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Abstract

Introduction

Protocol

Representative Results

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Materials

References

Biology

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Published: September 27th, 2018

DOI:

10.3791/58134

1Medical Department I, University Hospital Klinikum rechts der Isar, Technical University of Munich, 2German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, 3Beth Israel Deaconess Medical Center, Harvard Medical School
* These authors contributed equally

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) are an emerging tool to dissect molecular mechanisms of heart disease, to investigate novel therapies, and to screen for adverse cardiac drug effects1,2,3. Right from the start, arrhythmogenic diseases such as channelopathies have been an important focus of this research area4. Consequently, methods to investigate electrical phenotypes of CMs, such as arrhythmias or changes in action potential (AP) morphologies, are at the heart of this technology.

An i....

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1. Preparation of iPSC-derived Cardiomyocytes for Imaging

NOTE: Methods for iPSC culture and cardiac differentiation have been published before12,13,14 and are not discussed here in detail. The purification of iPSC-CMs by manual microdissection, magnetic cell separation, or lactate selection is recommended, depending on the differentiation protocol used. For the following protocol, microdissected explan.......

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In Figure 4a, a representative single iPSC-CM is depicted with the white dotted lines marking the ROI drawn during the imaging analysis in the RFP (left side) and the GFP (right side) channel. The signal from the RFP channel shows a periodic increase in fluorescent intensity during each action potential (Figure 4b, upper panel). As described in the Introduction, this is due to an increasing FRET caused by changes in the membrane .......

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The method described here enables an optical recording of APs from a specific subtype (i.e., ventricular-like cells) of CMs generated from human iPSCs. Human iPSC-CMs are an emerging tool to address a huge variety of biological and medical problems, and the differentiation to different CM subtypes is an important source of experimental variability. By using specific promoter elements, the expression of a GEVI is specifically achieved in CMs representing the subtype of interest, which are then optically imaged.

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This work was supported by grants from the German Research Foundation (Si 1747/1-1), the Else Kröner-Fresenius-Stiftung, and the Deutsche Stiftung für Herzforschung.

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Name Company Catalog Number Comments
ß-Mercaptoethanol Invitrogen 21985023
DMEM-F12 Medium Invitrogen 21331046
FBS(Fetal Bovine Serum) Invitrogen 16141079
MEM Non-Essential Amino Acids Invitrogen 11140050
GlutaMax-I Supplement Invitrogen 35050061 alternative L-Glutamine
Penicillin-Streptomycin Invitrogen 15140122
Fibronectin bovine plasma Sigma-Aldrich F1141 
Collagenase type II Worthington Biochem LS004174
Hexadimethrine Bromide (Polybrene) Sigma-Aldrich H9268 enhancing lentiviral infection
3.5 cm glass-bottom microdishes MatTek corporation, Ashland, MA, USA P35G-1.5-14-C
Microscope stand Leica Microsystems, Wetzlar, Germany DMI6000B
Microscope objective Leica Microsystems, Wetzlar, Germany HCX PL APO 63x/1.4-0.6 Oil
sCMOS camera Andor Technology, Belfast, UK Zyla V
Microscope filter cube: excitation filter Chroma Technology Corp, Bellows Falls, VT, USA ET480/40X bandpass 480/40
Microscope filter cube: dichroic mirror Chroma Technology Corp, Bellows Falls, VT, USA T505lpxr longpass 505 nm
Image splitter  Cairn Research, Faversham, UK OptoSplit II
Image splitter filter cube: dichroic mirror AHF Analysentechnik GmbH, Tübigen, Germany 568LPXR longpass 568 nm
Image splitter filter cube: emission filter 1 (GFP emission) AHF Analysentechnik GmbH, Tübigen, Germany 520/28 BrightLine HC bandpass 520/28 nm
Image splitter filter cube: emission filter 2 (RFP emission) AHF Analysentechnik GmbH, Tübigen, Germany 630/75 ET Bandpass bandpass 630/75 nm
Pacing inset Warner Instruments, Hamden, CT, USA RC-37FS

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