Fetal cardiac arrhythmias can lead to stillbirth, but direct studies on the human fetal heart are challenging. To address this, we developed a computational model of human fetal ventricular myocyte (hfVM) electrophysiology, focusing on early gestation (10 weeks). This model incorporates major ionic currents, including fetal-specific T-type calcium and funny currents, and is calibrated using mRNA expression data and experimental measurements. The hfVM model replicates key electrophysiological features, such as a shorter action potential duration and a more positive resting membrane potential compared to adult cells. Global sensitivity analysis reveals that the resting membrane potential is primarily influenced by the funny current and IK1, while action potential repolarisation depends mainly on IKr. Additionally, the sarcoplasmic reticulum contributes to calcium release, but less so than in adults; instead, the T-type calcium current and the sodium-calcium exchanger are more prominent in initiating calcium transients. This is the first human fetal ventricular myocyte model available for studying fetal cardiac physiology, pathology, and potential pharmacological interventions. It provides novel insights into the dominant ion channels governing fetal electrophysiology and calcium dynamics, offering a foundation for understanding arrhythmias and guiding therapeutic strategies.

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

BACKGROUND:Increased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated.METHODS:High-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes.RESULTS:TC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (-10.2±1.5 versus -5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (-22.3±1.1 versus -9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L.CONCLUSION:Our findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts.Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (K-ATP) channel subunits are expressed in cultured fibroblasts. K-ATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential.We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells.UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of K-ATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts in human fetal hearts. Hypoxia further increased the number of human fetal and rat neonatal myofibroblasts. However, chronically administered UDCA reduced the number of myofibroblasts and prevented hypoxia-induced depolarisation.In conclusion, our results show that the protective effect of UDCA involves both the reduction of fibroblast differentiation into myofibroblasts, and hyperpolarisation of myofibroblasts, most likely through the stimulation of potassium channels, i.e. K-ATP channels. This could be important in validating UDCA as an antifibrotic and antiarrhythmic drug for treatment of failing hearts and fetal arrhythmia. (C) 2016 Published by Elsevier Ltd.

Background: L-type calcium channels (LTCCs) play a key role in electrical remodeling during atrial fibrillation (AF). AF is associated with increased single LTCC activity and also accompanied by profound changes in myocyte microanatomy. However, it remains unknown how loss of structure affects the activity of LTCCs. Objective: To determine the microdomain-specific location, characteristics and structural composition of LTCCs in myocytes isolated from human right atrium. Methods: Scanning ion conductance and confocal microscopy were used to characterize surface topography and t-tubules (TTs) in myocytes collected from 22 patients with sinus rhythm (AF-) and 28 patients with AF (AF+). Super-resolution scanning patch-clamp was applied to identify LTCCs in subcellular microdomains. qPCR was used to determine mRNA levels of LTCCs forming subunits. Results: The surface topography of myocytes was less organized in AF+ compared to AF- patients with Z-groove index: 0.39± 0.04 vs 0.56 ± 0.02, P Open probability of LTCCs was 2.6-fold higher in AF+ cells. All α1C, β1, β2, β3 and β4 subunits showed transcriptional downregulation in AF+ samples. Conclusion: Significant surface and intercellular structural degradation was shown in human atrial cells collected from AF+ patients. Also single LTCCs located in TTs in AF+ myocytes showed increased open probability and decreased amplitude which correlated with transcriptional downregulation of channel subunits in samples collected from patients with AF. This could underlie their arrhythmogenic phenotype.

Infarct borderzone (BZ) expansion following myocardial infarction is accompanied by ventricular dilatation, remodeling, and heart failure. We showed that myofibroblasts (MFBs), which are present in the BZ, tend to form functional gap junctions with adjacent cardiomyocytes (CMs) in-vitro and such heterocellular pairing depolarizes CMs, induces discontinuity in conduction, and slows impulse propagation. Connexin-43 (C×43) plays an important role in establishing the electrical coupling, but its involvement in the dynamics of heterocellular pairing has never been investigated in live cells, due to the limit of the nanoscale resolution. We cultured neonatal rat CMs and MFBs as a culture model of BZ and followed the dynamics of contacts formed between these two cell types. We first seeded the CMs (0.5×103 cell /mm2) and, after 24 hrs, we seeded either wild type or C×43-deficient MFBs (0.15×103 cell/mm2). After another 24 hrs, preparations were mounted in a temperature controlled chamber and perfused with HBSS containing 1% serum. The mobility of the BZ was examined at 36°C using Scanning Ion Conductance Microscopy. We collected consecutive images of live-coupled areas (∼15 μm2) at 4 min. per scan over 60-70 min. The images were analysed in randomly selected points on the BZ to extract information on the movement of cell-cell contact points using a Matlab-based software.

Infarct borderzone (BZ) expansion after acute myocardial infarction is accompanied by functional and structural remodeling, ultimately leading to heart failure. We showed that in vitro myofibroblasts (MFBs) can form gap junctions with adjacent cardiomyocytes (CMs) contributing to CM depolarization, impulse propagation slowing and ectopic activity. Connexin43 (Cx43) plays an important role in establishing electrical coupling, but its involvement in the dynamics of heterocellular coupling has never been investigated in live cells, due to the limit of the nanoscale resolution. Scanning Ion Conductance Microscopy (SICM) can be used to overcome this limit. SICM is a non-invasive method capable of resolving the dynamics of live cell-to-cell contact between neonatal rat CMs and MFBs. We collected consecutive topographical images of live coupled areas (~15 μm2) at ~5 min/scan for 1h. Images were analysed by measuring the movement of randomly selected pixels in the contact area using Matlab-based software. We foun...

During mitotic entry, centrosomes separate to establish the bipolar spindle. Delays in centrosome separation can perturb chromosome segregation and promote genetic instability. However, interphase centrosomes are physically tethered by a proteinaceous linker composed of C-Nap1 ( also known as CEP250) and the filamentous protein rootletin. Linker disassembly occurs at the onset of mitosis in a process known as centrosome disjunction and is triggered by the Nek2-dependent phosphorylation of C-Nap1. However, the mechanistic consequences of C-Nap1 phosphorylation are unknown. Here, we demonstrate that Nek2 phosphorylates multiple residues within the C-terminal domain of C-Nap1 and, collectively, these phosphorylation events lead to loss of oligomerization and centrosome association. Mutations in non-phosphorylatable residues that make the domain more acidic are sufficient to release C-Nap1 from the centrosome, suggesting that it is an increase in overall negative charge that is required for this process. Importantly, phosphorylation of C-Nap1 also perturbs interaction with the core centriolar protein, Cep135, and interaction of endogenous C-Nap1 and Cep135 proteins is specifically lost in mitosis. We therefore propose that multisite phosphorylation of C-Nap1 by Nek2 perturbs both oligomerization and Cep135 interaction, and this precipitates centrosome disjunction at the onset of mitosis.