Bile acid signalling in the fetal heart and myometrium

Abstract

Intrahepatic Cholestasis of Pregnancy (ICP) is a liver disease that affects approximately 1% pregnancies in the United Kingdom. ICP is characterised by an increase in maternal serum bile acids (BA), as well as a change in bile acid composition, whereby the increase in primary bile acids, taurocholic acid (TC) and taurochenodeoxycholic acid (TCDCA) is most pronounced. ICP is characterised by maternal symptoms and can be complicated by adverse events for the fetus, the latter of which occur more commonly in pregnancies complicated by high maternal serum bile acid levels. Adverse events that may complicate ICP pregnancies include spontaneous preterm labour, fetal distress, fetal heart arrhythmia and even intrauterine death in the third term of pregnancy. The aetiology of the fetal death is still poorly understood and is thought to occur suddenly. This thesis aims to investigate the effect of bile acids on the models of the human fetal heart and elucidate the mechanism of action of the protective role of UDCA. In addition, interaction between cardiomyocytes and fibroblasts will be investigated. Potential mechanisms of protection against bile acid induced fetal arrhythmia were investigated in different fetal heart models. The different models of the fetal heart will be described first, which include previously used models and a novel model using cells isolated from human fetal tissue. The effect of bile acids on these models will be described subsequently, with the focus on prevention of TC induced arrhythmias using Ursodeoxycholic acid (UDCA) and NorUrsodeoxycholic acid (NorUDCA). Human fetal heart tissue was obtained from terminations of pregnancy and for the first time beating cardiomyocytes were obtained. Characterisation of cardiac markers such as α-Actinin in these cells shows that there are similarities between neonatal rat, human fetal cardiomyocytes and the induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from Cellular Dynamics (CDI). Upon addition of TC to human fetal cardiomyocytes and CDI adverse effects occurred, including decreased spontaneous beating and prolongation of calcium transients and action potentials. Both UDCA and NorUDCA were not toxic to neonatal rat cardiomyocytes and fibroblasts, as used in the model and prevented TC-induced adverse effects. It was previously shown that there was a transient appearance of myofibroblasts and that these myofibroblasts play a role in TC induced arrhythmias. This thesis investigates role of fibroblasts and myofibroblasts in bile acid mediated arrhythmias. Addition of TC to human fetal and neonatal rat fibroblasts led to further depolarisation of these cells which could potentially depolarise connected cardiomyocytes. UDCA and NorUDCA on the other hand hyperpolarised these cells, which may be a potential mechanism of the protection seen in the models. UDCA and NorUDCA may act via modulation of the Sulfonylurea Receptor (SUR), a subunit of the ATP-sensitive inward rectifying potassium channel (KATP channel). This channel is involved in the regulation of the membrane potential of the cell. The interaction between cardiomyocytes and fibroblasts and the role of gap junctions in this was also investigated. Page | 4 Maternal symptoms include pruritus of the skin. Pruritus is defined as itching without a visible rash, which can become very severe in women with ICP. The pruritus usually resolves quickly after delivery of the baby. In addition, high bile acid levels are associated with increased risk of spontaneous preterm labour. The mechanism by which bile acids induce spontaneous preterm labour is not well understood and this thesis aims to further elucidate bile acid associated receptors and labour associated proteins expression levels. Expression levels of nuclear and membrane-bound bile acid receptors were investigated in myometrium tissue of labouring and non-labouring patients at term. FXR, one of the main nuclear bile acid receptors was not expressed. LXRα, LXRβ, PPARα and PPARγ were expressed as were the bile acid G-protein coupled receptor TGR5 and muscarinic receptor M5, while some muscarinic receptors (M1, M3 and M4) were not. It is difficult to obtain human myometrial tissue from earlier gestation, as the tissue is obtained from caesarean sections usually of non-labouring term pregnancies. This makes studies into the causes of preterm labour more difficult, especially those where high bile acid levels are involved, as it is often not possible to obtain consent for tissue collection from patients undergoing an emergency caesarean section due to difficulties whilst in term or preterm labour. Therefore other models could help elucidate the mechanisms behind early labour. For this reason novel protocols using a combinatory method called CombiCult (Plasticell) were developed which led to the differentiation of human embryonic (ES) and induced pluripotent stem (iPS) cells into myometrial smooth muscle cells (SMCs) in collaboration with Plasticell and these protocols will be described in this thesis. The work presented in this thesis provides new insight into the effect of bile acids on the fetal heart, with the focus on the development of novel cellular models of the human heart. The characterisation of human fetal cardiomyocytes and CDI was carried out using immunofluorescence staining of cardiac markers, which show changes upon maturation of the heart. Both the CDI and human fetal cardiomyocytes show an immature phenotype of these markers, similar to the neonatal rat cardiomyocytes. Optical recording of human fetal cardiomyocytes containing naturally occurring myofibroblasts showed that, similar to the neonatal rat model of the fetal heart, TC leads to calcium transient duration prolongation which can be prevented by UDCA. It was shown that Cx43 is involved in this interaction and that the cardiomyocytes and myofibroblasts are functionally coupled by GJs. Furthermore, work presented in this thesis provides further insight into the expression bile acid receptors in the myometrium, by providing evidence of the expression of various bile acid sensitive receptors, including the GPCR TGR5. Finally, for the first time, novel protocols were developed for the differentiation of myometrial smooth muscle cells.