Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572257
Title: Inducible depletion of cardiac thymosin β4 : development and shortcomings of a popular technique in cardiac research
Author: Lexow, Jonas Maximilian
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
In reference to recent studies highlighting the importance of Thymosin beta 4 (Tβ4) during embryonic development and its therapeutic potential, an inducible cardiac-specific knockdown of Tβ4 was established to investigate the outcomes of loss of function in adult mammalian hearts. Tβ4shRNAflox/MerCreMer mice were generated with the aim of depleting cardiac Tβ4 expression by RNA interference in a tamoxifen-dependent fashion. In vivo, in vitro and ex vivo approaches showed that tamoxifen treatment of Tβ4shRNAflox/MerCreMer mice does not result in significant down-regulation of Tβ4 mRNA or protein in the heart. Interestingly, higher levels of Tβ4 were identified in cardiac fibroblasts compared to cardiomyocytes. The above analyses suggest that an inducible knockout of Tβ4 would be a more direct, more reliable and more effective means to study loss of function in the adult mammalian heart. In the course of the study, an adverse cardiac phenotype was observed in tamoxifen-treated MerCreMer-positive animals, consisting of perivascular and interstitial fibrosis, decreased cardiac function, a marked inflammatory response and increased expression of factors involved in cardiac remodelling and hypertrophy. This was not related to MerCreMer gene copy number (homo/heterozygosity) but found to be associated with tamoxifen dose, mode of delivery and genetic background. A thorough analysis of related literature revealed that a number of recent publications have failed to include tamoxifen-treated αMHC/MerCreMer controls and some of the presented data may be affected by Cre toxicity. In conclusion, αMHC/MerCreMer mice should be included as controls in the analysis of future studies or alternative inducible systems considered. Recent analyses revealed that IGF-1Ea overexpression in the heart improves the outcome of myocardial infarction in mice. This process has been described on the molecular level but a comprehensive analysis of the subcellular structure of αMHC/IGF-1Ea hearts had not previously been performed. A detailed transmission electron microscope analysis of αMHC/IGF-1Ea hearts revealed previously not described large electron-dense structures inside αMHC/IGF-1Ea cardiomyocytes. The structures resembled large autophagolysosomes although markers of autophagy were neither found to be associated with the structures nor upregulated in the hearts of αMHC/IGF-1Ea mice. Intriguingly lysosomes were identified in the proximity of the structures potentially implying a role in turnover of intracellular material. These results provide further insights into the diverse roles of IGF-1Ea in the heart.
Supervisor: Rosenthal, Nadia ; Santini, Maria Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.572257  DOI: Not available
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