183 - High resolution multiplexed gene expression mapping of intact hearts to reveal mechanisms of congenital heart disease

Publication Number: 183.5550

Junwoo Suh, Case Western Reserve University School of Medicine, Cleveland, OH, United States; Bosco Bakwatanisa, Case Western Reserve University School of Medicine, Cleveland, OH, United States; Andrew M. Rollins, Case Western Reserve University, Cleveland, OH, United States; Michael Jenkins, Case Western Reserve University School of Medicine, Cleveland, OH, United States; Michiko Watanabe, Case Western Reserve University, Kent, OH, United States; Stephanie Ford, UH Rainbow Babies & Children’s Hospital, Case Western Reserve University school of medicine, Cleveland, OH, United States

Background: Fetal Alcohol Spectrum Disorder (FASD) from prenatal alcohol exposure (PAE) is linked to congenital anomalies, including congenital heart disease (CHD), affecting up to 40% of children with FASD. These CHDs often involve abnormalities in the endocardial cushions and atrioventricular junction (AVJ), which are shaped by hemodynamic forces and gene regulation to create valves and septa. A study using multimodal imaging—Doppler OCT, RNA fluorescence in situ hybridization (FISH), and confocal microscopy—could uncover the complex interplay between function, anatomy, and molecular activity in heart development. However, studying the embryonic heart is challenging due to its small size, rapid morphological changes, and the variability in malformations.

Objective: Here, we used a novel combination of tissue clearing and multiplexed 3D RNA FISH imaging to investigate CHD mechanisms in quail hearts. Maintaining intact cleared hearts uniquely allows for structural imaging of the looping heart to be matched to precise 3D RNA expression. We then utilized sequential RNA FISH multiplexing to analyze key genes.

Design/Methods: Fertilized quail eggs were incubated at 38°C. At Hamburger and Hamilton (HH) stage 5 (gastrulation), eggs were injected with 40 µL of 50% ethanol to simulate binge drinking; control groups received 40 µL of 0.9% saline or no injection. Eggs were incubated for 3 days (HH 19–21), corresponding to human embryonic days 22–30, when tubular hearts begin looping and forming endocardial cushions. Six embryonic hearts from each group were stained with FISH probes for anatomical (MYH15, TBX18, TIE1), hemodynamic (KLF2, NOS3), and developmental (NOTCH1, HAS2, GJA5, BMP2) markers. Three rounds of FISH imaging were performed on the same hearts using confocal microscopy.

Results: We observed notable differences in gene expression patterns and heart morphology between the PAE and control groups. PAE embryos showed a less defined AVJ, with significant shifts in gene expression. For instance, BMP2, typically localized in the atrium, was expressed closer to the ventricle, and HAS2, essential for cushion formation, showed reduced expression. These abnormalities may be driven by irregular hemodynamics, activating shear stress pathways and triggering endocardial cushion remodeling.

Conclusion(s): The study revealed qualitative differences between control and PAE groups, underscoring the need for more samples to quantify gene expression. Future work with Doppler OCT will clarify the relationships between hemodynamics (i.e. cardiac output, doppler tracing analysis, and shear stress) and molecular expression.

Preliminary figure 1. Control (uninjected) vs. PAE quail heart
Confocal microscopy images of quail hearts showing atrium, AVJ, ventricle and outflow tract. The genes are list on the figure with the corresponding color. a. The heart of control (no injection) embryo. Two rounds of FISH images were registered to form a composite image. b. The heart of prenatal -alcohol exposed embryo. BMP2 expression in the atrium is greatly reduced while expression in AVJ is increased. HAS2 shows reduced fluorescence, suggesting decrease in expression level.