Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Summary

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Abstract

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Introduction

Small noncoding RNAs are important players in cellular gene regulation. These molecules are composed of 20-25 nucleotides that bind to a specific target mRNA, leading to the blockage of translation or to mRNA degradation^1,2. The gene regulatory process undertaken by small RNAs, such as miRNA and siRNA, is a highly conserved mechanism that has been found in many different species³. In particular, miRNA molecules are of crucial importance to a variety of physiological processes, including proliferation, differentiation, and regeneration^4,5. In addition, the dysregulation of miRNA expression is attributed to many pathological disorders. Correspondingly, miRNAs have been demonstrated to be suitable as biomarkers for diagnosis and as therapeutic agents for gene therapy^6,7.

Gap junctions (GJs) are specialized protein structures in the plasma membrane of two adjacent cells that allow the diffusional exchange of molecules with a molecular weight of up to 1 kD. They have been shown to be important to tissue development, differentiation, cell death, and pathological disorders such as cancer or cardiovascular disease^8,9,10. Several molecules have been described as being capable of crossing GJ channels, including ions, metabolites, and nucleotides. Interestingly, GJs were also found to provide a pathway for the intercellular movement of small RNAs^11,12. Thus, miRNAs can act not only within the cell in which they are produced, but also within recipient cells. This highlights the role of miRNAs in the intercellular signal transduction system. At the same time, the data demonstrate that gap junctional intercellular communication is closely linked to miRNA function. Because of the significant impact of miRNA and GJs on tissue homeostasis, pathology, and diagnosis, a comprehensive understanding of the function of GJs and the related intercellular dynamics of miRNA will help to clarify the mechanisms of miRNA-based diseases and to develop new strategies for miRNA therapies.

Depending on the extent of gap junctional coupling, the transfer of miRNA molecules between cells can be a very rapid process. Therefore, a methodology that allows the visualization and quantification of the fast intercellular movement of these regulatory signaling molecules is required. Commonly, flow cytometry and molecular biological techniques have been applied to demonstrate the shuttling of small RNAs¹¹,^12,13,14. However, as opposed to FRAP microscopy, these approaches lack high temporal resolution, which is mandatory when analyzing the exchange of miRNA via GJs. Moreover, FRAP microscopy is less invasive and therefore represents a powerful and novel live-cell imaging technique to evaluate the GJ-dependent exchange of molecules in several cell types^15,16,17.

Here, we present a detailed protocol describing the application of 3D-FRAP to assess miRNA shuttling between cardiomyocytes. For this purpose, cardiomyocytes were transfected with fluorescently labeled miRNA. A cell marked with this miRNA was photobleached, and the gap junctional miRNA re-influx from adjacent cells was recorded in a time-dependent manner. The high temporal resolution of FRAP experiments offers the possibility to perform kinetic studies for the precise evaluation of the intercellular transfer of miRNA and siRNA between living cells. Moreover, as small RNAs can be exchanged via different mechanisms with highly different kinetics, FRAP microscopy can help to clarify the extent to which GJs are involved in the respective shuttling processes¹⁸. In addition, 3D-FRAP can be used to investigate physiological and pathological alterations in GJ permeability and its impact on small RNA transfer^15,19.

Protocol

All steps in this protocol involving neonatal mice were performed per the ethical guidelines for animal care of the Rostock University Medical Centre.

1. Preparation of Cell Culture Dishes and the Medium for Cardiomyocyte Culture

1. Coat a cell culture plate with 0.1% gelatin in PBS and incubate at 37 °C for 4 h or at 4 °C overnight. Remove the gelatin and allow it to dry under sterile laminar air flow.
2. Prepare cell culture medium composed of 50 mL of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Pre-warm it to 37 °C.

2. Isolation of Neonatal Cardiomyocytes

1. Sacrifice neonatal mice (1-2 days old) by decapitation with sterile scissors and open the chest along the sternum. Remove the heart using forceps while slightly pressing the thorax together and transfer the heart into a 24-well plate containing ice-cold HBSS (without Ca²⁺ and Mg²⁺).
2. Remove non-cardiac tissue and larger vessels using sterile forceps. Transfer cleaned hearts into a 1.5 mL tube containing ice-cold HBSS. Use a maximum of 5 hearts per tube for enzymatic digestion.
3. Mince the hearts with small scissors into <0.5 to 1 mm³ pieces.
4. Wash the minced hearts with HBSS two times by aspiration/addition of HBSS using a 1-mL microliter pipette. Remove the HBSS completely before adding the enzymes.
5. For the enzymatic digestion of neonatal hearts, use a commercially available kit and follow the manufacturer's protocol (see the Table of Materials). Shake the tube containing the minced hearts every 5 min while incubating with enzymes at 37 °C for 35 min.
6. Seed suspended cells on non-coated cell culture dishes containing cell culture medium for 1.5-2 h to allow the adherence of the non-cardiomyocyte fraction. Repeat this step if a high purity of cardiomyocytes is needed. If desired, further culture the non-cardiomyocyte fraction.
   NOTE: For a cell suspension of 15 hearts, the pre-plating step can be performed on 75 cm² cell culture flasks. Usually, ~5 x 10⁵ cells are obtained from one neonatal heart.
7. Collect the supernatant in a 15 mL conical tube and centrifuge for 10 min at 300 x g. Resuspend the cells in 1 mL of cell culture medium.
8. Count the cells with a Neubauer chamber hemocytometer, or use an equivalent method.
9. Plate isolated cardiomyocytes on 6-well plates with a density of 3 x 10⁵ cells/cm². Incubate the cells overnight at 37 °C and 5% CO₂.
   NOTE: Optionally, cells can also be transfected at this point. However, increased viability was observed when the cells were cultured for one day before being subjected to electroporation.

3. Transfection with Fluorescently Labeled miRNA

1. Pre-warm cell culture medium (see step 1.2) to 37 °C in a water bath or an equivalent device.
2. Prepare a 20 µM stock solution of fluorescent miRNA in RNase-free sterile water.
3. Prepare electroporation buffer containing 90 mM Na₂HPO₄, 90 mM NaH₂PO₄, 5 mM KCl, 10 mM MgCl₂, and 10 mM sodium succinate and adjust the pH to 7.2; electroporation buffer can be stored at -20 °C for several months.
4. Detach the cells from the culture dish using 0.05% trypsin for 5 min. Inactivate the trypsin by adding cell culture medium.
5. Count the cells with a Neubauer chamber hemocytometer, or use an equivalent method. Centrifuge the cells at 300 x g for 10 min.
6. Resuspend the cells in electroporation buffer to obtain a concentration of 4 x 10⁵ cells per 100 µL.
7. Mix 100 µL of cell suspension with fluorescent miRNA (final concentration: 0.25 µM) in a tube and load the mixture into an electroporation cuvette.
   1. Empirically determine the appropriate amount of miRNA, depending upon cell type.
8. Perform electroporation using an electroporation device (see the Table of Materials), using the "G-009" program.
9. Add 500 µL of pre-warmed cell culture medium and transfer the whole cell suspension (4 x 10⁵ cells) into a well of a 4-well glass-bottom chamber slide. Culture the cells for 1 day at 37 °C in a 5% CO₂ atmosphere.
   NOTE: For FRAP analysis, a cell density of ~80% is optimal. The transfection of labeled miRNA should exclusively be performed using electroporation. Since the homogenous distribution of labeled miRNA molecules is beneficial for FRAP measurement, reagent-based transfection is not recommended.

4. Applying 3D-FRAP Microscopy (Day 3)

1. Warm up the microscope incubator to 37 °C and switch on the confocal microscope system at least 2 h before FRAP measurement to establish a thermal equilibrium and to decrease the possibility of drift. If possible, maintain a 5% CO₂ atmosphere.
2. Insert the chamber slide into the stage sample holder.
3. Find a cluster of transfected cardiomyocytes using a 1.4 N.A. oil objective (400X magnification) and 561-nm laser excitation light at low laser power, with a detection range of 570-680 nm.
   NOTE: The definition of FRAP settings, the image acquisition, and the analysis were done using microscope-specific software (see the Table of Materials).
4. Activate the "z-Stack," "Time Series," "Bleaching," and "Regions" buttons in the "Setup Manager."
5. Define the FRAP parameters.
   1. Define the image acquisition settings in the "Acquisition Mode" menu by setting the "frame size" to 512 x 512, the "line step" to 1, and the "scan time" to <1 s.
   2. In the "Channels" menu, adjust the laser power, offset, and gain settings to obtain maximal fluorescence from minimal laser excitation (e.g., laser power: 1-5%); adjust to avoid intensity saturation. Set the "pinhole size" to 2 µm.
   3. Next, in the "Regions" menu, select the "ROI drawing tool" and use the cursor to mark the target cell, the reference cell, and the background area. If required, select several target cells for photobleaching.
   4. Adjust the photobleaching settings in the "Bleaching" menu (e.g., iterations: 9-14, laser power for photobleaching: 100%, interval of image acquisition: 60 s, cycles: 15). Define the start of bleaching after 3 initial scans.
   5. In the "z-Stack" menu, define the limits for z-stack acquisition, depending on the thickness of the cells. Adjust the "number of z-layers" to 12 - 15.
   6. Start the FRAP experiment and record the fluorescence recovery.
      NOTE: Since the settings of a FRAP experiment depend upon the cell type, the fluorescent dye, and the microscope system, it is best to perform pilot experiments to determine the optimal parameters for FRAP. Bleaching should be sufficient to reduce the initial fluorescence intensity by at least 50%. Typically, fluorescence recovery should be recorded every 30-60 s until the plateau phase is reached.

5. Data Analysis

1. Create maximum projections of the acquired z-stacks and obtain fluorescence intensity values of the bleached target cells, the reference cell, and the background. Using microscope-specific software, click "Processing" → "Maximum intensity projection" → select file → "Apply."
   1. Alternatively, use an equivalent image analyzing tool (e.g., ImageJ).
2. Copy the fluorescence intensity data at all time points from the target cell, the background, and the reference cell into a spreadsheet.
   1. Subtract the background intensity and the intensity of the reference cell from the intensity values of the target cell to correct for photobleaching caused during the acquisition process. Perform background and reference cell corrections for each time point.
   2. Normalize the corrected FRAP data to the initial fluorescence intensity before bleaching by dividing each value by the initial fluorescence.
   3. To obtain FRAP curves, set the baseline to the fluorescence intensity immediately after the bleach by subtracting from the intensity values of each time point.

Results

Here, we present 3D-FRAP microscopy as a non-invasive technique to study the gap junctional shuttling of fluorescent miRNA within neonatal cardiomyocytes. The isolated cardiomyocytes revealed the typical striated α-actinin pattern and contained large plaques of Cx43 along the cell-cell borders (Figure 1A, white arrowheads), which allowed for the high intercellular flux of molecules. The purity of isolated cardiomyocytes was assessed by the microscopic quantif...

Discussion

miRNAs are key players in cellular physiology and were shown to act as signaling molecules by using—among others—GJs as a pathway for intercellular exchange^11,12,22. The current protocol presents an in vitro live-cell imaging technique to characterize this GJ-dependent shuttling using fluorescent miRNAs within cell clusters.

The protocol was developed on cardiomyocytes as a cell m...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
neonatal NMRI mice	Charles River
Gelatin	Sigma Aldrich	G7041	0.1% solution in PBS, sterilized
PBS	Pan Biotech	P04-53500
Dulbecco´s modified medium	Pan Biotech	P04-03550
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122	100 U/mL, 100µg/mL
Fetal bovine serum	Pan Biotech	P30-3306
Cell culture plastic	TPP
Primary Cardiomyocyte Isolation Kit	Thermo Fisher Scientific	88281
HBSS	Thermo Fisher Scientific	88281	included in primary cardiomyocyte isolation kit
Trypan blue	Thermo Fisher Scientific	15250061
miRIDIAN Dy547 labeled microRNA Mimic	Dharmacon	CP-004500-01-05	dissolve in RNAse free water, stock solution 20µM
Sodium phosphate monobasic	Sigma	S3139
Sodium phosphate dibasic	Carl Roth	T877.2
Potassium chloride	Sigma	P9333
Magnesium chloride	Serva	28305.01
Sodium succinate	Carl Roth	3195.1
0.05% Trypsin/EDTA solution	Merck	L2153
4-well- Glass bottom chamber slides	IBIDI	80827	coat with 0.1% gelatin solution
Amaxa Nucleofector II	Lonza		program G-009 was used for Electroporation
LSM 780 ELYRA PS.1 system	Zeiss
Excel software	Microsoft
α-actinin antibody	Abcam	ab9465	dilution 1:200
Connexin43 antibody	Santa Cruz	sc-9059	dilution 1:200
goat anti-mouse Alexa 594 antibody	Thermo Fisher Scientific	A-11005	dilution 1:300
goat anti-rabbit Alexa 488 antibody	Thermo Fisher Scientific	A-11034	dilution 1:300
Connexin43 siRNA	Thermo Fisher Scientific	AM16708 ID158724	final concentration 250 nM
Near-IR Live/Dead Cell Stain Kit	Thermo Fisher Scientific	L10119
CellTrace Calcein Red-Orange	Thermo Scientific	C34851
DAPI nuclear stain	Thermo Scientific	D1306