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    Cardiovascular Engineering

    Investigating the contractility of zebrafish cardiomyocyte by modulating myocardial Notch in vitro

    (G-259) Investigating the contractility of zebrafish cardiomyocyte by modulating myocardial Notch in vitro

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row G - Poster # 259

      Presenting Author(s)

    • Nguyen Phuc Nguyen, bachelor (he/him/his)

      Undergraduate student
      University of Texas at Arlington
      Arlington, Texas, United States

      • Co-Author(s)

    • AK

      Ayesha Khan

      PhD Student
      University of Texas at Arlington, United States

    • KN

      Kytai Nguyen

      Professor
      The University of Texas at Arlington, United States

    • BS

      Byung Ran So

      Professor
      University of Texas at Arlington, United States

      • Last Author(s)

    • JL

      Juhyun Lee

      Associate professor
      University of Texas at Arlington, United States

    Introduction:: Heart failure is characterized by the inability of the heart to pump enough blood to meet the body's needs. Heart failure arises due to cardiomyocyte damage following myocardial infarction. The regeneration potential of mammalian hearts is limited as cardiomyocytes lack the capability to proliferate. Consequently, the formation of non-contractile scar tissue post-heart attack can contribute to the progression of heart failure. Zebrafish present a valuable model for heart regeneration studies as they possess the remarkable ability to heal damaged heart muscles and regenerate contractility at the injury site. Utilizing notch1 CRISPR knockout plasmids to investigate the cellular mechanisms underlying regeneration in zebrafish cardiomyocytes holds significant promise as a potential therapeutic avenue for treating human heart failure.

    Materials and Methods:: The significance of this study lied in elucidating the indispensable role of Notch signaling, specifically the notch1b gene, as a critical signaling molecule in cardiac regeneration. Zebrafish cardiomyocytes were isolated and cultured in vitro to investigate the role of Notch signaling in cardiac regeneration. [1] Collagenases II and IV were used to digest the collected zebrafish heart, and the cardiomyocytes were purified through seven washing buffers containing increasing calcium chloride concentration, a process known as the calcium paradox, which helps to isolate cardiomyocytes from other cell types in the heart. After successful isolation, CRISPR knockout plasmids modulated the zebrafish notch1b gene. The plasmid contained a U6 promoter and GFP genes, which allowed us to track gene delivery and expression efficiency. Lipofection was employed to introduce the genes into the cardiomyocytes, with PCR amplification and Western blotting utilized to confirm gene expression. We transfected the CRISPR plasmid using the ViafectTM reagent for the successful transfection of the cardiomyocytes after cell sorting.

    Results, Conclusions, and Discussions:: Results:  We successfully isolated zebrafish cardiomyocytes and performed notch1b CRISPR knockout plasmid transfections maintained for up to 30 days. This long-term maintenance was beneficial for observing transfected cells and the effects of notch1b knock-out on cardiomyocyte contractility. We also successfully sub-passed cells from primary to secondary cells (Fig. 1). This sub-passing proved beneficial for cell sorting, PCR amplification, and Western blot results. Compared to non-transfected cells, unlike endocardial Notch, the notch1b CRISRP plasmid-transfected cardiomyocytes exhibited a higher rate of contraction and enhanced contractility, as quantified through cell tracking methods (2).
    Conclusion and Discussion: We used the CRISPR notch1b plasmid to knockout the Notch signaling pathway in zebrafish cardiomyocytes which is the opposite event of endocardial Notch signaling. We will pursue this by using nanoparticles to deliver the plasmid to the target cardiomyocytes to test in vivo experiments with the zebrafish model.


    Acknowledgements (Optional): :

    References (Optional): : [1] Sander V, Suñe G, Jopling C, Morera C, Izpisua Belmonte JC. Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts. Nat Protoc. 2013 Apr;8(4):800-9. doi 10.1038/nprot.2013.041. Epub 2013 Mar 28. PMID: 23538883
    [2] Tanveer Teranikar, Cameron Villarreal, Nabid Salehin, Toluwani Ijaseun, Jessica Lim, Cynthia Dominguez, Vivian Nguyen, Hung Cao, Cheng–Jen Chuong, Juhyun Lee. Scale-space detector for analyzing spatiotemporal ventricular contractility and nuclear morphogenesis in zebrafish. iScience, Volume 25, Issue 9, 2022, 104876, ISSN 2589-0042, https://doi.org/10.1016/j.isci.2022.104876