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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+myocardial+infarction.">Pathophysiology of myocardial infarction.</a> Comprehensive Physiology. 5 (4), 1841-1875 (2015).</li><li>Tian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC+inhibitor+valproic+acid+protects+heart+function+through+Foxm1+pathway+after+acute+myocardial+infarction.">HDAC inhibitor valproic acid protects heart function through Foxm1 pathway after acute myocardial infarction.</a> EBioMedicine. 39, 83-94 (2019).</li><li>Mohamed, T. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+ion+channels.">Cardiac ion channels.</a> Circulation: Arrhythmia and Electrophysiology. 2 (2), 185-194 (2009).</li><li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nature Methods. 6 (12), 875-881 (2009).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Generation+of+Induced+Cardiomyocytes+Using+a+Polycistronic+Construct+Expressing+Optimal+Ratio+of+Gata4,+Mef2c+and+Tbx5.">Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5.</a> Journal of Visualized Experiments: JoVE. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8211;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&#39;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&#160;&#181;m, PVDF, 33&#160;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&#8217;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&#160;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&#8211;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 &#181;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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3.3K Views.  The University of Michigan, Ann Arbor. We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-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...

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

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...