Intracellular Action Potential Recordings Research Articles

A jump in the atrioventricular conduction curve is not caused by a switch from fast pathway to slow pathway conduction.

Background: A jump in the atrioventricular (AV) conduction curve is the current clinical criterion of dual-pathway electrophysiology. However, the assumption that a jump indicates a switch from fast pathway (FP) to slow pathway (SP) conduction remains unconfirmed. This study was carried out to investigate whether a jump indeed indicates a transition from FP to SP conduction, and if not, what the potential cause is. Methods: Eighty-one experimental records from rabbit AV nodal preparations containing the following data were analyzed: 1) had at least one AV conduction curve and 2) had recording of His electrogram alternans (a validated new index of dual-pathway conduction). Most cases also had intracellular action potential recordings from the AV nodal fibers. Results: Of the 81 preparations, 11 (13%) showed a jump in the AV conduction curve. The jumps always occurred after the FP to SP transition. The FP-SP transition occurred at prematurity at 196 ± 39ms versus the jump at 114 ± 13ms (p < 0.001). The beat with a jump showed an SP-FP pattern in seven and an SP-SP pattern in four preparations. The jumps were always associated with and most likely caused by the formation of intranodal/nodal-atrial reentry and its subsequent conduction, rather than a switch from FP to SP conduction. Conclusion: Contrary to what has been assumed, a transition from FP to SP conduction does not produce a jump in the AV conduction curve. A jump in the AV conduction curve is most likely caused by the formation of intranodal/nodal-atrial reentry and its subsequent conduction.

Elevating intracellular action potential recording in cardiomyocytes: A precision-enhanced and biosafe single-pulse electroporation system

Action potentials play a pivotal role in diverse cardiovascular physiological mechanisms. A comprehensive understanding of these intricate mechanisms necessitates a high-fidelity intracellular electrophysiological investigative approach. The amalgamation of micro-/nano-electrode arrays and electroporation confers substantial advantages in terms of high-resolution intracellular recording capabilities. Nonetheless, electroporation systems typically lack precise control, and commonly employed electroporation modes, involving tailored sequences, may escalate cellular damage and perturbation of normal physiological functions due to the multiple or higher-intensity electrical pulses. In this study, we developed an innovative electrophysiological biosensing system customized to facilitate precise single-pulse electroporation. This advancement serves to achieve optimal and uninterrupted intracellular action potential recording within cardiomyocytes. The refinement of the single-pulse electroporation technique is realized through the integration of the electroporation and assessment biosensing system, thereby ensuring a consistent and reliable means of achieving stable intracellular access. Our investigation has unveiled that the optimized single-pulse electroporation technique not only maintains robust biosafety standards but also enables the continuous capture of intracellular electrophysiological signals across an expansive three-day period. The universality of this biosensing system, adaptable to various micro/nano devices, furnishes real-time analysis and feedback concerning electroporation efficacy, guaranteeing the sustained, secure, and high-fidelity acquisition of intracellular data, thereby propelling the field of cardiovascular electrophysiological research.

Abstract 12422: Does a Jump in the Atrioventricular Conduction Curve Really Indicate a Switch From Fast Pathway to Slow Pathway Conduction?

Introduction: A jump in the atrioventricular (AV) conduction curve (aka, a discontinuous curve) remains the current clinical criterion for diagnosing dual pathway AV nodal electrophysiology. However, it has never been proven that a jump in the AV conduction curve indicating a transition from fast pathway (FP) to slow pathway (SP) conduction, as it has been assumed. Hypothesis: A jump in the AV conduction curve may not reflect the transition from FP to SP conduction, as assumed it does by the current clinical criterion. Methods: Eighty-one experimental records from rabbit AV nodal preparations containing the following data were analyzed. 1. had at least one AV conduction curve, and 2. had intracellular action potential recordings from AV nodal fibers and/or recording of His electrogram alternans (a validated new index of dual pathway conduction). Results: There were 11 (13%) of 81 preparations showing a jump in the AV conduction curve. In these preparations, the transition from FP to SP conduction was smooth. The jumps always occurred after the FP to SP transition at a much shorter prematurity (Figure). On average, the FP-SP transition occurred at prematurity of 196±39ms versus the jump at 117±9ms (P&lt;0.001). Complex intranodal/nodal-atrial reentries were detected after the FP to SP transition. The jumps were always associated with and most likely caused by the intranodal/nodal-atrial reentry and its subsequent conduction to the His bundle, rather than a direct conduction by the premature beat itself. Conclusions: The transition from FP to SP conduction does not produce a jump in the AV conduction curve. A jump in the AV conduction curve is likely caused by induction of intranodal/nodal-atrial reentry by the premature beat and the subsequent conduction of the reentrant beat to the His bundle, rather than a direct conduction by the premature beat itself. Thus, a jump in the AV conduction curve as the current clinical criterion of dual pathway conduction should be invalidated.

Sensitive and prolonged intracellular electrophysiological recording by three‐dimensional nanodensity regulation

AbstractWith the advancement of micro/nanotechnologies, multielectrodes arrays (MEAs) with different three‐dimensional (3D) micro/nanostructures have been developed to achieve intracellular action potential recording of cardiomyocytes. However, the effect of the 3D micro/nanostructures density on the intracellular recording has not been fully investigated. In this work, 3D tunable nanodensity electrode arrays (TNDEA) are fabricated by hydrothermal synthesis and standard microfabrication to explore the effect of nanodensity regulation on intracellular biosensing. By low‐voltage electroporation, the signal quality of intracellular potentials recorded by the low‐nanodensity TNDEA showed was significantly improved compared to those recorded by the high‐nanodensity TNDEA. The low‐nanodensity TNDEA improved the amplitude (up to 7.7 mV), signal‐to‐noise ratio (SNR) (up to 69.46 dB), recording duration (up to 83 min), and recording yield (∼100%). The 3D nanodensity regulating strategy has achieved sensitive and prolonged intracellular biosensing of action potentials and is expected to be a powerful electrophysiological research tool in the biomedical field.

Direct recording of action potentials of cardiomyocytes through solution processed planar electrolyte-gated field-effect transistors

To achieve intracellular recording of action potentials by using simple devices that can be easily fabricated and processed is crucial in cardiology and neuroscience. Present tools and technology include invasive patch clamp technique, 3D nanostructures often combined with electro/opto poration methods and nanodevices such as nanowire field-effect transistors. However, these approaches mostly require complex manufacturing processes or are invasive. In this work, we report the spontaneous intracellular-like recording of cardiac cells using a cost-effective, planar Electrolyte-Gated Field-Effect Transistor (EGFET) based on solution-processed polymer-wrapped monochiral semiconducting single-walled carbon nanotubes (SWCNTs). By simply turning on the transistor, spontaneous recordings of intracellular-like action potentials of human induced pluripotent stem cells derived cardiomyocytes are enabled. In addition, we demonstrate that the same planar EGFET can also be employed as a platform for electroporation with significant device performance and cell viability. The simplicity of the device combined with the high signal to noise ratio opens up new opportunities for low-cost, reliable, and flexible biosensors and arrays for high quality parallel recording of cellular action potentials.

PO-01-146 PRO-ARRHYTHMIC REMODELLING OF THE PULMONARY VEIN AND LEFT ATRIAL APPENDAGE JUNCTION IN AN ANIMAL MODEL OF ENDURANCE EXERCISE

An increased incidence of atrial fibrillation (AF) is observed in endurance athletes but the mechanism underlying this is not fully known. Electrophysiological remodelling of pulmonary vein sleeve myocytes (PV), and the PV to left atrial junction may contribute to arrhythmogenesis. PV-left atrial appendage (LAA) preparations were isolated from male C57BL/6J mice after 6 weeks of treadmill exercise (ExT, running for 2 hours per day at 60-80% maximal oxygen uptake) and compared to age-matched sedentary mice (Sed). Intracellular action potential recordings and high density multielectrode array mapping was performed. Sections were stained for Masson’s trichrome to assess fibrosis. Data are expressed as mean ± standard error. In both groups there was spontaneous activity with burst firing in the PV, that significantly increased on superfusion with 10 micromolar noradrenaline (NA). There was more frequent burst activity in ExT PV (n=8/10; inter-burst time Sed 14.7 ± 8.4 s vs. ExT 4.9 ± 1.2 s; p<0.01) and programmed electrical stimulation from the right superior PV (rsPV) ostium at 20 Hz induced PV firing bursts with a shorter cycle length in ExT (n=5/5, Sed 313 ± 53 ms vs. ExT 223 ± 37 ms, p=0.05). NA induced a shift in the site of earliest activation from rsPV towards PV-LAA junction in 50% of ExT preparations vs. 25% in Sed (n=8/9). Conduction velocity (CV) at the PV-LAA junction was slowed by 65% in ExT (n=8/9, Sed 1.10 ± 0.26 m/s vs. ExT 0.39 ± 0.08 m/s, p<0.05) and CV from the site of earliest activation toward LAA within the PV was slowed (Sed 0.46 ± 0.10 m/s vs. ExT 0.20 ± 0.03 m/s, p<0.05) in ExT. Increased collagen deposition was seen the ExT PV-LAA junction vs. Sed (n =3/5; Sed 23 ± 4% vs. ExT 37 ± 1%, p<0.01). Exercise training caused alterations to the PV electrical susbtrate with an observed increase in burst frequency and reduction in cycle length in trained mice. There was also conduction slowing in the PVs and PV-LA junction of trained animals with evidence of increased collagen deposition. These data provide new insight into the electroanatomical substrate that predisposes athletes to AF.

Integrated Cardiomyocyte-Based Biosensing Platform for Electroporation-Triggered Intracellular Recording in Parallel with Delivery Efficiency Evaluation.

Electroporation is a proven technique that can record action potential of cardiomyocytes and serve for biomolecular delivery. To ensure high cell viability, micro-nanodevices cooperating with low-voltage electroporation are frequently utilized in research, and the effectiveness of delivery for intracellular access is typically assessed using an optical imaging approach like flow cytometry. However, the efficiency of in situ biomedical studies is hampered by the intricacy of these analytical approaches. Here, we develop an integrated cardiomyocyte-based biosensing platform to effectively record action potential and evaluate the electroporation quality in terms of viability, delivery efficiency, and mortality. The ITO-MEA device of the platform possesses sensing/stimulating electrodes which combines with the self-developed system to achieve intracellular action potential recording and delivery by electroporation trigger. Moreover, the image acquisition processing system analyzes various parameters effectively to assess delivery performance. Therefore, this platform has the potential for drug delivery therapy and pathology research for cardiology.

Scalable and Robust Hollow Nanopillar Electrode for Enhanced Intracellular Action Potential Recording.

Electrophysiology is a unique biomarker of the electrogenic cells that can perform a disease investigation or drug assessment. In the recent decade, vertical nanoelectrode arrays can successfully achieve a high-quality intracellular electrophysiological study in electrogenic cells and their networks. However, a high success rate and high-quality and long-term intracellular recording using low-cost nanostructures is still a considerable challenge. Herein, we develop a scalable and robust hollow nanopillar electrode to achieve enhanced intracellular recording of cardiomyocytes. The template-based synthesis of vertical hollow nanopillars is compatible with large-scale and efficient microfabrication processes and is convenient to regulate the geometry of hollow nanopillars. Compared with the conventional same-size planar electrode, the regulating height of a hollow nanopillar can achieve high-quality and prolonged intracellular recordings, which can improve the cell-electrode interface for tight coupling and effective electroporation. It is demonstrated that the geometry regulation of a nanostructure is a powerful strategy to enhance intracellular recording.

Porous Polyethylene Terephthalate Nanotemplate Electrodes for Sensitive Intracellular Recording of Action Potentials.

New strategies for intracellular electrophysiology break the spatiotemporal limitation of the action potential and lead a notable advance in the investigation of electrically excitable cells and their network. Although successful applications of intracellular recording have been achieved by 3D micro/nanodevices, complex micro/nanofabrication processes preclude the progress of extensive applications. We address this challenge by introducing porous polyethylene terephthalate (PET) membrane to develop a new type of nanotemplate electrode. This nanotemplate electrode is manufactured following a fabrication process on a porous PET membrane by atomic layer deposition. The 3D nanotemplate electrodes afford intracellular access to cardiomyocytes to report intracellular-like action potentials. These controllable nanotemplate electrodes exhibit sensitive and prolonged intracellular recordings of action potentials compared with free-growing 3D nanoelectrodes. This study indicates that the optimized structure of the nanoelectrode significantly promotes the performance of intracellular recording to assess electrophysiology in the fields of cardiology and neuroscience at an action potential level.

A universal, multimodal cell-based biosensing platform for optimal intracellular action potential recording

Intracellular recording of action potentials is an essential mean for studying disease mechanisms, and for electrophysiological studies, particularly in excitable cells as cardiomyocytes or neurons. Current strategies to obtain intracellular recordings include three-dimensional (3D) nanoelectrodes that can effectively penetrate the cell membrane and achieve high-quality intracellular recordings in a minimally invasive manner, or transient electroporation of the membrane that can yield temporary intracellular access. However, the former strategy requires a complicated and costly fabrication process, and the latter strategy suffers from high dependency on the method of application of electroporation, yielding inconsistent, suboptimal recordings. These factors hinder the high throughput use of these strategies in electrophysiological studies. In this work, we propose an advanced cell-based biosensing platform that relies on electroporation to produce consistent, high-quality intracellular recordings. The suggested universal system can be integrated with any electrode array, and it enables tunable electroporation with controllable pulse parameters, while the recorded potentials can be analyzed in real time to provide instantaneous feedback on the electroporation effectiveness. This integrated system enables the user to perform electroporation, record and assess the obtained signals in a facile manner, to ultimately achieve stable, reliable, intracellular recording. Moreover, the proposed platform relies on microelectrode arrays which are suited for large-scale production, and additional modules that are low-cost. Using this platform, we demonstrate the tuning of electroporation pulse width, pulse number, and amplitude, to achieve effective electroporation and high-quality intracellular recordings. This integrated platform has the potential to enable larger scale, repeatable, convenient, and low-cost electrophysiological studies.

An organic transistor matrix for multipoint intracellular action potential recording

Electrode arrays are widely used for multipoint recording of electrophysiological activities, and organic electronics have been utilized to achieve both high performance and biocompatibility. However, extracellular electrode arrays record the field potential instead of the membrane potential itself, resulting in the loss of information and signal amplitude. Although much effort has been dedicated to developing intracellular access methods, their three-dimensional structures and advanced protocols prohibited implementation with organic electronics. Here, we show an organic electrochemical transistor (OECT) matrix for the intracellular action potential recording. The driving voltage of sensor matrix simultaneously causes electroporation so that intracellular action potentials are recorded with simple equipment. The amplitude of the recorded peaks was larger than that of an extracellular field potential recording, and it was further enhanced by tuning the driving voltage and geometry of OECTs. The capability of miniaturization and multiplexed recording was demonstrated through a 4 × 4 action potential mapping using a matrix of 5- × 5-μm2 OECTs. Those features are realized using a mild fabrication process and a simple circuit without limiting the potential applications of functional organic electronics.

Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes.

Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.

Cutaneous Aβ-Non-nociceptive, but Not C-Nociceptive, Dorsal Root Ganglion Neurons Exhibit Spontaneous Activity in the Streptozotocin Rat Model of Painful Diabetic Neuropathy in vivo.

Diabetic peripheral neuropathic pain (DPNP) is the most devastating complication of diabetes mellitus. Unfortunately, successful therapy for DPNP remains a challenge because its pathogenesis is still elusive. However, DPNP is believed to be due partly to abnormal hyperexcitability of dorsal root ganglion (DRG) neurons, but the relative contributions of specific functional subtypes remain largely unknown. Here, using the strepotozotocin (STZ) rat model of DPNP induced by a STZ injection (60 mg/kg, i.p), and intracellular recordings of action potentials (APs) from DRG neurons in anesthetized rats, we examined electrophysiological changes in C-and Aβ-nociceptive and Aβ-low threshold mechanoreceptive (LTM) neurons that may contribute to DPNP. Compared with control, we found in STZ-rats with established pain hypersensitivity (5 weeks post-STZ) several significant changes including: (a) A 23% increase in the incidence of spontaneous activity (SA) in Aβ-LTMs (but not C-mechanosensitive nociceptors) that may cause dysesthesias/paresthesia suffered by DPNP patients, (b) membrane hyperpolarization and a ∼85% reduction in SA rate in Aβ-LTMs by Kv7 channel activation with retigabine (6 mg/kg, i.v.) suggesting that Kv7/M channels may be involved in mechanisms of SA generation in Aβ-LTMs, (c) decreases in AP duration and in duration and amplitude of afterhyperpolarization (AHP) in C-and/or Aβ-nociceptors. These faster AP and AHP kinetics may lead to repetitive firing and an increase in afferent input to the CNS and thereby contribute to DPNP development, and (d) a decrease in the electrical thresholds of Aβ-nociceptors that may contribute to their sensitization, and thus to the resulting hypersensitivity associated with DPNP.

Sinus node-like pacemaker mechanisms regulate ectopic pacemaker activity in the adult rat atrioventricular ring

In adult mammalian hearts, atrioventricular rings (AVRs) surround the atrial orifices of atrioventricular valves and are hotbed of ectopic activity in patients with focal atrial tachycardia. Experimental data offering mechanistic insights into initiation and maintenance of ectopic foci is lacking. We aimed to characterise AVRs in structurally normal rat hearts, identify arrhythmia predisposition and investigate mechanisms underlying arrhythmogenicity. Extracellular potential mapping and intracellular action potential recording techniques were used for electrophysiology, qPCR for gene and, Western blot and immunohistochemistry for protein expression. Conditions favouring ectopic foci were assessed by simulations. In right atrial preparations, sinus node (SN) was dominant and AVRs displayed 1:1 impulse conduction. Detaching SN unmasked ectopic pacemaking in AVRs and pacemaker action potentials were SN-like. Blocking pacemaker current If, and disrupting intracellular Ca2+ release, prolonged spontaneous cycle length in AVRs, indicating a role for SN-like pacemaker mechanisms. AVRs labelled positive for HCN4, and SERCA2a was comparable to SN. Pacemaking was potentiated by isoproterenol and abolished with carbachol and AVRs had abundant sympathetic nerve endings. β2-adrenergic and M2-muscarinic receptor mRNA and β2-receptor protein were comparable to SN. In computer simulations of a sick SN, ectopic foci in AVR were unmasked, causing transient suppression of SN pacemaking.

Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays.

The ability to monitor electrogenic cells accurately plays a pivotal role in neuroscience, cardiology and cell biology. Despite pioneering research and long-lasting efforts, the existing methods for intracellular recording of action potentials on the large network scale suffer limitations that prevent their widespread use. Here, we introduce the concept of a meta-electrode, a planar porous electrode that mimics the optical and biological behaviour of three-dimensional plasmonic antennas but also preserves the ability to work as an electrode. Its synergistic combination with plasmonic optoacoustic poration allows commercial complementary metal-oxide semiconductor multi-electrode arrays to record intracellular action potentials in large cellular networks. We apply this approach to measure signals from human-induced pluripotent stem cell-derived cardiac cells, rodent primary cardiomyocytes and immortalized cell types and demonstrate the possibility of non-invasively testing a variety of relevant drugs. Due to its robustness and easiness of use, we expect the method will be rapidly adopted by the scientific community and by pharmaceutical companies.

Multi-Modal 16,384-Electrode CMOS MEA with 16 Independent Multi-Well Assays for Physiological Studies of Different Cellular Models

Event Abstract Back to Event Multi-Modal 16,384-Electrode CMOS MEA with 16 Independent Multi-Well Assays for Physiological Studies of Different Cellular Models Beatrice Miccoli1, 2, Carolina Mora Lopez1, Ho Sung Chun1, Shiwei Wang1, Jan Putzeys1, Carl Van Den Bulcke1, 2, Andrea Firrincieli1, Nick Van Helleputte1, Veerle Reumers1 and Dries Braeken1* 1 Interuniversity Microelectronics Centre (IMEC), Belgium 2 KU Leuven, Belgium Passive and active multi-electrodes arrays (MEAs) stand out as powerful platforms for characterizing and unveiling complex electrophysiological dynamics of cells and tissues (Huys et al., 2012). Among them, active complementary metal oxide semiconductors (CMOS) based MEAs allow for a higher spatial resolution due to the possibility of reducing the electrodes size down to the single-cell scale, noise reduction due to reduced external interconnections, and higher automatization and portability, which strongly widen the applicability range (Huys et al., 2012). Mainly focused on electrogenic cells, these technologies impact very different fields ranging from high-throughput cardiotoxicity assays, fundamental for drug development or personalized-medicine applications, to advanced neuroscience studies (Chi et al., 2015). Despite the high versatility of CMOS MEAs, most of them are single-modal systems, therefore limiting the actual range of physiological parameters, that can be monitored on the same chip (Chi et al., 2015). Nevertheless, cellular physiological processes are extremely complex and multi-faceted therefore requiring either the simultaneous monitoring of multiple parameters or multiple types of electrophysiological measurements, on the same chip (Chi et al., 2015). Moreover, being able to access on the same chip both measurements at the cellular and network level is another key requirement, fundamental e.g., to understand how specific cellular processes influence the overall network dynamics in neuronal populations (Müller et al., 2015). This is also translated in the possibility of concurrently performing extra-cellular and intra-cellular action potential recordings in situ, on the same platform. This not only eliminates the need for external patch-clamp instrumentation, hence allowing long-term intracellular measurements along with extracellular recording but, also, it results in higher portability and versatility (Braeken et al., 2012). Therefore, we developed a multimodal 16,384 titanium nitride (TiN) electrode CMOS MEA chip with 1,024 parallel recording channels for physiological studies of different types of cells. The MEA allows both intracellular and extracellular recording, current and voltage stimulation, as well as impedance measurements, i.e. impedance spectroscopy (10 Hz to 1 MHz) or fixed frequency impedance monitoring (1 and 10 kHz), which significantly broaden the applicability range, also to non-electrogenic cells (Lopez et al., 2018). The 16,384 TiN electrodes are grouped on the chip surface in 16 independent wells, each one including 256 pixels, in a 16 x 16 matrix configuration. Each pixel then contains 4 electrodes thus leading to 1,024 electrodes per well that can be connected to 1,024 channels. The 6 different functionalities can be independently and simultaneously assessed on the 16 different wells leading to a potential 16 multi-well assays. It is important to underline that, while the electrodes pitch is fixed at 15 µm, 4 different electrodes sizes are implemented ranging from 2.5 x 3.5 µm^2 up to 10.5 x 11.0 µm^2. The presence of electrodes of different sizes and a large range of amplifier gain settings are crucial not only to widen the applicability of the chip for cells of different types and size, but, also, to accurately assess different physiological phenomena, at slightly different dimensional scales. The detailed circuit architecture of the presented chip was reported in (Lopez et al., 2018). Here, we present several assays demonstrating the multi-modality of the platform. At first, rat cardiomyocytes extracellular and intracellular action potentials were recorded, both independently and simultaneously, by exploiting the internal stimulation and recording circuitry of the chip. The amplifier gain for each channel can be independently adjusted to accommodate signals ranging from hundreds of µV (extra-cellular) to tens of mV (intra-cellular). Specifically, a peak-to-peak voltage amplitude of 1.43 mV ± 0.007 mV was measured on a 3 DIV rat cardiomyocytes culture showing a beating rate of 65.78 ± 0.027 bpm. The intracellular measurements revealed, instead, a spike peak-to-peak amplitude of 27.77 mV. Further, we used the fixed frequency impedance measurement mode (at 1 kHz) to assess cardiomyocyte contractility. Moreover, large ‘pacing’ electrodes were implemented to apply defined beating frequency to the cell culture. The 16 different wells feature independent on-chip reference electrodes thus 16 different measurement conditions can be applied on a single silicon die. All above-described measurements can be performed simultaneously on the array and therefore multi-parametric information from the same cell culture experiment can be obtained. By taking advantage of the diverse possibilities both in terms of electrode fabrication and of multi-modal operation, complex physiological phenomena as well as multi-parametric experiments can be characterized and deeply investigated, not only with a tunable resolution down to subcellular processes but, also, with high throughput thanks to the 16 independently controlled wells. References Braeken, D., Jans, D., Huys, R., Stassen, A., Collaert, N., Hoffman, L., et al. (2012). Open-cell recording of action potentials using active electrode arrays. Lab Chip 12, 4397. doi:10.1039/c2lc40656j. Chi, T., Park, J. S., Butts, J. C., Hookway, T. A., Su, A., Zhu, C., et al. (2015). A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening. IEEE Trans. Biomed. Circuits Syst. 9, 801–814. doi:10.1109/TBCAS.2015.2504984. Huys, R., Braeken, D., Jans, D., Stassen, A., Collaert, N., Wouters, J., et al. (2012). Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip. Lab Chip 12, 1274. doi:10.1039/c2lc21037a. Lopez, C. M., Chun, H. S., Berti, L., Wang, S., Bulcke, C. Van Den, Weijers, J., et al. (2018). A 16384-Electrode 1024-Channel Multimodal CMOS MEA for High-Throughput Intracellular Action Potential Measurements and Impedance Spectroscopy in Drug- Screening Applications. in 2018 International Solid-State Circuits Conference, 4–6. Müller, J., Ballini, M., Livi, P., Chen, Y., Radivojevic, M., Shadmani, A., et al. (2015). High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab Chip 15, 2767–2780. doi:10.1039/C5LC00133A. Keywords: multi-modal CMOS MEA, in vitro electrophysiology, CMOS multi-well assay, Intracellular action potential, impedance spectroscopy Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Microelectrode Array Technology Citation: Miccoli B, Mora Lopez C, Chun H, Wang S, Putzeys J, Van Den Bulcke C, Firrincieli A, Van Helleputte N, Reumers V and Braeken D (2019). Multi-Modal 16,384-Electrode CMOS MEA with 16 Independent Multi-Well Assays for Physiological Studies of Different Cellular Models. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00033 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Dries Braeken, Interuniversity Microelectronics Centre (IMEC), Leuven, Belgium, Dries.Braeken@imec.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Beatrice Miccoli Carolina Mora Lopez Ho Sung Chun Shiwei Wang Jan Putzeys Carl Van Den Bulcke Andrea Firrincieli Nick Van Helleputte Veerle Reumers Dries Braeken Google Beatrice Miccoli Carolina Mora Lopez Ho Sung Chun Shiwei Wang Jan Putzeys Carl Van Den Bulcke Andrea Firrincieli Nick Van Helleputte Veerle Reumers Dries Braeken Google Scholar Beatrice Miccoli Carolina Mora Lopez Ho Sung Chun Shiwei Wang Jan Putzeys Carl Van Den Bulcke Andrea Firrincieli Nick Van Helleputte Veerle Reumers Dries Braeken PubMed Beatrice Miccoli Carolina Mora Lopez Ho Sung Chun Shiwei Wang Jan Putzeys Carl Van Den Bulcke Andrea Firrincieli Nick Van Helleputte Veerle Reumers Dries Braeken Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease

The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.

A Sexy Approach to Pacemaking

Introduction: The sinoatrial node (SAN) is the primary pacemaker of the heart. Clinical evidence shows that SAN function differs between sexes. Males have slower resting (extrinsic and intrinsic) heart rates, and longer corrected SAN recovery time compared with females. Hypothesis: Sex based differences may be due to SAN ion channel differences. Therefore, we investigated functional behaviour and molecular makeup of SANs in male and female rats. Methods: Intracellular action potential recordings were obtained using sharp microelectrode technique (n=5 each). Gene expression was measured with quantitative PCR (n=6 each). Results: The amplitude of the action potential was significantly higher in male SANs (45.3 ± 2.4) compared with female (31.7 ± 2.5). The upstroke velocity (dV/dtmax) was significantly faster in female SANs (16.4 ± 1.6) compared with male (9.1 ± 1.5). Out of 96 transcripts investigated, two were significantly different between the sexes. L-Type Ca2+ channel, Cav1.3, and acetylcholine activated K+ channel, Kir3.1, mRNA levels were significantly higher in female SANs (66.6% and 45.6% respectively) compared with male. Immunolabelling for Cav1.3 showed levels were significantly higher in female SANs compared with male (18.6%, n=6 each). Inhibition of Cav1.3 with nifedipine (5μM) showed greater effect on the beating rate of male SANs compared with female (43%). A non-coding RNA, miR-139-3p (predicted by Ingenuity IPA software to affect Cav1.3) showed greater effect on the beating rate of male SAN preparations compared with female. Mathematical modelling showed that a higher density of Cav1.3 (as in the case of females) has a protective role from sinoatrial node arrhythmias. Conclusion: This study identified functional and molecular differences in key pacemaker ion channels in SAN between sexes. These differences advance our understanding of gender differences in cardiac electrophysiology and may have implications for gender specific design of anti-arrhythmic drugs and biological pacemakers.

Iridium oxide nanotube electrodes for sensitive and prolonged intracellular measurement of action potentials

Intracellular recording of action potentials is important to understand electrically-excitable cells. Recently, vertical nanoelectrodes have been developed to achieve highly sensitive, minimally invasive, and large scale intracellular recording. It has been demonstrated that the vertical geometry is crucial for the enhanced signal detection. Here we develop nanoelectrodes made up of nanotubes of iridium oxide. When cardiomyocytes are cultured upon those nanotubes, the cell membrane not only wraps around the vertical tubes but also protrudes deep into the hollow center. We show that this geometry enhances cell-electrode coupling and results in measuring much larger intracellular action potentials. The nanotube electrodes afford much longer intracellular access and are minimally invasive, making it possible to achieve stable recording up to an hour in a single session and more than 8 days of consecutive daily recording. This study suggests that the electrode performance can be significantly improved by optimizing the electrode geometry.

Extracellular magnesium and calcium reduce myotonia in ClC-1 inhibited rat muscle

Loss-of-function mutations in the ClC-1 Cl(-) channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg(2+) and Ca(2+) ([Mg(2+)]o and [Ca(2+)]o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg(2+)]o or [Ca(2+)]o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2 mM [Mg(2+)]o and 82-fold larger at 0.3 than at 1.27 mM [Ca(2+)]o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4-6 mV when [Mg(2+)]o was elevated from 0.6 to 3 mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na(+) channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl(-) channel function to disappearing in the absence of Cl(-) channel function when Na(+) channel activation was depolarized by 6 mV. In conclusion, variation in serum Mg(2+) and Ca(2+) may contribute to phenotypic variation in myotonia congenita patients.