Introduction: Heart disease is the most common cause of death worldwide. That is due to insufficient regeneration capacity of the adult myocardium, which leads to a poor prognosis in cases of severe heart injury. Interestingly, neonatal mice at postnatal day 1 (P1) have the ability to regenerate the heart but this capacity is lost shortly after birth at P7. In addition, our group has recently shown that neonatal heart at P1 have the ability to adapt to pressure overload (POL). This adaptive response of P1 mice is characterised by increased cardiomyocyte (CM) proliferation, enhanced angiogenesis, no interstitial fibrosis and preserved cardiac function. In contrast, POL in P7 mice induces CM hypertrophy and leads to interstitial fibrosis, which is associated with reduced cardiac function as early as 7 days after injury. Given the role of interstitial fibrosis in increasing myocardial stiffness and reducing contractility of the heart, which was only observed in P7 mice, we speculated that P1 and P7 fibroblasts (FB) have different features, which have to be further explored. Therefore, in this project we aimed to understand the differences of FBs in P1 versus P7 hearts and their crosstalk with CM to further discover their role in the adaptive response of P1 mice and lack of it in P7.

Methods: POL was induced by performing nTAC at P1 and P7. Cardiac function was analyzed by echocardiography 14 or 7 days after nTAC (14dpTACP1, 7dpTACP7). After sacrificing the mice at P14, body weight (BW) and heart weight (HW) were determined. Furthermore, protein expression of periostin (Postn) in the heart was determined by western blot analysis at P1, P7, P14, P21 and nTAC. In addition, by employing linage tracing mouse models (TCF21 x mTmG) we performed genetic fate mapping of TCF21+ FBs to determine their identity, fate and changes upon nTAC at P1 and P7. To determine changes in FB number and proliferation after induced POL immunohistological analysis were performed at P14. To assess transcriptomic changes of FBs and investigate their role in the adaptive response to POL, GFP+ FBs were sorted after nTAC and bulk RNAseq was performed.

Results: 14 days of POL induced at P1 shows significantly increased HW/BW ratio and LV wall thickness. Cardiac function was preserved demonstrated by no changes in EF. Even one week of pressure overload induced at P7 leads to impaired heart function observed by significantly reduced EF. Genetic fate mapping of FBs after nTAC using TCF21 transgenic mice showed no increase in FB number and proliferation in response to 14 days of POL. However, only 7 days of POL at P7 led to an increase in number of FBs. RNAseq analysis of sorted TCF21+ FBs after 14 days of nTAC at P1 revealed overexpression of cardioprotective genes in the LV FB but no fibrosis related genes. Although our western blot analysis demonstrated high expression of Postn in the LV of P1 hearts, which decreased with age, 14 days of POL enhanced Postn expression in the LV FB.

Conclusion: Our results confirmed that P1 cardiac FBs, unlike P7 FBs, do not enhance their number and proliferation in response to POL but they exert a cardioprotective role. Additionally, we are aiming to further characterize and investigate the role and contribution of P1 FBs to adaptive response of the neonatal heart to POL e.g. by secreting cytokines.