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 via calcium (Ca2+) flux.
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)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.
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% (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.
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)26Sortm38(CAG-GCaMP3)Hze /J (referred to as Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3) mouse strain (Figure 1)
2. Isolation and selection of neonatal Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mice hearts.
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.
4. Production of retrovirus encoding polycistronic MGT vector for cardiac reprogramming
5. Reprogramming NCFs to iCMs with MGT encoding retrovirus infection
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.
7. Statistical analysis and data presentation
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 (Figure 2, Video 1, and Video 2). Isolated NCFs will attach to the well within 2 h and show an oval to round shape 1 day after the seeding (Figure 3). The functional maturity and the reprogramming efficiency of iCMs were evaluated by Ca2+ flux 14 days after MGT introduction. The reprogrammed cells can be assessed under a fluorescence microscope to measure the Ca2+ flux. GCaMP3+ cells could be found in both IMAP and MGT groups, while the IMAP group shows significantly more GCaMP3+ cells and cells with Ca2+ oscillation patterns closer to normal CMs (Videos 3–8). As shown in Figure 4A, a representative cell in the IMAP group with Ca2+ oscillation will show GCaMP3 fluorescence change between the maximum (middle panel) and minimum (right panel), and the Ca2+ oscillation of such cells is periodically changed (Figure 4B). After the introduction of IMAP, the number of beating clusters was significantly higher than that in the control group, as the number of GCaMP3+ cells with Ca2+ flux per high-power field (HPF, 20x objective lens) was increased in the IMAP-medium-treated group (Figure 5).
Figure 1: Generating Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain. Illustration of Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain generation and the gene structure of the transgenic mice. Please click here to view a larger version of this figure.
Figure 2: Ca2+ flux of the beating heart. GCaMP3 fluorescence was synchronized with heart beating in Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 hearts (upper panel), while no fluorescence was observed in control hearts (lower panel). Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 3: Isolated NCFs attached to a 6-well plate. (A) NCFs under low power field (LPF, 10x objective, scale bar = 100 µm). (B) NCFs under high power field (20x objective, scale bar = 50 µm). Please click here to view a larger version of this figure.
Figure 4: Ca2+ flux of reprogrammed cells. (A) IMAP-treated NCFs reprogrammed to iCMs under GFP channel at high power field (20x objective). Ca2+ flux of iCMs was visualized as GCaMP3 fluorescence, in which cells with Ca2+ flux show repeated flashing between basic fluorescence (Ca2+ min, middle panel) and bright fluorescence (Ca2+ max, right panel) synchronized with beating. (B) Ca2+ trace curve of Ca2+ oscillation+ cells in IMAP group. Scale bar = 50 µm. F/F0: relative fluorescence intensity. Please click here to view a larger version of this figure.
https://www.jove.com/files/ftp_upload/62643/Zhaokai_Li_-_Video_1_GCaMP3+_heart.mp4 Figure 5: Evaluation of Ca2+ flux under IMAP medium. Number of GCaMP3+ cells with Ca2+ flux per HPF 2, 3, 4 weeks after MGT induction. Please click here to view a larger version of this figure.
Video 1: A beating heart isolated from pups with αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 genotype under scanning objective lens (4x objective) in the GFP channel. The heart is GCaMP3+ and flashing between basic fluorescence (Ca2+ min) and bright fluorescence (Ca2+ max) synchronized with beating. Please click here to download this Video.
Video 2: A beating heart isolated from pups with control genotype under scanning objective lens in the GFP channel. The heart is GCaMP3- and does not show fluorescence flashing synchronized with beating. Please click here to download this Video.
Video 3: iCMs in IMAP group under LPF in the bright field (BF) channel. A cell with significant beating in the center of the field can be seen. Both Video 3 and Video 4 focus on the same field. Please click here to download this Video.
Video 4: iCMs in IMAP group under LPF in the GFP channel. Multiple cells with flashing fluorescence, including the beating cell seen in the BF channel can be observed. Both Video 3 and Video 4 focus on the same field. Please click here to download this Video.
Video 5: iCMs in IMAP group under HPF in the BF channel. Center of the field of Video 3 and Video 4 was observed under HPF. A cell with significant beating in the center of the field can be observed. Both Video 5 and Video 6 focus on the same field. Please click here to download this Video.
Video 6: iCMs in IMAP group under HPF in the GFP channel. Center part of the field of Video 3 and Video 4 was observed under HPF. Multiple cells with flashing fluorescence, including the beating cell seen in the BF channel can be observed. Both Video 5 and Video 6 focus on the same field. Please click here to download this Video.
Video 7: iCMs in MGT group under HPF in the BF channel. In contrast of significant beating cells observed in IMAP group, there are a few beating cells under the BF channel in the MGT group, which has lower reprogramming efficiency. Both Video 7 and Video 8 focus on the same field. Please click here to download this Video.
Video 8: iCMs in MGT group under HPF in the GFP channel. Several cells with mild flashing fluorescence can be observed. Both Video 7 and Video 8 focus on the same field. Please click here to download this Video.
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 for successfully reprogramming and evaluating with this method. First, NCFs should be freshly prepared and healthy after isolation. A rapid procedure of heart isolation and cutting is essential. Most importantly, it is crucial to follow the incubation time to avoid over-digestion and reduction in cell viability and condition. Second, among all procedures, virus infection efficiency often introduces high variation in the results. Virus infection efficiency is majorly influenced by two factors. On the one hand, the virus titer should be constant among different attempts, which requires consistency in transfected plasmids quantity and similar Plat-E cell condition and density. Researchers following this protocol should evaluate the optimal seeding density and time before those procedures. The virus should be used immediately to avoid titer attenuation due to virus sensitivity to freeze-thaw cycles. Additionally, it is important to keep NCFs in a healthy condition and suitable density at the time of infection. Researchers should be familiar with the growth characteristics of NCFs. While the reprogramming efficiency variation should be limited, frequent monitoring could be helpful. This protocol can be easily modified to co-infect NCFs with different viruses or treat them with chemicals of interest. Thus, it is applicable as a universal method for cardiac reprogramming research. Besides points mentioned here, common issues with this method include low fluorescence observed after the reprogramming. This may be due to several reasons. First, the infection may not be as effective as desired, which leads to a low reprogramming efficiency and limits the number of iCMs. Second, the exposure condition may need to be adjusted optimally for the observation of GCaMP3 fluorescence of iCMs. Using neonatal cardiomyocytes isolated from the same pups as positive control will help to identify the potential reason.
Ca2+ flux has been widely used to assess cell activities, including in neuron cells16, mammary gland17, fat tissues18, etc. In this study, Ca2+ flux was used to assess the functional maturation of iCMs. Previously, it has been reported that Ca2+ flux can be measured by specific chemicals named small molecule calcium-sensitive dyes that can be used to evaluate the function of reprogrammed cells19. However, such a method has several limitations: the chemical introduced to the cells may have potential toxicity and influence the cellular processes, making the results less reliable than those obtained with the method presented here. Besides, the staining process is complicated and time-consuming while also preventing further evaluations of those cells. The method presented here, on the other hand, overcomes those limitations. GCaMP3 is non-invasive for the cells, which minimizes the influences on cell activities and enables further evaluation of the cells. Since the fluorescence of iCMs only depends on their identity, i.e., Myh6 expression and local Ca2+ concentration change, the Ca2+ flux fluorescence of cells becomes visible as long as NCFs are reprogrammed, which enables frequent monitoring of the reprogramming process without a time-consuming strategy. While Ca2+ flux can be monitored and recorded easily under an inverted fluorescence microscope, the repeated beating and related electronic activity across the cell membrane, i.e., Ca2+ flux, can be further temporally quantified20. As shown in Figure 4B, such quantification can provide more information about the maturity of iCMs and illustrate more detailed structures of Ca2+ flux change during cardiac reprogramming.
This method has several advantages. First, the Ca2+ flux is exclusively observed in iCMs. Because Myh6 is very specific to CMs but not CFs, only the successfully reprogrammed cells will express the GCaMP3 reporter and become fluorescent. Second, Ca2+ flux provides a way to evaluate the functional maturation of iCMs besides the method of monitoring the expression of CM-specific genes. Due to the relatively long experimental procedure and variation linked to this, the function of iCMs is not always acceptable for further study and potential clinical usage. While the CM-specific gene expression only reveals a part of the characteristics of the reprogrammed cell, Ca2+ flux provides another aspect of the cardiac reprogramming field to evaluate the reprogrammed cell quality and efficiency. Furthermore, functional maturation is more related to heart function, which can be a better indicator to evaluate efficiency. Widely used methods in this field include flow cytometry, a technique that necessitates trypsin digestion of all the cell groups. While digestion can influence cell functions and characteristics, it introduces variation to the system, decreasing the potential to reproduce the results observed and further evaluating those cells. Compared with those methods, the transgenic mouse strain shown here has limited the potential influence from chemicals or experimental procedures needed for the evaluation. With those advantages, this mouse strain simplifies the evaluation procedures needed for cardiac reprogramming and enhances the reproducibility of results in this field.
However, there are some limitations to this study. First, the mouse strain establishment is time-consuming. Inquiries of the mouse strain from colleagues in this field are welcomed to shorten the time needed for the strain establishment. Second, the cardiac reprogramming to iCMs with this protocol involves multiple factors and steps, which introduce relatively high variation to the system. Proficiency in this field will help to overcome this issue. Finally, because the GCaMP3 becomes fluorescent only under Ca2+ flux condition, the current evaluation method cannot directly be used for FACS as cardiac reprogramming with Myh6-GFP strain7. However, while the current strain has more and different applications compared with Myh6-GFP strain, such an inconvenience can be overcome.
Overall, as the protocol has described above, the Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain and following evaluation of iCMs maturation provide a strategy to monitor the whole process of cardiac reprogramming. This GCaMP3-mediated Ca2+ flux measuring strategy can be performed in live cells without harming cell viability. Because GCaMP3 fluorescence is driven by myocardial-specific gene expression, the acquired GCaMP3 fluorescent data can be further quantified to reveal the reprogramming efficiency and iCMs activity.
The authors have nothing to disclose.
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.
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 |