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

Summary

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.

Abstract

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.

Introduction

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 effects^1,2,3. 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....

Protocol

1. Preparation of iPSC-derived Cardiomyocytes for Imaging

NOTE: Methods for iPSC culture and cardiac differentiation have been published before^12,13,14 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 explants from beating areas, generated using a monolayer differentiation protocol¹³, were taken on day 15 of a cardiac differentiati....

Results

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 .......

Discussion

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.

Materials

List of materials used in this article

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