Analyzing the α-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

Summary

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Abstract

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the α-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Introduction

Cardiovascular diseases (CVD) such as myocardial infarction or cardiomyopathy remain the major cause of death in the western world¹. As the human heart possesses only poor regenerative capacity, there is a need for strategies to promote the recovery from CVDs. This includes cell replacement therapies to replenish lost cardiomyocytes (CM), as well as the development of new anti-arrhythmic drugs for efficient and safe pharmaceutical intervention. Induced pluripotent stem cell (iPSC) have been shown to be a promising cell source for the unlimited generation of human CM in vitro, suitable for regenerative therapies, disease modeling, and for the development of drug screening assays^2,3,4.

Although many different cardiac differentiation protocols exist, iPSC-derived CM still lack certain phenotypical and functional aspects that impede the in vitro and in vivo application^5,6. Beside electrophysiologic, metabolic, and molecular changes, the cardiac maturation process involves the structural organization of sarcomeres, which are the fundamental units required for force generation and cell contraction⁷. While adult CMs exhibit a well-organized contractile apparatus, iPSC-derived CMs commonly demonstrate disarranged sarcomere filaments, associated with a reduced contraction ability and altered contraction dynamics^8,9. In contrast to mature CM that show uniaxial contraction pattern, the disoriented structures in immature CM results in a radial contraction of the whole cell or promote the appearance of contraction focal points^9,10.

For improving cardiac maturation, multiple approaches have been applied, including 3D cell culture methods, electrical and mechanical stimulation, as well as the use of extracellular matrices mimicking in vivo conditions^11,12,13. To evaluate the success and efficiency of these different culture conditions, techniques are needed to monitor and estimate the degree of the structural maturation of iPSC CM, e.g., by microscopic techniques. In contrast to conventional confocal imaging, the resolution in case of photoactivated localization microscopy (PALM) is approximately 10x higher. This technique in turn allows for a more accurate analysis, detecting even subtle alterations of cellular structures¹⁴. Considering the high resolution of PALM-based imaging, the overall goal of this method was the microscopic evaluation of sarcomere maturity in iPSC-derived CMs by precise determination of z-Disc thickness and sarcomere length. In previous studies, these structural features have been shown to be appropriate parameters to assess cardiac maturity¹⁵. For example, diseased iPSC-CM lacking full length dystrophin exhibit reduced sarcomere length and z-band width when compared to wild type cells¹⁶. Likewise, the length of individual sarcomeres was measured to investigate the impact of topographic cues on cardiac development¹⁶. Hence, we applied this approach to evaluate the structural maturation of the sarcomere network in iPSC-CM by quantitatively measuring the sarcomere length and z-disc thickness.

Protocol

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

1. Cultivation and dissociation of iPSC-derived cardiomyocytes

1. Differentiate hiPSC-CMs for 25 days using a 2D monolayer method as described previously¹⁷.
2. Prewarm dissociation medium to 37 °C and support medium to room temperature.
3. Wash cells twice with PBS.
4. Add prewarmed dissociation medium to cells and incubate for 12 min at 37 °C and 5% CO₂.
5. Add support medium to the cells.
   NOTE: The volume of added support medium needs to be twice the volume of dissociation medium used in step 1.4.
6. Dislodge the cells using a 5 mL serological pipette.
7. Centrifuge cells at 200 x g for 5 min and resuspend the pellet in 3 mL of hiPSC-CM culture medium¹⁷.
8. Seed cells in 8 well glass bottom chambers at a cell density of 75,000 cells/well and culture for 3 days.

2. Adult cardiomyocyte isolation

1. Isolation and cultivation of adult cardiomyocytes from NMRI mice was performed as reported previously¹⁸.
2. Seed cells in 8 well glass chamber slide and culture for one day.

3. Isolation and cultivation of neonatal cardiomyocytes

1. Isolation procedure of neonatal cardiomyocytes, obtained from NMRI mice, was performed as described previously¹⁹.
2. Seed isolated cell in 8 well glass chamber slide at a cell density of 75,000 cells/well and culture for 3 days in neonatal CM culture medium¹⁹.

4. Immunofluorescence labeling of the α-actinin network

NOTE: For optimal results, cells are cultured in 8 well glass bottom chambers. Labeling should be performed one day before imaging.

1. Prewarm 4% PFA at 37 °C.
2. Fix iPSC-CM by adding 4% paraformaldehyde directly into the culture media (1:1 dilution) and incubate at 37 °C for 15 min. The final concentration of PFA for fixation is 2%.
3. Incubate fixed cells in 0.2% Triton-X, diluted in PBS, for 5 min at room temperature.
4. Wash cells twice with PBS, 5 min each.
5. Add 1% BSA solution (diluted in PBS) and incubate for 60 min at room temperature.
6. Prepare 150 µL of primary antibody solution by diluting α-actinin antibody 1:100 in 1% BSA, containing 0.05% Triton-X. Add to cells and incubate at room temperature for 60 min.
7. Wash cells twice with 0.2% BSA solution, 5 min each.
8. Prepare 150 µL of secondary antibody solution by diluting goat-anti-mouse Alexa647 antibody 1:100 in 1% BSA, containing 0.05% Triton-X. Add to the cells and incubate at room temperature for 40 min.
9. Wash cells twice with 0.2% BSA solution, 5 min each.
10. Wash cells twice with PBS, 5 min each. Keep labeled cells in the dark at 4 °C until PALM imaging.

5. Preparation of the PALM imaging buffer

NOTE: It is critical to freshly prepare the PALM imaging buffer for each experiment.

1. Prepare 50% glucose solution by dissolving 25 g glucose in 50 mL of distilled water.
2. Prepare basic buffer containing 50 mM Tris-HCl, 10% glucose and 10 mM sodium chloride.
   1. Adjust the pH level to ~8.0 using hydrochloric acid.
3. Prepare pyranose oxidase solution by dissolving 0.6 mg pyranose oxidase in 316 µL of basic buffer.
4. Prepare catalase solution by dissolving 7 mg of catalase in 500 µL of basic buffer. Mix thoroughly and centrifuge at 10,000 x g for 3 min. Keep the supernatant for further use. Catalase solution can be kept at 4 °C for several days.
5. Prepare 500 µL of PALM imaging buffer by mixing 316 µL of pyranose oxidase solution, 25 µL of catalase solution, 100 µL of 50% glucose solution, 50 µL of cysteamine, 5 µL of cyclooctatetraene and 3.5 µL of β-Mercaptoethanol. The final catalytic activity of pyranose oxidase and catalase need to be 7.5 U, 35,00U respectively.
   NOTE: The PALM imaging buffer provides optimal imaging conditions for 3-5 h. If blinking capability of the fluorescent dye decreases, prepare a new buffer aliquot.

6. PALM image acquisition

1. Switch on the microscope at least 3 h before use and bring the sample to room temperature before imaging to allow thermal equilibration. If the microscope is equipped with an incubation chamber, adjust temperature to 30 °C.
2. Clean the objective and bottom of the chamber slide using an appropriate cleaning solvent.
3. Add 300 µL of PALM imaging buffer into one well of labeled cells and insert the chamber slide into the stage holder of the microscope.
4. Set the PALM image acquisition parameters.
   1. Select 1.57 N.A. 100x oil objective for acquisition.
   2. Select PALM mode and activate the TIRF settings.
   3. Adjust the number of frames to be acquired. Usually 5, 000-10, 000 frames are sufficient to obtain optimal results. However, the number of acquired frames strongly depends on the labeling efficiency and the blinking capability of the fluorescent dye and may be adjusted by the user.
   4. Set UV laser power to 0.1% and 647 laser to 0.2%.
   5. Set the gain level to 50-100.
   6. Switch on the laser illumination and select a target cell. The gain level can be increased if signal intensity is to low (depending on fluorescence labeling).
   7. Switch off the laser illumination
5. PALM image acquisition
   1. Reduce gain to 0 and increase the laser power (ex 647) to 100%.
   2. Bleach the target cell for ~5 s.
   3. Increase gain to 50 and start PALM image acquisition.
      NOTE: Gain needs to be adjusted to get sufficient signal intensity while oversaturated pixels should be avoided. If signal intensity reduces, increase the gain level.
6. Optionally, steadily enhance the UV laser power (0.1%-10%) to increase signal intensity and to promote blinking of the fluorophore.

7. Reconstruction of PALM data

1. Open the Image J software and import PALM data.
2. Open Thunderstorm Plugin and “Run analysis”
   1. In the “Camera setup” menu enter pixel size and EM gain.
      NOTE: When using 100x objectives and 1.6x magnification lens, 100 nm pixel size is appropriate. However, as the pixel size depends on the hardware features of the microscope and camera used for PALM imaging, users need to carefully check and adapt this parameter. EM gain values can be obtained from the metadata.
   2. In the “Run analysis menu” set parameters as follows: B-spline order: 3, B-spline scale: 2.0, peak intensity threshold: stf(Wave.F1), fitting radius: 3, initial sigma: 1.6, magnification: 5.0, update frequency: 50, lateral shifts: 2. Confirm by clicking the “Ok” button.
3. Post processing of reconstructed PALM image
   1. In the “Plot histogram” menu select the “Sigma” parameter.
   2. Use the “Rectangle” tool to select a ROI, excluding possible artefacts and apply ROI to the filter. ROI values will appear in the filter command box.
   3. Add “& uncertainty <25” to the ROI values. A possible filter command will look like this: “(sigma > 48.6821 & sigma < 1117.40) & uncertainty <25”. Apply selected sigma values.
   4. In the “Remove duplicates” menu, enter a distance threshold of “10 nm” and apply.
   5. In the “Merging menu”, set maximum distance to “20”, maximum frames per molecule to “0” and maximum off frames to “1”. Apply settings.
   6. In the “Drift correction menu”, select cross correlation and set “Number of bins” to “5” and “Magnification” to “5.0”. Apply drift correction settings.
   7. Save the final PALM image and export post processed data if desired.

8. Analysis of sarcomere filaments

1. Analysis of sarcomere length
   1. Open Image J software and import the reconstructed PALM image.
   2. Draw a line between selected sarcomere structures perpendicular to the z-disc to measure the shortest distance between actinin filaments.
   3. Select “Plot profile” in the “Analyze” menu and acquire the length between two peaks. As sarcomere length may vary within one cell, a minimum of 20 sarcomeres should be measured in different areas of the target cell.
2. Analysis of z-Disc thickness
   1. Open Image J software and import the reconstructed PALM image.
   2. Convert the reconstructed PALM image into an 8-bit mode image.
   3. Open the ridge detection plugin and enter the following parameters: line width: 20, high contrast: 230, low contrast: 10, sigma: 0.79, lower threshold: 25.84, minimum line length: 20.
   4. Set “Estimate width”, “Extend line” and “Display results”.
   5. Click “Ok” and use “Mean line width” from results table for further analyses.

Results

For estimating the degree of structural maturation of CM, neonatal, fully mature adult, and iPSC CM were initially labeled the CM with α-actinin antibody to visualize the sarcomere network. Following PALM acquisition, images were reconstructed, and z-disc thickness was measured using plugin-based image processing software for the automatic detection of the width of individual filaments. Sarcomere length was calculated by measuring the distance between two adjacent intensity peaks, corresponding to neighboring filaments. ...

Discussion

The generation of functional iPSC-derived CM in vitro is important for regenerative therapies, disease modeling and the development of drug-screening platforms. However, insufficient maturity of these CM is a major obstacle in cardiovascular research²⁰. In this regard, high resolution imaging techniques are needed that enable monitoring of the structural maturation state of iPSC-derived CM. At the same time, super resolution microscopy can be a valuable tool to precisely analyze the function of sp...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
human iPSC cell line	Takara	Y00325
µ-Slide 8 Well Glass Bottom	ibidi	80827
0.5ml eppendorf tube	Eppendorf	30121023
Bovine serum albumin	Sigma Aldrich	A906
Cardiomyocyte Dissociation Kit	Stem Cell Technologies	05025
Catalase	Sigma Aldrich	C40-1G
Cyclooctatetraene	Sigma Aldrich	138924-1G
Cysteamine	Sigma Aldrich	30070-10g
Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+	Thermo Fisher	14190169
F(ab')2-Goat anti-Mouse IgG Alexa Fluor 647	Thermo Fisher	A-21237
Fiji image processing software (Image J)
Glucose	Carl Roth	X997.2
Hydrochloric acid	Sigma Aldrich	H1758
LSM 780 ELYRA PS.1 system	Zeiss
Paraformaldehyde	Merck	8187150100
Pyranose oxidase	Sigma Aldrich	P4234-250UN
sarcomeric α-actinin antibody [EA-53]	Abcam	ab9465
Sodium chloride	Sigma Aldrich	S7653
sterile water	Carl Roth	3255.1
Triton X-100	Sigma Aldrich	X100
Trizma base	Sigma Aldrich	T1503
β-Mercaptoethanol	Sigma Aldrich	63689