Background
Exposure to cadmium (Cd) is associated with cardiovascular diseases. Maternal Cd exposure is a significant risk factor for congenital heart disease. However, mechanisms of Cd on developmental cardiotoxicity are not well defined. Objectives: We evaluated the effects of Cd on the different stages (mesoderm, cardiac induction, cardiac function) of cardiac development using an early embryo development in vitro model and two- or three-dimensional (2- or 3D) cardiomyocyte and cardiac organoid formation models mimicking early cardiac development.
Discussion
In conclusion, using a human ESC-derived 2D/3D in vitro differentiation model system and cardiac organoids, we demonstrated that low-dose Cd suppressed mesoderm formation through mesoderm gene histone modification, thus inhibiting cardiomyocyte differentiation and cardiac induction. The studies provide valuable insights into cellular events and molecular mechanisms associated with Cd-induced congenital heart disease. https://doi.org/10.1289/EHP11208.
Methods
Embryonic stem cells (ESCs) form 3D aggregates, called embryoid bodies, that recapitulate events involved with early embryogenesis (e.g., germ layer formation). This model was used for early germ layer formation and signaling pathway identification. The 2D cardiomyocyte differentiation from the <math><mrow><mi>N</mi><mi>K</mi><mi>X</mi><mi>2</mi><mo>-</mo><msup><mrow><mi>5</mi></mrow><mrow><mrow><mi>e</mi><mi>G</mi><mi>F</mi><mi>P</mi></mrow><mo>/</mo><mi>w</mi></mrow></msup></mrow></math> human ESCs model was used to explore the effects of Cd exposure on cardiomyocyte formation and to model mesoderm differentiation and cardiac induction, allowing us to explore different developmental windows of Cd toxicity. The 3D cardiac organoid model was used in evaluating the effects of Cd exposure on contractility and cardiac development.
Results
Cd (0.6μM<math><mrow><mn>0.6</mn><mspace></mspace><mi>μ</mi><mi>M</mi></mrow></math>; 110 ppb<math><mrow><mn>110</mn><mtext> ppb</mtext></mrow></math>) lowered the differentiation of embryoid bodies to mesoderm via suppression of Wnt/β-catenin<math><mrow><mtext>Wnt</mtext><mo>/</mo><mi>β</mi><mtext>-catenin</mtext></mrow></math>-signaling pathways. During early mesoderm induction, the mesoderm-associated transcription factors MESP1 and EOMES showed a transient up-regulation, which decreased later in the cardiac induction stage. Cd (0.15μM<math><mrow><mn>0.15</mn><mspace></mspace><mi>μ</mi><mi>M</mi></mrow></math>) lowered mesoderm formation and cardiac induction through suppression of the transcription factors and mesoderm marker genes HAND1, SNAI2, HOPX, and the cardiac-specific genes NKX2-5, GATA4, troponin T, and alpha-actinin<math><mrow><mtext>alpha</mtext><mtext>-actinin</mtext></mrow></math>. In addition, Cd-induced histone modifications for both gene activation (H3K4me3) and repression (H3K27me3), which play vital roles in regulating mesoderm commitment markers. The effects of Cd inhibition on cardiomyocyte differentiation were confirmed in 3D cardiac organoids.