Zebrafish (Danio rerio): a model to study in-vivo and ex-vivo cardiac
Author(s)
Missinato, Maria Azzurra
Date Issued
February 18, 2010
Type
Doctoral Thesis
Abstract
Despite continuing progress in medical therapies and in revascularization strategies at coronaries level, heart diseases remains a leading cause of mortality throughout industrialized
countries. Mammals have an extremely limited capacity to repair damaged heart tissue after a myocardial infarction. Regeneration, the replacement of damaged or lost body parts, is a primary goal of stem cell research. In fact, during this process, differentiated cells reenter the
cell cycle and proliferate to generate a mass of undifferentiated cells. Several vertebrates display different ability to regenerate organs and tissues, thus encouraging biologists to seek
out models for heart regeneration. The robust capacity of zebrafish (Danio rerio) to
regenerate a variety of tissues, position it as an ideal genetic model system for understanding the molecular and cellular events governing regeneration. Numerous works have shown that adult zebrafish can effectively regenerate in-vivo injured hearts submitted to partial surgical
amputation of the ventricle area. In fact, during this process, new cardiomyocytes limit the scar formation and form new muscular tissue. However the molecular mechanisms that regulate this process remains unclear and resolving the genes, the microRNAs and the proteomic alterations that control these changes can illuminate how heart regeneration is naturally optimized. Activation of the regenerative potential of human tissue implicates a novel therapeutic approach to supplement, or replace, conventional pharmacotherapy and
mechanical intervention.
In this thesis, using qRT-PCR experiments, it was demonstrated that some microRNAs
(miRNAs), as miR1 and miR-133, were down-regulated during the in-vivo regeneration of
adult zebrafish hearts submitted to amputation of around 20% of the ventricle apex. Also, similar changing in microRNAs levels were observed during hypertrophic conditions induced ex-vivo. The adult zebrafish heart, containing numerous cardiac progenitors, resulted a good model to approach the general mechanisms of adult cardiac stem cell maintenance and
cardiogenesis. Here, zebrafish was also used as model to test for a correlation between
Fibroblast Growth factors (FGFs) signaling and cell proliferation in adult injured hearts. With In Situ Hybridization (ISH) experiments, here were reported the time and space expression of FGFs targets genes (erm, etv5, pea3, dusp6, sef sprouty4, and raldh2), involved in in-vivo
heart regeneration. With the aim to optimize the media to reproduce the regeneration process, it was also tested the ability of zebrafish heart to survive in ex-vivo cultures after the amputation of ventricular apex. Regenerating hearts in ex-vivo conditions were able to survive
4 and make contractions, and surprisingly showed different degree of cell replication,
incorporating BrdU, in dependence to the culture media.
So far, there is no study regarding the proteomic aspect the heart regeneration in
zebrafish. Proteomic offers an innovative approach to integrate the genomic study that alone is not sufficient to understand the all the cellular mechanisms. Here, for the fist time, it is reported a proteomic study of adult zebrafish heart, that allowed the identification of more than one hundred proteins, belonging to different biological classes as, enzymes, signal
transduction, growth factors, cytoskeletal components, globin, and structural proteins. The identification of the proteins naturally expressed in the adult zebrafish heart is not only important as basic biological knowledge, but also, because it constitute a reference point to compare proteins expression during cardiac regeneration. In fact, applying innovative and
sensible two-dimensional fluorescence difference gel electrophoresis (DiGE), combined with MALDI-TOF/TOF tandem mass spectrometry, here it was possible to identify proteins differentially expressed at 3 days after amputation of around 20% of the ventricle area, compared with hearts not injured. Among more than one hundred proteins spots detected in
2D-gels, three (ATP synthase, hyaluronan mediated motility receptor, and desmuslin) were up-regulated in injured hearts. Information concerning the global alteration protein pattern during heart regeneration will be helpful for a better formulation of new diagnostic and therapeutic markers. Also, due to the similarities between the fish and mammalian genomes,this experimental system should also provide clues to understanding human pathophysiology
and the new frontier of stem cell regeneration system.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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