Biology subjects

Christini, D. J.

Publications and source records attributed to Christini, D. J..

3 recordsLinked to original sources

bioRxiv2024

Resolving artefacts in voltage-clamp experiments with computational modelling: an application to fast sodium current recordings

Cellular electrophysiology is the foundation of many fields, from basic science in neurology, cardiology, oncology to safety critical applications for drug safety testing, clinical phenotyping, etc. Patch-clamp voltage clamp is the gold standard technique for studying cellular electrophysiology. Yet, the quality of these experiments is not always transparent, which may lead to erroneous conclusions for studies and applications. Here, we have developed a new computational approach that allows us to explain and predict the experimental artefacts in voltage-clamp experiments. The computational model captures the experimental procedure and its inadequacies, including: voltage offset, series resistance, membrane capacitance and (imperfect) amplifier compensations, such as series resistance compensation and supercharging. The computational model was validated through a series of electrical model cell experiments. Using this computational approach, the artefacts in voltage-clamp experiments of cardiac fast sodium current, one of the most challenging currents to voltage clamp, were able to be resolved and explained through coupling the observed current and the simulated membrane voltage, including some typically observed shifts and delays in the recorded currents. We further demonstrated that the typical way of averaging data for current-voltage relationships would lead to biases in the peak current and shifts in the peak voltage, and such biases can be in the same order of magnitude as those differences reported for disease-causing mutations. Therefore, the presented new computational pipeline will provide a new standard of assessing the voltage-clamp experiments and interpreting the experimental data, which may be able to rectify and provide a better understanding of ion channel mutations and other related applications.

systems biology↗

bioRxiv2022

Leak current, even with gigaohm seals, can cause misinterpretation of stem-cell derived cardiomyocyte action potential recordings

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have become an essential tool to study arrhythmia mechanisms. Much of the foundational work on these cells, and the computational models built from the resultant data, has overlooked the contribution of seal-leak current on the immature and heterogeneous phenotype that has come to define these cells. Here, we use in silico and in vitro studies to demonstrate how seal-leak current depolarises action potentials (APs), substantially affecting their morphology, even with seal resistances (Rseal) above 1 G{Omega}. We show that compensation of this leak current is difficult due to challenges with recording accurate measures of Rseal during an experiment. Using simulation, we show that Rseal measures: 1) change during an experiment, invalidating the use of pre-rupture values, and 2) are polluted by the presence of transmembrane currents at every voltage. Finally, we posit the background sodium current in baseline iPSC-CM models imitates the effects of seal-leak current and is increased to a level that masks the effects of seal-leak current on iPSC-CMs. Based on these findings, we make three recommendations to improve iPSC-CM AP data acquisition, interpretation, and model-building. Taking these recommendations into account will improve our understanding of iPSC-CM physiology and the descriptive ability of models built from such data. Key pointsO_LIHuman induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an essential tool in the study of cardiac arrhythmia mechanisms. C_LIO_LITheir immature and heterogeneous action potential phenotype complicates the interpretation of experimental data, and has slowed their acceptance in industry and academia. C_LIO_LIWe suggest that a leak current caused by an imperfect pipette-membrane seal during single-cell patch-clamp experiments is partly responsible for inducing this phenotype. C_LIO_LIUsing in vitro experiments and computational modelling, we show that this seal-leak current affects iPSC-CM AP morphology, even under ideal experimental conditions. C_LIO_LIBased on these findings, we make recommendations that should be considered when interpreting, analysing and fitting iPSC-CM data. C_LI

physiology↗

bioRxiv2021

A model-guided pipeline for drug cardiotoxicity screening with human stem-cell derived cardiomyocytes

New therapeutic compounds go through a preclinical drug cardiotoxicity screening process that is overly conservative and provides limited mechanistic insight, leading to the misclassification of potentially beneficial drugs as proarrhythmic. There is a need to develop a screening paradigm that maintains this high sensitivity, while ensuring non-cardiotoxic compounds pass this phase of the drug approval process. In this study, we develop an in vitro-in silico pipeline using human induced stem-cell derived cardiomyocytes (iPSC-CMs) to address this problem. The pipeline includes a model-guided optimization that produces a voltage-clamp (VC) protocol to determine drug block of seven cardiac ion channels. Such VC data, along with action potential (AP) recordings, were acquired from iPSC-CMs before and after treatment with a control solution or a low-, intermediate-, or high-risk drug. We identified significant AP prolongation (a proarrhythmia indicator) in two high-risk drugs and, from the VC data, determined strong ion channel blocks that led to the AP changes. The VC data also uncovered an undocumented funny current (If) block by quinine, which we confirmed with experiments using a HEK-293 expression line. We present a new approach to cardiotoxicity screening that simultaneously evaluates proarrhythmia risk (e.g. AP prolongation) and mechanism (e.g. channel block) from iPSC-CMs.

bioengineering↗