This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.
The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.
The heart is the first organ formed during embryonic development. This process involves the spatiotemporal coordination of various populations of progenitor cells from distinct areas of the embryo. All this occurs while the developing heart continues to beat and function, emphasizing the remarkable coordination required for its formation1,2,3. Given the crucial role of the heart, tight regulation at the cellular and molecular levels is essential for its proper formation4,5. Identifying the mechanisms that control heart development has been of great interest, as they are crucial for unraveling congenital heart disorders, which impact a substantial number of patients worldwide6. Furthermore, comprehending heart development is pivotal in deciphering cardiac regeneration, as postnatal mammalian hearts retain a regenerative capacity that is lost or hindered in adulthood7,8. Consequently, dissecting molecular regulators of heart development is imperative to advance research efforts on congenital heart disease and cardiac regeneration.
In pursuit of this objective, there has been a growing focus on investigating the role of the epicardium in cardiac development and regeneration9. The epicardium is a thin layer of mesothelial tissue that comprises the outermost layer of the mammalian heart (Figure 1). Recent studies have shown the importance of the epicardium during cardiac injury, revealing that this tissue is able to send proliferation signals to cardiomyocytes in the affected area to mitigate the damage10,11. Despite the importance of the epicardium, conducting further molecular investigations has been challenged by its immense heterogeneity. Single-cell RNAseq experiments have revealed the epicardium's heterogeneity, housing multiple cell subpopulations with distinct transcriptomic signatures12,13,14,15,16. Thus, a strategy to screen potential regulators of cardiac development should accommodate the diversity of epicardial progenitor cells.
In this sense, the mouse model's amenability to genetic modification has facilitated the identification of numerous genes crucial for heart development, allowing the generation of mutant lines with gain-of-function (GOF) or loss-of-function (LOF) of specific genes. However, these approaches imply a considerable investment of time and experimental resources; therefore, they are impractical when assessing the roles of a large number of candidate genes. Besides, developmental genes often exert pleiotropic functions in different tissues or are required for early embryonic development, hampering the interpretation of their contribution to development in a specific process. While it is possible to target gene function at specific structures or developmental time points, this usually requires the use of more complex genetic constructions, which can be difficult to generate or are generally unavailable.
To overcome these limitations, we present a methodology to electroporate mouse embryonic hearts for transient transgenesis (Figure 2). Paired with ex vivo culture and fluorescence-activated cell sorting (FACS), this strategy demonstrates its capabilities through transient GOF of Meis1, a well-characterized gene implicated in heart development and regeneration17,18,19. In this article, other potential applications of this methodology are also explored, and its advantages and limitations are discussed, as well as compared to existing protocols for transiently modulating gene expression. We believe the framework and visual examples presented will enhance the understanding of epicardium biology during development and disease.
Figure 1: Mouse embryonic heart layers. Schematic diagram of a coronal view of an E13-14 mouse embryonic heart. The three main cellular layers of the heart are represented in yellow (endocardium), red (myocardium), and blue (epicardium). The pericardium is represented in a brown line. The four chambers of the heart are abbreviated as LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. Please click here to view a larger version of this figure.
Figure 2: Schematic overview of the heart electroporation protocol. Please click here to view a larger version of this figure.
All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee and conformed to current legislation, including EU Directive 2010/63EU and Recommendation 2007/526/EC, as enforced by Spanish Law under Real Decreto 53/2013. For this protocol, female wild-type CD-1 mice aged 15-21 weeks were employed. Details regarding the animals, reagents, and equipment used are listed in the Table of Materials.
1. Plasmid and tool preparation
2. Embryo dissection and heart extraction
3. Heart electroporation and ex vivo culture
Figure 3: Electroporation setup. (A) Setup used for electroporation. Red arrows indicate the electroporator, electrodes, and Petri dish where hearts are electroporated. (A') Close detail of the electroporation needle as highlighted in (A) by the white rectangle. (B) Detail of the adjusted distance of the electrodes used in E12.5 hearts. (C) Schematic representation of the electroporation. Please click here to view a larger version of this figure.
4. Cell sorting and immunohistochemistry analysis
To demonstrate the effectiveness of this technique in performing gain-of-function (GOF) experiments for relevant heart developmental regulators, a construct was electroporated overexpressing the Meis1 transcription factor. To achieve this, RNA was extracted from E9.5 embryos, and reverse transcription was performed to obtain complementary DNA (cDNA). Using the cDNA as a template, the Meis1 coding sequence was cloned (Supplementary Table 1) into a pCAG expression plasmid (hereafter referred to as pCAG::Meis1), while a constitutive GFP-expressing plasmid (pCAG::GFP) was used as a control. Hearts were then electroporated with either pCAG::GFP only or in combination with pCAG::Meis1.
After 24 h post-electroporation (hpe), the hearts were beating and appeared to be in good condition (Figure 4A; Supplementary Movie 1; Supplementary Movie 2). To assess the viability of the electroporated cells, apoptosis was analyzed through immunohistochemical staining of caspase 3. Although some positive cells were detected within the heart, neither the myocardium nor the epicardium, where the majority of the electroporated cells are located, were found to be affected by apoptosis (Supplementary Figure 1C). When the fluorescence activity was assessed, a mosaic GFP signal was observed in almost all four chambers of the heart, indicating that this protocol is able to reach the majority of heart structures (Figure 4A). To determine which cardiac cell types were electroporated, pCAG::GFP-electroporated hearts were immunostained against markers of the epicardium, WT12, and myocardium, MF2023, and then imaged using a confocal microscope (Figure 4B). Most of the GFP+ cells were preferentially located in the external region of the heart, which largely corresponded with the WT1 signal, indicating that this method is most efficient in reaching epicardial cells (Figure 4B, white arrowheads).
On the other hand, pCAG::Meis1-electroporated or control (pCAG::GFP only) hearts were dissociated, and the cells were subjected to fluorescence-activated cell sorting (FACS) to isolate GFP+ cells from the whole organ, as described in step 4 of the protocol. RNA was extracted from the sorted cells, and reverse transcription quantitative PCR (RT-qPCR) was performed to assess the expression levels of Meis1 in the transfected cells (Figure 4C, Supplementary Table 1). Total Meis1 gene expression was normalized using the expression levels of the mouse housekeeping gene Eef224, and relative gene expression was calculated using the ΔΔCt method25. A significant upregulation of Meis1 RNA levels was observed in the hearts electroporated with the pCAG::Meis1 plasmid compared to those with only the pCAG::GFP plasmid (Figure 4C). Thus, this demonstrates the potential of the technique to overexpress genes of interest and address the molecular outcome.
Figure 4: Molecular analysis of electroporated E12.5 hearts. (A) GFP fluorescence of one CAG::GFP-electroporated heart 24 h after electroporation. The lower image shows GFP fluorescence, while the upper image shows a merge of GFP and a bright field of the same heart. (B) Immunofluorescence of electroporated E12.5 heart histological sections. MF20 marks cardiomyocytes, while WT1 marks epicardial cells. Images on the right correspond to zoom-ins of the white rectangle. White arrowheads indicate GFP+ cells located in the epicardium, roughly delimited by a dotted white line. (C) RT-qPCR analysis of hearts electroporated with pCAG::Meis1 or pCAG::GFP (control). Each dot represents a biological replicate obtained from the pooling of three hearts together. In total, qPCR analysis was performed in triplicates with three distinct biological replicates. Statistical analysis was performed using Student's t-test (*P < 0.05). Scale bars: 200 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Heart electroporation efficiency and cell viability. (A) Expression of GFP in two distinct E12.5 hearts electroporated with the control plasmid pCAG::GFP at 24 h or 48 h post-electroporation (hpe). The GFP signal image was superimposed over a bright field image of the same heart. (B) GFP+ cell quantification in 24 hpe and 48 hpe hearts electroporated with control pCAG::GFP. Dots indicate distinct samples; solid lines indicate the mean, while error bars indicate the standard deviation. No significant difference was observed between both conditions (non-parametric t-test; P > 0.9). (C) Confocal image of an E12.5 heart electroporated with pCAG::GFP immunostained against cleaved caspase 3 and MF20. The dotted line represents the myocardial layer of the heart. Images on the right are zoomed in from the white rectangle. Scale bars: 100 µm. Please click here to download this File.
Supplementary Table 1: Primer summary and sequences. Please click here to download this File.
Supplementary Movie 1: pCAG::GFP-electroporated heart after 24 h. Movie capturing one heart electroporated with pCAG::GFP control plasmid 24 h after the procedure. The heart continues to beat, and electroporated cells express GFP signals. Please click here to download this Movie.
Supplementary Movie 2: pCAG::GFP-electroporated heart after 48 h. Movie showing an electroporated heart with control pCAG::GFP, 48 h after electroporation. Please click here to download this Movie.
Overall, the methodology described here offers a robust framework for expressing transgenic constructs in the developing epicardium (Figure 4B), as demonstrated by Meis1 overexpression (Figure 4C). With the appropriate constructs, this protocol can be used to transiently assess the impact of either gain-of-function (GOF) or loss-of-function (LOF) of a specific gene. LOF can be implemented into the technique by transfecting a plasmid targeting a candidate gene through RNA interference26. The rapid assessment of gene GOF or LOF is particularly advantageous, especially considering the large transcriptomic heterogeneity of epicardial cells12, making it impractical to generate transgenic animals to evaluate each candidate gene individually.
Because this protocol utilizes ex vivo culture, it offers the possibility to investigate the fate of epicardial cells within a temporal window. The developing epicardium plays a pivotal role in shaping distinct regions within the adult heart13,27,28; thus, understanding its contributions to heart development is instrumental in dissecting the basis of congenital cardiac malformations. While this article does not delve into this application, the guidelines outlined in the protocol could potentially be helpful for future fate-mapping studies of the epicardium
Despite the practical utility of the current method, certain limitations need to be acknowledged. Since this methodology relies on transient transgenesis, plasmid expression can be lost or diminished over time, limiting the range of the protocol to study a specific developmental window29. However, in this study, GFP overexpression was maintained throughout the entire duration of the post-electroporation cultures (48 hpe). Additionally, potential limitations in current ex vivo culture technologies hinder the method's applicability for assessing late embryonic or postnatal developmental stages, especially where ex vivo culture after electroporation becomes challenging30. Although more sophisticated methods of ex vivo embryo culture exist31,32, they may require the use of equipment that is less accessible to researchers and may not guarantee the survival of the sample.
While the protocol presented here is designed to be as simple as possible to enhance reproducibility, it includes critical steps that are instrumental for success. Firstly, embryo dissection must be conducted carefully and swiftly to minimize heart damage, which could compromise the organ's survival after electroporation. Special care during this step, along with the use of optimal dissection equipment, is strongly recommended. Another critical aspect of the protocol is performing the electroporation itself, as inadequate plasmid injection may result in undesirable outcomes. Therefore, it is advisable to perform multiple injections on a single heart, and injecting the sample in a rather superficial manner could be beneficial to enhance organ survival. Finally, cell dissociation during heart processing needs to be rigorously performed, adhering to the times provided in the protocol to maximize cell viability.
In conclusion, identifying key genes in heart formation is instrumental in understanding the underlying mechanisms of cardiac congenital diseases and regeneration. The methodology established here allows for rapid screening of gene gain-of-function (GOF) and loss-of-function (LOF) effects in the developing heart. While acknowledging certain limitations, this method exhibits considerable potential for extrapolation to different stages of development or for mapping the fate of embryonic epicardial cells. Through the visual guidelines provided in this article, we aim to offer a valuable toolbox for assessing the immense complexity and potential of the developing epicardium.
The authors have nothing to disclose.
This study was supported by grant RTI2018-097617-J-I00 from the Spanish Ministerio de Ciencia e Innovación and Acción 9 from Universidad de Jaén to O.H.O. Grant PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovación and grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to M.T. JMG was supported by a PhD fellowship from the Spanish Ministry of Science and the Fundación Severo Ochoa (PRE2022-101884). Both the CNIC and CBMSO are supported by the Spanish Ministry of Science, and the CNIC is supported by the ProCNIC Foundation.
#55 Forceps | Dumont | 11295-51 | |
12-well Clear Flat Bottom Multiwell Cell Culture Plate | BD Falcon | 353043 | |
35 mm vise table | Grandado | SKU 8798771617573 | |
40 µm Cell Strainer | Fischer Scientific | 08-771-1 | |
50 mL tubes | BD Falcon | 352070 | |
70 µm Cell Strainer | Corning | CLS431751 | |
Anti-GFP Policlonal Antibody | Invitrogen | A10262 | 1:1000 dilution used |
Anti-Myosin 4 (MF20) Monoclonal Antibody | Invitrogen | 14-6503-82 | 1:500 dilution used |
CD1 Wild Type mice | Provided by Animalary Unit (CNIC) | ||
Cleaved Caspase-3 (Asp175) Antibody | Cell Signalling Technologies | 9661 | 1:400 dilution used |
DAPI | Cell Signalling Technologies | 4083 | 1:1000 dilution used |
Dispase/collagenase | Roche | 10269638001 | |
Distilled water | |||
DMEM – Dulbecco's Modified Eagle Medium | Gibco | 10313021 | |
Fetal Bovine Serum | Invitrogen | 10438-026 | |
Heracell 150i CO2 Incubator | Thermo Scientific | 51032720 | |
Leica Stereoscopic Microscope S8AP0 | Leica | 11524102 | |
Liberase | Roche | 5401119001 | |
Micropipette Puller Model P-97 | Sutter Instrument | SU-P-97 | |
pCAG expression plasmid | Addgene | #89689 | |
Penicillin-streptomycin | Invitrogen | 15070-063 | |
Petri dishes 35 × 10 mm | BD Falcon | 351008 | |
Petri dishes 60 × 15 mm | BD Falcon | 353002 | |
Phenol Red | Merck | P3532 | |
Pipette tips | Reused from old laboratory equipment | ||
Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD | Janvier Labs | 9979 | |
Recombinant anti-Wilms Tumor Protein 1 (WT1) Antibody | Abcam | ab89901 | 1:300 dilution used |
Square Wave Electroporator CUY21SC | Nepa Gene | CUY664-10X15 | |
Sterile PBS | Provided and autoclaved by technical unit | ||
Sucrose | Millipore | 84100 | |
Tweezer electrodes with variable gap | Nepa Gene | CUY650P5 |