Obstetric cholestasis is characterized by raised bile acids, and can be complicated by intrauterine death. We have shown that the bile acid taurocholate causes loss of synchronous beating, bradycardia and cessation of contraction in cultured rat cardiomyocytes [Williamson, Gorelik, Eaton, Lab, de Swiet and Korchev (2001) Clin. Sci. 100 , 363–369]. The aim of the present study was to investigate the effect of taurocholate on cardiomyocytes further. We demonstrated a reduced rate of contraction and proportion of beating cells when rat cardiomyocytes were exposed to increasing concentrations of taurocholate (0.1–3.0mM); more marked at higher concentrations ( P <0.001). Using scanning ion-conductance microscopy, we also demonstrated reduced amplitude of contraction and calcium transients with taurocholate. Our observations indicate that taurocholate affects calcium release from the sarcoplasmic reticulum and this parallels changes in contractile function. The relationship between the contraction amplitude and calcium transient is not linear, particularly at higher concentrations of taurocholate. We observed different effects in individual cultured neonatal cells; a reversible reduction in rate and amplitude of contraction in some, and irreversible oscillatory (fibrillatory) cessation of beating in others. The effects were more marked with higher concentrations. The contraction amplitude was also reduced in adult cardiomyocytes. The changes were reversible following removal of taurocholate in adult, but not in neonatal, cardiomyocytes exposed to higher concentrations (>0.3mM) ( P <0.001). In conclusion we have demonstrated that the bile acid taurocholate can cause different types of dysrhythmia in individual cardiomyocytes. These results provide further support for the hypothesis that obstetric cholestasis may produce cardiac-related sudden intrauterine death.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells ( P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

1. In order to gain information on the effect of protoporphyrin IX on changes in the properties of the canalicular plasma membrane, we studied the release of canalicular membrane constituents, namely phospholipids, cholesterol and 5′-nucleotidase, into bile in anaesthetized rats receiving saline or taurocholate (0.5 μmol min −1 100 g −1 body weight) with or without protoporphyrin IX infusion (10 or 20 μg min −1 100 g −1 body weight). 2. Protoporphyrin IX induced an impairment of spontaneous bile flow and of biliary secretion of cholesterol, phospholipids and bile acids. The taurocholate-induced increase in bile acid output was not significantly reduced by protoporphyrin IX at either of the doses used. However, when a cholestatic dose of protoporphyrin IX was infused, the taurocholate-induced bile flow and secretion of lecithin and cholesterol were significantly reduced. 3. Biliary output of phospholipid species other than lecithin did not counterbalance the protoporphyrin IX-induced reduction in biliary lecithin secretion. Biliary outputs of both total phospholipid and lecithin were inhibited by protoporphyrin IX to similar extents. 4. Protoporphyrin IX alone had no effect on the biliary release of 5′-nucleotidase, whereas when it was given with taurocholate, it increased the bile acid-induced biliary output of this enzyme markedly. 5. In summary, these results indicate that protoporphyrin IX impairs the biliary secretion of phospholipids and cholesterol but not that of bile acid. The release of canalicular membrane constituents other than lipids was also modified by protoporphyrin IX.

1. Isolated rat livers were perfused in a single pass with increasing doses of taurocholate with and without albumin in the perfusion media. 2. The kinetics of taurocholate uptake were thus evaluated. 3. In all the experiments, taurocholate uptake showed Michaelis-Menten kinetics. Increasing the albumin concentration in the medium resulted in an increase in the K m , without effect on the V max . When taurocholate and albumin were kept constant (20:1 molar ratio), the V max was significantly lower than in the other experiments. 4. These data suggest that taurocholate uptake shows saturation in the absence of albumin and that albumin reduces taurocholate uptake.

1. The interference between biliary phospholipid and bilirubin secretion was investigated in rats with bile fistulae, under conditions of normal and maximal bilirubin secretion. The enterohepatic circulation of bile salts was interrupted and the animals received infusions of sodium taurocholate, a micelle-forming physiological bile salt. 2. Sodium taurocholate infusion (0.19 μmol min −1 100 g −1 body weight) induced an increase in bile flow and phospholipid secretion, while basal bilirubin secretion was not increased. 3. Bilirubin infusion (0.26 μmol min −1 100 g −1 body weight) induced a decrease in basal and taurocholate-stimulated phospholipid secretion. Biliary mixed micelle formation was presumably altered during bilirubin infusion, although bile taurocholate concentration, taurocholate secretion rate and bile flow were not modified. 4. When sodium taurocholate was infused during bilirubin-decreased phospholipid secretion, this secretion was restored but maximal biliary bilirubin secretion was not increased. 5. These results provide circumstantial evidence for the hypothesis that mixed micelle formation is not an important determinant of maximal bilirubin transport into bile.