Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage

Abstract

Introduction: Gradual exposure to a chronic hypoxic environment leads to cardiomyocyte proliferation and improved cardiac function in mouse models through a reduction in oxidative DNA damage. However, the upstream transcriptional events that link chronic hypoxia to DNA damage have remained obscure.

Aim: We sought to determine whether hypoxia signaling mediated by the hypoxia-inducible factor 1 or 2 (HIF1A or HIF2A) underlies the proliferation phenotype that is induced by chronic hypoxia.

Methods and Results: We used genetic loss-of-function models using cardiomyocyte-specific HIF1A and HIF2A gene deletions in chronic hypoxia. We additionally characterized a cardiomyocyte-specific HIF2A overexpression mouse model in normoxia during aging and upon injury. We performed transcriptional profiling with RNA-sequencing on cardiac tissue, from which we verified candidates at the protein level. We find that HIF2A - rather than HIF1A - mediates hypoxia-induced cardiomyocyte proliferation. Ectopic, oxygen-insensitive HIF2A expression in cardiomyocytes reveals the cell-autonomous role of HIF2A in cardiomyocyte proliferation. HIF2A overexpression in cardiomyocytes elicits cardiac regeneration and improvement in systolic function after myocardial infarction in adult mice. RNA-sequencing reveals that ectopic HIF2A expression attenuates DNA damage pathways, which was confirmed with immunoblot and immunofluorescence.

Conclusion: Our study provides mechanistic insights about a new approach to induce cardiomyocyte renewal and mitigate cardiac injury in the adult mammalian heart. In light of evidence that DNA damage accrues in cardiomyocytes with aging, these findings may help to usher in a new therapeutic approach to overcome such age-related changes and achieve regeneration.

Keywords

Hypoxia / regeneration / myocardial infarction / DNA damage / cell division / HIF2A

References

[1] Nakada Y,Thet S.Hypoxia induces heart regeneration in adult mice.Nature2017;541:222-7

[2] Puente BN,Muralidhar SA.The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.Cell2014;157:565-79 PMCID:PMC4104514

[3] Nguyen NUN,Xiao F.A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.Nature2020;582:271-6 PMCID:PMC7670845

[4] McQuin C,Chernyshev V.CellProfiler 3.0: next-generation image processing for biology.PLoS Biol2018;16:e2005970 PMCID:PMC6029841

[5] Moslehi J,Shi J.Loss of hypoxia-inducible factor prolyl hydroxylase activity in cardiomyocytes phenocopies ischemic cardiomyopathy.Circulation2010;122:1004-16 PMCID:PMC2971656

[6] Ali SR,Saadat LV,Weissman IL.Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.Proc Natl Acad Sci USA2014;111:8850-5 PMCID:PMC4066522

[7] Scortegagna M,Oktay Y.Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1^-/- mice.Nat Genet2003;35:331-40

[8] Smith J,Xu N.Chapter 3 - the ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer.Adv Cancer Res2010;108:73-112

[9] Nakada Y,Xiao F.DNA damage response mediates pressure overload-induced cardiomyocyte hypertrophy.Circulation2019;139:1237-9 PMCID:PMC6467068

[10] Kinner A,Staudt C.Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.Nucleic Acids Res2008;36:5678-94 PMCID:PMC2553572

[11] Choudhury S,Kim J.Somatic mutations in single human cardiomyocytes reveal age-associated DNA damage and widespread oxidative genotoxicity.Nat Aging2022;2:714-25 PMCID:PMC9432807

[12] Fan Q,Angelini A.Depletion of endothelial prolyl hydroxylase domain protein 2 and 3 promotes cardiomyocyte proliferation and prevents ventricular failure induced by myocardial infarction.Circulation2019;140:440-2 PMCID:PMC6699770

[13] Batie M,Frost M,Schofield P.Hypoxia induces rapid changes to histone methylation and reprograms chromatin.Science2019;363:1222-6

[14] Chakraborty AA,Myllykoski M.Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate.Science2019;363:1217-22 PMCID:PMC7336390

[15] Koeppen M,Seo SW.Hypoxia-inducible factor 2-alpha-dependent induction of amphiregulin dampens myocardial ischemia-reperfusion injury.Nat Commun2018;9:816 PMCID:PMC5827027

[16] Wu JW,Li D.Hypoxia-inducible factor 2-alpha-dependent induction of IL-6 protects the heart from ischemia/reperfusion injury.Aging2021;13:3443-58 PMCID:PMC7906200

[17] Hölscher M,Krull S.Cardiomyocyte-specific prolyl-4-hydroxylase domain 2 knock out protects from acute myocardial ischemic injury.J Biol Chem2011;286:11185-94 PMCID:PMC3064173

[18] Pradeep SR,Thirunavukkarasu M.Protective effect of cardiomyocyte-specific prolyl-4-hydroxylase 2 inhibition on ischemic injury in a mouse MI model.J Am Coll Surg2022;235:240-54

[19] Hölscher M,Krull S.Unfavourable consequences of chronic cardiac HIF-1α stabilization.Cardiovasc Res2012;94:77-86