Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

A histologic hallmark of heart failure is the loss of cardiomyocytes (CMs), either following injury, senescence, or apoptosis¹. Replenishing lost or dysfunctional myocardium through tissue regeneration represents a potential therapeutic strategy for curing patients with heart failure. Over the past several decades, seminal advances in developmental and regenerative biology have unearthed a limited ability for the mammalian heart to replenish lost CMs^2,3,4,5. This exciting work has raised the possibility that innate growth mechanisms can be deployed for regeneration. Because innate regenerative responses are functionally absent in the adult mammalian heart, methods to improve the robustness of endogenous repair are needed for therapeutic heart regeneration to be realized.

The mechanisms for innate heart regeneration appear to be conserved across species. Following injury, pre-existing CMs proliferate to generate new CMs in zebrafish^6,7, newts^8,9, mice^4,10, rats¹¹, and pigs^12,13. Accordingly, many groups are seeking to identify mitogens capable of promoting cardiomyogenesis. However, such work is challenging. Not only is the task of getting adult mammalian CMs to proliferate daunting but being able to identify rare proliferative events is difficult^1,14. The challenge of identifying rare cycling CMs is compounded by the tendency of adult mammalian CMs to preferentially undergo endomitosis. For example, after injury to the mouse heart, almost 25% of CMs in the border zone re-enter the cell cycle, but only 3.2% of CMs divide⁴. Because most cycling CMs duplicate their genome but fail to undergo cytokinesis, simply assaying for an increase in the numbers of cycling CMs is ambiguous to bona fide cardiomyogenesis. Thus, assays for nucleoside incorporation by CMs or for the presence of proliferative markers on CMs may not entirely indicate regeneration. As more candidate factors for heart regeneration emerge, assays to better identify CM hyperplasia are needed.

Clonal analysis by lineage tracing is a valuable approach to assay for cardiomyogenesis because it allows for the direct visualization of cells and their progeny. Traditional approaches for clonal analysis involve rare labeling of single cells with a reporter gene. However, single-color lineage tracing of rare cells may be of limited value for infrequent events such as CM proliferation because the chances of labeling a proliferating CM are low¹⁵. Alternatively, multicolor lineage tracing can increase the sensitivity for clonal analysis¹⁶. Briefly, individual cells are genetically labeled with one of several fluorescent proteins at random, such that proliferating cells will generate homogeneously colored clusters of cells that can be resolved from neighboring fluorescent cells. This method has been used to trace growth across a variety of organs, and has been more recently applied to studies of mammalian heart regeneration^17,18. While multicolor lineage tracing can detect clonal expansion of CMs in embryonic and neonatal stages, innate regenerative responses are not easily detected in the adult mouse heart after cardiac injury^17,19. One approach to enhance the sensitivity of multicolor lineage tracing would be to increase the level of labeling and increase the probability for visualizing rare events. However, wider labeling comes at the cost of not being able to distinguish similarly labeled cells as rising from a common ancestor versus cells that were independently labeled with the same fluorophore. Presented here is a protocol that uses nearest-neighbor modeling to identify clonally related CMs in the neonatal mouse heart. This method is unbiased, quantitative, and works over a range of labeling conditions.