Hypoxia and HIF signalling in the epicardium during development and regeneration

Heart failure is a major cause of death worldwide due to the inability of the mammalian heart to regenerate after injury. The neonatal mouse heart however mounts a substantial regenerative response; though this capacity is lost one week after birth. The epicardium, which is activated in response to injury, becomes quiescent during this time-frame and may, therefore, be involved in the loss of regenerative potential. Another key change that occurs in the heart after birth is the transition to a post-natal circulation, meaning it becomes less hypoxic. Hypoxia inducible factor (HIF) is a transcription factor, which is activated in response to hypoxia and significantly, activated HIF can directly bind to and upregulate expression of Wt1, the master regulator of epicardial activation via epithelial-to-mesenchymal transition (EMT). I, therefore, sought to investigate a functional requirement for hypoxia and HIF-signalling in the activation of the epicardium during murine heart development and neonatal cardiac regeneration. In the developing heart, co-expression of WT1 with hypoxyprobe at embryonic stages (E)16.5 and E18.5 indicated the epicardium is hypoxic at later stages of embryogenesis. Epicardial-specific loss of Hif1-alpha or Hif2-alpha during development led to decreased epicardial activation and EMT which impacted on coronary vessel development and myocardial compaction. Furthermore, stabilisation of HIF in epicardial explant cultures, via genetic ablation of prolyl hydroxylase domain 2 gene (Phd2), or chemical inhibition of PHD enzymes via FG-4592 (also known as Roxadustat), resulted in enhanced EMT, implicating a role for HIF signalling in epicardial activation. Whilst the epicardium becomes quiescent within the first postnatal week, stabilisation of HIF signalling after birth through loss of Phd2 was sufficient to maintain activation of the epicardium. Moreover, genetic knock-down and pharmacological (treatment with FG-4592) inhibition of PHD2 to stabilise HIF augmented the epicardial response to injury and preliminary results suggested an improvement in the regenerative response. Further analysis into the full impact on neonatal cardiac regeneration, and transition to the injured adult heart, is required. Nevertheless, FG-4592, which is already in clinical trials for patients with anaemia, presents a potential novel therapeutic approach to improve cardiac repair in human patients following myocardial infarction.