profiles which were used to ascertain the myofiber cross-sectional area (C), which are expressed as means ± minimum and maximal measured areas. Nuclei are stained in blue by DAPI-counterstain; 40X objective. Masson’s trichrome staining (D) was used to highlight the myocardial extracellular matrix as a proxy for fibrosis (E); 40X objective. (F) Number of differentially expressed genes in the adult heart as a consequence of AHR disruption in utero, from Ahr ^-/- and TCDD-exposed Ahr ^+/+ right atrium (RA), left atrium (LA), and ventricle (V) tissue from males and females. (G) Compartment-specific number of persistent genes (i.e. differentially expressed in the embryo heart and in the adult heart).

Fig 3. Differentially expressed canonical pathways, toxicological functions, and upstream regulators underlie AHR disruption-driven altered structure and function.

(A) Fold change expression of differentially expressed Ingenuity Pathway Analysis (IPA) canonical pathways and toxicological functions from and Ahr ^-/- females and high and low dose TCDD-exposed females relative to naïve Ahr ^+/+. (B) IPA’s upstream regulators from and Ahr ^-/- and high and low dose TCDD-exposed mice relative to naïve Ahr ^+/+.

Fig 4. In utero disruption of AHR signaling modestly affects calcium-handling signaling.

Fold change mRNA expression of Nkx2-5 (A), Slc8a2 (B), Ryr2 (C), and Pln (D), and protein expression of NCX (E) from naïve Ahr ^+/+ and Ahr ^-/- and high and low dose TCDD-exposed mice at 10 months of age (PND 300) (mean ± SEM).

Fig 5. Mitochondrial dysfunction and altered oxidative phosphorylation genes in the adult heart as a result of in utero disruption of AHR signaling.

Fold change of expression of significantly altered genes in (A) functional mitochondrial pathways and (B) oxidative phosphorylation (B) at 10 months of age (PND 300) of high dose TCDD-exposed Ahr ^+/+ female adult hearts.

Fig 6. Uncoupling proteins are differentially expressed following in utero disruption of AHR signaling.

Fold change of expression of Ucp1 (A), Ucp2 (B), and Ucp3 (C) at 10 months of age (PND 300) of naïve Ahr ^+/+ and Ahr ^-/- and high and low dose TCDD-exposed mice.

Fig 7. Disruption of AHR signaling induces changes in mitochondria abundance and structure.

(A) Quantification of heart mitochondrial DNA relative to nuclear DNA, defined as the ratio of mtDNA to nDNA ratio at 10 months of age (PND 300) and expressed as the mean fold change relative to naïve Ahr ^+/+ hearts ± SEM; * p≤0.05. (B) Ultra-thin sections of embryonic hearts collected at 10 months of age from Ahr ^-/- and Ahr ^+/+, either naïve or exposed to high or low dose ligand in utero, were used for transmission electron microscopy evaluation of the embryonic heart ultrastructure. Illustrative photomicrographs showing the density of mitochondria (arrowheads) within the cardiomyocyte sarcoplasm in Ahr ^-/- and in high and low dose TCDD-exposed Ahr ^+/+ mice compared to naïve Ahr ^+/+. Uranyl acetate and lead citrate stain. Scale bar = 2 μm.