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Heart Regeneration: Lessons from the Red Spotted Newt
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Unlike mammals, adult salamanders possess an intrinsic ability to regenerate complex organs and tissue types, making them an exciting and useful model to study tissue regeneration. The aims of this thesis are two fold, (1) to develop and characterize a reproducible cardiac regeneration model system in the newt, and (2) to decipher the cellular and molecular underpinnings involved in regeneration.

In Paper I of this thesis we developed a novel and reproducible heart regeneration model system in the red-spotted newt and demonstrated for the first time the newt’s ability to regenerate functional myocardial muscle, following resection injury, without scarring. The observed findings coincide with an increase in several developmental cardiac transcription factors, wide-spread cellular proliferation of cardiomyocytes and non-cardiomyocyte populations in the ventricle and reverse-remodeling at later time points during regeneration. Of further interest was the identification of functionally active Islet1+ve and GATA4+ve cardiac precursor cells in regenerating areas. The observation of such cell types further compels the similarity between mammalian cardiac development and newt cardiac regeneration and justifies these animals as suitable model organisms for studying heart regeneration. In Paper II we wanted to decipher the molecular cues possibly driving cardiac regeneration in newts. Here we used qualitative and quantitative methods to delineate the function microRNAs (miRNAs) have in this process. One interesting candidate, miR-128, a known tumor suppressor miRNA and regulator of myogenesis, was found to have a regulatory role in controlling non-cardiomyocyte hyperplasia during newt cardiac regeneration. Of further interest was the discovery of a novel binding site of miR-128 in the 3’UTR of Islet1. We speculate that the natural increase in miR-128 expression levels during cardiac regeneration functions as a fine-tuning mechanism to control cellular proliferation of precursor cells. In Paper III of my thesis we sought to explore if a link exists between RNA editing, a wide-spread post-transcriptional process and regeneration. We observed that A-to-I editing enzymes (ADARs) are present in regenerating newt tissues and the localization of ADAR1 alternates between nuclear and cytoplasmic compartments during regeneration. This activity of ADAR1 during regeneration may be partly responsible for driving the cellular plasticity that is needed during multiple phases of tissue regeneration in the red-spotted newt.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2013. , 76 p.
Keyword [en]
Heart, Regeneration, Newt, microRNAs, RNA editing
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-93058ISBN: 978-91-7447-726-9 (print)OAI: oai:DiVA.org:su-93058DiVA: diva2:644438
Public defence
2013-10-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2013-09-12 Created: 2013-08-30 Last updated: 2013-09-04Bibliographically approved
List of papers
1. Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury
Open this publication in new window or tab >>Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury
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2011 (English)In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 354, no 1, 67-76 p.Article in journal (Refereed) Published
Abstract [en]

Urodele amphibians, like the newt, are the champions of regeneration as they are able to regenerate many body parts and tissues. Previous experiments, however, have suggested that the newt heart has only a limited regeneration capacity, similar to the human heart. Using a novel, reproducible ventricular resection model, we show for the first time that adult newt hearts can fully regenerate without any evidence of scarring. This process is governed by increased proliferation and the up-regulation of cardiac transcription factors normally expressed during developmental cardiogenesis. Furthermore, we are able to identify cells within the newly regenerated regions of the myocardium that express the LIM-homeodomain protein Istet1 and GATA4, transcription factors found in cardiac progenitors. Information acquired from using the newt as a model organism may help to shed light on the regeneration deficits demonstrated in damaged human hearts.

Keyword
Bromodeoxyuridine, Echocardiogram, Islet1, Resection, Reverse remodeling
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-68122 (URN)10.1016/j.ydbio.2011.03.021 (DOI)000290550500007 ()
Note

authorCount :5

Available from: 2012-01-03 Created: 2012-01-03 Last updated: 2017-12-08Bibliographically approved
2. miR-128 regulates non-myocyte hyperplasia, deposition of extracellular matrix and Islet1 expression during newt cardiac regeneration
Open this publication in new window or tab >>miR-128 regulates non-myocyte hyperplasia, deposition of extracellular matrix and Islet1 expression during newt cardiac regeneration
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cardiovascular disease is a global scourge to society, with novel therapeutic approaches required in order to alleviate the suffering caused by sustained cardiac damage. MicroRNAs (miRNAs) are being touted as one such approach in the fight against heart disease, acting as possible post-transcriptional molecular triggers responsible for invoking cardiac regeneration. To further ones understanding of miRNAs and cardiac regeneration, it is prudent to learn from organisms that can intrinsically regenerate their hearts following injury. Using the red-spotted newt, an adult chordate capable of cardiac regeneration, we decided to delve deeper into the role miRNAs play during this process.  RNA isolated from regenerating newt heart samples, was used in a microarray screen, to identify significantly expressed candidate miRNAs during newt cardiac regeneration. We performed quantitative qPCR analysis on several conserved miRNAs and found one in particular, miR-128, to be significantly elevated when cardiac hyperplasia is at its peak following injury. In-situ hybridisation techniques revealed a localised expression pattern for miR-128 in the cardiomyocytes and non-cardiomyocytes in close proximity to the regeneration zone and in vivo knockdown studies revealed a regulatory role for miR-128 in proliferating non-cardiomyocyte populations and extracellular matrix deposition. Finally, 3’UTR reporter assays revealed Islet1 as a biological target for miR-128, which was confirmed further through in vivo Islet1 transcriptional and translational expression analysis in regenerating newt hearts. From these studies we conclude that miR-128 regulates both cardiac hyperplasia and Islet1 expression during newt heart regeneration and that this information could be translated into future mammalian cardiac studies.

Keyword
Heart, Hyperplasia, Islet1, miR-128, Newt, Regeneration
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-93065 (URN)
Available from: 2013-08-30 Created: 2013-08-30 Last updated: 2013-09-02Bibliographically approved
3. ADAR-Related Activation of Adenosine-to-Inosine RNA Editing During Regeneration
Open this publication in new window or tab >>ADAR-Related Activation of Adenosine-to-Inosine RNA Editing During Regeneration
2013 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 22, no 16, 2254-2267 p.Article in journal (Refereed) Published
Abstract [en]

Urodele amphibians possess an amazing regenerative capacity that requires the activation of cellular plasticity in differentiated cells and progenitor/stem cells. Many aspects of regeneration in Urodele amphibians recapitulate development, making it unlikely that gene regulatory pathways which are essential for development are mutually exclusive from those necessary for regeneration. One such post-transcriptional gene regulatory pathway, which has been previously shown to be essential for functional metazoan development, is RNA editing. RNA editing catalyses discrete nucleotide changes in RNA transcripts, creating a molecular diversity that could create an enticing connection to the activated cellular plasticity found in newts during regeneration. To assess whether RNA editing occurs during regeneration, we demonstrated that GABRA3 and ADAR2 mRNA transcripts are edited in uninjured and regenerating tissues. Full open-reading frame sequences for ADAR1 and ADAR2, two enzymes responsible for adenosine-to-inosine RNA editing, were cloned from newt brain cDNA and exhibited a strong resemblance to ADAR (adenosine deaminase, RNA-specific) enzymes discovered in mammals. We demonstrated that ADAR1 and ADAR2 mRNA expression levels are differentially expressed during different phases of regeneration in multiple tissues, whereas protein expression levels remain unaltered. In addition, we have characterized a fascinating nucleocytoplasmic shuttling of ADAR1 in a variety of different cell types during regeneration, which could provide a mechanism for controlling RNA editing, without altering translational output of the editing enzyme. The link between RNA editing and regeneration provides further insights into how lower organisms, such as the newt, can activate essential molecular pathways via the discrete alteration of RNA sequences.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-93062 (URN)10.1089/scd.2013.0104 (DOI)000322595500003 ()23534823 (PubMedID)
Available from: 2013-08-30 Created: 2013-08-30 Last updated: 2017-12-06Bibliographically approved

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