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The cardiac regeneration and development fields lack low-barrier-to-entry techniques that distinguish cardiomyocyte division from alternative outcomes in vivo. Here, we present a protocol in rodents to determine if cardiomyocyte cell division has occurred. We describe thymidine analog administration, Langendorff procedure, immunofluorescent labeling, microscopy, and analysis of fluorescent images to assess ploidy, thereby allowing an investigator to retrospectively claim cell division. Finally, we provide instructions for additional metrics including quantification of total cardiomyocytes, total cycling cardiomyocytes, and cellular dimensions. For complete details on the use and execution of this protocol, please refer to Swift et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cardiomyocyte proliferation is a challenging metric to assess. Current methodologies have limitations in detecting the generation of new cardiomyocytes and technical challenges that reduce widespread applicability. Here, we describe an improved cell suspension and imaging-based methodology that can be broadly employed to assess cardiomyocyte cell division in standard laboratories across a multitude of model organisms and experimental conditions. We highlight additional metrics that can be gathered from the same cell preparations to enable additional relevant analyses to be performed. We incorporate additional antibody stains to address potential technical concerns of miscounting. Finally, we employ this methodology with a dual-thymidine analog-labeling approach to a post-infarction murine model, which allowed us to robustly identify unique cycling events, such as cardiomyocytes undergoing multiple rounds of cell division., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Somatic polyploidization, an adaptation by which cells increase their DNA content to support growth, is observed in many cell types, including cardiomyocytes. Although polyploidization is believed to be beneficial, progression to a polyploid state is often accompanied by loss of proliferative capacity. Recent work suggests that genetics heavily influence cardiomyocyte ploidy. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent mouse strains: C57BL/6J and A/J. Both strains are born and reach adulthood with comparable numbers of cardiomyocytes; however, the end composition of ploidy classes and developmental progression to reach the final state differ substantially. We expand on previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy and uncover a role for Runx1 in ploidy dynamics and cardiomyocyte cell division, in both developmental and injury contexts. These data provide novel insights into the developmental path to cardiomyocyte polyploidization and challenge the paradigm that hypertrophy is the sole mechanism for growth in the postnatal heart., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)