Myostatin in the human placenta: expression and potential functions

Abstract

The placenta acts as a life support system maintaining the fetus throughout gestation. Inadequate implantation and insufficient development of the placenta are known to be linked to preeclampsia (PE) and intrauterine growth restriction (IUGR) of the developing fetus. Throughout gestation the placenta needs to continually grow and differentiate to increase its functional capacity in order to meet the needs of the fetus during its growth. Myostatin was initially identified as a negative regulator of muscle development. Without functional myostatin a doubling of muscle tissue is observed. For example a young boy had increased muscle development and extraordinary strength due to a homozygous mutation (G to A transition in the first intron-exon region) of the myostatin gene leading to a truncated myostatin protein. Myostatin is a distinct member of the Transforming Growth Factor-b (TGF-b) superfamily. Members of this superfamily are known to function in the development of the placenta. In the human placenta, myostatin protein expression decreases with gestational age. Myostatin treatment alters glucose uptake of placental explants and cells. In complicated pregnancies, higher myostatin has only been reported in placentae and serum of pregnancies complicated with PE. No difference in the serum concentrations of myostatin was observed in gestational diabetes mellitus (GDM) and placental expression of myostatin in GDM is not reported. Due to the limited information and the apparent expression and function of myostatin in the placenta further investigations were needed to better understand the role of myostatin in the human placenta. Firstly, the localization of myostatin in first and third trimester human placentae was investigated using immunohistochemistry. Third trimester extravillous trophoblast (EVT) stained most strongly for myostatin. This led to the isolation of EVT (from first trimester tissues) and confirmation of myostatin by immunocytochemistry. Following this, the potential for myostatin to affect the proliferation and migration was investigated in an EVT cell line and isolated EVT. The findings of this research were the first to demonstrate that myostatin could positively affect both proliferation and migration of placental cells. Secondly, the ability of myostatin to modulate cytokine release from placental explants was investigated. First trimester placental explants were maintained under hypoxic (1%), physiologically relevant oxygen (3%) and standard culture conditions (21%) +/- myostatin treatment. Pro-inflammatory cytokine (IL-8, IFN-g, TNF-a) production was significantly lower in the presence of myostatin whereas anti-inflammatory IL-4 was higher. Thirdly, myostatin concentrations were measured by ELISA in the plasma of pre-symptomatic women (12-14 weeks gestation) who later developed PE, IUGR and in women with normal pregnancies. Higher myostatin concentrations were found in plasma of pre-symptomatic women who later developed PE compared to IUGR and normal pregnancies. Investigations by western blot identified higher active myostatin dimer protein expression in placental tissue of pregnancies complicated with PE, IUGR, PE with IUGR (PE-IUGR) compared to gestational age matched women (PTB control). In the fourth study, similar analyses as described above were performed in plasma of pre-symptomatic women who later developed GDM and in placentae of diet and insulin treated GDM women compared to women with normal glucose tolerance (NGT). No difference was observed in plasma of pre-symptomatic women who later developed GDM compared to NGT. In comparison to placentae of NGT women, higher myostatin precursor and lower dimer expression was observed. Furthermore, lower precursor and higher dimer expression were observed in placental tissues of the insulin treated compared to diet treated women with GDM. Finally, the possibility of developing a more sensitive and specific detection method for myostatin utilising mass spectrometry was investigated. Based on the digestion and discernment of recombinant myostatin proteins, the method developed can clearly distinguish specific peptides of the mature (dimerization of which yields the active protein) and pro-peptide regions of the myostatin protein (myostatin inhibitory protein). This method enables the detection of the myostatin forms independent of antibodies on which current gold standard protein methods heavily rely. The next step to the utility of this method is to develop a protocol for the preparation of biological samples for use with this method of detection. In summary, in this thesis I identified that myostatin is localised in EVT and that myostatin could increase the proliferative and migrative capacities of EVT. Myostatin can modulate cytokine production in a manner that would support normal pregnancy. In complicated pregnancies, higher myostatin concentrations were evident in plasma of pre-symptomatic PE women and myostatin protein expression in placentae of women with PE, IUGR, PE-IUGR complicated pregnancies. Significant differences of myostatin protein were also observed in placentae of GDM complicated pregnancies compared to placentae of normal pregnancies and depending on the treatment (insulin and diet) received for GDM. Furthermore, a new more sensitive method of myostatin detection is well under development. In all, I believe this thesis has advanced the knowledge of myostatinrs role in the human placenta and provides a necessary platform from which further studies can be implemented.