Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Summary

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Abstract

Maintenance of steady-state calcium (Ca²⁺) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca²⁺ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca²⁺ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca²⁺-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca²⁺ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca²⁺ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca²⁺. This tool is useful for investigation into the nuanced changes in SR Ca²⁺ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca²⁺ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca²⁺ indicator, D1SR, is used to detect Ca²⁺ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca²⁺ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca²⁺ movement.

Introduction

Ca²⁺ is a very important ion found in abundance within every single cell in the body. It has significant roles in many different cellular functions, including growth, proliferation, migration, and apoptosis^1-4. It is well established that Ca²⁺ can affect these processes through both direct and indirect actions, and therefore, changes to the normal intracellular concentrations of this ion can easily result in negative outcomes for affected cells. The SR is considered as the largest intracellular Ca²⁺ store within the cell⁵. Steady state levels of Ca²⁺ in both the SR and cytoplasm are normally maintained through constant flux into and out of the Ca²⁺-transporting channels of this organelle. The stressful conditions imposed upon cells due to any form of injury or disease are commonly associated with significant changes in SR Ca²⁺ that go on to have long-lasting effects on the health of the cell. In the most severe of cases, the inability of a stressed cell to recover and maintain steady state SR Ca²⁺ levels may even culminate in death by apoptosis^6-8.

Current research into cellular Ca²⁺ dynamics is limited by the fact that few studies test organelle Ca²⁺ store content directly^9-11. The most common practice instead involves measurement of cytoplasmic Ca²⁺ levels as indirect measurements of changes in SR Ca²⁺ content ^12-14. In these experiments, Ca²⁺ is commonly induced to be released from the SR through the use of pharmacological agents causing the organelle's depletion (e.g. thapsigargin). Conclusions are then drawn with regard to changes to SR Ca²⁺ based on fluctuations in the cytoplasmic Ca²⁺ concentrations. Despite the ability remaining for investigators to draw such conclusions in a roundabout manner, this method of SR Ca²⁺ measurement is clearly an indirect way to glean such information, with many limitations concerning the interpretation of collected data. In order to bypass this clear restriction, it is necessary to measure the amount of Ca²⁺ found directly within the SR luminal network.

Vital to the final outcome of being able to directly record intraluminal SR Ca²⁺ levels are the cell culture tools and the Ca ²⁺ indicator used. For the data referenced in the current manuscript, it is important to note that VSMCs used came from a frozen cell line. Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) + 10% newborn calf serum (NCS) over passages 22-26 for the outlined experiments, incubated at a constant 37 °C with 5% CO₂ supply. Using the current method, for example, cells have been very successfully grown on a protein mixture that simulates the extracellular environment of many different types of tissue¹⁵. Important for success along the vein of SR Ca²⁺ research is the type of protein mixture used; in this case, a low growth factor variety was necessary to avoid components of this tool from affecting the regular Ca²⁺ signaling and movements constantly occurring within the tested cells. Following successful growth of test cells, the Ca²⁺ indicator must also be effectively introduced to these cultured cells. This has become possible by using an adenoviral vector that carries the SR-residing Ca²⁺ indicator D1SR. To achieve a high transfection efficiency, cells must be incubated with viral vectors for at least 36-48 hr prior to imaging. This preparation provides a reliable tool to measure Ca²⁺ transients within the SR lumen with high accuracy and reproducibility.

D1SR indicator used in this protocol is a modified variant of the D1ER Ca²⁺ indicator that was originally created by Dr. Roger Y. Tsien's laboratory at the University of California, San Diego, USA^9,16. The original D1ER belongs to a second generation of genetically encoded Ca²⁺ indicators called cameleons that display Ca²⁺ sensitivities over a much broader range (0.5-160 µM) as compared to previous Ca²⁺ indicators^9,16. The new variant D1SR, a kind gift from Dr. Wayne Chen (University of Alberta, Canada), however, carries a mutant calsequestrin sequence (instead of a calreticulin sequence as in the original D1ER). The mutant calsequestrin has a reduced binding to Ca²⁺ that eliminates the issue of competing with endogenous calsequestrin in binding to Ca²⁺ within the SR lumen¹¹. The D1SR indicator carries a truncated enhanced cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) that bind by a linker protein containing modified calmodulin (CaM) and M13 (the 26-residue peptide of myosin light-chain kinase that binds to CaM) sequences. The CaM-M13 sequence has been modified to prevent M13 from binding to endogenous calmodulin. Also, to ensure SR retention, a calsequestrin sequence has been added on the 5´ end of CFP¹¹. When bound to Ca²⁺, the CaM-M13 domain goes through conformational changes that result in an increase in the energy transfer between the flanking CFP and YFP, which is recorded as an increase in the FRET signal intensity. On the other hand, when the concentration of SR luminal Ca²⁺ drops, the CaM-M13 domain goes through reverse conformational changes, resulting in a decrease in the energy transfer between the flanking CFP and YFP, and a significant drop in FRET signal intensity.

Protocol

Ethics statement: All experiments and procedures were conducted in agreement with the Laboratory Biosafety guidelines of the University of British Columbia, Vancouver, Canada.

1. Preparation of 35 mm Glass-bottom Culture Dishes for FRET-based Confocal Microscopy

1. Prepare plates using the following procedure 36-48 hr prior to experiments.
2. Retrieve chosen gelatinous protein mixture and 0.25% trypsin-EDTA from -20 °C storage and keep them in mobile ice bath until use.
   NOTE: The coating protein mixture should only be exposed to RT for a brief period of time to prevent the mixture from solidifying before use. Trypsin solution should also be keep chilled until needed; alternatively, trypsin can be retrieved from -20 °C storage following completion of step 1.5.
3. Prepare a 1:25 dilution of protein mixture: DMEM (no added serum, chilled). Transfer appropriate volume of protein mixture/DMEM to each plate being prepared. For 35 mm glass-bottomed plates, add approximately 200 µl to fully cover inner glass circle on bottom of plate.
4. Leave plates to sit for at least 30 min inside the safety cabinet and at RT. Warm DMEM with 10% NCS to 37 °C in water bath during this time period.
5. Warm trypsin in water bath to 37 °C immediately prior to next steps.
6. Remove DMEM that is currently in culture flask using a glass pipette and suction. Wash floor of the flask with warm sterile PBS to remove remaining DMEM residue.
7. Dislodge previously cultured smooth muscle cells from flask bottom:
   NOTE: Flask was established days to this step to allow cells to reach desired confluency (approximately 80%) before plate preparation. Cells were originally suspended in the flask in 20 ml of DMEM with 10% NCS and incubated at 37 °C with 5% CO₂ supply for 24 hr, after which DMEM supply was refreshed and cells were returned to incubator. DMEM was refreshed every 48 hr following these initial steps until cells reached confluency.
   1. Wash flask quickly with 1 ml of trypsin and then remove 1 ml trypsin using suction.
   2. Add in another 1 ml of trypsin and leave for longer period (approximately 30-60 sec), swishing flask to ensure the enzyme makes contact with the entire bottom of the flask.
   3. Observe how much cells have detached under a microscope until satisfied with separation from flask. Alternate swishing trypsin solution and checking cell detachment until satisfied with proportion of cells separated from flask.
      NOTE: The flask can be returned to the incubator for approximately 30-60 sec to speed up this process at 37 °C.
   4. Once cells have been dislodged using trypsin, add fresh DMEM with 10% NCS to the flask to stop the trypsin reaction (sufficient volume so that trypsin forms 1/8^th of total volume in flask).
      NOTE: For example, when using a 250 ml culture flask, this would be 7 ml DMEM added to the 1 ml of trypsin to result in 8 ml of cell solution.
8. Transfer resulting cell solution from the flask to a clean (labeled) 50 ml conical tube.
9. Add 1 ml fresh DMEM plus 10% NCS to each plate being prepared (sufficient medium to last 24 hr).
10. Gently invert tube containing cell solution multiple times to break up any pellet that may have formed.
11. Pipette desired volume of gently mixed cell solution into each plate.
    NOTE: The recommended SMCs number plated for this protocol is 0.5-0.8 x 10⁶ per 35 mm culture dishes. These numbers may vary depending on the cell type and should be tested and modified by each laboratory.
12. Swirl each plate thoroughly clockwise/counterclockwise to ensure equal distribution of cells across glass bottom.

2. Transient Transfection with Adenoviral Vector Carrying D1SR Ca²⁺ Indicator

1. Following addition of medium and cell solution to each plate, leave plates to incubate at 37 °C with 5% CO₂ supply for at least 1 hr to allow cells to attach properly to glass on bottom of dishes. Check plates under a microscope at 15 min intervals until cells are clearly attached.
2. Retrieve adenovirus-D1SR aliquot from -80 °C storage and keep in mobile ice bath to transport to biological safety cabinet.
3. Pipette desired dose (multiplicity of infection of 100) of chilled D1SR to each plate.
   NOTE: The calculation for the volume of adenoviral solution needed per plate depends on the virus titer in the original stock solution, which is presented as the plaque forming unit (PFU). Based on our experience, we recommend a multiplicity of infection (MOI) of 100, which is interpreted as the number or virus particles needed to infect one smooth muscle cell (SMC) in the culture plate.
4. Incubate the infected cells at 37 °C with 5% CO₂ supply O/N.
5. Next day, replenish cells with 1 ml of fresh DMEM plus 10% NCS.
6. Incubate plates in a humidified incubator at 37 °C in 5% CO₂ O/N.

3. Measurement of SR Luminal Ca²⁺ Using FRET-based Confocal Imaging

1. Following O/N incubation, remove the culture medium from the plate.
2. Wash the culture plate with 1 ml warm physiological HEPES-PSS buffer containing (in mmol·L⁻¹) NaCl 140, glucose 10, KCl 5, HEPES 5, CaCl₂ 1.5 and MgCl₂ 1 (pH 7.4) three times.
3. Add 1 ml fresh warm HEPES-PSS buffer immediately prior to initiation of recording.
4. Perform initial imaging using the FRET SE (Sensitized Emission) application (or similar feature) on the confocal microscope's associated software.
   1. Once application is open, click into the "Setup" tab to adjust settings to achieve desired experimental conditions.
   2. Manually change channel settings so that cells are excited in sequence at 440 nm (for the donor and FRET channels) and 513 nm (for the acceptor channel). Collect the emission wavelengths at 488 nm (for donor) and 535 nm (for FRET and acceptor).
   3. Set the intensity of light at 15% transmission, with 150 ms excitation exposure time of cells (recording time for three sequential CFP donor, FRET, and YFP acceptor channels) and 10 sec intervals between exposures.
5. Stabilize plate on the microscope stage.
6. Use the "Live" button to check image resolution. Tweak settings further until desired resolution is reached.
7. Still under the "Setup" tab, take an image of the sample using the "Capture image" button.
8. Moving on to the "Evaluation" tab, choose the appropriate method to calculate the FRET efficiencies for the experiments being done. Method 3, using the Ratiometric Calculation [E_A(i) = B/A], is recommended for experiments involving cameleons such as the D1SR indicator.
9. Switch over to the "Graph" tab to be able to observe the intensity values being recorded in graph form during the experiment.
10. Draw an ROI in the image viewer to observe the corresponding average intensity of this area of interest on the graph as the experiment proceeds.
    NOTE: Investigator may also choose to draw multiple ROIs and have corresponding intensities be recorded and displayed simultaneously on the graph. Alternatively, investigators may outline a single ROI for initial recording, and proceed to outline multiple ROIs at a later time by opening the experiment file on a data analysis-oriented version of the software.
    NOTE: Further detail on the steps required to set up FRET-based experiments can be found in FRET application wizard manuals. If the microscope is not equipped with the FRET Wizard, the following protocol can be used to set up the parameters for recording manually.
11. Start recording and allow data collection for at least 3 min to ensure a steady basal recording of the signal prior to introducing the agent of interest.
12. Introduce Ca²⁺-moving agent (e.g. 2 µM thapsigargin; 100 nm endothelin-1) as a tool for depleting SR Ca²⁺, by manually pipetting the predetermined dose directly into the plate at a spot as near to the region of interest being recorded as possible. Continue recording for a desired time post treatment.
13. Capture ratiometric FRET serial images (512 x 512 pixels) with a 63X oil-immersion objective of the confocal inverted microscope using the FRET SE application.
14. Collect data resulting from imaging from a cluster of 5-10 SMCs in each region of interest (ROI) and subsequently format and analyze using statistics-based software.
    1. To save data for later use, right-click over the recorded trace and choose the "Export" option to save the spreadsheet with recorded mean signal intensities on the investigator's chosen drive.
    2. Normalize data from each individual experiment by dividing each data point by the starting intensity value observed at time point 0 sec.
    3. Import normalized data from spreadsheets into a statistical program project file by manually copying and pasting relevant data rows from their original spreadsheet/sec.
    4. Automatically generate graphs corresponding to the data sets pasted into the program.
       NOTE: The program's default settings generate line graphs from imported data. Type of graph displayed can be modified by clicking the "Choose a different type of graph" button under the "Change" heading found within the main toolbar.
    5. Pool data representing multiple independent trials within a larger group of identical experiments into a single data sheet to produce an average trace for the specific test performed.

4. Preparation of the Cytoplasmic Ca²⁺ Indicator

1. Dissolve 0.0125 g Pluronic Acid (PA) in 1 ml of dimethyl sulfoxide (DMSO) in a black microcentrifuge tube (may have to warm the tube in mobile water bath to help dissolve PA).
2. Retrieve a tube (50 µg) of the cytoplasmic Ca²⁺ indicator dye (Fluo-4 AM) from -20 °C storage.
3. Wrap the tube of dye in foil to protect it as much as possible from contact with light.
4. Pipette 45 µl of the DMSO+PA solution into the tube containing the dye. Wrap DMSO+PA solution in foil and store at RT.
5. Mix contents of the tube thoroughly by pipetting solution up and down multiple times. Alternatively, sonicate the dye tube for 10 min.
   NOTE: Either method of thoroughly distributing the dye throughout the DMSO+PA solution will be sufficient; it is simply a matter of the investigator's preference. If investigator chooses to sonicate solution, it should be done in an isolated area/room where others will be unaffected by the noise. Investigator should also use heavy mufflers to protect ears.
   1. If sonicating solution, insert foil-wrapped tube containing the dye inside a foam tube holder and place inside the 2/3 full (with dH₂O) sonicator bath.
   2. Place sonicator bath in isolated area/room and power machine on (ensure protective headwear is in place prior to initiating sonication process). Leave room and leave machine running for 10 min before shutting it off and retrieving the tube (keep wrapped in foil to protect from light for as long as possible).
6. Aliquot the now thoroughly mixed solution into dark microcentrifuge tubes (5 µl per tube, should be able to make 10 tubes total).
7. Wrap each aliquot tube in foil. Label tubes and store in desiccant at -20 °C conditions until use.

5. Measurement of Cytoplasmic Ca²⁺

1. Follow steps in 1.1-1.12 to prepare culture plates of rat aortic SMCs.
2. Remove the culture medium from the plate at 48 hr post culture.
3. Remove the previously prepared aliquot of cytoplasmic Ca²⁺ indicator (5 µl) from -20 °C storage and maintain in mobile ice bath until use.
4. Wash the culture plate with 1 ml warm physiological HEPES-PSS buffer containing (in mmol·L⁻¹) NaCl 140, glucose 10, KCl 5, HEPES 5, CaCl₂ 1.5 and MgCl₂ 1 (pH 7.4) three times.
5. Mix the indicator with 1 ml of warm HEPES-PSS and load it onto the culture plate.
6. Incubate SMCs with the indicator for 1 hr at RT (24 °C).
7. Remove indicator from culture plate and wash with 1 ml warm physiological HEPES-PSS buffer three times.
8. Add 1 ml fresh warm HEPES-PSS buffer immediately prior to initiation of recording.
9. Capture serial images (512 x 512 pixels) with a 63X oil-immersion objective of the confocal inverted microscope.
   1. Manually change settings so that cells are excited at 488 nm, and the emission wavelengths are collected at 555 nm with a scanning speed of 700 Hz.
   2. Set the intensity of light at 15% transmission, with 150 msec excitation exposure time of cells (the amount of time that cells are exposed to light at designated time intervals during recording) and 5 sec intervals between exposures.
10. Stabilize plate on the microscope stage and start recording.
11. Record for at least 3 min to ensure a steady basal recording of the signal prior to introducing the agent of interest.
12. Introduce Ca²⁺-moving agent (e.g. 2µM thapsigargin) as a tool for depleting SR Ca²⁺ (as described in step 3.16) and continue recording for desired amount of time post treatment.
13. Record signal intensity using microscope-specific software by averaging fluorescence signals (F/F₀) from a cluster of 5-10 SMCs in each region of interest (ROI). Collect, format, and analyze data resulting from imaging using data analysis software. See Steps 3.18.1-3.18.5 for description of data collection and analysis using this software.

Results

This section provides examples of the results that can be obtained using the described method to capture SR luminal Ca²⁺ concentration data, compared to the commonly employed manner of indirectly measuring changes to organelle Ca²⁺ store concentrations by observing fluctuations in cytoplasmic Ca²⁺.

Figure 1A shows a snapshot of a region of interest (ROI) of the SMC culture transf...

Discussion

This protocol describes the transfection of VSMCs with the D1SR adenovirus to study Ca²⁺ concentrations within the lumen of the SR. This method allows for the direct measurement of Ca²⁺ within this organelle as well as its movement into/out of the SR as a result of the application of different calcium-moving agents. This method has many advantages for investigators working towards a comprehensive understanding of how changes to SR and cytoplasmic Ca²⁺ levels affect overall cellular health...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle's Medium (high glucose, pyruvate)	Life technologies	11995-065	Warm in 37 °C water bath before use
Matrigel Basement Membrane Matrix Growth Factor Reduced, 5 ml	Corning	356230	Keep at -20 °C until right before use
Newborn Calf Serum	Sigma-Aldrich		Keep at -20 °C until right before use
35 mm glass-bottomed plates	MatTek Corporation	P35G-1.5-14-C
250 ml 0.2 μM vented blue plug seal cap flask	BD Falcon	353136
Trypsin/EDTA Solution	Life technologies	25200056	Keep at -20 °C until right before use
50 ml Polypropylene Conical Tube	BD Falcon	352070
D1SR	N/A	N/A	Gift
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8779
Pluronic F-127	Sigma-Aldrich	P2443
Fluo 4-AM	Life technologies	F-23917	Keep at -20 °C until right before use
1.5 ml Black Microcentrifuge Tubes	Argos Technologies	T7100BK
Leica TCS SP5 confocal microscope	Leica Microsystems
Leica Application Suite 2.6.3 (with FRET SE Wizard application)	Leica Microsystems
GraphPad Prism 5.0	GraphPad Software, Inc.