Dr C. Williamson, Maternal and Fetal Disease Group, Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road London, W12 0NN, UK.
Obstetric cholestasis is associated with intrauterine death. In obstetric cholestasis, primary bile acids are more commonly conjugated with taurine than glycine, while glycoconjugates predominate in normal pregnancy. Using an in vitro model of rat cardiomyocytes, we compared the effect of tauro- and glycoconjugated cholate on cardiomyocyte rhythm, contraction amplitude and network integrity. We demonstrated that taurocholate had a more marked effect on all of these parameters, and the effects of the glycoconjugates were fully reversible while those of tauroconjugates were not. The increased proportion of tauroconjugated bile acids in obstetric cholestasis may contribute to the aetiology of the intrauterine death associated with the condition.
Obstetric cholestasis, also called intrahepatic cholestasis of pregnancy, causes maternal pruritus and hepatic impairment and can be complicated by fetal distress, prematurity and intrauterine death. The condition results in raised maternal liver transaminases and serum bile acids and raised fetal serum bile acids.
Serum bile acid levels do not change significantly throughout the course of normal pregnancy. In patients with obstetric cholestasis, the most sensitive bile acid for early diagnosis and follow up is cholic acid. Chenodeoxycholic acid is also raised, but to a lesser extent, and the cholic acid/chenodeoxycholic acid ratio also rises.1 After biosynthesis from cholesterol and before excretion from the hepatocyte, bile acids are conjugated with either glycine or taurine. This causes them to exist as charged bile salts to which membranes are impervious. In humans, the majority of bile acids are conjugated with glycine. In normal pregnancy, the glycine/taurine ratio is 1.4, and this is reduced to 0.8 in obstetric cholestasis pregnancies.1
Fetal serum bile acids are raised in obstetric cholestasis pregnancies but are lower than the corresponding maternal bile acids,2 suggesting that maternal bile acids cross the placenta and enter the fetal circulation and are then excreted from the fetus to the mother via the placenta. Raised bile acid levels have also been demonstrated in amniotic fluid2 and meconium3 in affected pregnancies.
The drug most commonly used to treat obstetric cholestasis is ursodeoxycholic acid. Administration of this hydrophilic bile acid to women with obstetric cholestasis results in a clinical and/or biochemical improvement in 87% of cases.4 Ursodeoxycholic acid treatment results in a reduction in the total serum bile acid level, and a normalisation of the glycine/taurine ratio, with an increase in the proportion of glycoconjugated bile acids.1 Ursodeoxycholic acid treatment has also been shown to improve bile acid levels measured in cord blood and amniotic fluid at the time of delivery,2 but there have been no studies of the effect of ursodeoxycholic acid on the glycine/taurine ratio of bile acids in fetal serum.
While ursodeoxycholic acid is an effective treatment for maternal features of obstetric cholestasis, it is not known whether it also improves the fetal outcome. We have previously used primary cultures of neonatal rat cardiomyocytes in order to model in vitro the fetal heart at term and have shown that addition of taurocholate, the main bile acid that is raised in obstetric cholestasis, results in abnormal contraction and calcium dynamics. We have therefore hypothesised that the cause of the intrauterine death in obstetric cholestasis is fetal dysrhythmia and cardiac arrest.5 We have also shown that addition of ursodeoxycholic acid and dexamethasone to the culture medium protects against the arrhythmogenic effect of taurocholate.6 As ursodeoxycholic acid treatment alters the glycine/taurine ratio of bile acids in maternal serum, the aim of this study was to investigate whether conjugation with either glycine or taurine influences the effect of cholic acid on the rate, rhythm and amplitude of contraction of cardiomyocytes using this in vitro model.
Ventricular myocytes were isolated from the hearts of one to two day old rats and cultured as described previously.5
Taurocholate or glycocholate was added to give final concentrations of 0.05, 0.1, 1.0 and 3.0 mM to the culture medium of single cardiomyocytes, each with its own independent rate of contraction, and to a network of synchronously beating cells. The rate of contraction and proportion of contracting cardiomyocytes was calculated as described previously.5
To investigate the changes of the rhythmicity of cardiomyocyte contraction caused by the addition of taurocholate, we recorded the vertical cell displacement of individual cells using a scanning ion conductance microscope as described previously.5
Results are expressed as mean and standard deviation (SD). Comparisons were made using the unpaired t test. The data were checked for normal distribution prior to parametric analyses.
Figure 1 shows the effect of different doses of taurocholate and glycocholate (0.05 to 3.0 mM) on the rate of contraction of single neonatal cardiomyocytes. Addition of both taurocholate and glycocholate to the culture medium to give a final concentration of 0.05 mM had no effect. Taurocholate at a concentration of 0.1 mM caused a 28% reduction in the mean rate of contraction (P < 0.001). These changes were completely reversible when the culture medium was substituted by taurocholate-free medium. Glycocholate at a concentration of 0.1 mM did not cause any effect.
Following taurocholate addition to give a concentration of 1.0 mM, the rate of cardiomyocyte contraction reduced by 46% (P < 0.001), and following addition of glycocholate, it reduced by 11% (these changes were completely reversible).
Taurocholate at a concentration of 3.0 mM caused a 59% reduction in the mean rate of contraction, whereas glycocholate at this concentration caused a 49% reduction. At these concentrations, the rate of contraction only partially recovered after the addition of bile acid-free medium, and did not reach control values (P < 0.001). Taurocholate and glycocholate had a similar effect on the proportion of beating cardiomyocytes in the single cell culture (data not shown).
Figure 2 shows the effects of taurocholate on the rhythm and amplitude of cardiomyocyte contraction recorded by the one-point measurement technique.
A typical measurement of cell vertical displacement prior to the addition of taurocholate is shown in Fig. 2A. The cell is contracting rhythmically. There is a slight reduction in the rate and amplitude of contraction when 0.1 mM taurocholate is applied to newborn rat cardiomyocytes in culture (Fig. 2B), and there is a strong reduction of rate and amplitude of contraction when 1 mM taurocholate is administered (Fig. 2C). One hour after addition of bile acid-free medium (recovery), the rate of contraction was slower than the rate before addition of taurocholate (Fig. 2D).
Figure 3 illustrates the effect of glycocholate on the rhythm and amplitude of cardiomyocyte contraction. Addition of glycocholate to give a concentration of 0.1 mM did not have any effect on contraction (Fig. 3B). However, there was a slight reduction in the rate of contraction when applying 1 mM glycocholate to newborn rat cardiomyocytes in culture (Fig. 3C). These changes were completely reversible when the culture medium was substituted by glycocholate-free medium for 1 hour (recovery) (Fig. 3D).
The effects of high concentrations (3 mM) of taurocholate and glycocholate on cardiomyocytes when they were cultured in a network are summarised in Fig. 4.
Addition of 3 mM taurocholate resulted in the disruption of network integrity, therefore cells ceased to beat synchronously. After the cells were transferred to taurocholate-free medium, they started to beat again, but at a slower rate.
Glycocholate at 3 mM caused only reduction in the rate of contraction (P < 0.001). These changes were reversible after transfer to glycocholate-free medium.
We have demonstrated that the addition of glycocholate to an in vitro culture of neonatal cardiomyocytes has a less marked arrhythmogenic effect than the addition of tauro-cholate at equivalent concentrations using an in vitro model of cardiomyocytes. As it is not possible to investigate the effect of bile acids on the human fetal heart at term, it is necessary to use models such as the one reported in this study. If this model is a reliable indicator of the effects of taurocholate and glycocholate on the fetal heart in obstetric cholestasis pregnancies, the findings reported in this study are of clinical interest because it has been shown that ursodeoxycholic acid treatment increases the proportion of maternal bile acids that are conjugated with glycine rather than taurine in obstetric cholestasis pregnancies.1 Therefore, ursodeoxycholic acid administration may also result in alterations in the fetal bile acid pool, and this in turn may protect the fetus from the arrhythmogenic effects of the raised bile acids in obstetric cholestasis. There have been no studies of the effect of ursodeoxycholic acid on the conjugation of fetal bile acids, but as most of the raised fetal serum bile acids in obstetric cholestasis are thought to be of maternal origin, an improvement in the maternal glycine/taurine ratio is likely to also result in a greater proportion of glycoconjugated bile acids in the fetal serum.
The main indication for ursodeoxycholic acid treatment in obstetric cholestasis is for relief of maternal symptoms and to treat raised serum transaminases and bile acids in the mother. It has not been established whether ursodeoxycholic acid protects against the fetal complications of the condition to date, and prospective clinical studies to address this question will be difficult to design as the reported prevalence of fetal distress and intrauterine death has reduced in recent studies where most cases are delivered by 37/38 weeks of gestation. However, there is accumulating evidence from in vitro studies that maternal ursodeoxycholic acid treatment may also protect the fetus in obstetric cholestasis. The results of this study and our previous report of the protective effect of ursodeoxycholic acid and dexamethasone using the rat cardiomyocyte model 6 are of relevance. In vitro studies have also shown that trophoblast vesicles from the placentas of women with obstetric cholestasis who were not treated with ursodeoxycholic acid have an impaired rate and affinity of bile acid transport in the basal membrane and that these changes are reversed in vesicles from placentas of women treated with ursodeoxycholic acid.7
One study has demonstrated that the total bile acid and cholic acid levels in meconium are increased in obstetric cholestasis.3 Ursodeoxycholic acid treatment did not decrease the bile acid levels in meconium despite a threefold reduction in maternal serum bile acid concentrations. This suggested that once bile acids have accumulated in meconium they are retained, and their levels cannot be significantly altered by subsequent ursodeoxycholic acid treatment, and the authors proposed that ursodeoxycholic acid treatment is started early in pregnancy to prevent accumulation of bile acids in meconium.3
In summary, we hypothesise that the increased proportion of tauroconjugated bile acids in obstetric cholestasis may explain the intrauterine death in the condition. If this is correct, the results of the study reported in this paper, and those of other clinical and in vitro investigations, provide accumulating evidence that ursodeoxycholic acid treatment may have beneficial effects on the fetal outcome of obstetric cholestasis.
Ventricular neonatal rat myocytes were kindly provided by Peter H. Sugden, National Heart and Lung Institute Division, Imperial College. This work was supported by grants from the Wellcome Trust, the Biotechnology and Biological Science Research Council and the British Heart Foundation.