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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session E

Poster H11 - Piezo1 Channel Activation Rescues the Hypoplastic Left Heart Syndrome in Avian Embryos

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Congenital heart defects (CHDs) are estimated to affect around 2% of live births. Only 20% of CHDs are attributed to genetic causes[1], implying that there may be other factors, such as cellular and environmental interactions, that influence development. Among these defects, hypoplastic left heart syndrome (HLHS) is particularly severe, accounting for 25–40% of all neonatal cardiac deaths if left untreated[2]. In HLHS, structures on the left side of the heart remain underdeveloped, which compromises the heart's ability to pump blood, leading to low and oxygen-poor blood flow[3].

Notably, hemodynamics plays a crucial role in heart formation. Hemodynamic changes activate several necessary mechanotransduction pathways in cardiac cells[4], though the exact relationship between hemodynamics and cardiac development remains unclear. We suspect a possible mechanism to improve clinical outlook of HLHS through modulating hemodynamically-mediated developmental pathways. To model HLHS, we conduct left atrial ligation (LAL) surgery on embryonic chicks, causing hemodynamics disturbances in the left ventricle[2]. Then, we investigated a pathway involving the Piezo1 channel, which is previously observed to respond to hemodynamic changes to determine cardiac activity[5]. We stimulated this pathway through the introduction of Yoda1, a Piezo1 channel agonist. We hypothesize that reduced blood flow on the left side due to LAL surgery leads to decreased Piezo1 activation, and therefore activating Piezo1 using Yoda1 could serve as a rescue measure against HLHS. We observe the effects of Yoda1 on heart development at various concentrations to analyze the morphology and expression of markers that indicate maturity and function of the ventricles.

Materials and

Methods:  2.1 Embryo Culture and Preparation
Fertilized eggs are cultured at 37.5°C, 60% humidity, and continuous rocking for 4 days. The eggs are then transferred to a stationary incubator, positioned with the air pocket facing upward. The top shell over the air pocket is exposed.

2.2 LAL Procedure
The LAL procedure is adapted from previously published work[2]. Notably, the surgery is conducted over the air pocket area instead of the side as previously reported. On the chicken embryos at Hamburger-Hamilton stage 21 (HH21), the chorionic and allantoic membranes are removed, and the left atrium is then tied using 10-0 nylon surgical sutures. The knot is tightened to reduce the left atrium volume by 75% and restrict blood flow through the left side of the AV canal.

2.3 Yoda1 Injection
The injection procedure is performed on post-ligation chicken embryos at HH23. Using the pre-pulled calibrated pipette tip, 5µL of Yoda1 concentrations at 5µM, 15µM, 20µM, 25µM, and 30µM is injected at the corner of the blood vessel where it bifurcates into smaller branches. The control group received HBSS (0µM).

2.4. Data Analysis
The data is collected at HH38 to record the viability at different concentrations. Morphological assessments are conducted using nano-CT imaging and quantitative cell/tissue immunohistochemistry (IHC) to identify potential changes in heart morphology or prevention of CHDs. Moreover, pipette aspiration (PA), previously reported from the lab[6], was performed to assess the mechanical properties of the muscle and tissue in the ventricles and septum walls.

Results, Conclusions, and Discussions: LAL chicken embryos injected with Yoda1 concentration were sacrificed at HH38. The percentage of viability (Figure 1), microscopic images (Figure 2), nano-CT imaging (Figure 3), IHC (Figure 4), and PA (Figure 5) were recorded.

Viability was calculated by dividing the number of surviving embryos at HH38 by the total number injected at HH23 (Figure 1). The viability in the experimental group is significantly higher than the control group. The viability peaks at 15µM, suggesting that Yoda1 increases the viability in a dose dependent manner and positively influences heart blood health optimally around 15µM. The decrease in viability above 15µM indicates adverse overdosage of Yoda1 on cardiac development.

Figure 2 captures the heart morphology at different Yoda1 concentrations under the microscope. Deeper morphological assessments were conducted through nano-CT imaging and IHC. In figure 3, the region on the left side of the heart is visibly smaller, confirming that LAL generates a phenotype resembling HLHS. We then compared the heart morphology with Yoda1 injected groups to investigate whether Yoda1 can increase the left side and help recover from HLHS. Figure 4 signifies an increase in the size of the left side with higher Yoda1 concentration, reversing the effect of LAL. It provides evidence that Yoda1 may serve as a potential mechanobiological gain-of-function rescue for HLHS.

Furthermore, PA analysis on the left ventricle wall was conducted to examine the impact of mechanical properties on cardiac development. Figure 5 demonstrates an increase in the rate of stiffness in the experimental group (5µM) compared to the control group (0µM). This finding reveals that Yoda1 contributes to Piezo1 activation and enhances the mechanical activity of the heart, building stronger and stiffer muscles essential for normal heart function.

We conclude that Yoda1 helps rescue LAL induced HLHS as it demonstrates the possibility of reversing HLHS progress through Piezo1 activation. Our data suggests the function of Piezo1 in the mechanobiology of the ventricle development, but more data analysis will be conducted to elucidate this process.

Acknowledgements (Optional):  [1] Blue, Gillian M, Edwin P Kirk, Gary F Sholler, Richard P Harvey, and David S Winlaw. “Congenital Heart Disease: Current Knowledge about Causes and Inheritance.” Medical Journal of Australia 197, no. 3 (August 2012): 155–59. https://doi.org/10.5694/mja12.10811.

[2] Salman, Huseyin Enes, Maha Alser, Akshay Shekhar, Russell A. Gould, Fatiha M. Benslimane, Jonathan T. Butcher, and Huseyin C. Yalcin. “Effect of Left Atrial Ligation-Driven Altered Inflow Hemodynamics on Embryonic Heart Development: Clues for Prenatal Progression of Hypoplastic Left Heart Syndrome.” Biomechanics and Modeling in Mechanobiology 20, no. 2 (April 2021): 733–50. https://doi.org/10.1007/s10237-020-01413-5.

[3] "Normal Heart Anatomy vs. Hypoplastic Left Heart Syndrome (HLHS) Anatomy." YouTube, uploaded by NationwideChildrens, 30 August 2017, https://www.youtube.com/watch?v=cC6fYCvCvsc&t=1s.

[4] Courchaine, Katherine, Graham Rykiel, and Sandra Rugonyi. “Influence of Blood Flow on Cardiac Development.” Progress in Biophysics and Molecular Biology 137 (September 2018): 95–110. https://doi.org/10.1016/j.pbiomolbio.2018.05.005.

[5] Blythe, Nicola M., Katsuhiko Muraki, Melanie J. Ludlow, Vasili Stylianidis, Hamish T.J. Gilbert, Elizabeth L. Evans, Kevin Cuthbertson, et al. “Mechanically Activated Piezo1 Channels of Cardiac Fibroblasts Stimulate P38 Mitogen-Activated Protein Kinase Activity and Interleukin-6 Secretion.” Journal of Biological Chemistry 294, no. 46 (November 2019): 17395–408. https://doi.org/10.1074/jbc.RA119.009167.

[6] Buskohl, Philip R., Russell A. Gould, and Jonathan T. Butcher. “Quantification of Embryonic Atrioventricular Valve Biomechanics during Morphogenesis.” Journal of Biomechanics 45, no. 5 (March 2012): 895–902. https://doi.org/10.1016/j.jbiomech.2011.11.032.