Introduction: In contrast to the insufficient regenerative capacity of the adult myocardium, neonatal mice at postnatal day 0 (P0) exhibit a capacity for cardiac regeneration that is rapidly lost after birth. Previous studies by our group have demonstrated that hearts at P0 are able to adapt to pressure overload (POL), a pathological stimulus that leads to heart failure in adults. This adaptive response in P0 hearts is characterized by enhanced cardiomyocyte (CM) proliferation and angiogenesis, while cardiac function remains preserved in the absence of interstitial fibrosis or hypertrophy. In contrast, POL at P7 triggers a maladaptive response characterized by CM hypertrophy, interstitial fibrosis and impaired cardiac function. As interstitial fibrosis was observed only after POL in P7 hearts, we hypothesized that cardiac fibroblasts (cFB) at P0 and P7 have different properties and respond differently to POL, which may determine whether the neonatal heart undergoes adaptive or maladaptive remodeling by directly influencing CM behavior. Therefore, here we investigated cFB to CM crosstalk to discover cFB-mediated mechanisms that drive the adaptive response to POL in P0 hearts. Ultimately, our goal is to uncover potential strategies to prevent maladaptive response of the hearts to POL at later stages.
Methods: POL was induced in CD1 mice by performing neonatal transverse aortic constriction (nTAC). cFB and CM were isolated 3 days after P0 and P7 nTAC. Bulk RNA sequencing was conducted to profile their transcriptomes and to identify potential ligand-receptor interactions between cFB and CM. To validate the expression of selected ligands in cFB and the corresponding receptor in CM, western blot and immunohistological analyses were performed. The functional impact of the identified ligand-receptor interaction on CM hypertrophy, proliferation and apoptosis was assessed in vitro by culturing neonatal primary CM on ligand-coated plates. To test the ligand's potential to prevent the maladaptive response to POL, it was administrated in vivo to neonatal mice from P0 to P3 followed by P7 nTAC. Cardiac function was assessed by echocardiography 7 and 14 days after nTAC. Hearts were then used for immunohistological analyses to quantify CM hypertrophy, proliferation, angiogenesis and interstitial fibrosis.
Results: Transcriptomic analysis identified a collagen-mediated signaling as a key underlying cFB to CM crosstalk. Functional testing of this crosstalk in vitro confirmed its anti-apoptotic, anti-hypertrophic, and pro-proliferative effects on CM, which likely contribute to the adaptive response upon P0 nTAC. Consistent with this, not only the ligand but also its corresponding receptor exhibited downregulation with age, which was further accentuated after P7 nTAC, coinciding with the loss of regenerative capacity. In vivo, administration of the ligand effectively prevented the maladaptive response of the heart upon P7 nTAC. Treated mice exhibited preserved cardiac function, enhanced CM proliferation and angiogenesis and no signs of hypertrophy or interstitial fibrosis.
Conclusion: We showed that cFB to CM crosstalk plays a crucial role in the adaptive response of neonatal mice following P0 nTAC and can serve as a preventative strategy to protect the heart against the maladaptive response upon P7 nTAC. We are currently investigating the underlying mechanism and signaling pathway that drives this regeneration and its therapeutic effect in adult mice.
