Study of midkine-a and caveolin-1 in zebrafish heart regeneration
Author
Grivas, DimitriosAdvisor
Pompa Mínguez, José Luis de laEntity
UAM. Departamento de Biología Molecular; Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC)Date
2019-12-19Funded by
This doctoral thesis was performed in the Intercellular Signaling in Cardiovascular Development and Disease laboratory directed by Dr. José Luis de la Pompa at Centro Nacional de Investigaciones Cardiovasculares (CNIC) in Madrid. This study was financed by different grants of Ministerio de Ciencia, Innovación y Universidades (SAF2013-45543-R, SAF2015-71863-REDT y SAF2016-78370-R), CIBER Cardiovascular. Instituto de Salud Carlos III. Ref.: CB16/11/00399, Red de Terapia Celular. Instituto de Salud Carlos III. Ref.: RD16/0011/0021, CardioNeT- International Training Network on the Cellular and Molecular Bases of Heart Homeostasis and Repair. Agency: European Union, ref. 28600, 2012-2015. Dimitrios Grivas holds a PhD fellowship linked to the grant [PITN-GA-2011-289600] (CardioNeT)Subjects
Tejidos (Biología) - Regeneración - Modelos animales - Tesis doctorales; Corazón - Enfermedades - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 19-06-2021
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Zebrafish have the ability to regenerate cardiac tissue after injury in contrast to mammals.
Cryoinjury of the heart triggers a regenerative program that includes inflammatory response, cell
proliferation and formation of a transient scar that eventually lead to complete tissue regeneration.
This thesis focused on understanding the molecular and mechanical properties governing zebrafish
heart regeneration. In particular, we studied the role of midkine-a (mdka) and caveolin-1 (cav1),
two genes that were upregulated in a microarray analysis of regenerating hearts performed in the
laboratory. Additionally, we studied the epigenetic regulation of mdka and tested in vivo different
mdka cis-regulatory elements with the goal to identify the minimum regulatory regions that drive
mdka expression in development and after injury.
Midkine-a (Mdka) is a neurite-growth factor that is involved in the formation of the media
floor plate in zebrafish. Here, we show that mdka expression was strongly induced after heart injury,
although it was not expressed in intact hearts. In injured hearts, the onset of mdka expression was
one-day post cryoinjury (dpci) in all the epicardial layer, whereas by 7dpci the expression became
restricted to the epicardial cells covering the injured area. To study the role of Mdka in heart
regeneration, we generated mdka-knock out (KO) zebrafish strains. In injured heart, mdka depletion
did not trigger upregulation of mdkb and ptn, the two other members of the midkine gene family,
which might compensate for the loss of Mdka. Analysis of 90dpci hearts showed that mdka deletion
resulted in impaired heart regeneration, with the injured area enriched in collagen deposition.
However, the proliferation ratio of cardiomyocytes (CM) and epicardial cells was not affected.
Analysis of extracellular matrix turnover, immune cells infiltration in the injured area and
revascularization of the regenerating tissue will clarify the reasons for the impaired cardiac
regeneration in mdka-KO hearts.
During development, mdka is expressed in neural tissues such us neural tube and brain, and
is not detected neither in developing hearts nor in adult intact hearts. However, mdka expression
was activated upon heart injury, having dynamic expression pattern. To investigate such a change
in the expression pattern, we used available ChIP-seq and ATAC-seq data to analyse the epigenetic
landscape of mdka. In mdka locus, there are three intronic enhancer sequences and two additional
enhancers located upstream of mdka. We tested these enhancers and the mdka promoter linked to
GFP reporter and studied their expression patterns in our transgenic lines. We found that the
promoter of mdka and the intronic enhancers were responsible for the spatio-temporal expression
pattern of mdka during development, in adult tissues and after injury of the heart and the fin.
Caveolin-1 (Cav1) is the structural protein of caveolae, small membrane invaginations that
are involved in signal transduction and mechanoprotection. Our expression analysis showed that
Cav1 was expressed in the endocardium, endothelium and epicardium of intact adult heart. After
heart cryoinjury, Cav1 was strongly expressed in epicardial cells covering the injury, and in the
endocardium invading the damaged tissue. We introduced mutations in cav1 sequence that resulted
in loss of Cav1 and caveolae. Analysis of the regeneration process revealed that the caveolae
depleted hearts regenerated normally, although cav1-KO hearts showed a transient defect on CM
proliferation. However, loss of Cav1 and caveolae affected heart function, since the ejection
fraction and the heart rate were significantly decreased in intact hearts. Using atomic force
microscopy, we found that epicardial cells and cortical cardiomyocytes lacking caveolae were stiffer than the control hearts explaining the decline of cardiac function
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Google Scholar:Grivas, Dimitrios
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