magnetic bead grafting in the zebrafish cardiac lumen for controlled force amplification: unraveling mechanotransduction in heart valve development

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

Since its introduction in the late 1970s1, the zebrafish (Danio rerio) has emerged as a powerful model system for studying the intricacies of cardiac development and congenital heart disorders. Unlike most vertebrates, including mouse and chick embryos, which rely on a functional cardiovascular system and cannot survive early heart defects, zebrafish provide a unique advantage by enabling the investigation of severe heart phenotypes. This is due to their small size, which facilitates sufficient oxygen supply through passive diffusion, allowing survival even in the absence of heart contraction and active blood circulation2,3,4. Furthermore, among the many significant features of zebrafish is the optical transparency of their embryos, which enables non-invasive monitoring of the developing heart5,6,7,8.
Mechanical forces continuously provide feedback to the heart valve morphogenetic programs9,10,11,12,13,14,15,16,17,18,19,20,21,22, and aberrant blood flow is widely acknowledged as a shared factor in various cardiovascular disorders23,24. In zebrafish, cardiac valve development relies on heart contraction and mechanical forces generated by the beating heart. Multiple zebrafish mutants have demonstrated the significance of heart-generated mechanical stimuli in valvulogenesis. Remarkably, the complete absence of heart contraction and, consequently, blood flow due to mutations of cardiac troponin T (tnnt2) in silent heart (sih) mutants results in the absence of tissue convergence and endocardial cell (EdC) clustering during early morphogenetic stages25.
Intracardiac hemodynamics and mechanical forces generated by the blood flow emerge as fundamental epigenetic components shaping the development of the zebrafish embryonic heart. Numerous studies suggest that proper cardiac morphogenesis in zebrafish requires distinct flow stimuli, and deviations from these physiological patterns lead to heart valve defects10,13,14,22,26. Here, we describe an effective method, adapted from Fukui et al.13, to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead within the developing zebrafish beating heart. The technique involves microsurgical insertion of a bead into the cardiac lumen of anesthetized larvae without perturbing heart function. The presence of the bead leads to the amplification of the mechanical forces experienced by EdCs, directly triggering mechanical stimulus-dependent calcium influx13. This approach enables the investigation of mechanotransduction pathways that regulate heart morphogenesis and offers a means to deepen our understanding of the role of mechanical forces in valve formation.

Author Spotlight: Understanding Mechanical Forces Involved in Shaping the Zebrafish Heart

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

We focus on understanding how mechanical forces shape the zebrafish heart using advanced imaging techniques. By combining our knowledge of biology, physics, microscopy, and computing, we develop tools for optical imaging and image analysis to study how mechanical stimuli influence the way the cardiovascular system develops. A beating heart generates several type of forces, such as pressure for the shear and contractor force.Although each of these forces has a function in vitro culture systems, it is difficult to separate these parameters in the in vivo heart. By developing our approach, we aim to tackle this challenging task. We have established a novel approach to assess, adapt biological output caused by external force stimulation, and we verified our force spin and constant signal in crosscutting the cardiac cells.As a result this approach enables us to impetrate other aspects of tissue neurogenesis in the world of mechanical biology. This method enables the study of heart function in zebrafish embryo via targeted mechanical stimulation. Unlike genetic, pharmacological, or optogenetic approaches, pit grafting offers a more direct way to influence heart physiology through mechanical means.Pit grafting is viable for examining the roles of mechanical forces and calcium influx in cardiac biological processes. It provides means to deepen our understanding of the mechanical transduction pathways involved in heart development and function.

Magnetic Bead Grafting in the Zebrafish Cardiac Lumen for Controlled Force Amplification: Unraveling Mechanotransduction in Heart Valve Development

To begin, place three to six dechorionated embryos in a Petri dish containing Danios medium supplemented with 0.02%tricaine. Once the embryos are anesthetized, transfer them to a 35 by 15 millimeter glass bottom dish with minimal medium. Quickly add 300 to 400 microliters of 1%low melting point agarose containing tricaine to create a dome.Using forceps, position the embryos so they lie straight in contact with the glass with the ventral side facing up. Next, deposit approximately 0.5 microliters of magnetic beads on top of the agarose. Then add a drop of Danios medium containing 0.02%tricaine on top of the agarose and the beads.Based on the sample and heart size, select the appropriate bead and use tweezers to place it on top of the embryo yolk. Create a yolk lesion or hole in the center of the yolk using one arm of the tweezers. Place the bead into the lesion and gently push it anteriorly until it reaches the venous pole.If suction alone is insufficient to move the bead, gently push it toward the atrium, applying enough pressure to guide it into the pericardial cavity. After grafting the magnetic beads into the embryos, add one to two milliliters of Danios medium containing PTU to the glass bottom dish. Then using tweezers, gently break the low melting point agarose and carefully remove the embryos.Finally, using a Pasteur pipette, transfer the embryos to a new Petri dish containing Danios medium with PTU and allow them to recover and develop at 28.5 degrees Celsius. The magnetic bead was correctly positioned within the atrium of the zebrafish heart, allowing for unobstructed blood flow and no observed hemorrhage with the heart walls maintained.

magnetic-bead-grafting-zebrafish-cardiac-lumen-for-controlled-force

Research

JoVE Journal

Developmental Biology

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...