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

Summary

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sor^{tm38(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 via calcium (Ca²⁺) flux.

Abstract

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 αMHC-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)26Sor^{tm38(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.

Introduction

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 2019^1,2. 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 function^5,6. 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 function^7,8.

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% (αMHC-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.

Protocol

All experimental procedures and practices involving animals were approved by Institutional Animal Care & 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)26Sor^{tm38(CAG-GCaMP3)Hze} /J (referred to as Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3) mouse strain (Figure 1)

1. Prepare Tg(Myh6-cre)1Jmk/J mouse strain (Jackson lab stock 009074, referred to as Myh6-Cre) and Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}/J mouse strain (Jackson lab stock 014538, referred to as Rosa26A-Flox-Stop-Flox-GCaMP3), respectively.
2. Breed each strain upto 8 weeks old to obtain adult Myh6-Cre and Rosa26A-Flox-Stop-Flox-GCaMP3 mice, respectively.
3. Crossbreed the adult Myh6-Cre and the Rosa26A-Flox-Stop-Flox-GCaMP3 mice.
   ​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.

2. Isolation and selection of neonatal Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mice hearts.

1. Obtain P0-P2 pups. Ensure that 8-10 pups are present to isolate 10 million NCFs with this protocol.
2. Deeply anesthetized the pups by hypothermia. Place pups in a latex glove and immerse up to the neck in crushed ice and water (2°C - 3°C).
3. Briefly sanitize the pups with 75% ethanol.
4. Sacrifice the pups by decapitation with sterile scissors.
5. Make a horizontal incision near the heart, squeeze the heart, and then isolate it by separating at the root of the aorta with scissors.
6. 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).

3. Isolation of neonatal cardiac fibroblasts (NCFs)

NOTE: For this part, protocol from Dr. Li Qian's Lab¹⁴ was adopted with minor optimizations when applicable to this study.

1. After isolation of the αMHC-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.
2. Transfer the hearts to a 15 mL conical tube.
3. Digest the hearts with 8 mL of warm 0.25% Trypsin-EDTA at 37 °C for 10 min.
4. Discard the trypsin supernatant and add 5 mL of warm type-II collagenase (0.5 mg/mL) in HBSS.
5. Vortex the mixture thoroughly, and incubate at 37 °C for 5 min.
6. After the incubation, vortex thoroughly and let the undigested tissue settle down by gravity.
7. Collect the supernatant in a 15 mL conical tube with 5 mL cold fibroblast (FB) medium (IMDM with 20% FBS and 1% penicillin/streptomycin).
8. Repeat steps 3.4-3.7 for the undigested tissue 4-5 times.
9. Collect all the supernatant together and filter the supernatant with a 40 µm strainer.
10. Centrifuge at 200 x g for 5 min at 4 °C and discard the supernatant.
11. Resuspend the cells in 10 mL of Magnetic-activated cell sorting buffer (MACS buffer; 1x PBS with 2 mM EDTA and 0.5% BSA).
12. Determine the viable cell number by trypan blue staining.
    1. Take 10 µL of cells out from 10 mL of cell suspension in step 3.11.
    2. Mix with 10 µL of 0.4% trypan blue solution and incubate for 5 min at room temperature.
    3. 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.
13. Centrifuge the cells at 200 x g for 5 min at 4 °C and discard the supernatant.
14. Resuspend the cells with 10 µL of Thy1.2 microbeads in 90 µL 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 °C for 30-60 min.
15. Add 10 mL of MACS buffer and mix well.
16. Centrifuge at 200 x g for 5 min, discard the supernatant.
17. Repeat steps 3.15-3.16 once.
18. Resuspend the cells and beads with 2 mL of MACS buffer.
19. 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.
20. When the LS column is equilibrated, pass the cells through the column.
21. Wash the LS column with 2 mL of MACS buffer three times.
22. 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.
23. Centrifuge at 200 x g for 5 min and discard the supernatant.
24. Resuspend the cells with 5 mL of FB media.
25. Determine the cell number with a hemocytometer.
26. 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).

4. Production of retrovirus encoding polycistronic MGT vector for cardiac reprogramming

1. Maintain Plat-E with Plat-E culture media (DMEM supplemented with 10% FBS, 1 µg/mL of puromycin and 10 µg/mL of blasticidin) at 37 °C with 5% CO₂.
2. On day 1, split Plat-E to a 6-well plate at approximately 4-5 x 10⁵ cells/well density.
3. 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.
   1. Dilute 2 µg of pMX-puro-MGT polycistronic retrovirus expression plasmid vector (Addgene 111809) to 500 ng/µL with TE buffer.
   2. Prepare the transfection mixture by mixing 10 µL of Lipofectamine with 150 µL of reduced serum medium. Carefully pipette to mix well and incubate at room temperature for 5 min. Be careful to avoid bubbles when pipetting.
   3. Meanwhile, prepare a plasmid mixture by mixing the plasmid with 150 µL of reduced serum medium. Carefully pipette to mix well and incubate at room temperature for 5 min. Be careful to avoid bubbles when pipetting.
   4. Carefully mix the two mixtures and incubate at room temperature for 5 min. The solution may appear cloudy.
   5. Add the mixture drop by drop to the cells to be transfected.
   6. Incubate the cells at 37 °C overnight.
4. On day 3, change the medium to a fresh complete cell culture medium lacking puromycin and blasticidin.
5. On day 4, 48 h after the transfection, collect the supernatant that contains retrovirus and store it in 4 °C.
6. On day 5, 72 h after the transfection, collect the supernatant that contains retrovirus.
7. Filter both the 48 h and 72 h supernatant with a 0.45 µm filter, precipitate overnight at 4 °C by adding 1/5 volume of 40% Poly (ethylene glycol) (PEG) solution to make a final concentration of 8% PEG.
8. Centrifuge at 4,000 x g for 30 min to precipitate the virus.
   NOTE: PEG8000-virus forms small white precipitation.
9. Resuspend the virus with iCM medium containing 8 µg/mL polybrene as desired. Use the retrovirus immediately.

5. Reprogramming NCFs to iCMs with MGT encoding retrovirus infection

1. Grow or passage NCFs before virus infection.
   NOTE: Usually, NCF can be passaged twice.
2. On day 0, seed NCF to the density around 1-2 x 10⁴ cells/cm² in FB medium.
3. 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.
4. Incubate at 37 °C overnight.
5. On day 2, 24 h after the virus infection, replace the virus-containing medium to a regular iCM medium.
6. 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.
7. Replace the medium every 2-3 days during the reprogramming. If necessary, perform a positive selection for MGT retrovirus infected cells by adding 2 µg/mL of puromycin to the culture medium for 3 days and maintaining it at 1 µg/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.
8. After 14 days of infection, replace the medium with B27 medium to further induce iCM maturation.

6. Evaluation of iCM functional maturation and reprogramming efficiency by Ca²⁺ flux

NOTE: Add 1 µM isoproterenol to the cells to be evaluated before assessment, if necessary.

1. Assess the Ca²⁺ flux with an inverted fluorescence microscope at room temperature.
2. In GFP channel, observe the GCaMP3+ cells under 10x objective. Ensure that it shows spontaneous cell beating in the bright field channel.
3. 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 3, Video 4, Video 5, Video 6, Video 7, Video 8).
4. Manually quantify the cells with Ca²⁺ flux.

7. Statistical analysis and data presentation

1. 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 ± S.E with p < 0.05 regarded as statistically significant. Each experiment was performed at least three times.

Results

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' 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 (

Discussion

Evaluating iCMs function is necessary for the cardiac reprogramming field. In this manuscript, the protocol describes a Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sor^{tm38(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...

Materials

List of materials used in this article

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