Exposure to low-dose trichloroethylene alters shear stress gene expression and function in the developing chick heart

Abstract

Trichloroethylene is an organic solvent used as an industrial degreasing agent. Due to its widespread use and volatile nature, TCE is a common environmental contaminant. Trichloroethylene exposure has been implicated in the etiology of heart defects in human populations and animal models. Recent data suggest misregulation of Ca2+ homeostasis in H9c2 cardiomyocyte cell line after TCE exposure. We hypothesized that misregulation of Ca2+ homeostasis alters myocyte function and leads to changes in embryonic blood flow. In turn, changes in cardiac flow are known to cause cardiac malformations. To investigate this hypothesis, we dosed developing chick embryos in ovo with environmentally relevant doses of TCE (8 and 800 ppb). RNA was isolated from control and treated embryos at specific times in development for real-time PCR analysis of blood flow markers. Effects were observed on Endothelin-1 (ET-1), Nitric Oxide Synthase-3 (NOS-3) and Krüppel-like Factor 2 (KLF2) expression relative to TCE exposure and consistent with reduced flow. Further, we measured function in the developing heart after TCE exposure by isolating cardiomyocytes and measuring half-width of contraction and sarcomere lengths. These functional data showed a significant increase in half-width of contraction after TCE exposure. These data suggest that perturbation of cardiac function contributes to the etiology of congenital heart defects in TCE-exposed embryos.

Figure 1

A pooled sample of 20 Whole Hearts were collected after 24hr exposure of TCE at 8 ppb and 800 ppb. Real-time PCR results for Endothelin-1 (ET-1), Nitric Oxide Synthase 3 (NOS3) and Krüppel Like Factor 2 (KLF2). ET-1 was unchanged while both NOS3 and KFL2 where significantly reduced at 8 ppb TCE exposure but not at 800 ppb (*** = p-value < 0.001).

Figure 2

HH24 Whole Hearts were collected after 48hr exposure of TCE at 8 ppb and 800 ppb. Real-time PCR results for Endothelin-1 (ET-1), Nitric Oxide Synthase 3 (NOS3) and Krüppel Like Factor 2 (KLF2). ET-1 was unchanged while NOS3 was significantly reduced at both 8 ppb and 800 ppb TCE exposure. Additionally KLF2 was significantly reduced only after 8 ppb TCE exposure (** = p-value < 0.01; * = p-value < 0.05).

Figure 3

KLF2 Antibody Expression in HH17 Whole Hearts collected after approximately 24hr exposure of 8 ppb TCE. KFL2 Antibody expression can be observed in both the endothelium and myocardium of the developing chick heart in both the control and 8 ppb TCE treated samples. Staining in TCE-treated endothelia appears to show an increase in extra-nyclear distribution of the protein.

Figure 4

Half Width of isolated E18 cardiomyocytes exposed to 8 ppb TCE relative to contol cardiomyocytes. Half-width of TCE exposed cardiomyoctes is significantly increased when compared to control cardiomyoctes (*** = p-value < 0.001).

Figure 5

Sarcomere Length of isolated E18 cardiomyocytes exposed to 8 ppb TCE relative to control cardiomyocytes. Sarcomere length is unchanged by TCE exposure.