Showing papers by "Niels Voigt published in 2022"
TL;DR: In this article , a broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC.
Abstract: Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp (APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.
9 citations
TL;DR: In this article , a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used to examine the cellular basis of arrhythmogenesis in DCM patients with a mutation in cardiac troponin T (R173W).
Abstract: Abstract Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca 2+ ] i ) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca 2+ ] i -alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca 2+ ] i -alternans. Myofilaments are known to play a role as intracellular Ca 2+ buffers and here we show increased Ca 2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca 2+ . Similarly, EMD57033, a myofilament Ca 2+ sensitiser, replicated the abnormal [Ca 2+ ] i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca 2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca 2+ dynamics, normalising Ca 2+ transient profile and minimising the occurrence of Ca 2+ alternans at physiological frequencies. This finding suggests that increased Ca 2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca 2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.
7 citations
TL;DR: In this article , a confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to ryanodine receptor (RyR2) in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions.
6 citations
TL;DR: This work has developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals and demonstrates this method using hERG expressed in HEK cells, as well as NaV 1.5, NaV1.7, and KCa3.1 expressed in CHO cells.
Abstract: Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.
3 citations
TL;DR: This work has shown that not only the structure of the mitochondria but also the number of ‘excitable’ cells in a ‘spatially aggregating’ network is determined by the type of molecule used in the manufacturing of DNA.
Abstract: Funsho E. Fakuade1,2,4, Jeremy Fauconnier3 and Niels Voigt1,2,4 1Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany 2DZHK (German Centre for Cardiovascular Research), Partner Site Göttingen, Germany 3PhyMedExp, INSERM, CNRS, Université de Montpellier, Montpellier, France 4Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’ (MBExC), University of Göttingen, Germany