Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes

Abstract

Background: Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and results: Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2) receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2) receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion: We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2) receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

Figure 1. Fxr interaction with TC in NRCM.

(a) The 3H TC influx assay showed low uptake of 3H TC in NRCM (closed circle) compared to primary human hepatocytes (open circle). While hepatocytes showed uptake of 200000 units of TC in the first 20 minutes, cardiomyocytes did not take up more than 50 units of radioactive TC. fxr in neonatal cardiomyocytes (b-d). (b) Graph quantifying the relative expression of fxr protein from western blots performed using NRCM and the human hepatocyte cell line Huh7 treated with TC 0.1 mmol/L at 0, 5, 30 and 60 minutes. (c) Representative western blot showing expression of fxr protein in rat neonatal cardiomyocytes with and without addition of TC. (d) Control human hepatocyte cell line Huh7 stained for fxr; pre (i) and post-bile acid treatment (ii). NRCM stained for fxr; control (iii) and after 1 incubation with TC 1 mmol/L (iv). FITC (green) = fxr; DAPI (blue) =  nucleus; yellow arrow fxr nuclear region staining. (n = 3 observations).

Figure 2. Involvement of TGR5 and fxr in TC induced arrhythmia in NRCM.

(a) Lack of expression of TGR5 in cardiac cells. Top panels: TGR5 immunolocalisation in a rat liver section (left) and cultured rat Kupffer cells (right) Scale bar 10 μm. Bottom panels. Immunostaining for TGR5 in a cluster of NRCM (left and right panels, red colour). Nuclei are stained with Hoechst dye (left panel, blue colour) (b) Graph demonstrating that the TGR5 agonist Oleanolic acid (0.1 mmol/L) and the fxr agonist GW4064 (0.01 mmol/L) do not protect NRCM from TC-induced arrhythmia. (* Control vs P<0.05); n = 3 observations).

Figure 3. TC is a partial agonist of the M₂ muscarinic receptor.

(a) Specific binding of TC to muscarinic receptors in cardiomyocyte membranes. Competitive displacement of ³H-NMS with TC is shown (K _d values were 17.2±7.3 µmol/L, n = 3). (b) FRET-based cAMP measurements in NRCM upon stimulation with TC show its partial agonistic effect compared to the full agonist carbachol (CCh). Isoproterenol (Iso) was used a positive control to stimulate cAMP production. Representative experiment (n = 5), quantification is shown in (c).

Figure 4. The muscarinic receptor is involved in TC-induced arrhythmia.

Contraction of NRCM expressed as beat per minutes (bpm). (a) Treatment with TC or the muscarinic agonist carbachol in the presence or absence of the Gi protein blocker PTX. (b) Dose dependent effect of carbachol (CCh) and taurocholate (TC). (c) Pharmacological inhibition of muscarinic receptors, treatment with M₁, M₂ and M₃ muscarinic receptor antagonists. (d) Scramble (non-targeting) siRNA and M₂ siRNA knockdown of cells. (* Control vs P<0.001); n≥3 observations).

Figure 5. Muscarinic M₂ receptor mediates TC effects of cardiac contraction.

Representative measurement of the amplitude contraction of NRCM using SICM.(a-b) Scramble (non-targeting) siRNA NRCM showed regular contraction and alterations in rhythm and amplitude following TC treatment.(c-d) cardiomyocytes showed regular contraction following siRNA knockdown of M₂ and TC treatment did not have an effect on rhythm of contraction. (e) Graph demonstrating the influence of adding 0.2 mmol/L and 1.0 mmol/L TC on the amplitude of contraction of non-targeting siRNA (grey bar) and siRNA-M₂ knockdown (black bar) of NRCM. This is represented as a percentage (%) of the amplitude of contraction in cells prior to the addition of TC (designated controls). The extent to which the amplitude of contraction returns to normal after transfer of cells into TC-free medium is also shown. (* Control vs P<0.05); n = 6 observations).

Figure 6. Functional involvement of the muscarinic M₂ receptor in the TC-induced calcium desynchronization.

(a) Overview of a cluster loaded with 5 µg/ml Fluo-4 AM (scale bar  = 10 µm). Representative of intracellular Calcium dynamics recorded in NRCM pre and post-treatment with 0.2 mmol/L TC. (b) Ca ²⁺ transients recorded in untreated (left panel) and treated (right panel) of control cells. (c) Ca ²⁺ transients recorded in untreated (left panel) and treated (right panel) of scramble (non-targeting) siRNA knockdown cells. (d) Ca ²⁺ transients recorded in untreated (left panel) and treated (right panel) of siRNA M₂ knockdown cells. n>5 observations.