Session: 16-01-01: Government Agency Student Poster Competition

Paper Number: 150524

150524 - Cyclopamine Dosing in an Embryonic Chicken Model Induces Decreased Blood Flow Velocity and Conformational Changes in the Developing Heart Outflow Tract 

INTRODUCTION:

Interactions between signaling pathways and biophysical forces are critical for embryonic heart development, and developmental defects are associated with signaling disruptions and changes in biophysical forces in cardiac tissue. How genetic programs and biophysics synergistically interact during heart formation is not completely understood.

We explore the quantitative changes in blood flow velocity and heart morphology associated with cyclopamine-induced signaling disruption in a chicken embryo model of heart development. Early during development, the heart has a tubular structure containing the outflow tract (OFT) and its cushions, which later develops into the left and right ventricular outlets. Hamburger Hamilton (HH) Staging is the accepted method to stage chick embryos throughout development. The Hedgehog signaling pathway, including the protein Sonic Hedgehog (Shh), mediates crucial cell migration during heart development, and Shh knockdown is known to result in heart defects. Cyclopamine is a natural steroidal alkaloid which induces developmental defects such as Double Outlet Right Ventricle (DORV), Tetrology of Fallot (TOF), and Patent Ductus Arteriosus (PTA) by inhibiting the Hedgehog signaling pathway. Blood flow and morphology of the OFT in chick embryos treated with cyclopamine is measured to explore the relationship between heart defects and changes in biophysical forces. 

METHODS:

 

Chicken embryos provide a useful model for the investigation of early changes in embryonic development and their association with heart defects. 

Cyclopamine dissolved in a vehicle of EtOH/ePBS at a concentration of 0.8 μg/μL was applied to windowed chick embryos at HH14. Control embryos were either left untreated, dosed with ePBS alone, or dosed with the EtOH/ePBS vehicle solution. After dosing, embryonic hearts were imaged in vivo with optical coherence tomography at HH18, and then again with ultrasound at HH31. 

 

RESULTS:

Blood velocity in the outflow tract was reduced in cyclopamine-treated embryos at HH18 in comparison to control groups. Cyclopamine-dosed embryos (n=46) at HH18 had a mean peak OFT velocity of 31.24 +/- 0.75 mm/s, while untreated embryos (n=16), ePBS-dosed embryos (n=20), and vehicle-dosed embryos (n=12) had mean peak velocities of 35.67 +/- 1.28, 36.45 +/- 1.14, and 38.42 +/- 1.47 mm/s, respectively. Student’s t-tests revealed significant differences between cyclopamine-dosed embryos and the control groups (p=0.0186, p=0.0014, p=0.002). Mean peak velocities measured at HH31 were not significantly different between groups.

Ventral cushion thickness in untreated embryos was larger in comparison to vehicle-dosed and cyclopamine-dosed embryos. Cyclopamine-dosed embryos (n=27) at HH14 had an average ventral cushion thickness of 0.12079 +/- 0.024 mm, untreated embryos (n=7) 0.14552 +/- 0.026 mm, ePBS-dosed embryos (n=9) 0.136 +/- 0.0199 mm, and vehicle-dosed embryos (n=7) 0.11633 +/- 0.0278 mm. Student’s t-tests comparing untreated ventral OFT thickness to vehicle-dosed and cyclopamine-dosed groups returned significant differences (p=0.0289 and p=0.0201).

 

DISCUSSION/CONCLUSIONS:

These experiments correlate hedgehog pathway inhibition with quantitative mechanical changes early in OFT development. Because biophysical forces exerted by abnormal blood flow on cardiac tissues independently leads to congenital heart defects, our work shows how inhibition of the Shh signaling pathway leads to altered blood flow and, ultimately, the interaction of both abnormal signaling and biophysics drives abnormal heart development. Thus our work continues to elucidate the mechanism by which heart defects such as VSD, TOF, and DORV develop. In future experiments, positive identification of TOF, DORV, PTA in later stages of embryonic development via microCT imaging will be used to further confirm the correlation between decreased OFT velocity at HH18 and the development of heart defects. Determining the early-stage mechanical changes by which heart defects form paves the way for future research toward earlier diagnosis and treatment of congenital heart diseases.

Presenting Author: Makena Phillips Oregon Health and Science University

Presenting Author Biography: Makena Phillips is a first year Ph.D. student at OHSU in the Biomedical Engineering program, working under the Principal Investigator, Dr. Sandra Rugonyi. She received her bachelor's degree from Lewis & Clark College, graduating with a degree in Biochemistry and Molecular Biology.

Authors:

Nathan Petrucci Oregon Health and Science University
Rachel Opferman Oregon Health and Science University
Makena Phillips Oregon Health and Science University
Sandra Rugonyi Oregon Health and Science University