Axl improves cardiac microvascular injury via mitochondrial Ca2+ and Fe2+ homeostasis in cardiac ischemia-reperfusion injury

Background Despite primary percutaneous coronary intervention (PCI), up to 60% of acute myocardial infarction (AMI) patients experience the no-reflow phenomenon driven by ischemia-reperfusion (I/R) injury1-2. AXL is a prominent member of the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases family. With its strong kinase activity, AXL regulates cell fate by directly phosphorylating downstream targets. However, the precise function and underlying mechanisms of AXL in vascular endothelial cells during I/R injury remain elusive3. Aims We aim to investigate the role of AXL in cardiac microvascular reperfusion injury, specifically focusing on its regulation of intracellular calcium homeostasis and ferroptosis in endothelial cells. Method Bulk mRNA sequencing was performed on mouse heart tissues and primary endothelial cells to identify transcriptomic changes at various reperfusion time points. These results were then integrated with clinical data from patients with ischemic cardiomyopathy (ICM) and a re-analysis of single-cell datasets from mouse and human hearts. Endothelial cell (EC) specific conditional AXL knockout mice and mice treated with AXL degrader were subjected to cardiac ischemia-reperfusion (I/R) injury. Downstream interacting proteins of AXL were identified using immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS). Results The expression of AXL was significantly downregulated in endothelial cells following cardiac I/R injury(Fig.1A-C,E-F). Linear correlation analysis demonstrated that AXL was negatively correlated with the extent of myocardial fibrosis(Fig.1D). Endothelial-specific knockout of AXL or treatment of an AXL degrader reduced microvascular perfusion and expanded the no-reflow area and infarction sizes in the acute phase, and aggravated cardiac fibrosis and cardiac dysfunction in the long-run(Fig.1G-L). KEGG enrichment analysis of DEGs between AXL-positive (AXL+) and AXL-negative (AXL-) endothelial cells identified calcium overload, mitochondrial dysfunction, and ferroptosis as significantly enriched pathological processes in the MI group(Fig.2 A).Deficiency of AXL in HCMECs leads to severe calcium overload and ferroptosis after OGD.R injury(Fig.2 C-G). Mechanistically, AXL directly bound to and inhibited ITPR3, thereby restoring mitochondrial calcium and iron homeostasis and ultimately attenuating endothelial ferroptosis(Fig.2 H-I). Conclusion In conclusion, we reveal the protective role and molecular mechanism of AXL in maintaining endothelial homeostasis after I/R injury. Our study suggests that for cardiovascular diseases characterized by mitochondrial calcium and iron overload, targeting endothelial AXL may be a valuable strategy.