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.

First to determine how the proportion of FB/CM changes in the postnatal heart we performed immunohistological analysis by staining the heart tissues against PDGFRα (FB marker) and WGA at several postnatal days such as P1, P7, P14. We quantified the ratio of FBs to CMs at each time points. Furthermore, to determine changes in protein expression level of periostin (Postn) in the heart, we performed Western Blot analysis at P1, P7, P14 and P21. In addition, by employing a linage tracing mouse model (Postn x mTmG) we performed genetic fate mapping of Postn+ cells and to determine their identity, fate and changes during postnatal development we stained against PDFGRα, Postn and β-Tubulin (nerve marker). Moreover, in vitro experiments were performed to investigate FB-CM crosstalk and the effect of FBs on CMs. For this purpose, FBs and CMs from neonatal mouse hearts (P1) were isolated and cultured for 48h. Immunohistological analysis were performed against troponin T and PDGFRα and CM size were studied in each condition.

Our study showed that FB to CM ratio increases significantly during the first postnatal week and then remains unchanged from P7 to P14. Western Blot analysis demonstrated high expression of Postn in P1 hearts, which decreased significantly at P7 with further reduction to a lower level until P21. Genetic fate mapping of Postn expressing cells revealed loss of Postn expression in almost 30 % of the labeled cells from P1-P3 to P7. Further analysis revealed 85 % of Postn+ cells are FB, 15 % of Postn+ cells were co localized with nerve cells and density of GFP+ cells revealed higher percentage in the right ventricle. In vitro experiments showed P1 left ventricular FBs have the potential to reduce CM hypertrophy.

Our result confirmed changes in the features of FB in the heart from P1 to P7 such as changes in number and expression of Postn and their influence on CM hypertrophy. This indicates that FBs could play a role in regeneration capacity of the heart at P1 and/or loss of it at P7. Thus, further analyses are needed to explore the role of FBs and Postn+FB in regenerative and adaptive ability of P1 mice to POL.