Abstract
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.
Highlights
In recent years there has been an accumulation of evidence challenging the supposition that the heart is a terminally differentiated, post-mitotic organ[1,2]
Using a BrdU based assay, we previously demonstrated that the mammalian heart responds to chronic cardiomyocyte loss, in the mdx mouse model of Duchenne muscular dystrophy, with the regeneration of new cardiomyocytes
To accurately quantify cardiomyocyte turnover and regeneration assays must distinguish between true cardiomyocyte generation and nonproductive DNA division
Summary
In recent years there has been an accumulation of evidence challenging the supposition that the heart is a terminally differentiated, post-mitotic organ[1,2]. The protocol described here enables the accurate and sensitive identification of neo cardiomyocyte nuclei generation in the mouse heart, irrespective of the cellular origins (Figure 1A and B) while simultaneously excluding nucleoside labeling due to polyploidization from the analysis (Figure 1C and D). This method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neocardiomyocyte nuclei which is required for the accurate quantification of cardiomyocyte turnover. All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes
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