We would like to acknowledge all the lab members in the Division of Regenerative Medicine at the Jichi Medical University for the helpful discussion and technical assistance. This study was supported by the grants from the Japan Agency for Medical Research and Development (AMED; JP18bm0704012&#160;and JP20bm0804018), the Japan Society for the Promotion of Science (JSPS; JP19KK0219), and the Japanese Circulation Society (the Grant for Basic Research) to H.U.

<ol>
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H.U. has filed a patent related to this manuscript.

PSC-CMs have great potential to be utilized as an&#160;in vitro platform to model heart disease and to test the effects of drugs. Nevertheless, an accurate, unified method to assess PSC-CMs functions must first be established. Most of functional tests work with PSC-CMs, e.g., electrophysiology, calcium transient, and metabolism26, and one of the first patient-derived PSC-CM studies was about long-QT syndrome27. However, contractility, one of the most important functions of a cardiomyocyte, is still difficult to assess. With adult cardiomyocytes, sarcomere shortening is widely used. In contrast, due to the underdeveloped and disorganized sarcomere of PSC-CMs, the standard method for sarcomere shortening does not work with these cells. Therefore, we have presented an alternative method for examining sarcomere shortening in PSC-CMs using fluorescent-tagged sarcomere proteins. As demonstrated, proteins localized to the M-line (MYOM2) or Z-line (ACTN2, TCAP, and PDLIM3) fused with fluorescent proteins can be used for this approach. We have also shown that fluorescent-tagged proteins can be expressed from endogenous loci or by AAVs. AAVs provide more flexibility for expressing fluorescent-tagged proteins than do endogenous loci, as AAVs can be applied to any type of patient-derived PSC-CMs. In contrast, expressing proteins from endogenous loci may have a lesser effect on sarcomere function, as the expression level of the genes is tightly regulated and can also be used for monitoring the maturation of PSC-CMs18.
Even though Myom2-TagRFP, ACTN2-mCherry, and Lifeact were all used to examine the sarcomere shortening16,18,19, it is still unclear if these proteins interfere with sarcomere function. Recently, Lifeact was reported to disturb actin organization and cellular morphology28. It is also important to note that fusion patterns (i.e., the GFP fusion site at N-term or C-term of target protein) also affect the sarcomere function29. Therefore, before being used widely, it is important to thoroughly assess whether fluorescent-tagged sarcomere proteins interfere with sarcomere function and whether protein-protein interactions occur in sarcomeres in vitro, in vivo, and/or in adult cardiomyocytes. This repertoire of fluorescent-tagged sarcomere proteins provides a good starting point for future protein-engineering options (i.e., shortening the sarcomere proteins to only localization signals). Selecting proteins to tag is another key to success. We have tagged another Z-line protein with GFP, however, this protein displayed only a cytoplasmic distribution rather than localizing to the sarcomere. For live-imaging, protein stability may also play a role, as, for example, if a tagged protein is unstable, the signal level will be lower. The photostability of the fluorescent protein is also important, as unstable protein signals will be easily quenched during imaging.
To examine the contractility of PSC-CMs using methods other than described, indirect measurements of force generated by PSC-CMs (e.g., micro-post arrays, traction force microscopy) or motion (e.g., high-resolution motion detection using SI8000)11,12,13,14 can be used. Our method can be combined with these methods or with dye-based action potential/calcium transient measurements. The combinatorial approach may provide further information on how a disease causes dysfunction in patient-derived PSC-CMs.
One of the challenges in sarcomere shortening in PSC-CMs is to find a good sarcomere that moves linearly, otherwise, the cells may easily come off the line for sarcomere detection of SarcOptiM and cause unstable sarcomere shortening results. Here, we demonstrate that using a patterned culture generated with PDMS stamps may provide a more stable and linear sarcomere movement. A patterned culture is also known to support the maturation of PSC-CMs16, which is important for sarcomere function.

Cardiovascular diseases are the leading cause of mortality worldwide1 and cardiomyopathy represents the third cause of cardiac-related deaths2.Cardiomyopathy is a collective group of diseases that affect cardiac muscles. The recent developments of induced pluripotent stem (iPS) cells and the directed-differentiation of iPS cells toward cardiomyocytes (PSC-CMs) have opened the door for studying cardiomyocytes with patient genome as an in vitro model of cardiomyopathy. These cells can be used to understand the pathophysiology of cardiac diseases, to elucidate their molecular mechanisms, and to test different therapeutic candidates3. There is a tremendous amount of interest, thus, patient-derived iPS cells have been generated (e.g., hypertrophic cardiomyopathy [HCM]4,5, arrhythmogenic right ventricular cardiomyopathy [ARVC]6, dilated cardiomyopathy [DCM]7, and mitochondrial-related cardiomyopathies8,9). Because one of the characteristics of cardiomyopathy is the dysfunction and disruption of sarcomeres, a valid tool that uniformly measures sarcomere function is needed.
Sarcomere shortening is the most widely used technique to assess sarcomere function and the contractility of adult cardiomyocytes derived from animal models and humans. To perform sarcomere shortening, well-developed sarcomeres that are visible under phase-contrast are required. However, PSC-CMs cultured in vitro display underdeveloped and disorganized sarcomeres and, therefore, are unable to be used to properly measure sarcomere shortening10. This difficulty to properly assess the contractility of PSC-CMs hinders their usage as a platform to assess cardiac functions in vitro. To assess PSC-CMs contractility indirectly, atomic force microscopy, micro-post arrays, traction force microscopy, and impedance measurements have been used to measure the effects of the motion exerted by these cells on their surroundings11,12,13. More sophisticated and less invasive video-microscopy recordings of actual cellular motion (e.g., SI8000 from SONY) can be used to alternatively assess their contractility, however, this method does not directly measure sarcomere motion or force generation kinetics14.
To directly measure sarcomere motion in PSC-CMs, new approaches, such as fluorescent-tagging to sarcomere protein, are emerging. For example, Lifeact is used to label filamentous actin (F-actin) to measure sarcomere motion15,16. Genetically modified iPS cells are another option for tagging sarcomere proteins (e.g., &#945;-actinin [ACTN2] and Myomesin-2 [MYOM2]) by fluorescent protein17,18,19.
In this paper, we describe how to perform time-lapse imaging for measuring sarcomere shortening using Myom2-TagRFP (mouse embryonic stem [ES] cells) and ACTN2-mCherry (human iPS cells). We also show that a patterned culture facilitates sarcomere alignment. In addition, we describe an alternative method of sarcomere labeling, using adeno-associated viruses (AAVs), which can be widely applied to patient-derived iPS cells.

<table><tbody><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M6145-25</td><td></td></tr><tr><td>2-Mercaptoethanol (55mM)</td><td>Thermo Fisher Scientific</td><td>21985-023</td><td></td></tr><tr><td>2-methacryloyloxyethyl phosphorylcholine (MPC) polymer,</td><td>NOF Corp.</td><td>LIPIDURE-CM5206</td><td></td></tr><tr><td>2-Propanol</td><td>Fujifilm wako</td><td>166-04836</td><td></td></tr><tr><td>35-mm imaging dish with a polymer coverslip (&amp;micro;-Dish 35 mm, high)</td><td>ibidi</td><td>81156</td><td></td></tr><tr><td>AAVproR Helper Free System (AAV6)&lt;br/&gt; (vectors; pHelper, pRC6, pAAV-CMV-Vector)</td><td>Takara</td><td>6651</td><td></td></tr><tr><td>ACTN2-mCherry (AR12, AR21) hiPSCs</td><td>N.A.</td><td></td><td>We inserted IRES-puromycin resistant casette to 3&#039; UTR of TNNT2 locus and mCherry around the stop codon of ACTN2 in 610B1 hiPSC line, following a method describe elsewhere (Anzai, Methods Mol Biol, in press)</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>Thermo Fisher Scientific</td><td>17504-044</td><td></td></tr><tr><td>B-27 Supplement, minus insulin</td><td>Thermo Fisher Scientific</td><td>A18956-01</td><td></td></tr><tr><td>B27 supplement (50X), minus Vitamin A</td><td>Thermo Fisher Scientific</td><td>12587-010</td><td></td></tr><tr><td>Benzonase (25 U/&amp;micro;L)</td><td>Merck Millipore</td><td>70746</td><td></td></tr><tr><td>Blasticidin S Hydrochloride</td><td>Fujifilm wako</td><td>029-18701</td><td></td></tr><tr><td>BMP-4, Human, Recombinant,</td><td>R&amp;amp;D Systems, Inc.</td><td>314-BP-010</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4503-100g</td><td></td></tr><tr><td>C59, Wnt Antagonist (WntC59)</td><td>abcam</td><td>ab142216</td><td></td></tr><tr><td>CAD drawing software,</td><td>Robert McNeel and Associates, WA, USA</td><td>Rhinoceros 6.0</td><td></td></tr><tr><td>Centrifugal ultrafiltration unit (100k MWCO), Vivaspin-20</td><td>Sartorius</td><td>VS2042</td><td></td></tr><tr><td>CHIR99021</td><td>Cayman</td><td>13122</td><td></td></tr><tr><td>Chromium etchant</td><td>Nihon Kagaku Sangyo Co., Ltd., Japan</td><td>N14B</td><td></td></tr><tr><td>Chromium mask coated with AZP1350</td><td>Clean Surface Technology Co., Japan</td><td>CBL2506Bu-AZP</td><td></td></tr><tr><td>Dr. GenTLE Precipitation Carrier (20mg/mL Glycogen, 3 M Sodium Acetate (pH 5.2))</td><td>Takara</td><td>9094</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM) - high glucose</td><td>Sigma-Aldrich</td><td>D6429-500</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM) - high glucose, without sodium pyruvate</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Ethanol (99.5)</td><td>Fujifilm wako</td><td>057-00456</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Moregate</td><td>59301104</td><td></td></tr><tr><td>FGF-10, Human, Recombinant,</td><td>R&amp;amp;D Systems, Inc.</td><td>345-FG-025</td><td></td></tr><tr><td>Fibroblast Growth Factor(basic), human, recombinant</td><td>Fujifilm wako</td><td>060-04543</td><td></td></tr><tr><td>Gelatin from porcine skin powder</td><td>Sigma-Aldrich</td><td>G1890-100g</td><td></td></tr><tr><td>Glasgow Minimum Essential Medium (GMEM)</td><td>Sigma-Aldrich</td><td>G5154-500</td><td></td></tr><tr><td>GLASS BOTTOM culture plates</td><td>MatTek</td><td>P24G-1.5-13-F/H</td><td></td></tr><tr><td>Ham&amp;rsquo;s F-12</td><td>Thermo Fisher Scientific</td><td>11765-062</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Thermo Fisher Scientific</td><td>12440-061</td><td></td></tr><tr><td>L-alanine-L-glutamine (GlutaMAX Supplement, 200mM)</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td></td></tr><tr><td>L(+)-Ascorbic Acid Sodium Salt</td><td>Fujifilm wako</td><td>196-01252</td><td></td></tr><tr><td>Laminin-511 E8 fragment (LN511-E8, iMatrix-511)</td><td>Nippi</td><td>892012</td><td></td></tr><tr><td>Mask aligner</td><td>Union Optical Co., Ltd., Japan</td><td>PEM-800</td><td></td></tr><tr><td>Maskless lithography tool</td><td>NanoSystem Solutions, Inc., Japan</td><td>D-Light DL-1000</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td></td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45 &amp;micro;m, PVDF (0.45-&amp;micro;m filter)</td><td>Merck Millipore</td><td>SLHVR33RS</td><td></td></tr><tr><td>Myom2-RFP (SMM18)</td><td>N.A.</td><td></td><td>Developed in our previous paper (Chanthra, Sci Rep, 2020)</td></tr><tr><td>N-2 Supplement (100X)</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>ORCA-Flash4.0 V3 digital CMOS camera</td><td>Hamamatsu</td><td>C13440-20CU</td><td></td></tr><tr><td>PD0325901</td><td>Stemgent</td><td>04-0006-10</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Petri dish</td><td>Sansei medical co. Ltd</td><td>01-004</td><td></td></tr><tr><td>Phenol/Chloroform/Isoamyl alcohol (25:24:1)</td><td>Nippon Gene</td><td>311-90151</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) elastomer</td><td>Dow Corning Corp., MI, USA</td><td>SILPOT 184</td><td></td></tr><tr><td>polyethylenimine MAX (MW. 40,000)</td><td>Polyscience</td><td>24765-1</td><td></td></tr><tr><td>Positive photoresist developer</td><td>Tokyo Ohka Kogyo Co., Ltd., Japan</td><td>NMD-3</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>Thermo Fisher Scientific</td><td>A25742</td><td></td></tr><tr><td>Proteinase K</td><td>Takara</td><td>9034</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>Fujifilm wako</td><td>166-23153</td><td></td></tr><tr><td>Recombinant Human/Mouse/Rat Activin A Protein</td><td>R&amp;amp;D Systems, Inc.</td><td>338-AC-050</td><td></td></tr><tr><td>Recombinant trypsin-like protease (rTrypsin; TrypLE express)</td><td>Thermo Fisher Scientific</td><td>12604-039</td><td></td></tr><tr><td>RPMI1640 Medium</td><td>Thermo Fisher Scientific</td><td>11875-119</td><td></td></tr><tr><td>Silicon wafer</td><td>Matsuzaki Seisakusyo Co., Ltd., Japan</td><td>N.A.</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Thermo Fisher Scientific</td><td>11360-070</td><td></td></tr><tr><td>Spin-coater</td><td>Mikasa Co., Ltd., Japan</td><td>MS-A100</td><td></td></tr><tr><td>Spininng confocal microscopy</td><td>Oxford Instruments</td><td>Andor Dragonfly Spinning Disk System</td><td></td></tr><tr><td>StemSure LIF, Mouse, recombinant, Solution (10^6U)</td><td>Fujifilm wako</td><td>195-16053</td><td></td></tr><tr><td>SU-8 3010</td><td>Kayaku Advanced Materials, Inc., MA, USA</td><td>SU-8 3010</td><td></td></tr><tr><td>SU-8 developer</td><td>Kayaku Advanced Materials, Inc., MA, USA</td><td>SU-8 developer</td><td></td></tr><tr><td>Tris-EDTA</td><td>Nippon Gene</td><td>314-90021</td><td></td></tr><tr><td>Vascular Endothelial Growth Factor-A165(VEGF), Human, recombinant</td><td>Fujifilm wako</td><td>226-01781</td><td></td></tr></tbody></table>

sarcomere shortening of pluripotent stem cell-derived cardiomyocytes using fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

Measuring sarcomere shortening using knock-in PSC-CMs reporter lines. Sarcomere-labeled PSC-CMs were used to measure sarcomere shortening. The lines express Myom2-RFP and ACTN2-mCherry from endogenous loci. TagRFP was inserted to Myom2, coding M-proteins that localize to the M-line, while mCherry was knocked-in to ACTN2, coding &#945;-Actinin, which localizes to the Z-line18,25. Time-lapse images were obtained and used to determine sarcomere shortening as presented in Figures 1 and 2 and Movie 1-3.
To overcome the disorganized sarcomere of PSC-CMs, specific PDMS stamps were used to culture PSC-CMs in the stripe pattern. This patterned culture promoted an elongated cell shape and a more organized sarcomere pattern compared to cells cultured in the non-pattern area (Figures 2B and 2C). With this advantage, the patterned culture promoted better contraction of the cells and provided a smooth sarcomere length profile as shown in Movies 2 and 3 and Figure 2D.
Fluorescent tagging of Z-line protein using AAV vectors. To visualize the Z-line of PSC-CMs without generating knock-in iPS cells, fluorescent-tagged Z-line proteins were expressed using AAV transduction. Two of small Z-line proteins, Telethonin (TCAP) and Actin-associated LIM protein (PDLIM3) with GFP, were tagged and packaged using the AAV6 capsid (Figure 3A). Once PSC-CMs were differentiated and purified, AAVs were transduced to PSC-CMs (Figure 3B). The transduced PSC-CMs expressed sarcomeric GFP signals along the PSC-CMs as early as three days post-transduction (Figures 3C and 3D). Typically, the transduction of AAV at an MOI of 105 vg/cell is sufficient to visualize fluorescent-tagged sarcomere proteins and a higher titer may cause non-specific localization of GFP to cytoplasm though it increases overall GFP intensity.
Purification of PSC-CMs using AAV vectors. Current methods rely on the drug selection cassette that is already on the genome of PSC-CMs, either transgenic or knock-in line. However, it is labor-intensive to produce such a line from patient-derived iPS cells. As AAV vectors have been demonstrated to drive the expression of fluorescent-labeled Z-line proteins without the need for knock-in, we sought to establish the purification method without knock-in&#160;as well (Figure 4). To this end, a new AAV vector, which encodes blasticidin-resistant gene under the control of cTNT promoter, was constructed (Figure 4A). The AAV (MOI of 105 vg/cell) was transduced to differentiating human iPS cells at day 4. Then cells were treated with 2.5-10 &#956;g/mL of blasticidin (need to titrate for each cell line) between days 7 and 9 (Figure 4B). At day 14, the purity of PSC-CMs was more than 90% (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62129/62129fig01.jpg" /> 
Figure 1: Sarcomere shortening of the mouse PSC-CMs derived from the Myom2-TagRFP cell line. A. The timeline for mouse PSC-CM differentiation. B. Representative images for sarcomere shortening in different time points with measuring regions as indicated by yellow bars. Scale bar = 10 &#956;m. C. Sarcomere length profile during contraction of the cardiomyocytes that was stimulated with electricity at 1 Hz. The framerate was 50 frames per second. The pixel size was 0.26 &#956;m. <a href="https://www.jove.com/files/ftp_upload/62129/62129fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62129/62129fig02.jpg" /> 
Figure 2: Representative data showing sarcomere shortening of the human PSC-CMs derived from the ACTN2-mCherry cell line in non-patterned and patterned culture. A. The timeline for human PSC-CM differentiation. B. and C. Cardiomyocytes cultured in non-patterned cultures show disorganized sarcomere patterns (B)&#160;while patterned cultures promote a good alignment of the sarcomere (C). Measuring regions presented by yellow bars. D. Corresponding sarcomere length profiles obtained during cell contraction induced by electrical stimulation at 0.5 Hz and a 100 frames per second frame rate. Pixel size = 0.26 &#956;m, scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62129/62129fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62129/62129fig03.jpg" /> 
Figure 3: Mouse PSC-CMs after AAV transduction for 3 days. A. Schematic vector map of AAV for sarcomere labeling. A sarcomere protein (gene of interest, GOI) is linked to GFP with a Gly-Gly-Gly-Ser linker (L) and expressed under the control of cardiac troponin T (cTNT) promoter. B. The timeline for mouse PSC-CM differentiation and AAV transduction. C. and D. Representative images showing clear sarcomere localization and the corresponding sarcomere length profile of TCAP-GFP (C) and PDLIM3-GFP (D) after 3 days of transduction into PSC-CMs generated from the Myom2-TagRFP cell line. Scale bar = 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/62129/62129fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62129/62129fig04.jpg" /> 
Figure 4: Blasticidin Purification of human PSC-CMs without knock-in.&#160; 
A. Schematic vector map of AAV, in which a blasticidin-resistance gene cassette (BSR) is inserted downstream to cTNT promoter. B. The timeline of human PSC-CMs differentiation, transduction, and blasticidin selection. C. Representative data showing percentage of cTNT + cells in human PSC-CMs (transduced 105 vg/cell AAV6 on day 4 then treated with 2.5 &#181;g/mL blasticidin on days 7 and 9).&#160;<a href="https://www.jove.com/files/ftp_upload/62129/62129fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: Fluorescent time-lapse video of mouse PSC-CMs generated from the Myom2-TagRFP cell line. RFP signals showed a sarcomere pattern after PSC-CM culture for 28 days. The cells showed beating synchronously when stimulated with electricity at 1 Hz. The time-lapse images were acquired every 20 ms with a 100X lens. Scale bar = 5 &#956;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62129/Movie1.mp4" target="_blank">Please click here to download this movie.</a>
Movie 2: Fluorescent time-lapse video of the human PSC-CMs with ACTN2-mCherry cultured on a non-patterned culture dish. The PSC-CMs expressing ACTN2-mCherry on a non-patterned culture dish not only showed disorganization of sarcomere but also presented a waving contraction, for which it is difficult to determine sarcomere shortening. The cells were stimulated with electricity at 0.5 Hz and images acquired every 10 ms with a 100X lens. Scale bar = 10 &#956;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62129/Movie2.mp4" target="_blank">Please click here to download this movie.</a>
Movie 3: Fluorescent time-lapse video of the human PSC-CMs with ACTN2-mCherry cultured on a patterned culture dish. The patterned culture promoted alignment of the sarcomere and forced the cells to a rod shape. This method allowed the sarcomere shortening in PSC-CMs to be determined more easily. The video was obtained by stimulating the cells with electricity at 0.5 Hz. The framerate was 100 frames per second. Scale bar = 10 &#956;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62129/Movie3.mp4" target="_blank">Please click here to download this movie.</a>
Supplemental CAD files. CAD files for creating stamps with strips of 200 &#956;m width and grooves of 10 &#956;m (Stamp_200x10.dxf), 25 &#956;m (Stamp_200x25.dxf), and 50 &#956;m (Stamp_200x50.dxf).&#160;<a href="https://www.jove.com/files/ftp_upload/62129/stamp_200.zip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins. at JoVE.com

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

sarcomere-shortening-pluripotent-stem-cell-derived-cardiomyocytes

Nanyang Technological University

Research

JoVE Journal

Medicine

4.4K Views.  Jichi Medical University. Het is moeilijk om de sarcomerefunctie in de pluripotente stamcel-afgeleide cardiomyocyten te beoordelen vanwege hun onderontwikkelde en ongeorganiseerde sarcomere. Met fluorescerende sarcomere-eiwitten hebben we nu toegang tot sarcomere verkorting. We kunnen sarcomen visualiseren en hun functie in vitro beoordelen met behulp van stamcel-afgeleide cardiomyocyten en fluorescerende sarcomere-eiwitten.Met behulp van AAV-gebaseerde transductie kunnen we de methode uitbreiden naar alle door...

Video: Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Developmental Biology

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Biology

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Bioengineering

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

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