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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 38 - ChRmine optogenetic cardiac pacing in Drosophila melanogaster depends on the expression level

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • Vivian Wang

    High School Student
    John Burroughs School
    Clayton, Missouri, United States

    • Co-Author(s)

  • EG

    Elena Gracheva, PhD

    Staff Scientist
    Department of Biomedical Engineering, Washington University in St. Louis, Missouri, United States

  • Fei Wang

    Graduate Student
    Washington University in St. Louis
    University City, Missouri, United States

    • Primary Investigator(s)

  • CZ

    Chao Zhou

    Professor
    Washington university in St. louis, United States


Introduction: Drosophila melanogaster (fruit fly) is an effective model organism for cardiovascular research. Recently, optogenetics started playing a significant role in this field. Optogenetic methods include expression of light sensitive proteins, opsins, in spatiotemporal manner, illumination of the tissue of interest with light of specific wavelength, and detection of the resulting effects using high resolution optical systems, e.g. Optical Coherence Microscopy (OCM). In previous studies, an integrated optogenetic light stimulation and OCM imaging system (Gracheva et al., 2022) was utilized for non-invasive cardiac pacing; this was achieved by using red light (617 nm) to illuminate transgenic Drosophila expressing red-activatable channelrhodopsin (ReaChR) in the heart. Light was delivered in short (20 ms) pulses with a frequency exceeding the normal heart rate. The resulting heart rates followed the frequencies of the light pulses. To move towards the application of optical pacing technology in patients with heart disorders, we need to find sensitive opsins that can respond to limited light penetration through layers of tissue and ensure accurate pace making. ChRmine (Kishi et al., 2022) has successfully stimulated mouse neocortex (Marshel et al., 2019) and can potentially be used in cardiovascular applications. To model heart pacing, we employed Drosophila versatile UAS/GAL4 genetic system to express the recently developed transgenic ChRmine opsin in the heart tissue. In this study, we determined the impact of two distinct heart specific GAL4 drivers on ChRmine’s pacing efficiency. With further research, optogenetic pacing can provide millions of patients with a reliable and non-invasive alternative to surgically implanted electrical pacers.

Materials and

Methods: Drosophila Genetics:
We crossed virgin females (w;P{〖y^+ w〗^+,Hand-GAL4}/ TM6 Tb or y[1]w[*];Mi{Trojan-GAL4.0}Hand[MI04106-TG4.0]/SM6a ) from the Bloomington Drosophila Stock Center at the University of Indiana (stock 48397 and 66795 respectively) with transgenic males (yw;P{w^+,UAS-ChRmine-Scarlet}/ TM6 Tb) developed in the Zhou lab at Washington University in St. Louis. Non-tubby progeny were selected for OCM imaging. The TM6 Tb (tubby) marker allows for distinction of progeny that do not carry both the ChRmine transgene and the GAL4 driver. Animals expressing ChRmine were also verified based on visual detection of Scarlet fluorescent marker.
Procedures:
• Females and males were placed and kept in ATR-containing media vials in the dark at 25°C, 70% humidity. We followed crossing procedures outlined in this previous study. (Gracheva et al., 2022)
• Early stage (white) pupae were collected 7 days after egg laying and OCM imaging data was collected as described previously. (Gracheva et al., 2022)
• Imaging data was processed using FlyNet3.0 software. (Ouyang et al., 2024)

Results, Conclusions, and Discussions: Results
We have demonstrated the ability of ChRmine opsin to mediate Drosophila heart tachypacing. We obtained animals having different expression levels of ChRmine using two different GAL4 drivers, Hand-GAL4 and Trojan-Hand-GAL4. We acquired dynamic heart images and evaluated pacing probabilities at various 617 nm light power levels, then compared the efficiency of Hand>ChRmine and Trojan-Hand>ChRmine. Our results suggest that higher expression level plays a significant role in successful heart pacing.
Discussion
Based on this study, ChRmine appears to be able to successfully pace Drosophila heart; however, it’s pacing ability in comparison to other opsins has not been assessed previously. Trojan-Hand-GAL4 drives higher expression than Hand-GAL4 when crossed with ChRmine, resulting in higher sensitivity. The research conducted suggests that the Trojan-Hand-GAL4 and UAS-ChRmine genetic combination warrants further study and development. Optogenetic pacing has proven to be an effective model system for developing non-invasive, highly precise pacing therapies and studying heart diseases and critical conditions such as heart failure. Further research will allow for progression towards applications in mammals.

Acknowledgements (Optional): Acknowledgements
Many thanks to the Zhou lab members at Washington University in St. Louis for providing scientific advice and enriching discussions, and to Hongwu Liang for technical assistance.

References (Optional): References
Gracheva, E., Wang, F., Matt, A., Liang, H., Fishman, M., & Zhou, C. (2022). Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function. Journal of Visualized Experiments, 186, 63939. https://doi.org/10.3791/63939
Kishi, K. E., Kim, Y. S., Fukuda, M., Inoue, M., Kusakizako, T., Wang, P. Y., Ramakrishnan, C., Byrne, E. F. X., Thadhani, E., Paggi, J. M., Matsui, T. E., Yamashita, K., Nagata, T., Konno, M., Quirin, S., Lo, M., Benster, T., Uemura, T., Liu, K., … Kato, H. E. (2022). Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine. Cell, 185(4), 672-689.e23. https://doi.org/10.1016/j.cell.2022.01.007
Marshel, J. H., Kim, Y. S., Machado, T. A., Quirin, S., Benson, B., Kadmon, J., Raja, C., Chibukhchyan, A., Ramakrishnan, C., Inoue, M., Shane, J. C., McKnight, D. J., Yoshizawa, S., Kato, H. E., Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science, 365(6453), eaaw5202. https://doi.org/10.1126/science.aaw5202
Ouyang, X., Matt, A., Wang, F., Gracheva, E., Migunova, E., Rajamani, S., Dubrovsky, E. B., & Zhou, C. (2024). Attention LSTM U-Net model for Drosophila melanogaster heart tube segmentation in optical coherence microscopy images. Biomedical Optics Express, 15(6), 3639. https://doi.org/10.1364/BOE.523364