Poster T20 - In vitro study of the isolated zebrafish cardiomyocytes to understand mechanoresponsiveness of Notch1 signaling

Introduction: Cardiac morphogenesis in mechanobiology is significant important process in both clinical and basic science. Zebrafish has been a popular animal model to study in this field due to many advantages including optical transparency in embryonic stages. In this project, we aim to focus on the mechanistic roles of the Notch1 signaling pathway in cardiac contraction for ventricular development. Notch1b in zebrafish is a crucial regulator of cardiomyocytes proliferation and differentiation. By understanding these processes, we aim to use isolated zebrafish cardiomyocytes for in vitro study to regulate notch1b expression levels by modulate fluid shear stress using the flow channel to understand the relationship between fluid shear stress and notch1b in zebrafish cardiomyocytes.

Materials and

Methods: Part 1: Isolating Cardiomyocyte: Following established protocols, we dissected zebrafish hearts and subjected them to low-calcium perfusion to minimize cellular stress and optimize enzymatic digestion at 32°C, ensuring complete tissue breakdown and maintaining cellular integrity. [1] Reintroducing calcium during the wash phase helped mitigate cellular trauma and improve cardiomyocyte survival. Our culture medium, enriched with 5% fetal bovine serum, supports vital cardiomyocyte functions such as contraction and proliferation. Additionally, poly-L-lysine coatings were used to enhance cardiomyocyte adhesion and structural integrity. Cardiomyocyte morphology and contractility, along with viability indicators including live/dead assays, proliferation markers, and cell counts, were assessed over a four-week period to analyze both structural and functional dynamics.
Part 2: Free DNA Transfection Guide RNAs (gRNAs) targeting the Notch1 gene are designed using tools like CHOPCHOP to maximize specificity and reduce off-target effects. gRNAs are incorporated into vectors for genomic editing. Our transfection protocol involves coating wells with poly-L-lysine, trypsinizing, and transfecting with plasmids using Viafect in Opti-MEM. Post-transfection, cells are monitored for GFP expression, followed by genomic DNA isolation and sequencing to confirm editing.
Part 3: PLGA Nanoparticle Encapsulated Plasmid DNA PLGA nanoparticles encapsulating the PX458 plasmid are synthesized using a double emulsion solvent evaporation method. Parameters like size, charge, and encapsulation efficiency are optimized and characterized by dynamic light scattering and gel electrophoresis. The effectiveness of these nanoparticles in delivering CRISPR components is tested under simulated dynamic blood flow conditions in zebrafish, assessing cytotoxicity with MTT assays and potential inflammatory responses through cytokine assays and immunofluorescent staining.

Results, Conclusions, and Discussions:

Results:
The successful isolation of cardiomyocytes from zebrafish was confirmed by the restoration of contractile function within four to five days post-isolation. These cardiomyocytes demonstrated viability for up to 30 days, facilitating extended observations of transfected cells. Nonetheless, the transfection efficiency observed in these experiments was relatively low, and a noticeable reduction in contractility was observed in the transfected cells. Conversely, the incorporation of nanoparticle encapsulation and myl7 binding antibody techniques, along with the employment of the ibidi system to control environmental conditions, significantly improved transfection efficiency. Maintaining controlled environmental conditions is crucial for acquiring data devoid of physiological artifacts, thereby ensuring the robustness of the results. To be specific, the nanoparticles were optimized for molecular weight, improving their compatibility and stability in medium effectively delivering bioactive plasmid DNA to cardiomyocytes with minimal adverse effects. The optimization of the molecular weight of poly(lactic-co-glycolic acid) (PLGA) nanoparticles significantly enhanced their cytocompatibility, hemocompatibility, and stability. Specifically, higher molecular weight (HMW) PLGA nanoparticles (55–65 kDa) demonstrated superior performance compared to lower molecular weight (LMW) versions in various biological environments. The optimized HMW nanoparticles showed greater stability in saline and serum, exhibited less aggregation, and maintained high viability rates in zebrafish cardiomyocyte. Additionally, these nanoparticles effectively delivered the plasmids into cardiomyocyte, resulting in bioactive gene expression with minimal adverse effects, indicating their potential for safe and efficient gene therapy applications.
Discussion and Conclusion
Our study demonstrates the profound potential of the modulation of Notch1 signaling to rescue cardiac trabeculation from lack of mechanics in zebrafish. Through our methods of isolating cardiomyocytes and enhancing their viability, along with precise genomic editing strategies, we successfully manipulated Notch1 signaling. The significant restoration of contractility in isolated zebrafish cardiomyocytes highlights the developmental role in cardiac trabeculation process in early stage. Moreover, the use of higher molecular weight PLGA nanoparticles optimized for cytocompatibility, and stability has proven effective in delivering therapeutic genes without adverse effects.

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