The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium1, and the adult heart is capable of modest self-renewal2,3. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage4,5. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.
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We thank the McDermott Center Sequencing and Bioinformatics Cores for sequencing and analysis, J. Richardson and J. Shelton for assistance with histology, G. Jay for assistance with the hypoxia protocol, K. Nham for help with coronary imaging and A. Darehshouri for assistance with electron microscopy. This work is supported by the NIH (grant 1R01HL115275-01) (H.A.S.), and Center for Regenerative Science and Medicine. C.X.S. was partially supported by NIH grant UL1TR001105. A.M.S. is supported by the British Heart Foundation (CH/1999001/11735).
The authors declare no competing financial interests.
Reviewer Information Nature thanks R. Lee and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Food consumption over the course of hypoxia exposure demonstrating a gradual decline (n = 9). b, Blood gas and pH analysis (n = 14 for normoxia control group, and n = 9 for hypoxia group). c, Two days of exposure to 7% O2 showed a significant increase in number of tdTomato+ cardiomyocytes in αMHC-CreERT2-ODD;R26/tdTomato double reporter mice, indicating an enhancement of Hif1α stabilization (n = 4 each). Scale bars, 50 μm. d, Comprehensive quantitative mass spectrometry analysis of enzymes involved in Krebs cycle and fatty acid β oxidation (n = 3 each). e, Dihydrorhodamine 123 staining on fresh cryosections revealed a significant decrease in ROS levels in hypoxia-treated hearts (n = 3 each). Scale bars, 100 μm. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01.
Extended Data Figure 2 A relative abundance of metabolites extracted from quintuplicated samples of control and hypoxic heart demonstrating dysregulation of numerous pathways involved in hypoxic response (n = 5 each).
a, Principal component analysis (PCA) plot for the 10 extracts shown in Extended Data Fig. 2. b, The list of differentially regulated metabolic pathways in hypoxic heart compared with control normoxic hearts.
a, Heart weight, body weight (n = 6 each), right ventricular weight to body weight ratio, and left ventricular and septum weight to body weight ratio (n = 4 for normoxia, and 5 for hypoxia) after 2 weeks of exposure to 7% oxygen. b, Wall dimension of right ventricular, intraventricular septum and left ventricular wall were all increased after 2 weeks of exposure to 7% oxygen (n = 3 each). Cardiomyocyte size in the right ventricle showed a significant increase after 2 weeks exposure to 7% oxygen (n = 3 each). c, Complete dissociation of cardiomyocytes yields a larger pellet of digested cardiomyocyte. d, Two weeks of exposure to hypoxia results in an increased mononucleated cardiomyocytes and reduced binucleated cardiomyocytes. Cardiomyocytes were isolated from whole ventricle (n = 3 each).
a–h, Ontology analysis was performed using DAVID Functional Annotation Tool. The heat maps show a number of dysregulated pathways including metabolism, cell cycle, angiogenesis and hypertrophy. Red and green colours represent upregulated and downregulated genes, respectively (n = 2 each duplicate).
a, Expansion of tdTomato+ cardiomyocytes in αMHC-CreERT2-ODD;R26/tdTomato indicates enhancement of cell division in ODD+ cardiomyocytes (n = 4 for normoxia, and 3 for hypoxia). Scale bars, 100 μm. b, The number of pH3S10/tdTomato double-positive cardiomyocytes showed an increase in hypoxic αMHC-CreERT2-ODD;R26/tdTomato transgenic mice (rate of tdTomato+/pH3S10+ double-positive cardiomyocytes to tdTomato+ single-positive cardiomyocytes, n = 3 each; rate of tdTomato+/pH3S10+ double-positive cardiomyocytes to all pH3S10+, n = 6 for all pH3S10+, n = 3 for tdTomato+/pH3S10+ double-positive). Scale bars, 10 μm. c, Distribution of proliferating cardiomyocytes in the border and remote zones. The majority of pH3S10+ and BrdU+ cardiomyocytes were localized to the remote zone rather than the MI border zone (n = 3 each).
a, Schematic of 3 week hypoxia protocol. Mice were exposed to gradual FiO2 reduction to 7% (one week following MI), which was then maintained for 3 weeks. Mice were then either euthanized, or subjected to gradual FiO2 increase back to normoxia. b, Cumulative survival curve (Kaplan–Meier survival plot) of MI and sham-operated mice after reaching 7% of oxygen (n = 8 for sham-operated mice, and n = 14 for MI mice). c, Left ventricular function for sham and MI groups exposed to 3 weeks of 7% FiO2 (longer duration hypoxia) (n = 8 for normoxia sham-operated mice, and n = 7 for hypoxia sham-operated mice; n = 9 for normoxia MI mice, and n = 6 for hypoxia MI mice). d, PVacceleration time after return to normoxia showed no evidence of pulmonary hypertension (manifested by shortened PV acceleration time) (n = 3 each).
Extended Data Figure 8 Co-immunostaining using anti-SM22α (smooth muscle cells) or FITC-conjugated isolectin B4 (endothelial cells) with an anti-BrdU antibody reveals a significant increase in BrdU incorporation in vascular lineages.
Scale bars, 20 μm.
a, Schematic of gradual induction of mild hypoxaemia. b, Heart weight to body weight ratio showed a slight increase in the mild hypoxia group (n = 5 each). c, d, Cell size (c) and pH3S10 quantification (d) showed no significant difference between mild hypoxia and normoxia groups (n = 3 each). e, Schematic of MI followed by 10% hypoxia protocol. f, Echocardiography did not show significant improvement in left ventricular ejection fraction following exposure to 10% hypoxia (n = 7 for sham-operated mice, and n = 13 for hypoxia MI mice).
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Nakada, Y., Canseco, D., Thet, S. et al. Hypoxia induces heart regeneration in adult mice. Nature 541, 222–227 (2017). https://doi.org/10.1038/nature20173
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