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Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact tha...

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Autor principal: Richardson, Gavin D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927713/
https://www.ncbi.nlm.nih.gov/pubmed/27285379
http://dx.doi.org/10.3791/53979
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author Richardson, Gavin D.
author_facet Richardson, Gavin D.
author_sort Richardson, Gavin D.
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description Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.
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spelling pubmed-49277132016-07-12 Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover Richardson, Gavin D. J Vis Exp Developmental Biology Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. MyJove Corporation 2016-05-23 /pmc/articles/PMC4927713/ /pubmed/27285379 http://dx.doi.org/10.3791/53979 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by/3.0/us/ This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. To view a copy of this license, visithttp://creativecommons.org/licenses/by/3.0/us/
spellingShingle Developmental Biology
Richardson, Gavin D.
Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title_full Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title_fullStr Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title_full_unstemmed Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title_short Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
title_sort simultaneous assessment of cardiomyocyte dna synthesis and ploidy: a method to assist quantification of cardiomyocyte regeneration and turnover
topic Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927713/
https://www.ncbi.nlm.nih.gov/pubmed/27285379
http://dx.doi.org/10.3791/53979
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