Name: in vitro assessment of cardiac reprogramming by measuring cardiac specific calcium flux with a gcamp3 reporter
Uploaded: 2022-02-22T13:00:00
Description: 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

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. ...

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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.

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			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
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		<tr>
			<td>50mL Conical Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
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			<td>6 Well Cell Culture Plates</td>
			<td>Alkali Scientific</td>
			<td>TP9006</td>
			<td></td>
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			<td>A83-01</td>
			<td>Stemgent</td>
			<td>04&#8211;0014</td>
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			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X800E</td>
			<td>Inverted fluorescence microscope</td>
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			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
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			<td>Blasticidin S HCl (10 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113903</td>
			<td></td>
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			<td>Bovine Serum Albumin (BSA) DNase- and Protease-free Powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP9706100</td>
			<td></td>
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			<td>CD90.2 MicroBeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-101</td>
			<td>Thy1.2 microbeads</td>
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			<td>Collagenase, Type 2</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9693955</td>
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			<td>Counting Chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-671-51B</td>
			<td>Hemocytometer</td>
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			<td>DMEM, high glucose, no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>11960069</td>
			<td></td>
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			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-040-133</td>
			<td></td>
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			<td>Ethanol, 200 proof (100%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
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			<td>Ethylenediamine Tetraacetic Acid (Certified ACS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>E478-500</td>
			<td></td>
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			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
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			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td></td>
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			<td>IMDM media</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td></td>
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			<td>IX73 Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX73P2F</td>
			<td>Inverted fluorescence microscope</td>
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			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-668-019</td>
			<td></td>
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			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
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			<td>Medium 199, Earle&#39;s Salts</td>
			<td>Thermo Fisher Scientific</td>
			<td>11150059</td>
			<td></td>
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		<tr>
			<td>MidiMACS Separator and Starting Kits</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
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			<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>
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		<tr>
			<td>MM589</td>
			<td></td>
			<td></td>
			<td>Obtained from Dr. Shaomeng Wang&#8217;s lab in University of Michigan</td>
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			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31-985-070</td>
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			<td>PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-010-049</td>
			<td></td>
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			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
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			<td>Platinum-E (Plat-E) Retroviral Packaging Cell Line</td>
			<td>Cell&#160;Biolabs</td>
			<td>RV-101</td>
			<td></td>
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			<td>pMx-puro-MGT</td>
			<td>Addgene</td>
			<td>111809</td>
			<td></td>
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			<td>Poly(ethylene glycol)</td>
			<td>Millipore Sigma</td>
			<td>P5413-1KG</td>
			<td>PEG8000</td>
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			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Millipore Sigma</td>
			<td>TR-1003-G</td>
			<td></td>
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		<tr>
			<td>PTC-209</td>
			<td>Sigma</td>
			<td>SML1143&#8211;5MG</td>
			<td></td>
		</tr>
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			<td>Puromycin Dihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113803</td>
			<td></td>
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			<td>Recombinant Human IGF-I</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
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			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
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			<td>ST 16 Centrifuge Series</td>
			<td>Thermo Fisher Scientific</td>
			<td>75-004-381</td>
			<td></td>
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			<td>Sterile Cell Strainers</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-363-547</td>
			<td>40 &#181;m strainer</td>
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			<td>Surface Treated Tissue Culture Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
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			<td>TE Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090015</td>
			<td></td>
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		<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>
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		<tr>
			<td>Vortex Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>02215365</td>
			<td></td>
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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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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 ...

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 ...

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 ...

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

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 ...

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 ...

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 ...

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) ...