alexa Arrhythmia Research| Assays on Stem Cell-| cellular cardiac electrophy
ISSN: 2161-0398
Journal of Physical Chemistry & Biophysics
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Novel Automated Patch-clamp Assays on Stem Cell-derived Cardiomyocytes: Will they Standardize In Vitro Pharmacology and Arrhythmia Research?

Amuzescu B*, Scheel O and Knott T
Cytocentrics Bioscience GmbH, Rostock, Germany
Corresponding Author : Amuzescu B
Cytocentrics Bioscience GmbH
Joachim Jungius Str. 9, Rostock 18059, Germany
Tel: +49 381 440 388-0
Fax: +49 381 440 388-47
E-mail: [email protected]
Received July 21, 2014; Accepted August 15, 2014; Published August 22, 2014
Citation: Amuzescu B, Scheel O, Knott T (2014) Novel Automated Patch- Clamp Assays on Stem Cell-Derived Cardiomyocytes: Will they Standardize In Vitro Pharmacology and Arrhythmia Research?. J Phys Chem Biophys 4:153. doi:10.4172/2161-0398.1000153
Copyright: © 2014 Amuzescu B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Journal of Physical Chemistry & Biophysics

Abstract

Recent progress in embryonic stem cell and human induced pluripotent stem cell technology allowed effective generation of cultured cardiomyocyte preparations with over 99% purity, rendering them suitable for automated patchclamp approaches. Compared to current high-throughput drug screening methods, such as fluorescence assays using calcium-sensitive or transmembrane potential-sensitive dyes, or field potential recordings and activation mapping using multi-electrode arrays, patch-clamp experiments offer the possibility to combine action potential recordings in current-clamp mode with detailed characterization of drug effects on multiple ion current components with carefully designed voltage-clamp protocols, leading to an in-depth understanding of arrhythmogenesis conditions and mechanisms, especially when combined with cellular electrophysiology computerized models. The recently issued Comprehensive in vitro ProArrhythmia Assay (CiPA) guidelines emphasize the importance of pharmacological tests on multiple cardiac ion channels, including at least Nav1.5 (early and late), Cav1.2, hERG1, Kv7.1/minK, and Kir2.1, via voltage-clamp protocols, instead of simple hERG screening, combined with computer modeling, in order to determine the proarrhythmic liability of a drug candidate. In addition, patch-clamp assays on patient-specific induced pluripotent stem cell-derived cardiomyocytes will enhance current molecular diagnosis methods in cardiac channelopathies by identification of the faulty current component and individualized screening of drug sensitivity of mutant channels, a step forward for personalized medicine approaches.

Keywords
Automated patch-clamp; Cardiac cell electrophysiology model; Cardiac channelopathy; Drug safety high-throughput screening; Cipa; MICE; Human embryonic stem cell-derived cardiomyocyte; Human induced pluripotent stem cell-derived cardiomyocyte; Patientspecific induced pluripotent stem cell-derived cardiomyocyte; Calciumsensitive dye; Transmembrane voltage-sensitive dye; Multielectrode array; Field potential; Action potential
Introduction
The first significant achievements in cellular cardiac electrophysiology date back to the beginning of the 1950s, when the two-microelectrode technique was applied to a variety of preparations, most notably “false tendons” of dog, kid, or lamb heart [1]. The Purkinje cells contained in these tissue samples present several important features, including a larger diameter (40-100 μm) and length and a lower contractility compared to ordinary ventricular fibres, enhancing multiple impalement and long stable recordings (over 10 minutes), an almost complete insensitivity to parasympathomimetic agents, and higher densities of voltage-dependent ion channels, required to achieve a speed of action potential propagation of several m/s. Beyond the remarkable contribution brought to the discovery of different ion current components and modeling the cardiac cell electrophysiology [2,3], they remained for decades a preferred system for in vitro cardiac pharmacology trials. It has been shown that, compared to papillary muscles or ventricular trabeculae, isolated rabbit Purkinje fibers are far more sensitive in detecting changes in action potential (AP) duration (APD90 – duration from the point of maximum upstroke velocity to 90% return from the peak depolarization to the resting potential) and presence of early afterdepolarizations (EAD) for a variety of drugs, including erythromycin, dofetilide, sertindole, and sparfloxacin [4]. Recently, this classical “golden standard” has been challenged by rapid progress in the field of embryonic stem cell (ESC) and especially induced pluripotent stem cell (iPSC) research. Since the first report in 2006 by the group of Shinya Yamanaka of iPSC generation via transfection of four “pluripotency” factors genes using retroviral vectors [5], multiple technological breakthroughs allowed for substantially higher yields of stem cells derived from differentiated cells like skin fibroblasts, peripheral blood lymphocytes, adipocytes or hair follicle keratinocytes, lower risks of tumorigenesis, as well as increasingly sophisticated protocols for redifferentiation of these stem cells into mature cardiomyocytes [6]. The painstaking in vitro redifferentiation process requires a stepwise application of conditioning media including three families of extracellular growth factors required for cardiac development in mammals: bone morphogenetic (BMP) factors like BMP4 and activin A, inhibitors of Wnt (wingless) signaling like DKK1 (dickkopf 1), and fibroblast growth factors, like βFGF [7]. However, once obtained, these cardiomyocytes are suitable for a variety of applications, including regenerative medicine approaches, in vitro models of diseases, and cardiac safety drug screening.
Multiple pharmacology assays using hiPSC-CM
More than a decade has passed since the first report describing spontaneous APs and contractility in cardiomyocytes within embryoid bodies grown from human embryonic stem cells [8], and recently we have witnessed an emergence of studies using either human embryonic stem cell-derived (hESC-CM) and/or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as standardized preparations for in vitro experiments. Recent progresses include refinement of cell culture protocols [9], use of antibiotic resistance tags to increase homogeneity of cell preparations [10], use of retro/lentiviral-free transfection methods in view of clinical applications [6]. Commercial stem cell-derived cardiomyocyte products are available from several suppliers, e.g. hESC-CMs from GE Healthcare (Piscataway, NJ; Cytiva™) or hiPSC-CMs from Cellular Dynamics International (Madison, WI; iCell® Cardiomyocytes), Axiogenesis (Cologne, DE; Cor.4U®), Pluriomics (Leiden, NL; StemCARDcells) or ReproCell (Yokohama, JP; ReproCardio2).
This scientific development led to novel high-throughput pharmacology screening equipments and techniques, based either on optical methods [11], using wide dynamic range Ca2+-sensitive dyes and improved cell loading with extracellular dye fluorescence quenching (e.g. FLIPR® Calcium 6 assay) and transmembrane voltage-sensitive dyes (e.g. FLIPR® Membrane Potential Assay Kit), or multi-electrode array (MEA) methods, allowing extracellular recordings of field potentials (FP), the cellular equivalent of the surface electrocardiogram, impedance measurements [12], and even two-dimensional local activation maps and conduction velocity studies [13]. Both optical and FP MEA-based screening assays present certain advantages, such as high throughput and simplicity of use, the possibility to assess AP propagation and effects of gap junction modulators due to use of confluent monolayers of mature highly differentiated iPSC-CM, and the ability to monitor “subacute” or “chronic” drug exposure effects. However, these systems suffer several shortcomings in exploring the mechanism of action and proarrhythmic effects of certain pharmacological compounds. This is related to the fact that arrhythmogenesis represents an emergent property at cell level, involving complex imbalances between multiple ion current components and intracellular ion handling and modulatory mechanisms [14], and optical/MEA-based iPSC-CM assays report only the sum of all drug-induced effects on the cell. The whole-cell patch clamp adds to this the proper reporting and control of the membrane potential. Recent modeling studies in combination with experimental approaches such as isolated heart preparations have evidenced new arrhythmogenesis mechanisms, such as synchronization of chaotic EADs in the genesis of torsades-des-pointes (TdP) and polymorphic ventricular tachycardia (PVT), a fascinating example of “deterministic chaos” at cellular level [15]. Moreover, the use of detailed cardiomyocyte cell electrophysiology models and advanced mathematical methods like dynamical systems linear stability and bifurcation analysis contributed to a detailed understanding of the mechanisms of cellular pace-making and ectopic focal arrhythmic activity in terms of damped or sustained oscillatory regimes achieved for certain combinations of parameters of a dynamical system, including a steady externally applied current [16-19], which is experimentally possible with whole-cell patch-clamp, but not with extracellular methods like MEA.
Performance of automated patch-clamp platforms for current-clamp AP recordings in hiPSC-CM
Thus, evidence of a pharmacological effect at the level of AP (FP or whole-cell) in spontaneous or induced pacing conditions represents only the first step in a complex process of arrhythmogenic mechanism assessment for that compound. In a simulation study performed with the LuoRudy dynamic model [20], using a large database of 45,000 APs generated with variable combinations of ion current surface densities, in the range of 0.25-2 times the default value, scanned against a test action potential obtained by modifying a single current component, we have shown that the simple AP shape matching criterion does not lead to a unique unambiguous identification of the modified current component [21]. The whole-cell patch-clamp approach offers superior versatility compared to both optical and MEA-based screening assays, because for each tested cell, beyond AP recordings in various pacing conditions, pharmacological effects on different current components can be thoroughly assessed via adequately designed voltage-clamp protocols. There are a few recent reports of hiPSC-CM experiments using planar automated patch-clamp platforms, consisting either in AP recordings in current-clamp mode [22] or voltage-clamp recordings of INa, ICa and IKr [10]. Our own experiments with the CytoPatch™ 2 equipment on iCell® Cardiomyocytes (CDI, Madison, WI) indicate a good stability of AP duration and shape in the classical whole-cell configuration using physiological external and internal solutions, as shown in Figure 1, and the possibility to combine current-clamp and voltage-clamp protocols in complex assays. Previous studies have emphasized the capacity of automated patch-clamp platforms to use very small samples of cell suspension [23], and our special cytocentering technique [24] allows experiments with as few as 150 cells in a 3-μl sample, offering over 80% capture rates and high yields of stable tight seals, with both seal and membrane resistance over 1 GΩ. In addition, using special culture conditions, we obtained routinely high percentages of viable mature cardiomyocytes with predominantly ventricular phenotype (APD50/90>0.7).
iPSC-CMs represent however imperfect electrophysiology models of mature cardiomyocytes of different types, due to variations in the levels of expression of multiple ion channel subunits and other morphological or functional features, such as a low density of T tubules, which leads to a completely different dynamics of Ca2+ between subcellular compartments during an AP [25]. Some of these unwanted features can be corrected using advanced stimulus protocols, e.g. a steady negative externally applied current can override the effects of excessive inward ion currents, bringing the resting potential from a depolarized state back to a value of -80 to -90 mV, specific for mature ventricular cardiomyocytes, and suppressing spontaneous APs, thus allowing pharmacological screening in ventricular-like settings using defined external pacing. Furthermore, a specific current component can be pharmacologically suppressed, while controlling the number of APs executed by the cell, to unravel the repolarization reserve [26], which is also very important in cardiac channelopathies [27].
Multiple ion channel effects and the CiPA paradigm
A paradigm that gains more and more support in the field of drug safety high-throughput screening is that of Multiple Ion Channel Effects (MICE), relying on assessment of pharmacological effects on three ion channels playing the major role in shaping the ventricular AP (Nav1.5, Cav1.2, and hERG1), and then evaluating the risks for different types of arrhythmias (e.g. the “torsadogenic” risk) [28]. Using a receiver operating characteristic (ROC) analysis for different risk prediction models, these authors have convincingly proved that the MICE approach offers a better sensitivity/specificity compromise compared to hERG screening alone, the classical paradigm in cardiac drug safety assessment. The new Comprehensive in vitro ProArrhythmia Assay (CiPA) guidelines, issued by a consortium of regulatory agencies and international research institutions, drive home the paramount importance of patch-clamp methods for cardiac safety pharmacology screening, proposing a combination of automated high-throughput or manual voltage-clamp assays and computer simulations with cellular cardiac electrophysiology models, in view of a complex proarrhythmic liability assessment of a drug candidate [29].
The importance of patch-clamp electrophysiology and pharmacology trials is even more obvious when it comes to in vitro studies using patient-derived iPSC-CMs as a diagnostic tool in cardiac channelopathies and other cardiac diseases with genetic inheritance [30-33]. Since the clinical picture is often unspecific, an accurate molecular diagnosis currently relies on extensive gene sequencing assays. The use of patch-clamp experiments on patient-derived iPSCCMs could change the diagnostic and therapeutic flowchart in the future, by pinpointing the faulty ion current component based on significant changes in cell surface density and/or kinetics, thus focusing and limiting the gene screening approach. In addition, patient-derived iPSC-CMs can be used in extensive patch-clamp pharmacology trials to explore in detail the arrhythmogenesis mechanisms, to generate an extended list of drugs incurring arrhythmia risks in an individual case, a list of well-tolerated drugs, and, in certain instances, even a patienttailored pharmacological treatment for arrhythmia prevention. A summary of our preliminary experiments with patient-specific hiPSCCM preparations can be found in an application note available at http://www.cytocentrics.com/en-us/newsmedia/downloadinformation.aspx.
Conclusion
In the context of extended electrophysiology and pharmacology trials on human (including patient-specific) iPSC-CMs, automated patch-clamp equipments, such as the CytoPatch™ 2, commercially available at Cytocentrics [24,34], in combination with advanced experimental protocols and computerized modeling studies, may prove invaluable tools in improving the prognosis and life quality of patients with such rare but severe diseases, as well as in routine quality assurance of batch production and daily use of cardiomyocytes in CiPA safety assays.
Acknowledgements
The authors gratefully acknowledge the entire Cytocentrics team for support, and Dr. Blake Anson (Cellular Dynamics International) for instructive discussions.
References

Figures at a glance

 

Figure
Figure 1
Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Article Usage

  • Total views: 11672
  • [From(publication date):
    August-2014 - Oct 24, 2017]
  • Breakdown by view type
  • HTML page views : 7876
  • PDF downloads :3796
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

[email protected]

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

[email protected]

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

[email protected]

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

[email protected]

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

[email protected]

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

[email protected]

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

[email protected]

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

[email protected]

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

[email protected]

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

[email protected]

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

[email protected]

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

[email protected]

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

[email protected]

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

[email protected]

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

[email protected]

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

[email protected]

1-702-714-7001 Extn: 9042

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords