We appreciate the efforts of Leo Gnatovskiy in editing the English text of this manuscript. Figure 1 was created with BioRender.com. This study was supported by the National Institutes of Health (NIH) of the United States (1R01HL109054) grant to Dr. Wang.

All experimental procedures and practices involving animals were approved by Institutional Animal Care &#38; Use Committee at the University of Michigan. All experimental procedures and practices involving cell culture must be performed BSL2 Biological Safety Cabinet under sterile conditions. For the procedures and practices involving viruses, the guideline of the proper disposal of transfected cells, pipette tips, and tubes to avoid the risk of environmental and health hazards was followed.
1. Establishment of a Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze /J (referred to as Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3) mouse strain (Figure 1)
<ol>
	<li>Prepare Tg(Myh6-cre)1Jmk/J mouse strain (Jackson lab stock 009074, referred to as Myh6-Cre) and Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J mouse strain (Jackson lab stock 014538, referred to as Rosa26A-Flox-Stop-Flox-GCaMP3), respectively.</li>
	<li>Breed each strain upto 8 weeks old to obtain adult Myh6-Cre and Rosa26A-Flox-Stop-Flox-GCaMP3 mice, respectively.</li>
	<li>Crossbreed the adult Myh6-Cre and the Rosa26A-Flox-Stop-Flox-GCaMP3 mice. 
	&#8203;NOTE: Set up Myh6-Cre male/ Rosa26A-Flox-Stop-Flox-GCaMP3 female or vice versa. There is no significant difference between their descendants. Typically, the female mice will give birth to 8-10 pups 19-21 days after the crossbreeding.</li>
</ol>
2. Isolation and selection of neonatal Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mice hearts.
<ol>
	<li>Obtain P0-P2 pups. Ensure that 8-10 pups are present to isolate 10 million NCFs with this protocol.</li>
	<li>Deeply anesthetized the pups by hypothermia. Place pups&#160;in a latex glove and immerse&#160;up to the neck in crushed ice and water (2&#176;C - 3&#176;C).</li>
	<li>Briefly sanitize the pups with 75% ethanol.</li>
	<li>Sacrifice the pups by decapitation with sterile scissors.</li>
	<li>Make a horizontal incision near the heart, squeeze the heart, and then isolate it by separating at the root of the aorta with scissors.</li>
	<li>Observe the heart beating under a fluorescence microscope. Ensure the hearts with Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 genotype shows Ca2+ flux indicated by GCaMP3 with heart beating. The other genotypes do not show fluorescence (Figure 2, Video 1, and Video 2).</li>
</ol>
3. Isolation of neonatal cardiac fibroblasts (NCFs)
NOTE: For this part, protocol from Dr. Li Qian&#39;s Lab14 was adopted with minor optimizations when applicable to this study.
<ol>
	<li>After isolation of the &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 hearts, cut them into four pieces that are loosely connected. Wash them in ice-cold DPBS in a 6 cm plate thoroughly several times to limit the blood cell pollution in the isolated cells.</li>
	<li>Transfer the hearts to a 15 mL conical tube.</li>
	<li>Digest the hearts with 8 mL of warm 0.25% Trypsin-EDTA at 37 &#176;C for 10 min.</li>
	<li>Discard the trypsin supernatant and add 5 mL of warm type-II collagenase (0.5 mg/mL) in HBSS.</li>
	<li>Vortex the mixture thoroughly, and incubate at 37 &#176;C for 5 min.</li>
	<li>After the incubation, vortex thoroughly and let the undigested tissue settle down by gravity.</li>
	<li>Collect the supernatant in a 15 mL conical tube with 5 mL cold fibroblast (FB) medium (IMDM with 20% FBS and 1% penicillin/streptomycin).</li>
	<li>Repeat steps 3.4-3.7 for the undigested tissue 4-5 times.</li>
	<li>Collect all the supernatant together and filter the supernatant with a 40 &#181;m strainer.</li>
	<li>Centrifuge at 200 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells in 10 mL of Magnetic-activated cell sorting buffer (MACS buffer; 1x PBS with 2 mM EDTA and 0.5% BSA).</li>
	<li>Determine the viable cell number by trypan blue staining.
	<ol>
		<li>Take 10 &#181;L of cells out from 10 mL of cell suspension in step 3.11.</li>
		<li>Mix with 10 &#181;L of 0.4% trypan blue solution and incubate for 5 min at room temperature.</li>
		<li>Add the mixture to a hemocytometer and determine the viable cell number. The dead cells are stained in blue, while the viable cells are unstained.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 200 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells with 10 &#181;L of Thy1.2 microbeads in 90 &#181;L of chilled MACS buffer for less than 10 million viable cells. Add more beads proportionally if there are more than 10 million viable cells. Pipette the mixture well and incubate at 4 &#176;C for 30-60 min.</li>
	<li>Add 10 mL of MACS buffer and mix well.</li>
	<li>Centrifuge at 200 x g for 5 min, discard the supernatant.</li>
	<li>Repeat steps 3.15-3.16 once.</li>
	<li>Resuspend the cells and beads with 2 mL of MACS buffer.</li>
	<li>Set up a MACS separator in the hood. Insert an LS column to the separator and equilibrate the column with 3 mL of MACS buffer.</li>
	<li>When the LS column is equilibrated, pass the cells through the column.</li>
	<li>Wash the LS column with 2 mL of MACS buffer three times.</li>
	<li>Take the column off the separator, elute it with 2 mL of MACS buffer three times, and then collect the elution to a 50 mL tube.</li>
	<li>Centrifuge at 200 x g for 5 min and discard the supernatant.</li>
	<li>Resuspend the cells with 5 mL of FB media.</li>
	<li>Determine the cell number with a hemocytometer.</li>
	<li>Dilute the cells with FB media and seed the cells to dishes or plates as desired. Ensure that the cell seeding density is around 2-2.5 x 104 cells/cm2 (optimize the density based on individual experiments). Ensure that the attached fibroblasts have an oval to round shape on the second day after seeding (Figure 3).</li>
</ol>
4. Production of retrovirus encoding polycistronic MGT vector for cardiac reprogramming
<ol>
	<li>Maintain Plat-E with Plat-E culture media (DMEM supplemented with 10% FBS, 1 &#181;g/mL of puromycin and 10 &#181;g/mL of blasticidin) at 37 &#176;C with 5% CO2.</li>
	<li>On day 1, split Plat-E to a 6-well plate at approximately 4-5 x 105 cells/well density.</li>
	<li>On day 2, Plat-E typically reaches 80% confluency. Transfect the cells with the following procedures. Adjust the volume and quantity of each element present here based on each well in a 6-well plate.
	<ol>
		<li>Dilute 2 &#181;g of pMX-puro-MGT polycistronic retrovirus expression plasmid vector (Addgene 111809) to 500 ng/&#181;L with TE buffer.</li>
		<li>Prepare the transfection mixture by mixing 10 &#181;L of Lipofectamine with 150 &#181;L of reduced serum medium. Carefully pipette to mix well and incubate at room temperature for 5 min. Be careful to avoid bubbles when pipetting.</li>
		<li>Meanwhile, prepare a plasmid mixture by mixing the plasmid with 150 &#181;L of reduced serum medium. Carefully pipette to mix well and incubate at room temperature for 5 min. Be careful to avoid bubbles when pipetting.</li>
		<li>Carefully mix the two mixtures and incubate at room temperature for 5 min. The solution may appear cloudy.</li>
		<li>Add the mixture drop by drop to the cells to be transfected.</li>
		<li>Incubate the cells at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>On day 3, change the medium to a fresh complete cell culture medium lacking puromycin and blasticidin.</li>
	<li>On day 4, 48 h after the transfection, collect the supernatant that contains retrovirus and store it in 4 &#176;C.</li>
	<li>On day 5, 72 h after the transfection, collect the supernatant that contains retrovirus.</li>
	<li>Filter both the 48 h and 72 h supernatant with a 0.45 &#181;m filter, precipitate overnight at 4 &#176;C by adding 1/5 volume of 40% Poly (ethylene glycol) (PEG) solution to make a final concentration of 8% PEG.</li>
	<li>Centrifuge at 4,000 x g for 30 min to precipitate the virus. 
	NOTE: PEG8000-virus forms small white precipitation.</li>
	<li>Resuspend the virus with iCM medium containing 8 &#181;g/mL polybrene as desired. Use the retrovirus immediately.</li>
</ol>
5. Reprogramming NCFs to iCMs with MGT encoding retrovirus infection
<ol>
	<li>Grow or passage NCFs before virus infection. 
	NOTE: Usually, NCF can be passaged twice.</li>
	<li>On day 0, seed NCF to the density around 1-2 x 104 cells/cm2 in FB medium.</li>
	<li>On day 1, replace the culture medium with a virus-containing medium for each well as desired. Use the virus from one well Plat-E in a 6-well plate to infect two wells in a 24-well plate. Titer the virus to determine the optimal virus concentration. 
	NOTE: Viruses containing other reprogramming factors of interest could be introduced along with MGT retrovirus.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
	<li>On day 2, 24 h after the virus infection, replace the virus-containing medium to a regular iCM medium.</li>
	<li>To monitor the GCaMP3 expression, plate it under an inverted fluorescence microscope. Under 10x at GFP channel, observe the mild basal GCaMP3 fluorescence of a portion of cells as early as day 5.</li>
	<li>Replace the medium every 2-3 days during the reprogramming. If necessary, perform a positive selection for MGT retrovirus infected cells by adding 2 &#181;g/mL of puromycin to the culture medium for 3 days and maintaining it at 1 &#181;g/mL. 
	NOTE: Introduce chemicals of interest (e.g., IGF-1, MM589, A83-01, and PTC-209, referred to as IMAP as we previously reported15) along with medium change.</li>
	<li>After 14 days of infection, replace the medium with B27 medium to further induce iCM maturation.</li>
</ol>
6. Evaluation of iCM functional maturation and reprogramming efficiency by Ca2+ flux
NOTE: Add 1 &#181;M isoproterenol to the cells to be evaluated before assessment, if necessary.
<ol>
	<li>Assess the Ca2+ flux with an inverted fluorescence microscope at room temperature.</li>
	<li>In GFP channel, observe the GCaMP3+ cells under 10x objective. Ensure that it shows spontaneous cell beating in the bright field channel.</li>
	<li>Randomly select three fields under 20x and record the Ca2+ flux of the iCMs for 3 min for each field. 
	NOTE: Here, the Ca2+ flux was synchronized with spontaneous cell beating (Figure 4, Figure 5, and Video&#160;3, Video 4, Video 5, Video 6, Video 7, Video&#160;8).</li>
	<li>Manually quantify the cells with Ca2+ flux.</li>
</ol>
7. Statistical analysis and data presentation
<ol>
	<li>Analyze the differences among groups one-way analysis of variance (ANOVA) and perform the Student-Newman-Keuls multiple comparison tests. 
	NOTE: Results are as mean &#177; S.E with p &#60; 0.05 regarded as statistically significant. Each experiment was performed at least three times.</li>
</ol>

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The authors have nothing to disclose.

Evaluating iCMs function is necessary for the cardiac reprogramming field. In this manuscript, the protocol describes a Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J mouse strain that has been established, how to use the NCFs isolated from the neonatal mice in this strain for the reprogramming to iCMs, and the evaluation of iCMs function by Ca2+ flux. This is a de novo method to evaluate iCMs functional maturation.
Several critical steps are important fo...

Myocardial infarction (MI) is a severe disease worldwide. Cardiovascular diseases (CVDs) are the leading cause of death worldwide and account for approximately 18.6 million deaths in 20191,2. The total mortality of CVDs has decreased during the past half a century. However, this trend has been slowed or even reversed in some undeveloped countries1, which calls for more effective treatments of CVDs. As one of the fatal manifestations of CVD, MI accounts for about half of all deaths attributed to CVDs in the United States2. During the ischemia, with the blocking of coronary arteries and limited supply of both nutrients and oxygen, the myocardium suffers severe metabolic changes, impairs the systolic function of cardiomyocytes (CMs), and leads to CM death3. Numerous approaches in cardiovascular research have been explored to repair heart injury and restore the function of the injured heart4. Direct cardiac reprogramming has emerged as one promising strategy to repair the damaged heart and restore its function5,6. By introducing Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed to iCMs in vitro and in vivo, and those iCMs can reduce the scar area and improve the heart function7,8.
Though cardiac reprogramming is a promising strategy for MI treatment, there remain a number of challenges. First, the reprogramming efficiency, purity, and quality are not always as high as expected. MGT inducement can only achieve 8.4% (cTnT+) or 24.7% (&#945;MHC-GFP+) of the total CFs to be reprogrammed to iCMs in vitro7, or up to 35% in vivo8, which limits its application. Even with more factors induced in the system, such as Hand29 or Akt1/PKB10, the reprogramming efficiency is still barely satisfactory to be used in a clinical setting. Thus, more studies focused on improving the reprogramming efficiency are needed in this field. Second, the electrical integrity and contraction characteristics of iCMs are important for the efficient improvement of heart function, yet these are challenging to evaluate. Currently, widely used evaluation methods in the field, including flow cytometry, immunocytochemistry, and qPCR of some key CMs genes expression, are all focused on the similarity of iCMs and CMs, but not directly related to the functional characteristics of iCMs. Furthermore, those methods have relatively complicated procedures and are time-consuming. While reprogramming studies usually involve a screening of potential reprogramming factors that promotes iCMs maturation11, cardiac reprogramming calls for a quick and convenient method based on iCMs function.
CMs open the voltage-gated calcium ion channels on the cytomembrane during each contracting cycle, which leads to a transient influx of calcium ion (Ca2+) from the intercellular fluid to the cytoplasm to participate in the myofilament contraction. Such a Ca2+ influx and outflux cycle is the fundamental trait of myocardial contraction and constitutes the normal function of CMs12. Thus, a method that detects Ca2+ influx could be a potential way to measure the function of CMs and CM-like cells, including iCMs. Furthermore, for iCMs, such a method provides another way to evaluate reprogramming efficiency.
Genetically encoded calcium indicators (GECIs) have been developed and widely used to indicate cell activities, especially action potentials. Generally, GECIs consist of a Ca2+ binding domain such as calmodulin, and a fluorescent domain such as GFP, and GCaMP3 is one with high affinity and fluorescence intensity. The fluorescence domain of GCaMP3 will be activated when the local calcium concentration is changed13. In this paper, a mouse strain that specifically expresses a GCaMP3 reporter in Myh6+ cells is described. By introducing MGT to the isolated NCFs from neonates of this strain, the reprogramming can be monitored by fluorescence, which successfully reprogrammed iCMs will exhibit. Such a mouse strain and method will provide a valuable platform to investigate cardiac reprogramming.

<table><tbody><tr><td>15 mL Conical Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>50mL Conical Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>6 Well Cell Culture Plates</td><td>Alkali Scientific</td><td>TP9006</td><td></td></tr><tr><td>A83-01</td><td>Stemgent</td><td>04&amp;ndash;0014</td><td></td></tr><tr><td>All-in-One Fluorescence Microscope</td><td>Keyence</td><td>BZ-X800E</td><td>Inverted fluorescence microscope</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>Blasticidin S HCl (10 mg/mL)</td><td>Thermo Fisher Scientific</td><td>A1113903</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) DNase- and Protease-free Powder</td><td>Thermo Fisher Scientific</td><td>BP9706100</td><td></td></tr><tr><td>CD90.2 MicroBeads, mouse</td><td>Miltenyi Biotec</td><td>130-049-101</td><td>Thy1.2 microbeads</td></tr><tr><td>Collagenase, Type 2</td><td>Thermo Fisher Scientific</td><td>NC9693955</td><td></td></tr><tr><td>Counting Chamber</td><td>Thermo Fisher Scientific</td><td>02-671-51B</td><td>Hemocytometer</td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Thermo Fisher Scientific</td><td>11960069</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14-040-133</td><td></td></tr><tr><td>Ethanol, 200 proof (100%)</td><td>Thermo Fisher Scientific</td><td>04-355-451</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid (Certified ACS)</td><td>Thermo Fisher Scientific</td><td>E478-500</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14025092</td><td></td></tr><tr><td>IMDM media</td><td>Thermo Fisher Scientific</td><td>12440053</td><td></td></tr><tr><td>IX73 Inverted Microscope</td><td>Olympus</td><td>IX73P2F</td><td>Inverted fluorescence microscope</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11-668-019</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Medium 199, Earle&#039;s Salts</td><td>Thermo Fisher Scientific</td><td>11150059</td><td></td></tr><tr><td>MidiMACS Separator and Starting Kits</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45&amp;nbsp;&amp;micro;m, PVDF, 33&amp;nbsp;mm, gamma sterilized</td><td>Millipore Sigma</td><td>SLHV033RB</td><td></td></tr><tr><td>MM589</td><td></td><td></td><td>Obtained from Dr. Shaomeng Wang&amp;rsquo;s lab in University of Michigan</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31-985-070</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Thermo Fisher Scientific</td><td>10-010-049</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Platinum-E (Plat-E) Retroviral Packaging Cell Line</td><td>Cell&amp;nbsp;Biolabs</td><td>RV-101</td><td></td></tr><tr><td>pMx-puro-MGT</td><td>Addgene</td><td>111809</td><td></td></tr><tr><td>Poly(ethylene glycol)</td><td>Millipore Sigma</td><td>P5413-1KG</td><td>PEG8000</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>Millipore Sigma</td><td>TR-1003-G</td><td></td></tr><tr><td>PTC-209</td><td>Sigma</td><td>SML1143&amp;ndash;5MG</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>Thermo Fisher Scientific</td><td>A1113803</td><td></td></tr><tr><td>Recombinant Human IGF-I</td><td>Peprotech</td><td>100-11</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Thermo Fisher Scientific</td><td>11875093</td><td></td></tr><tr><td>ST 16 Centrifuge Series</td><td>Thermo Fisher Scientific</td><td>75-004-381</td><td></td></tr><tr><td>Sterile Cell Strainers</td><td>Thermo Fisher Scientific</td><td>22-363-547</td><td>40 &amp;micro;m strainer</td></tr><tr><td>Surface Treated Tissue Culture Dishes</td><td>Thermo Fisher Scientific</td><td>FB012921</td><td></td></tr><tr><td>TE Buffer</td><td>Thermo Fisher Scientific</td><td>12090015</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Millipore Sigma</td><td>T8154</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Fisher Scientific</td><td>25300054</td><td></td></tr><tr><td>Vortex Mixer</td><td>Thermo Fisher Scientific</td><td>02215365</td><td></td></tr></tbody></table>

in vitro assessment of cardiac reprogramming by measuring cardiac specific calcium flux with a gcamp3 reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

The experimental workflow to generate Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain and the gene structure of the transgenic mice is shown in Figure 1. While the mouse strain is established, the pups&#39; hearts were isolated and observed under a reverse fluorescence microscope to confirm the genotype. Hearts with correct genotype show Ca2+ flux synchronized with beating, visualized as GCaMP3 fluorescence, while no fluorescence was observed in control hearts (<strong class="...

Watch this Scientific Journal Video about In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter at JoVE.com

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

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Research

JoVE Journal

Medicine

3.3K Views.  The University of Michigan, Ann Arbor. Methoden die in dit protocol worden beschreven, zullen een waardevol platform bieden om de functionele rijping van ICM's voor cardiale herprogrammeringsstudies te beoordelen. De calciumflux wordt uitsluitend waargenomen in ICM's en biedt een manier om de functionele rijping van ICM te evalueren. Deze muizenstam vereenvoudigt de evaluatieprocedures en verbetert de reproduceerbaarheid van de resultaten.Dit protocol is een nieuwe methode om de functionele rijping van ICM's in het veld van...

Video: In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

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 ...

Developmental Biology

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 ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Biology

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Neuroscience

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

Bioengineering

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

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