Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts1,2, mediated primarily by proliferation of pre-existing cardiomyocytes3,4,5. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers6. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function7,8,9,10. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.
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We thank W. Kaelin and M. Safran for providing the ODD plasmid, K. Luby-Phelps and A. Budge for help with confocal microscopy. We thank E. Olson and J. McAnally as well as R. Hammer and the UT Southwestern transgenic core facility for microinjections. We also thank the McDermott Center Sequencing and Bioinformatics Cores for sequencing and analysis.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 tdTomato+ cardiomyocytes in creERT2-ODD;R26R/tdTomato transgenice mice were pimonidazole+ at 3 days after tamoxifen administration.
Scale bars indicate 50 μm.
Extended Data Figure 2 Counting total number of cardiomyocyte included in four-chamber view section at the level of aortic valve.
Sections stained with anti-WGA was analysed with ImageJ the number of cardiomyocytes in ventricles were manually counted. Circles indicate manually counted cardiomyocytes.
Extended Data Figure 3 Hypoxic cardiomyocytes share characteristics of proliferative cardiomyocytes.
a, tdTomato+ cardiomyocytes in CAG-CreERT2–ODD;R26R/tdTomato mice at 1 week after tamoxifen administration were juxtaposed to fewer capillary blood vessels compared with surrounding non-labelled cardiomyocytes. b, Oxidative DNA damage indicated by immunostaining with an anti-8-oxoG antibody was lower in tdTomato+ hypoxic cardiomyocytes compared with surrounding non-labelled cardiomyocytes at 1 week after tamoxifen administration. c, Immunostaining of cryosections with anti-8-oxoG (red) and anti-α-actinin (green) antibodies and quantification of nuclear foci in cardiomyocytes showed significantly decreased oxidative DNA damage after single exposure to 6% O2 for 6 h. Scale bars indicate 10 μm. d, Cell fusion does not contribute mainly to the increase in number of tdTomato+ cardiomyocytes. Schematic diagram shows the experiment using CAG-CreERT2–ODD;R26R/mTmG reporter mice. The number of eGFP+/tdTomato− cardiomyocytes significantly increased at 1 month after tamoxifen pulse compared with 1 week after tamoxifen pulse, whereas the number of eGFP+/tdTomato+ cardiomyocytes did not. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01. A two-tailed unpaired t-test was used for statistical analysis.
Extended Data Figure 4 Hypoxic cells of non-cardiomyocyte lineage at 1 week and 1 month after tamoxifen administration to CAG-creERT2-ODD;R26R/tdTomato trandgenic mice.
tdTomato+ stabilized-Hif-1α cells were detected in endothelia in large vessels and capillaries (stained with anti-PECAM), vascular smooth muscle (stained with anti-SM22α) and interstitial fibroblasts (stained with anti-vimentin). Scale bars indicate 50 μm.
Extended Data Figure 5 Co-immunostaining with anti-Cre and anti-Hif-1α antibodies showed co-localization of these two proteins in αMHC-creERT2-ODD;R26R/tdTomato transgenic hearts.
Scale bars indicate 50 μm.
Extended Data Figure 6 RNA-seq analysis of Hif-1α-related signalling pathway and cardiomyocyte cell cycle.
a, Laser microdissection of tdTomato+ cardiomyocytes. Hearts of αMHC-CreERT2–ODD;R26R/tdTomato mice were harvested 3 days after three doses of tamoxifen for three consecutive days. tdTomato+ cardiomyocytes were collected from fresh cryosections. Scale bars indicate 10 μm. b, A table showing negative regulators of Hif-1α which are downregulated in tdTomato+ cardiomyocytes. The log2 fold change of gene expression in tdTomato+ cardiomyocytes compared with tdTomato− cardiomyocytes is shown. c, Positive regulators of Hif-1α which are upregulated in tdTomato+ cardiomyocytes. d, A table showing known Hif-1α target genes upregulated in tdTomato+ cardiomyocytes. e, Cyclin/CDKs upregulated in tdTomato+ cardiomyocytes. A known cardiomyocyte cell cycle regulator cyclin D1 (Ccnd1) was consistently upregulated in tdTomato+ stabilized-Hif-1α cardiomyocytes. f, Negative regulators of cell cycle including p21 (Cdkn1a), p57 (Cdkn1c), and DNA damage response related factors such as p53 (Trp53), Chek1, Chek2 and Brca2. g, Fold changes in Hif α and β subunits. h, All Meis family genes were downregulated in tdTomato+ cardiomyocytes. i, A list of hypertrophy-related genes which are downregulated in tdTomato+ cardiomyocytes.
Extended Data Figure 7 Selected heat maps showing differentially expressed genes involved in the ten most significantly altered pathways based on ingenuity pathway analysis.
Extended Data Figure 8 Hypoxic cardiomyocytes undergo cell cycle progression during normal cardiomyocyte turnover and in response to injury.
a, WGA co-staining showed clusters of tdTomato+ cardiomyocytes 1 month after tamoxifen pulse, indicating clonal expansion of cardiomyocytes. Scale bars indicate 10 μm. b, A marker for cycling cells, Ki67, was detected significantly more frequently in the nuclei of tdTomato+ cardiomyocytes compared with that of tdTomato− cardiomyocytes. Scale bars indicate 10 μm. c, Acute myocardial infarction induces proliferation of hypoxic cardiomyocytes. Both the number of tdTomato+ cardiomyocytes and BrdU+ tdTomato+ cardiomyocytes were increased within 1 week after coronary occlusion. Scale bars indicate 50 μm. *P < 0.05, **P < 0.01. A two-tailed unpaired t-test was used for statistical analysis.
a, The intra-cardiac localization of tdTomato+ cardiomyocytes is depicted at six different levels: right ventricular lateral wall (RV), ventricular apex (Apex, a third of the ventricular myocardium from the apex of the heart), septum (Sep), and left ventricular lateral wall (LW); the septum and lateral wall are further divided into base (a third of the ventricular myocardium from the base of the heart) and middle (the region between base and apex. The graphs show the distribution of tdTomato+ cardiomyocytes according to this classification. Upper graph shows the results from transgenic mice administered tamoxifen at 1 month of age, and lower graph shows the results from transgenic mice pulsed at 2 months of age. b, The localization of tdTomato+ cardiomyocytes is depicted at three levels: subendo (a third of ventricular myocardium from endocardium), subepi (a third of ventricular myocardium from epicardium) and middle (the region between subendo and subepi). The graphs show the distribution of tdTomato+ cardiomyocytes according to this classification. Upper graph shows the results from transgenic mice administered tamoxifen at 1 month of age, and lower graph shows the results from transgenic mice pulsed at 2 months of age. *P < 0.05, **P < 0.01. A two-tailed unpaired t-test was used for statistical analysis.
Extended Data Figure 10 Cell fusion does not contribute significantly to the increase in number of hypoxic cardiomyocytes.
a, Schematic diagram shows the experiment using αMHC-CreERT2–ODD;R26R/mTmG reporter mice. b, The number of eGFP+/tdTomato− cardiomyocytes significantly increased at 1 month after tamoxifen pulse compared with the 1 week after tamoxifen pulse, whereas the number eGFP+/tdTomato+ cardiomyocytes did not. *P < 0.05, **P < 0.01. A two-tailed unpaired t-test was used for statistical analysis.
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Kimura, W., Xiao, F., Canseco, D. et al. Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart. Nature 523, 226–230 (2015). https://doi.org/10.1038/nature14582
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