1,720,981 research outputs found
High throughput methods for cardiac cellular electrophysiology studies: The road to personalized medicine
No full textPersonalized medicine refers to the tailored application of medical treatment at an individual level, taking into account the specific genotype or phenotype of each patient for targeted therapy. In the context of cardiovascular diseases, implementing personalized medicine is challenging due to the high costs involved and the slow pace of identifying the pathogenicity of genetic variants, deciphering molecular mechanisms of disease and testing treatment approaches. Scalable cellular models such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as useful in vitro tools which reflect individual patient genetics and retain clinical phenotypes. High throughput functional assessment of these constructs is necessary to rapidly assess cardiac pathogenicity and test new therapeutics if personalized medicine is to become a reality. High throughput photometry recordings of single cells coupled with potentiometric probes offer cost effective alternatives to traditional patch-clamp assessments of cardiomyocyte action potential characteristics. Importantly, automated patch-clamp (APC) is rapidly emerging in the pharmaceutical industry and academia as a powerful method to assess individual membrane-bound ionic currents and ion channel biophysics over multiple cells in parallel. Now amenable to primary cell and hiPSC-CM measurement, APC represents an exciting leap forward in the characterization of a multitude of molecular mechanisms that underlie clinical cardiac phenotypes. This review provides a summary of state-of-the-art high throughput electrophysiological techniques to assess cardiac electrophysiology and an overview of recent works which successfully integrate these methods into basic science research which could potentially facilitate future implementation of personalized medicine at a clinical level.Deutsche Forschungsgemeinschaft 10.13039/501100001659Deutsche Forschungsgemeinschaft 10.13039/501100001659Deutsche Forschungsgemeinschaft 10.13039/501100001659Deutsche Forschungsgemeinschaft 10.13039/501100001659German Center for Cardiac ResearchGerman Center for Cardiac Researc
Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
No full textSingle-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
No full textNanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy
No full textRecording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions
No full textBackground:
The rapid delayed rectifier potassium current (I
Kr
) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting I
Kr
, hERG channel, is not only permeable to K
+
ions but also to Cs
+
ions when present in equimolar concentrations inside and outside of the cell.
Methods:
In this study, I
hERG
was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs
+
or K
+
as the charge carrier. Equimolar Cs
+
has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs
+
- and K
+
-based currents.
Results:
Using equimolar Cs
+
solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs
+
- and K
+
-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs
+
solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs
+
-based conductance is larger compared to the known K
+
-based conductance, and the Cs
+
hERG conductance can be inhibited similarly to the K
+
-based conductance.
Conclusion:
Using equimolar Cs
+
instead of K
+
for I
hERG
measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small I
Kr
current densities in combination with small membrane capacitances.ZonMw http://dx.doi.org/10.13039/501100001826Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Deutsches Zentrum für Herz-Kreislaufforschung http://dx.doi.org/10.13039/10001044
Blebbistatin reduces calcium buffering in cardiomyocytes: Consequences for cellular electrophysiology
No full textAbstract Blebbistatin is an excitation–contraction uncoupling agent commonly used in cardiac optical mapping; however, it has been reported to influence cardiac myofilament Ca 2+ sensitivity. As primary contributors to Ca 2+ buffering within cardiomyocytes, cardiac myofilaments play a critical role, and even minor disruptions in intracellular Ca 2+ buffering significantly alter the free Ca 2+ concentration. In this study, we investigated the effect of blebbistatin, a myosin II ATPase inhibitor, on intracellular Ca 2+ buffering and cellular electrophysiology in induced pluripotent stem cell‐derived atrial cardiomyocytes. Simultaneous whole‐cell ruptured patch‐clamp and fluorescence microscopy techniques were used to assess intracellular Ca 2+ handling, in addition to automated high‐throughput patch‐clamp to investigate ion channel function. Comprehensive analysis of Ca 2+ buffering revealed that blebbistatin (10 µmol/l) causes a significant increase in buffer dissociation constant, suggesting decreased affinity of Ca 2+ buffers. Furthermore, systolic and diastolic Ca 2+ levels, sarcoplasmic reticulum Ca 2+ leak and the incidence of spontaneous Ca 2+ release events were significantly higher upon blebbistatin treatment. Although there was lack of impact on I Na and I Ca,L peak density, Ca 2+ ‐dependent inactivation of I Ca,L was significantly enhanced, and I K1 density was significantly smaller after blebbistatin. Importantly, these effects were reversed after chelation of intracellular Ca 2+ with EGTA. Our observations indicate that blebbistatin reduces Ca 2+ buffering, which, in turn, causes changes in cellular electrophysiology in cardiomyocytes. image Key points Intracellular Ca 2+ buffering plays an important role in determining Ca 2+ dynamics in cardiomyocytes. Blebbistatin, an excitation–contraction uncoupling agent widely used in experimental studies, decreases the affinity of intracellular Ca 2+ buffers, as indicated by an increased buffer dissociation constant. Blebbistatin leads to higher systolic and diastolic Ca 2+ levels and increased sarcoplasmic reticulum Ca 2+ leak. Blebbistatin enhances L‐type Ca 2+ current ( I Ca,L ) Ca 2+ ‐dependent inactivation and reduces inward rectifier potassium current ( I K1 ) density, while I Na and I Ca,L remain unchanged. The effects of blebbistatin are mitigated by chelation of intracellular Ca 2+ with EGTA, showing that they are secondary to altered Ca 2+ buffering
High throughput strategies for electrophysiological characterisation of cardiomyocytes
Full text linkPersonalised medicine describes the customised application of medical treatment at
a patient-specific level. This type of precision medicine stratifies patients based on
their specific genotype or phenotype for targeted therapy. For treatment of
cardiovascular disorders, this is not yet clinically feasible due to high monetary costs
and the slow rate at which molecular disease mechanisms are determined and
treatment modalities can be tested. Scalable cellular models such as induced
pluripotent stem cell derived cardiomyocyte technology (iPSC-CM) are required for
widespread use, along with a concomitant increase in high throughput measurement
strategies for robust assessment of cardiac function. This work aims to demonstrate
how state-of-the-art high throughput electrophysiological methods can be successfully
implemented into basic science research and could aid future implementation
strategies for patient-specific care. Herein, three peer-reviewed and published articles
deeply analyse cardiomyocyte function using novel high throughput
electrophysiological methods. Using specialised voltage and calcium sensitive dyes in
single cells, Article I elegantly identifies and targets a novel arrhythmogenic
mechanism in iPSC-CM derived from a patient with genetic dilated cardiomyopathy.
Such fluorescent dyes provide accurate and non-invasive readouts of cellular
membrane voltage and cytosolic calcium concentration, respectively. Article II
contains the first known measurements of primary cardiomyocytes using a market-
leading high throughput automated patch-clamp (APC) device. Revolutionary in
automating the typically complex and experimenter dependent patch-clamp technique,
APC is only now beginning to migrate into academic institutions with wider user
applications. Article III utilises multiple measurement modalities including APC and in
silico techniques to model age-related variability in iPSC-CM technology and assess
the suitability of the construct for mechanistic studies and compound screening in the
development of personalised medicine paradigms.2024-06-0
Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development
No full textAbstract Aims Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP). Methods and results First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM. Conclusions Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies
A modern automated patch-clamp approach for high throughput electrophysiology recordings in native cardiomyocytes
No full textCrucial 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
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