In this study, we characterised the levels of hypoxia in the heart during mid-to-late gestation when the epicardium is fully formed. At E12.5, hypoxia was mainly observed in areas of compact myocardium, most significantly in the atrioventricular (AV) groove, close to where the coronary plexus first develops (Chen et al., 2014). By E14.5, hypoxic regions localised to the interventricular septum (IVS), which is perfused later than the muscle of the free wall (Tian et al., 2013). Finally, by E16.5 and E18.5, when the heart is adequately perfused by the coronary vessels, hypoxia was primarily restricted to the epicardium. We further analysed the expression and distribution analysis of HIF-1 isoforms, finding a predominance of the HIF-1α isoform in WT1+ cells, whereas HIF-2α was more localised in the myocardium. Given hypoxia’s known role in promoting EMT (Scully et al., 2016), we confirmed a functional role for HIF-1α in promoting morphological changes associated with EMT by direct regulation of Wt1 expression (Figures 2 and 3, Figure 2—figure supplement 3). Moreover, epicardial-specific deletion of Hif1a significantly reduced the number of WT1-expressing cells in both epicardial and myocardial compartments, along with impaired coronary vessel development in E16.5 embryos. Collectively, this suggests that HIF-1α induces epicardial EMT and plays an essential role in coronary vessel formation during development. While Cre-mediated deletion of HIF-1α, induced at E9.5 and E10.5, predominantly targeted epicardial cells, it is worth noting that expression of WT1 in the coronary endothelium has been reported as early as E11.5 (Lupu et al., 2020), although the biological significance of endothelial WT1 remains elusive. Thus, aberrant HIF signalling in WT1 +endothelial cells cannot be completely excluded as contributing to the observed defects on the expansion of the coronary vasculature. However, stabilisation of HIF signalling in epicardial explant cultures, under physiological oxygen levels, either via genetic ablation of Egln1 or chemical inhibition of PHD enzymes with Molidustat, resulted in enhanced EMT and WT1 expression, further confirming a role for HIF signalling in epicardial activation.
To gain insight into the post-natal molecular pathways regulating the epicardium, we performed single cell RNA sequencing analysis comparing P1 and P7 stages, focusing on the specific gene expression signature of the epicardial cell cluster. GO terms analysis showed an enrichment of hypoxia-related pathways in P1 hearts, consistent with the expression of well-known HIF target genes. Importantly, Egln1 expression was enriched in P7 hearts, potentially contributing to epicardial quiescence during the first week of life, concurrent with a decrease in HIF signalling. Complete regeneration following MI requires both the replacement of lost cardiomyocytes and the formation of new blood vessels. Given the role of the epicardium during development in promoting coronary vessel formation and myocardial growth, the observed epicardial quiescence after birth likely contributes to the loss of regenerative capacity. Genetic and pharmacological stabilisation of HIF signalling beyond P7 proved to be effective in extending the regenerative window after LAD surgery, resulting in reduced pathological remodelling and preserved cardiac function against a background of fibrotic repair. Interestingly, genetic stabilisation of HIF signalling specifically in WT1-expressing cells significantly reduced scar size post-injury (Figure 5). Reduced fibrosis was also observed in PHD-inhibitor treated P7 mice (Figure 6). This is important since necrotic cardiomyocytes can trigger the activation of fibroblasts and promote fibrosis (Prabhu and Frangogiannis, 2016). The epicardium is a key source of mitogens in the embryonic heart (Simões and Riley, 2018) and provides paracrine signals targeting cardiomyocytes during regeneration (Zhou and Pu, 2011; Wills et al., 2008). In our study, we did not observe any change in either cardiomyocyte proliferation or hypertrophy, strengthening the hypothesis of an epicardium-mediated role in cardiomyocyte protection from apoptosis via paracrine signalling (Zhou et al., 2011).
It is noteworthy that despite persistent scarring (albeit reduced) following either genetic or pharmacological perturbation of PHDs, significant functional improvements were observed, in contrast to the prevailing view that fibrotic repair after MI is a barrier to effective tissue regeneration (Liang et al., 2019; Simões et al., 2020; Koth et al., 2020). Previous studies have suggested the involvement of HIF-1α in myocardial remodelling after injury. Mice with constitutive overexpression of HIF-1α in the myocardium showed enhanced angiogenesis, attenuation of infarct size, and improved cardiac performance after MI (Kido et al., 2005). Furthermore, cardiac-specific PHD2 inactivation and consequent HIF activation play a causal role in the pathogenesis of ischaemic cardiomyopathy (Moslehi et al., 2010). In our study, we focused on the stabilisation of HIF signalling as a therapeutic approach to improve outcomes after heart injury. The mean half-life of Molidustat ranges from 4 to 10 hours (Böttcher et al., 2018), ensuring prolonged stabilisation of HIF signalling to enable therapeutic benefit. Molidustat is an orally administered small molecule used for the treatment of anaemia in patients with non-dialysis-dependent chronic kidney disease (CKD) (Macdougall et al., 2019) and could potentially be repurposed to treat ischaemic heart disease.
In summary, we show that the epicardium is hypoxic at later stages of development, with HIF-1α playing a crucial role in epicardial activation and EMT, necessary for supporting coronary vessel development. The epicardium becomes quiescent after birth, coinciding with decreased HIF-signalling. However, maintaining HIF signalling after birth can extend epicardial activation, leading to a significant improvement in cardiac remodelling and function after injury, thus representing a potential novel therapeutic target to improve cardiac regeneration following MI.