Development and validation of dual-cardiotoxicity evaluation method based on analysis of field potential and contractile force of human iPSC-derived cardiomyocytes / multielectrode assay platform

2021 ◽  
Vol 555 ◽  
pp. 67-73
Author(s):  
Seul-Gi Lee ◽  
Jin Kim ◽  
Min-Seok Oh ◽  
Bokyeong Ryu ◽  
Kyu-Ree Kang ◽  
...  
Keyword(s):  
Evaluation Method ◽  
Field Potential ◽  
Contractile Force ◽  
Development And Validation ◽  
Human Ipsc

Abstract 15558: Characterization of Sarcolemmal K Atp Channels in Human Ipsc-derived Cardiomyocytes

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Robert Geiger ◽  
Naheed Fatima ◽  
Michael Klein ◽  
Robert E Goldstein ◽  
Mark C HAIGNEY ◽  
...  
Keyword(s):  
Katp Channel ◽  
Field Potential ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Levels ◽  
Typical Composition ◽  
Human Ipsc ◽  
Action Potential Shortening

Background: The ATP-sensitive potassium channel (KATP) plays a key role in protecting heart muscle during metabolic challenges such as ischemia. KATP activation causes action potential shortening that reduces calcium entry and contraction thus reducing calcium overload induced damage and preserving energy reserves. Cardiomyocytes derived from human inducible pluripotent stem cell (hiPSC) have emerged as a model to study cardiac function, however there are few studies that have focused on KATP. Methods: In the present study, cardiomyocytes were either generated from hiPSC using heparin- based chemically defined media or purchased from Cellular Dynamics (iCells2). Expression of the pore-forming (Kir6.2) and regulatory (SUR1 & SUR2) subunits of the KATP channel during differentiation were assessed using western blot. KATP function was assessed by measuring the field potential duration (FPD) and spontaneous beat rate in a confluent monolayer using the Axion Maestro multielectrode array system. Cells were probed using the KATP activators P1075 and diazoxide, specific for SUR2 and SUR1, respectively. Results: We found that the pore-forming subunit of the sarcolemmal KATP channel (Kir6.2) was expressed in iPSC and maintained throughout the course of differentiation. Consistent with the typical composition of sarcolemmal KATP, we observe a significant increase of SUR2 but little SUR1 protein following Wnt inhibition. Functionally, the FPD is markedly reduced by P1075 in a concentration-dependent manner, with 24% reduction at 100 nM and 92 % reduction at 100 μM. Moreover, glibenclamide 10μM reduces FPD shortening confirming a role for KATP. Finally, we observe little change in FPD when cells are exposed to diazoxide (100 μM) consistent with reduced SUR1 protein levels. Conclusion: These results indicate that cardiomyocytes derived from human iPSC express the KATP channel composed of primarily the SUR2 isoform and suggest that iPSC derived cardiomyocytes would be an effective model for studying the role of KATP during metabolic challenges.

Abstract 14803: Activin A Directly Impaired Human IPSC-cardiomyocyte Contractility and Electrophysiology

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Jake S Megna ◽  
Scott MacDonnell ◽  
Qin Ruan ◽  
Lori G MORTON
Keyword(s):  
Gene Expression ◽  
Cardiac Dysfunction ◽  
Field Potential ◽  
Activin A ◽  
Calcium Flux ◽  
Calcium Handling ◽  
Direct Role ◽  
Expression Of Genes ◽  
Cardiomyocyte Contractility ◽  
Human Ipsc

Activin A is a homo-dimeric TGF-β family member involved in embryonic development, tissue morphogenesis, and cellular differentiation. Levels of activin A correlate with NYHA functional classification and age dependent cardiac dysfunction; however, no data are available to demonstrate a direct role for activin A in regulating human cardiomyocyte dysfunction. The purpose of this study was to characterize the functional impact of chronic activin A on human cardiomyocyte electrophysiology, contractility, calcium handling, and gene expression. Methods: Human iPSC-cardiomyocytes (iPSC-CM) were supplied by Fujifilm Cellular Dynamics. Calcium transients were captured using the FLIPR Tetra imager and impedance/electrophysiology by the Nanion CardioExcyte96. Cells were plated (50k/well), allowed to reach a stable synchronous monolayer, and dosed with 1nM of activin A (A) at each media change alongside a media control (C) or in the presence of inhibitory anti-activin A antibodies. Gene expression was determined using Taqman. Results: Activin A reduced contractile impedance amplitude (C=9.82±0.30, A=6.82±0.21 Ohms, p<0.01) and slowed relaxation velocity kinetics (C=48.78±2.65, A=35.07±1.07 Ohms/sec, p<0.01). Elongated extracellular field potential durations were observed with activin A treatment compared to control (C=0.49±0.02, A=0.56±0.01 sec, p<0.01). Impaired calcium handling peak amplitude (C=609±99, A= C=447±33 RFU, p<0.05) was also observed alongside slower calcium flux falling times (C=0.52±0.05, A=0.70±0.04 sec, p<0.01). An inhibitory activin-A antibody restored all parameters to control values. Chronic exposure of iPSC-CM to activin A reduced expression of calcium handling genes RYR2 (C=1.01±0.07, A=0.75±0.03, p<0.01) and ATP2A2 (C=1.00±0.05, A=0.89±0.05, p<0.05). Conclusions: Chronic activin A treatment reduced cardiomyocyte contractile amplitude, slowed contractile kinetics, impaired cardiomyocyte calcium handling, elongated the action potential, and reduced expression of genes regulating key calcium handling proteins. These data demonstrate that elevated levels of activin A can directly act on cardiomyocytes and therefore may contribute to cardiac dysfunction in heart failure and aged populations.

scholarly journals Applying automated patch-clamp to disease modeling: recapitulate phenotypes of Brugada syndrome using iPSC-CMs

2021 ◽  
Author(s):  
Wener Li ◽  
Xiaojing Luo ◽  
Ying Ulbricht ◽  
Kaomei Guan
Keyword(s):  
Patch Clamp ◽  
Brugada Syndrome ◽  
Disease Modeling ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Quality Data ◽  
High Quality Data ◽  
Automated Patch Clamp ◽  
Induced Pluripotent ◽  
Human Ipsc

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of manual patch-clamp (MPC) such as labor intensive and low output. However, it was not clear whether the data generated by using the APC could be reliably used for iPSC-CM disease modeling. In this study, we improved the iPSC-CM preparation method by applying blebbistatin (BB, an excitation-contraction coupling uncoupler) in the whole APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology, however, BB-supplement leads to significant improvements in morphology and INa recording. We observe no effects of BB on action potential and field potential. Furthermore, APC faithfully recapitulates the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high quality data with higher throughput.

scholarly journals Optimizing Rate Correction of Field Potential Duration, a Biomarker for QT Risk Assessment, in Human Ipsc-Cardiomyocytes

2014 ◽  
Vol 106 (2) ◽  
pp. 720a ◽  
Author(s):  
Greg Luerman ◽  
Carlos Obejero-Paz ◽  
Arthur M. Brown ◽  
Andrew Bruening-Wright
Keyword(s):  
Risk Assessment ◽  
Field Potential ◽  
Human Ipsc

Integrated Ca2+ flux and AFM force analysis in human iPSC-derived cardiomyocytes

2020 ◽  
Vol 402 (1) ◽  
pp. 113-121
Author(s):  
Andrey V. Malkovskiy ◽  
Nadezda Ignatyeva ◽  
Yuanyuan Dai ◽  
Gerd Hasenfuss ◽  
Jayakumar Rajadas ◽  
...  
Keyword(s):  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Contractile Force ◽  
Force Microscopy ◽  
Force Dynamics ◽  
Sarcomeric Protein ◽  
Atomic Force ◽  
Analysis Strategy ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractWe developed a new approach for combined analysis of calcium (Ca2+) handling and beating forces in contractile cardiomyocytes. We employed human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dilated cardiomyopathy (DCM) patients carrying an inherited mutation in the sarcomeric protein troponin T (TnT), and isogenic TnT-KO iPSC-CMs generated via CRISPR/Cas9 gene editing. In these cells, Ca2+ handling as well as beating forces and –rates using single-cell atomic force microscopy (AFM) were assessed. We report impaired Ca2+ handling and reduced contractile force in DCM iPSC-CMs compared to healthy WT controls. TnT-KO iPSC-CMs display no contractile force or Ca2+ transients but generate Ca2+ sparks. We apply our analysis strategy to Ca2+ traces and AFM deflection recordings to reveal maximum rising rate, decay time, and duration of contraction with a multi-step background correction. Our method provides adaptive computing of signal peaks for different Ca2+ flux or force levels in iPSC-CMs, as well as analysis of Ca2+ sparks. Moreover, we report long-term measurements of contractile force dynamics on human iPSC-CMs. This approach enables deeper and more accurate profiling of disease-specific differences in cardiomyocyte contraction profiles using patient-derived iPSC-CMs.

scholarly journals Simultaneous Measurement of Contractile Force and Field Potential of Dynamically Beating Human iPS Cell-Derived Cardiac Cell Sheet-Tissue with Flexible Electronics

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Takashi Ohya ◽  
Haruki Ohtomo ◽  
Tetsutaro Kikuchi ◽  
Daisuke Sasaki ◽  
Yohei Kawamura ◽  
...  
Keyword(s):  
Functional Assessment ◽  
Flexible Electronics ◽  
Cell Sheet ◽  
Field Potential ◽  
Contractile Force ◽  
Cardiac Cell ◽  
Ips Cell ◽  
Cardiac Tissues ◽  
Induced Pluripotent

Human induced pluripotent stem (iPS) cell-derived cardiomyocytes are used for in vitro pharmacological and pathological studies worldwide. In particular, the functional assessment of cardiac tissues created from iPS cell-derived cardiomyocytes...

Morphological Basis of the Disparity Between Muscle Length and Sarcomere Length in the Myocardium

1973 ◽  
Vol 31 ◽  
pp. 422-423
Author(s):  
G.E. Adomian ◽  
L. Chuck ◽  
W.W. Pannley
Keyword(s):  
Cardiac Muscle ◽  
Sarcomere Length ◽  
Muscle Length ◽  
Contractile Force ◽  
Direct Function ◽  
Morphological Basis ◽  
Tension Curve

Sonnenblick, et al, have shown that sarcomeres change length as a function of cardiac muscle length along the ascending portion of the length-tension curve. This allows the contractile force to be expressed as a direct function of sarcomere length. Below L max, muscle length is directly related to sarcomere length at lengths greater than 85% of optimum. However, beyond the apex of the tension-length curve, i.e. L max, a disparity occurs between cardiac muscle length and sarcomere length. To account for this disproportionate increase in muscle length as sarcomere length remains relatively stable, the concept of fiber slippage was suggested as a plausible explanation. These observations have subsequently been extended to the intact ventricle.

Sign in / Sign up

Export Citation Format

Share Document