Abstract Body: Introduction
Adult mammals are unable to regenerate their heart muscle after an ischemic event, creating a fibrotic scar that cannot meet the contractile needs of the organ. However, other vertebrates can regenerate their heart throughout their adult life, like the axolotl (Ambystoma mexicanum). The question of whether we can apply any strategy from axolotls to mammals is still unresolved. In this work, we aimed to characterize the heart regeneration process in the axolotl by generating a contextualized single-cell atlas with advanced bioinformatic tools.
Results
Using single-nucleus multi-omics and spatial transcriptomics, we mapped axolotl heart regeneration and identified a pro-regenerative border-zone CM population. Ligand–receptor analysis revealed AXL enrichment in injury-responsive CMs and its ligand Gas6 in endothelial cells. Our functional studies show that AXL is required for CM activation and regeneration, though it has not been reported in mammalian CMs.
In cultured neonatal mouse ventricular CMs (NMVCs), AXL overexpression via an AAV vector caused prominent phenotypic alterations, most notably sarcomere disassembly as detected by staining of alpha-actinin, cTnT and titin. We also observed this phenomenon in both P7 in vitro CMs and adult heart sections upon AAV-AXL administration. However, EdU incorporation in NMVCs did not differ between AXL-overexpressing and GFP-expressing CMs, suggesting a lack of proliferation induction despite the sarcomeric disassembly.
Interestingly, bulk RNA-Seq analysis of NMVCs revealed a more immature transcriptomic profile in AXL-overexpressing cells compared to GFP controls, showing upregulation of glycolytic enzymes, downregulation of OXPHOS and a switch in sarcomeric components. Functional metabolic assays demonstrated that AXL-transduced CMs exhibited increased glycolytic capacity in basal conditions and in the presence of glucose.
Conclusions
These findings show the first proof to our knowledge of a molecule translating from axolotl to mammals. In summary, we showed that AXL signaling induces CM dedifferentiation in mice and uncouples it from cell cycle re-entry, revealing a mechanism to isolate proliferation drivers. We hypothesize that AXL activation could prime CMs for proliferation by promoting sarcomeric disassembly. Future experiments to determine whether AXL triggers a proliferative and regenerative response in adult mice upon injury are warranted.