Name: Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins. (Video) | JoVE Journal
Uploaded: 2021-03-03T13:00:00.000+00:00
Duration: 8 min 37 s
Description: Learn to assess sarcomere shortening in pluripotent stem cell-derived cardiomyocytes using fluorescent-tagged sarcomere proteins. This method enables visualization of sarcomere function in vitro for cardiology research.

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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.

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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 func...

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.

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 α-Actinin, which localizes to the Z-line18,25. Time-lapse images were...

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

Research

JoVE Journal

Medicine

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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.

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

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

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.

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

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.

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

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.

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

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated 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.

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

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.

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

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

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

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Learn to assess sarcomere shortening in pluripotent stem cell-derived cardiomyocytes using fluorescent-tagged sarcomere proteins. This method enables visualization of sarcomere function in vitro for cardiology research.

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

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