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Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters.

ABSTRACT: Discovery of small molecules promoting cardiomyocyte proliferation is important for heart regeneration and related heart disease. Here, we describe a protocol to isolate neonatal rat and mouse cardiomyocytes, infect cardiomyocytes with Tnnt2-mAG-hGeminin (1/110) or Tnnt2-Cre adenovirus, and identify small molecules that promote cardiomyocyte proliferation by high-content microscopy. This protocol can be modified to investigate other pro-proliferation factors in cardiomyocytes and other cell types. For complete details on the use and execution of this protocol, please refer to Du et al. (2022).¹.

SUBMITTER: Zheng L

PROVIDER: S-EPMC9732407 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters.

Zheng Lixia L Wang Zihao Z Du Jianyong J Zhu Xiaojun X Xiong Jing-Wei JW

STAR protocols 20221205 4

Discovery of small molecules promoting cardiomyocyte proliferation is important for heart regeneration and related heart disease. Here, we describe a protocol to isolate neonatal rat and mouse cardiomyocytes, infect cardiomyocytes with Tnnt2-mAG-hGeminin (1/110) or Tnnt2-Cre adenovirus, and identify small molecules that promote cardiomyocyte proliferation by high-content microscopy. This protocol can be modified to investigate other pro-proliferation factors in cardiomyocytes and other cell type ...[more]

PMID: 36595956

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Project description:RationaleUnderstanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed.ObjectiveTrack cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes.Methods and resultsCardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium.ConclusionsCardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

Project description:BackgroundThe adult mammalian heart is incapable of meaningful regeneration after substantial cardiomyocyte loss, primarily due to the inability of adult cardiomyocytes to divide. Our group recently showed that mitochondria-mediated oxidative DNA damage is an important regulator of postnatal cardiomyocyte cell cycle arrest. However, it is not known whether mechanical load also plays a role in this process. We reasoned that the postnatal physiological increase in mechanical load contributes to the increase in mitochondrial content, with subsequent activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes.ObjectivesThe purpose of this study was to test the effect of mechanical unloading on mitochondrial mass, DDR, and cardiomyocyte proliferation.MethodsWe examined the effect of human ventricular unloading after implantation of left ventricular assist devices (LVADs) on mitochondrial content, DDR, and cardiomyocyte proliferation in 10 matched left ventricular samples collected at the time of LVAD implantation (pre-LVAD) and at the time of explantation (post-LVAD).ResultsWe found that post-LVAD hearts showed up to a 60% decrease in mitochondrial content and up to a 45% decrease in cardiomyocyte size compared with pre-LVAD hearts. Moreover, we quantified cardiomyocyte nuclear foci of phosphorylated ataxia telangiectasia mutated protein, an upstream regulator of the DDR pathway, and we found a significant decrease in the number of nuclear phosphorylated ataxia telangiectasia mutated foci in the post-LVAD hearts. Finally, we examined cardiomyocyte mitosis and cytokinesis and found a statistically significant increase in both phosphorylated histone H3-positive, and Aurora B-positive cardiomyocytes in the post-LVAD hearts. Importantly, these results were driven by statistical significance in hearts exposed to longer durations of mechanical unloading.ConclusionsProlonged mechanical unloading induces adult human cardiomyocyte proliferation, possibly through prevention of mitochondria-mediated activation of DDR.

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Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters.

Publications

Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters.

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