This work was supported by grants from the German Research Foundation (Si 1747/1-1), the Else Kr&#246;ner-Fresenius-Stiftung, and the Deutsche Stiftung f&#252;r Herzforschung.

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...

<ol>
	<li>Sinnecker, D., Laugwitz, K. L., Moretti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+development+and+toxicity+testing.">Induced pluripotent stem cell-derived cardiomyocytes for drug development and toxicity testing.</a> Pharmacology &amp; Therapeutics. 143 (2), 246-252 (2014).</li><li>Goedel, A., My, I., Sinnecker, D., Moretti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+and+Challenges+of+Pluripotent+Stem+Cells+in+Cardiac+Arrhythmia+Research.">Perspectives and Challenges of Pluripotent Stem Cells in Cardiac Arrhythmia Research.</a> Current Cardiology Reports. 19 (3), 23 (2017).</li><li>Rocchetti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+arrhythmogenic+mechanisms+of+long-QT+syndrome+CALM1-F142L+mutation+in+patient-specific+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.</a> Cardiovascular Research. 113 (5), 531-541 (2017).</li><li>Sinnecker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+long-QT+syndromes+with+iPS+cells.">Modeling long-QT syndromes with iPS cells.</a> Journal of Cardiovascular Translational Research. 6 (1), 31-36 (2013).</li><li>Talkhabi, M., Aghdami, N., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiomyocyte+generation+from+pluripotent+stem+cells:+A+state-of-art.">Human cardiomyocyte generation from pluripotent stem cells: A state-of-art.</a> Life Sciences. , 98-113 (2016).</li><li>Ben-Ari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+electrophysiological+characteristics+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes.">Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.</a> Heart Rhythm. 13 (12), 2379-2387 (2016).</li><li>Den Hartogh, S. C., Passier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+Fluorescent+Reporters+in+Human+Pluripotent+Stem+Cells:+Contributions+to+Cardiac+Differentiation+and+Their+Applications+in+Cardiac+Disease+and+Toxicity.">Concise Review: Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity.</a> Stem Cells. 34 (1), 13-26 (2016).</li><li>Schweizer, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-specific+differentiation+of+cardiac+pacemaker+cell+clusters+from+human+induced+pluripotent+stem+cells.">Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.</a> Stem Cell Research &amp; Therapy. 8 (1), 229 (2017).</li><li>Moretti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+induced+pluripotent+stem-cell+models+for+long-QT+syndrome.">Patient-specific induced pluripotent stem-cell models for long-QT syndrome.</a> The New England Journal of Medicine. 363 (15), 1397-1409 (2010).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-specific+promoter-driven+action+potential+imaging+for+precise+disease+modelling+and+drug+testing+in+hiPSC-derived+cardiomyocytes.">Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.</a> European Heart Journal. 38 (4), 292-301 (2017).</li><li>Lam, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nature Methods. 9 (10), 1005-1012 (2012).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (5), 424-429 (2011).</li><li>Burridge, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature Methods. 11 (8), 855-860 (2014).</li><li>Bhattacharya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+differentiation+of+human+pluripotent+stem+cells+to+cardiomyocytes+and+characterization+by+flow+cytometry.">High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.</a> Journal of Visualized Experiments. (91), e52010 (2014).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> Journal of Visualized Experiments. (32), e1499 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Jung, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dantrolene+rescues+arrhythmogenic+RYR2+defect+in+a+patient-specific+stem+cell+model+of+catecholaminergic+polymorphic+ventricular+tachycardia.">Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.</a> EMBO Molecular Medicine. 4 (3), 180-191 (2012).</li><li>Lemoine, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+cardiomyocytes+cultured+in+3D+engineered+heart+tissue+show+physiological+upstroke+velocity+and+sodium+current+density.">Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density.</a> Scientific Reports. 7, 5464 (2017).</li><li>Dorn, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+nkx2-5+transcriptional+repression+of+isl1+controls+cardiomyocyte+subtype+identity.">Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.</a> Stem Cells. 33 (4), 1113-1129 (2015).</li><li>Kaestner, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+Encoded+Voltage+Indicators+in+Circulation+Research.">Genetically Encoded Voltage Indicators in Circulation Research.</a> International Journal of Molecular Sciences. 16 (9), 21626-21642 (2015).</li></ol>

The authors have nothing to disclose.

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.</...

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 important consideration in the application of iPSC-CMs is that current cardiac differentiation protocols do not result in a homogenous population of cells. Instead, they are rather a mixture of cells resembling sinus node, atrial, and ventricular CMs at different levels of maturation5,6,7,8. This heterogeneity can be a relevant source of experimental variability, especially if parameters such as AP duration (APD) are investigated, which intrinsically differ between CM subtypes (e.g., the APD is shorter in atrial than in ventricular CMs). The conventional approach to address this problem is to investigate single iPSC-CMs using the patch clamp method and to classify each cell as nodal-, atrial-, or ventricular-like, based on its AP morphology9. Any subsequent analysis can be then restricted to the cells representing the CM subtype of interest. The major drawback of this strategy is its limited throughput and lack of scalability. Moreover, the invasive nature of patch clamp electrophysiology does not allow the imaging of the same cells sequentially over extended time periods.
Here, we provide experimental details on a method10 developed to optically image APs in specific subtypes of iPSC-CMs. This overcomes the problem of subtype heterogeneity and dramatically increases the throughput as compared to conventional methods, allowing the rapid phenotyping of iPSC-CMs carrying genetic variants or being exposed to pharmacological agents.
Overview of the subtype-specific optical imaging approach
A genetically-encoded voltage indicator (GEVI), whose fluorescence properties change upon depolarization and repolarization of the cell membrane, is used to optically image changes of the membrane potential of CMs. The GEVI applied here is the voltage-sensing fluorescent protein VSFP-CR11, which consists of a voltage-sensing transmembrane domain fused to a pair of a green (Clover) and a red (mRuby2) fluorescent protein (Figure 1A). Due to the close proximity of the two fluorophores, the excitation of the green fluorescent protein results in a fraction of the excitation energy being transferred to the red fluorescent protein via F&#246;rster resonance energy transfer (FRET). Therefore, the excitation of the green fluorescent protein results in an emission from both the green and the red fluorescent proteins (Figure 1A, upper panel). When the cell depolarizes, a structural rearrangement of the voltage sensor occurs that translates into a reorientation of the two fluorescent proteins, increasing the FRET efficiency. Thus, even more of the excitation energy is transferred from the green to the red fluorescent protein (Figure 1A, lower panel). As a result, in a depolarized cell, the green fluorescence emission is dimmer, and the red fluorescence emission is brighter than in a cell at resting membrane potential (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58134/58134fig1.jpg" /> 
Figure 1: Optical imaging of membrane potential with VSFP-CR. (A) A schematic depicting the action of the voltage-sensitive fluorescent protein VSFP-CR is shown. Upon the depolarization of the cell membrane, a structural rearrangement in the voltage-sensing transmembrane domain translates into a reorientation of the green (GFP) and red (RFP) fluorescent protein, increasing the efficiency of the intramolecular F&#246;rster resonance energy transfer (FRET). (B) The emission spectra of a VSFP upon excitation of the GFP in cells at the resting membrane potential (upper panel) and in depolarized cells (lower panel) are depicted. The spectral change upon depolarization is exaggerated for clarity. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The changes in the FRET efficiency mirroring the fluctuations of the membrane potential are imaged using a fluorescence microscope equipped with an image splitter, which separates red and green fluorescence emissions and projects them onto two adjacent areas of the chip of an sCMOS camera (Figure 2). With this set-up, the fluorescence emission at two different wavelength bands can be recorded simultaneously, which allows the calculation of a ratio of red-to-green fluorescence to reflect the membrane potential in every image of a time-lapse series.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58134/58134fig2.jpg" /> 
Figure 2: Configuration of the imaging system. The principal components of the imaging system used to image the spectral changes of the voltage-sensitive fluorescent protein mirroring the membrane potential changes at a high temporal resolution are depicted. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The expression of VSFP-CR in CMs is achieved by lentiviral transduction. To direct expression to the CM subtype of interest, the lentivirus contains a promoter element (the MLC2v enhancer) that specifically drives transcription in ventricular-like iPSC-CMs10. When the iPSC-CMs that represent a mixture of atrial-like, nodal-like, and ventricular-like cells are transduced with this lentivirus, VSFP-CR is expressed only in the ventricular-like cells. Since the optical action potential imaging depends on this fluorescent sensor, the recorded action potentials exclusively represent the CM subtype of interest (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58134/58134fig3.jpg" /> 
Figure 3: Promoter-driven VSFP expression for subtype-specific membrane potential imaging. (a)&#160;This schematic shows how cardiomyocyte subtype-specific optical action potential recordings are achieved. (b) iPSC-CMs infected with a VSFP under the control of the ventricular-specific MLC2v-enhancer are shown. The expression of the voltage sensor is observed only in ventricular-like CMs in the GFP channel (left panel). The phase contrast (middle panel) and overlay image (right panel) are also provided. The white dotted lines mark cell boundaries. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>&#223;-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 Medium</td>
			<td>Invitrogen</td>
			<td>21331046</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS(Fetal Bovine Serum)</td>
			<td>Invitrogen</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Invitrogen</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax-I Supplement</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>alternative L-Glutamine</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Worthington Biochem</td>
			<td>LS004174</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>enhancing lentiviral infection</td>
		</tr>
		<tr>
			<td>3.5 cm glass-bottom microdishes</td>
			<td>MatTek corporation, Ashland, MA, USA</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope stand</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>DMI6000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope objective</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td colspan="2">HCX PL APO 63x/1.4-0.6 Oil</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor Technology, Belfast, UK</td>
			<td>Zyla V</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope filter cube: excitation filter</td>
			<td>Chroma Technology Corp, Bellows Falls, VT, USA</td>
			<td>ET480/40X</td>
			<td>bandpass 480/40</td>
		</tr>
		<tr>
			<td>Microscope filter cube: dichroic mirror</td>
			<td>Chroma Technology Corp, Bellows Falls, VT, USA</td>
			<td>T505lpxr</td>
			<td>longpass 505 nm</td>
		</tr>
		<tr>
			<td>Image splitter</td>
			<td>&#160;Cairn Research, Faversham, UK</td>
			<td>OptoSplit II</td>
			<td></td>
		</tr>
		<tr>
			<td>Image splitter filter cube: dichroic mirror</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>568LPXR</td>
			<td>longpass 568 nm</td>
		</tr>
		<tr>
			<td>Image splitter filter cube: emission filter 1 (GFP emission)</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>520/28 BrightLine HC</td>
			<td>bandpass 520/28 nm</td>
		</tr>
		<tr>
			<td>Image splitter filter cube: emission filter 2 (RFP emission)</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>630/75 ET Bandpass</td>
			<td>bandpass 630/75 nm</td>
		</tr>
		<tr>
			<td>Pacing inset</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>RC-37FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtype-specific optical action potential recordings in human induced pluripotent stem cell-derived ventricular cardiomyocytes

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.

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 ...

Watch this Scientific Journal Video about Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes at JoVE.com

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

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 ...

Cardiomyocytes generated from Induced Pluripotent Stem or IPS cells are increasingly used to answer a variety of biological questions in the field of cardiac electrophysiology. The main advantages of this technology are that it allows a dramatic increase in throughput as well as subtype specific action potential recordings in ventricular like myocytes, using a ventricular specific promoter element. The introduction of genetically encoded voltage sensors through lentiviral transduction allows the optical imaging of human-induced pluripotent stem cells-derived cardiomyocytes action potentials.Begin by mixing lentivirus-containing cell culture supernatant with cardiomyocyte maintenance medium, at a one to one ratio. Add hexadimetherine bromide at a final concentration of 8 micrograms per milliliter, to enhance the infection efficiency. And replace the cardiomyocyte maintenance medium in the IPS cell-derived cardiomyocytes culture dish, with the infection medium.Culture the infected cardiomyocytes for 12 hours at 37 degrees Celsius, and replace the infection medium with fresh cardiomyocyte maintenance medium. Before imaging, exchange the cell culture medium with Tyrode's solution, supplemented with calcium. Then place the cardiomyocytes in an imaging chamber on the stage of an inverted epifluorescence microscope equipped with an image splitter.If electrical pacing is required, install pacing electrodes in the imaging chamber and connect the electrodes to a stimulus generator. Bring the cells into focus, and locate a cell expressing the genetically encoded voltage indicator in the center of the viewing field. Set the optical path so that the emitted light is routed to the image splitter and switch the filter cube in the microscope to the one to be combined with the filters in the image splitter.In the software controlling the camera, set the acquisition setting so that high-speed imaging is possible, and set up the image splitter according to the manufacturer's instructions. The two images representing the two different emission wavelength bands should be adjacent to each other, each occupying one half of the image. Check and adjust the focus of the image produced by the camera as necessary.And adjust the brightness of the illumination light so that pixel saturation is avoided. Set the acquisition of the time series, and dim the light in the room to avoid stray light influencing the measurement. Then open the excitation light shutter and begin the acquisition of the image series, starting the electrical pacing at the appropriate time point, as experimentally appropriate.When the acquisition is finished, close the excitation light shutter as necessary and save the recording to the hard drive. Then, leaving the recording setting unchanged, record the background over a region of the coverslip without cells. For analysis of the optical membrane potential recordings, open the tiff stack containing the cells in ImageJ, and use the free-hand selection tool to draw a region of interest over a fluorescent cardiomyocyte, in the red channel, taking care that the area is large enough that the cell will not move out of the region while contracting.Next, select Analyze, Tools, Region Of Interest Manager and select Add T, to add the region as Region Of Interest 1. Drag the region of interest over the same cell in the green channel, and add this region as Region Of Interest 2. Under the Analyze and Set Measurements menus, unselect all of the options except for the Mean gray value.In the Region Of Interest Manager, click More and Multi Measure to select the Measure all slices and One row per slice options and click OK.A window will open containing a spreadsheet with the three rows. Use File and Save As to save the spreadsheet into a comma separated value file. Close the window with the image stack and the spreadsheet window without closing the Region of Interest Manager window, and open the tiff stack containing the background measurement.In the Region Of Interest Manager, click the More and Multi Measure to select the Measure all slices and One row per slice options and click OK.Then save the background data in a separate csv file. Here, a representative single IPS cell-derived cardiomyocyte is depicted with the white dotted lines marking the regions of interest, drawn during the imaging analysis in the RFP and GFP channels is shown. The signal from the RFP channel demonstrates a periodic increase in fluorescent intensity during each action potential due to an increasing forced or resonance energy transfer caused by changes in the membrane potential.Concordantly, the GFP signal exhibits a periodic decrease in fluorescent intensity. The RFP/GFP ratio is the biological signal used in downstream analyses, such as action potential duration 50 and 90 measurements. For example, using this method, 30-day old ventricular like IPS cell-derived cardiomyocytes placed at 0.5 Hertz, showed a mean action potential duration 50 of 439 milliseconds, and a mean action potential duration 90 of 520 milliseconds.Electrical stimulation of the IPS cell-derived cardiomyocytes at increasing stimulation rates every five beats, demonstrated a progressive shortening of the action potentials as the beating rate increased. Moreover, treatment of the spontaneously beating IPS cell-derived cardiomyocytes with isoproternol, a nonselective beta adreno receptor agonist, led to a prompt increase in the beating rate, that could be further increased with increasing doses of the agonist. While this method provides superior throughput compared to classical electrophysiological measurements, one must be aware of its limitations, including a temporal resolution that is limited by the intrinsic kinetic properties of the fluorescent voltage sensor.

subtype-specific-optical-action-potential-recordings-human-induced

Research

JoVE Journal

Biology

7.1K visualizações.  Technical University of Munich. Cardiomiócitos gerados a partir de células-tronco pluripotentes induzidas ou células IPS são cada vez mais usados para responder a uma variedade de questões biológicas no campo da eletrofisiologia cardíaca. As principais vantagens desta tecnologia são que ela permite um aumento dramático no throughput, bem como gravações potenciais de ação específicas do subtipo em miócitos ventriculares, usando um elemento promotor específico ventricular. A introdução de sensores de tensão...