Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo.

Hessel Honkoop,Katharina F Sonnen,Veronique E M Van Der Velden,Phong D Nguyen,Jeroen Bakkers

doi:10.1242/dev.199740

Hessel Honkoop, Katharina F Sonnen + Show 3 more

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https://doi.org/10.1242/dev.199740

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Abstract

ABSTRACTZebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.

Highlights

Cardiomyocyte turn-over in the adult mammalian heart is typically very low (Bergmann et al, 2009; Senyo et al, 2013), which hampers cardiac regeneration after injury
Cardiomyocyte proliferation in the adult zebrafish heart can be induced by activation of Nrg1, vitamin D or Klf1 signaling and involves changes in energy metabolism (Gemberling et al, 2015; Han et al, 2019; Honkoop et al, 2019; Fukuda et al, 2020; Ogawa et al, 2021), very little is known about cellular processes and mechanisms within proliferating adult cardiomyocytes
Cardiac slices retain characteristics of the regenerating heart Slices of mammalian hearts have been invaluable tools for pre-clinical drug screening, as they retain electrophysiological properties of the intact heart (Kang et al, 2016; Watson et al, 2017; Perbellini et al, 2018). Based on this premise we aimed to establish a cardiac slice culture system of the injured adult zebrafish heart to study the molecular processes of heart regeneration

Summary

Introduction

Cardiomyocyte turn-over in the adult mammalian heart is typically very low (Bergmann et al, 2009; Senyo et al, 2013), which hampers cardiac regeneration after injury. Zebrafish hold a remarkable capacity to regenerate their hearts after injury (Poss, Wilson and Keating, 2002). This is achieved by a process in which surviving cardiomyocytes located in a region close to the injury, called the border zone, proliferate to restore the damaged myocardium (Jopling et al, 2010a; Kikuchi et al, 2010). Cardiomyocyte proliferation in the adult zebrafish heart can be induced by activation of Nrg, vitamin D or Klf signaling and involves changes in energy metabolism (Gemberling et al, 2015; Han et al, 2019; Honkoop et al, 2019; Fukuda et al, 2020; Ogawa et al, 2021), very little is known about cellular processes and mechanisms within proliferating adult cardiomyocytes. Current knowledge on zebrafish heart regeneration is based on snapshots taken at key timepoints after injury, thereby losing temporal information that is paramount for an in-depth understanding of the cellular events underlying proliferation

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