Identification of Growth Factors and Regulatory Cytokines during Postnatal Cell Cycle Exit in Cardiomyocytes

Abstract

During the embryonic development, the cardiac growth is mainly promoted by cardiomyocyte proliferation. In mice the cardiomyocyte proliferation declines continuously after birth, such that in adults cardiac myocytes are largely unable to divide. If an exposure to an extrauterine environment induces postanal cell cycle arrest, cardiomyocyte numbers per heart might be reduced following preterm birth compared to term deliveries. This might affect cardiovascular health later in life. Thus, in this thesis, we evaluated the onset of cell cycle withdrawal in mouse hearts in the period immediately after birth. We performed independent immunofluorescence (IF) staining for Ki67, and phospho-Histone H3 together with Caveolin 3 as a cardiomyocyte marker in murine ventricular myocardium at two latest fetal stages (E17.5 and E18.5), shortly after birth (NB18.5 and NB19.5), a day after delivery (NB18.5+1 and NB19.5+1), and two days after birth (NB19.5+2). Both E18.5 and NB18.5 mice share an identical gestational age, however, only the NB18.5 mice were briefly exposed to the extrauterine environment. Thus, comparing E18.5 and NB18.5 hearts allows for the evaluation of the birth influence on cardiac growth regulation. Moreover, in the entire analytical steps, the mouse gestational age and sex were precisely monitored. Our IF data revealed no remarkable variations in the cardiac cell cycle activity between E17.5 and E18.5 stages. Nonetheless, shortly after birth, the cell cycle activity rate (Ki67) in cardiac cells, as well as mitosis rate in cardiomyocyte and non-myocyte (phospho-Histone H3 and Caveolin 3) were noticeably reduced in mouse ventricular myocardium at NB18.5 and NB19.5 compared to E18.5 (p<0.05). Comparing the cardiac proliferation rate in NB18.5 and NB19.5 hearts, out data suggested that the cell cycle activity was gender and gestational age-independent. Furthermore, at one day after birth compared to immediately after delivery, the mitosis rate in cardiomyocytes and non-myocytes dropped significantly, whereas the cardiac cell cycle activity remained unchanged during the early postnatal stages. Notably, within the first week after birth factors such as the metabolic switch, oxidative stress and DNA damage, as well as the activity of the cardiac growth regulating signalling pathways and the availability of cell cycle regulators influence the murine postnatal cardiac cell cycle arrest. However, assessing the availability of glycolysis-involved enzymes (HK-II, ALDOA, PKM1/2, LDHA, and Eno-II), levels of antioxidative enzymes (SOD2, TRX2 and hyperoxidized PRDX-SO3), and the degree of oxidative DNA-damage (using IF staining for 8´-Oxo-7,8-dihydroguanine and MEF2A/C) in perinatal mouse ventricular myocardium, we observed no noticeable modifications in the level of these factor at NB18.5 compared to E18.5. Thus, it is very unlikely that these factors would be associated in the cell cycle arrest in CMs shortly after birth. Interestingly, via western blot and IF approaches, we revealed that coincidence with the birth-associated cell cycle arrest in CMs, the levels of type-D Cyclins and activity of MAP-kinase, AKT, and mTORC1 were significantly reduced in mouse ventricular myocardium at NB18.5 compared to E18.5. These pathways are partially regulated by growth factors and cytokines. Accordingly, we hypothesized that immediately after a separation from an intrauterine environment, an alteration in the availability of growth factors and cytokines promote the postnatal cardiac cell cycle arrest. Indeed, our in silico approaches revealed that out of 161 studied growth factors and cytokines, 68% exhibited an altered RNA expression level in neonatal compared to fetal mouse and human hearts. Via proteomics analyses (antibody-array screenings and ELISA experiments), we unveiled that concomitant with the birth-associated cardiac cell cycle arrest, the level of the growth factors and cytokines including Angptl3, IGF-1, IGFBP6, and PDGFAA declined remarkably, whereas Adiponectin, CRP, IGFBP1, Osteopontin, and Resistin levels were noticeably accelerated in mouse hearts at NB18.5 compared to shortly before delivery (at E18.5). Consequently, to promote the cell cycle activity in cultured primary cardiomyocytes isolated from neonatal mouse hearts, the cells were incubated with either medium (negative control), or Angptl3, or PDGFAA, and thereby the BrdU incorporation rates were assessed. While the Angptl3 treatment did not influence the cell cycle activity in cardiomyocytes, an incubation of primary cardiomyocytes with PDGFAA enhanced the rate of cardiomyocytes exhibiting a BrdU-incorporation to twice its value observed in negative controls. Collectively, it seems that PDGFAA might promote proliferation in cardiomyocyte, which needs to be elucidated in future. In this context, the outcome of this thesis can have beneficial clinical implications in preterm-born humans as well as cardiac regeneration studies in humans.