More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients' cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.
To date more than 700 RYR1 variants have been identified in the human population and linked to various neuromuscular disorders including malignant hyperthermia susceptibility (MHS), exercise induced rhabdomyolysis, central core disease (CCD), multi-minicore disease (MmD), centronuclear myopathy (CNM)1,2,3; nevertheless, studies to characterize their functional effects are lagging and only approximately 10% of mutations have been tested functionally. Different experimental approaches can be used to assess the impact of a given RyR1 variant, including transfection of heterologous cells such as HEK293 and COS-7 cells with plasmid encoding for the WT and mutant RYR1 cDNA4,5, transduction of dyspedic mouse fibroblasts with plasmids and vectors encoding for the WT and mutant RYR1 cDNA, followed by transduction with myo-D and differentiation into myotubes6, generation of transgenic animal models carrying mutant RyR1s7,8,9, characterization of cells from patients expressing the RYR1 variant endogenously10,11,12. Such methods have helped established how different mutations functionally impact the RyR1 Ca2+ channel.
Here, methods developed to assess the functional effects of RYR1 mutations are described. Various parameters of intracellular calcium homeostasis are investigated in human cells endogenously expressing the RyR1 calcium channel, including myotubes and Epstein Barr Virus (EBV) immortalized B-lymphocytes. Cells are obtained from patients, expanded in culture and loaded with ratiometric fluorescent calcium indictors such as Fura-2 or indo-1. Parameters which have been reported to be altered because of pathogenic RYR1 mutations including the resting [Ca2+], the sensitivity to different pharmacological agonists and the size of the intracellular Ca2+ stores are measured either at the single cell level, using fluorescence microscopy, or in cell populations using a fluorimeter. Results obtained in cells from mutation carriers are then compared to those obtained from healthy control family members. This approach has demonstrated that: (i) many mutations linked to MHS lead to an increase in the resting [Ca2+] and a shift to the left in the dose response curve to either KCl-induced depolarization or pharmacological RyR1 activation with 4-chloro-m-cresol10,11,12,13; (ii) mutations linked to CCD lead to a decrease in the peak [Ca2+] released by pharmacological activation of the RyR1 and decreased size if the intracellular Ca2+ stores12,13,14,15; (iii) some variants do not impact Ca2+ homeostasis13. Advantages of this experimental approach are: the RyR1 protein is not over-expressed and physiological levels are present, cells can be immortalized (both muscle cells and B-lymphocytes) providing cell lines containing mutations. Some disadvantages relate to the fact that patients may carry mutations in more than one gene encoding proteins involved in calcium homeostasis and/or excitation contraction coupling (ECC) and this may complicate experimental conclusions. For example, two JP-45 variants were identified in the MHS and control population and their presence were shown to impact the sensitivity of the dihydropyridine receptor (DHPR) to activation16. Patients need to be available, biological material needs to be freshly collected and ethical permits need to be obtained from the local ethical boards.
The protocols described below comply with the ethics guidelines of the Ethikkommission Nordwest- und Zentralschweiz EKNZ.
1. Preparation of Epstein Barr immortalized B-lymphocyte cell lines11
- After informed consent, collect 30 mL of whole blood in EDTA-treated sterile tubes from the proband carrying a RYR1 mutation and from healthy family members with no mutation.
NOTE: Keep all solutions sterile and work in a tissue culture hood.
- Isolate mononuclear cells from whole blood by density gradient centrifugation media (e.g., Ficoll-Hypaque, .077 g/L).
- Place 30 mL of sterile blood in a 50 mL conical sterile tube.
- Place the tip of a Pasteur pipette containing the density gradient centrifugation media at the bottom of tube and layer 20 mL sterile of density gradient centrifugation media solution slowly underneath the blood.
- Centrifuge for 30 min at 900 x g at 18°-20°C, with no break.
NOTE: The mononuclear cell layer appears as a cloudy ring at the interphase between the density gradient centrifugation media layer and the top layer containing platelet enriched plasma.
- With a sterile pipette gently remove the interphase layer containing mononuclear cells (approximately 3-5 mL) and transfer the solution to a clean 50 mL sterile conical tube.
- Add 20 mL of phosphate buffer saline (PBS) to rinse cells, centrifuge for 10 min at 600 x g at room temperature and resuspend the pellet in PBS. Repeat for a total of three times; this ensures that all the media is removed.
- After the last wash, resuspend cells in 1-2 mL of tissue culture medium (RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 units of penicillin and streptomycin). Place mononuclear cells (approximately 1 x 106 cells) in a T125 tissue culture flask containing 20 mL of tissue culture medium.
- Infect mononuclear cells with Epstein-Barr virus.
- Use supernatants from B95.8 cell line cultures (containing 102-103 transforming units/mL stocked at -80 °C) as a source of EBV.
- Resuspend 1 x 106 mononuclear cells from step 1.5 in 20 mL of tissue culture medium and expose them to 2 mL of supernatant from the B95.8 cell line in the presence of cyclosporin A (0.2 µg/mL final concentration) for infection.
- Place the flask in a 37 °C cell culture incubator and allow the cells to grow. After one week change the culture medium.
NOTE: Once B-cells start proliferating they form recognizable clumps and grow rapidly so that the cultures can be expanded and frozen.
- Extract the genomic DNA from the EBV immortalized B-lymphocyte cell lines11 to confirm the presence or absence of the given mutation.
2. Intracellular Ca2+ measurements
NOTE: Changes in the intracellular calcium concentration of the EBV-transformed B-lymphocyte cell lines can be monitored in cell populations, with a spectrofluorometer equipped with a magnetic stirrer and cuvette holder set to 37 °C. Alternatively Ca2+ changes can be monitored in single cells by fluorescence microscopy. In both cases cells are removed from the tissue culture flask, washed twice with Krebs Ringer's solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES, 1 mM NaHPO4, 5.5 mM glucose, pH 7.4 containing 1 mM CaCl2) and counted .
- For experiments in cell populations using a spectrofluorometer11,13,14
- Resuspend cells at a final concentration of 1 x 107 cells/ mL in Krebs Ringer's solution and incubate at 37°C for 30 min with a final concentration of 5 µM Fura-2/AM.
- Centrifuge cells at 900 x g for 10 min and resuspend them in Krebs Ringer's solution at a concentration of 2 x 106 cells/mL.
- Measure fluorescence changes (ratio 340/380 nm) using a spectrofluorometer equipped with a magnetic stirrer set at maximal velocity and set to 37 °C.
- Just before the experiment spin cells at 900 x g for 5 min in a microcentrifuge and quickly resuspend the pellet in 1.5 mL of Krebs Ringer's solution with 0.5 mM EGTA but no added Ca2+.
- Place cells in a 3 mL glass spectrofluorometer cuvette and record the fluorescence ratio (340 nm/380 nm excitation, 510 nm emission).
- Achieve a stable base line (approximately 30 seconds), add the selected concentration of RyR1 agonist (4-chloro-m-cresol, or 4-cmc) and record the calcium transient.
NOTE: A 300 mM stock solution of 4-cmc made in DMSO is use used as a starting reagent. This solution can be made in advance, aliquoted and stored at -20 °C for several months.
- Perform experiments for different 4-cmc concentrations.
NOTE: Include 75 µM, 150 µM, 300 µM, 450 µM, 600 µM, 750 µM to 1 mM to generate a dose response curve of agonist versus change in [Ca2+]. The different 4-cmc concentrations are obtained by adding the appropriate volume of 4-cmc from the stock solution, directly into the cuvette containing the Fura-2 loaded cells. For example, for a final concentration of 300 µM 4-cmc, 1.5 µL of the stock solution are added to the cuvette containing 1.5 mL of cells in Krebs Ringer's solution. For lower agonist concentrations, the 300 mM stock solution should be diluted to 75 mM with DMSO and the appropriate volume added to the cuvette containing 1.5 mL of cells in Krebs Ringer's solution.
- Add 400 nM thapsigargin to cells to calculate the total amount of Ca2+ present in intracellular stores. Record the peak Ca2+.
- Plot the peak calcium induced by a given 4-cmc concentration versus the peak calcium induced by thapsigargin, which is considered 100% and construct a 4-cmc dose response curve comparing cells from a proband and healthy relative
- For experiments on single cells13
- Dilute poly-L-lysine 1:10 in sterile H2O and pre-treat the glass coverslips for 30 min. Allow to air-dry under a sterile tissue culture hood.
- Re-suspend EBV-transformed B-lymphocytes to a final concentration of 1 x 106 cells/mL in Krebs Ringer's solution containing 1 mM CaCl2 and add a final concentration of 5 µM Fura-2/AM.
- Place 1 mL of cells on the poly-L-lysine treated coverslips and incubate at 37 °C in a humidified cell culture incubator for 30 min to allow the EBV cells to stick to the glass coverslip during loading.
- Place the coverslip in the perfusion chamber and start perfusion (at a rate of 2 mL/min) with Krebs Ringer's solution containing 1 mM Ca2+.
- Use an inverted fluorescent microscope (equipped with a 40x oil-immersion objective (0.17 numerical aperture), filters (BP 340/380, FT 425, BP 500/530) to record on-line measurements, with a software-controlled charge coupled device (CCD) camera attachment.
- Acquire Images at 1 s intervals at a fixed exposure time (100 ms for both 340- and 380-nm excitation wavelengths. Use imaging software to analyse changes in fluorescence. Measure the average pixel value for each cell at excitation wavelengths of 340 and 380 nm13.
- To achieve cell stimulation, use a cell perfusion stimulator with 12 valves and add different concentrations of 4-cmc. The flush valve contains Krebs Ringer's solution with no added Ca2+ plus 100 µM La3+ to monitor calcium release from intracellular stores only.
- Construct a dose response curve of 4-cmc versus change in [Ca2+], as described above.
3. Preparation of human myotubes from muscle biopsies10,12,15
NOTE: Different methods have been used by different laboratories to obtain satellite cell-derived myoblasts and myotubes. Below is the description of the method used in Basel.
- Rinse muscle biopsy with sterile PBS to remove excess blood and cut into small fragments of about 0.5-1 mm.
- Prepare 6 well tissue culture dishes with insert. Add 1.5 mL of human muscle growth medium to each well and 0.5 mL of human muscle growth medium to each insert.
NOTE: The growth medium is made up as follows: 500 mL of Dulbecco's modified Eagle's medium with high glucose, or DMEM (4.5 mg/mL), containing 10% horse serum, 5 ng/mL insulin, 3 mM glutamine, 600 ng/mL penicillin G and streptomycin, and 7 mM HEPES, pH 7.4. Commercially available skeletal muscle growth medium can also be used.
- Place 2-3 small muscle fragments into each insert (Figure 1A) and place culture dishes into the cell culture incubator (5% CO2, 37 °C). After approximately 8-10 days satellite cells can be seen growing out of and surrounding the muscle biopsy, attached to the insert (Figure 1, B and C, arrows).
- Release a sufficient number of cells from the biopsy (after approximately 10-14 days), trypsinize as follows:
- Remove all culture medium, rinse the cells once with 1 mL of PBS, add 0.5 mL of trypsin/EDTA solution (0.025% trypsin and 0.01% EDTA) and incubate at 37 °C for 5 min.
- Add 1 mL of growth medium to the cells to neutralize the effect of trypsin and transfer the satellite cells into a new T25 cell culture flask; add 3 mL of growth medium and place the cells in a cell culture incubator (5% CO2, 37 °C).
- Change the growth medium the next day to remove the EDTA and subsequently change the medium once per week.
- When myoblasts are approximately 75% confluent, trypsinize and transfer them onto laminin-treated glass coverslip.
NOTE: As a proportion, cells growing in one T25 flask should be transferred onto one 43 mm diameter laminin-treated glass coverslip. The glass coverslip should be placed within a 60 mm diameter tissue culture plate containing 3 mL of growth medium.
- Grow cells on the glass coverslip in growth medium in a cell culture incubator (5% CO2, 37 °C) changing the medium once per week. At 90% confluency, switch to differentiation medium made up as follows: high glucose DMEM (4.5 mg/mL), 0.5% bovine serum albumin, 10 ng/mL epidermal growth factor, 0.15 mg/mL creatine, 5 ng/mL insulin, 200 mM glutamine, 600 ng/mL penicillin G and streptomycin, and 7 mM HEPES, pH 7.4). Commercially available differentiation medium may also be used. Change the differentiation medium once per week.
- After 7-10 days in differentiation medium, multinucleated myotubes are visible. Assess for changes in [Ca2+] within one week, as described below.
4. [Ca2+]i ratio measurements determined with Fura-2
- Load glass coverslip grown myotubes with Fura-2/AM (final concentration of 5 µM) diluted in DMEM for 30 min at 37 °C. Briefly, remove the differentiation medium from the glass coverslip grown cells and add 2 mL fresh differentiation medium. Add 10 µL of Fura-2 AM from a stock solution of 1 mM and incubate 30 min in a cell culture incubator (5% CO2, 37 °C).
- Transfer glass coverslip to the perfusion chamber and rinse cells with Krebs Ringer's solution containing 2 mM CaCl2.
- Perform on-line [Ca2+] measurements as described above in the EBV single cell section with the use a 20x water immersion FLUAR objective (0.17 numerical aperture).
[Ca2+]i measurements in populations of EBV-immortalized B lymphocytes
Primary B-lymphocytes express the RyR1 isoform that functions as a Ca2+ release channel during B cell antigen receptor stimulated signaling processes17. Immortalization of B-cells with EBV, a procedure routinely used by geneticists to obtain cell lines containing genomic information of patients, provides the advantage of generating cell lines that express mutant RyR1 Ca2+ channels in patients harboring RYR1 mutations11,13. [Ca2+]i changes brought about by the addition of specific RyR1 agonists such as 4-chloro-m-cresol18 and caffeine can be easily monitored in order to establish whether a given RYR1 mutation alters the sensitivity to an agonist, the amount of calcium released, the resting [Ca2+], or other parameters that have shown to be impacted by mutations. [Ca2+]i changes can be monitored either in populations of Fura-2 loaded cells in suspension with a spectrofluorometer or on groups of cells attached to glass coverslips and examined by epifluorescence. Figure 2 shows a representative experiment carried out on cell suspensions. Immediately before being placed in the cuvette, cells were spun to remove Fura-2 that may have leaked out; cells were then resuspended to a final concentration of 1 x 106 cells/mL in warm (37°C) Krebs Ringer's solution containing no additional Ca2+ plus 0.5 mM EGTA and placed in the spectrofluorometer. The magnetic stirrer was switched on to position 4 (the highest position) to keep cells in suspension and fluorescence was recorded. After a stable trace was obtained, the selected agonist was added (in Figure 2A this was 300 µM 4-chloro-m-cresol) leading to a rapid Ca2+ increase which then slowly declined back to resting levels. The transient nature of the change in fluorescence is important as it indicates (i) that it is not an artefact caused for by the addition of a fluorescent or quenching compound, (ii) that it is not due to calcium binding to extracellular Fura-2 and (iii) that the cells are healthy and can actively remove calcium from their cytoplasm.
The same experiment needs to be repeated several times to be analyzed statistically. For each cell line and each day, the experiments are carried out and the total amount of rapidly releasable calcium in the stores needs to be determined by adding the SERCA inhibitor thapsigargin11,13,14. As shown in Figure 2B, the addition of 400 nM thapsigargin causes a large calcium transient reaching a peak fluorescence value of 2.4 arbitrary units (a.u.); thus the total amount of calcium that can be released from the intracellular stores of the EBV-immortalized B- cells shown in Figure 2B equals 2.4 a.u. (thapsigargin peak) - 1.45 a.u. (resting ratio) or 0.95 This fluorescence value was considered 100% when constructing the dose response curve shown in Figure 2C.
Single cell [Ca2+]i measurements in EBV-immortalized B lymphocytes
This second approach relies on the availability of a fluorescence microscope and microperfusion set up allowing the stimulation of a single cell or small groups of cells with a given concentration of agonist and the simultaneous recording of the fluorescence changes. The syringes of the microperfusion system are loaded with different concentrations of the selected RyR1 agonist (either caffeine or 4-chloro-m-cresol) which will be used to generate dose response curves. In the example shown in Figure 3, cells were stimulated with 0.5-10 mM caffeine dissolved in Krebs Ringer's solution containing no added calcium plus 100 µM La3+ in order to monitor Ca2+ release from intracellular stores. Glass coverslips on which Fura-2 loaded EBV-immortalized B lymphocytes were allowed to attach are placed in the perfusion chamber and perfused with Krebs Ringer's solution containing 1 mM Ca2+. Most of the cells will have adhered to the poly-L-lysine treated coverslip and small groups of cells should be identified and checked for Fura-2 loading. The tip of the perfusion system is placed close to the cells to bathe them (and not all the cells on the coverslip) with caffeine. Normally cells are stimulated starting from the lowest to the highest concentration of caffeine; for each concentration, a new cell or group of cells are selected. Ratiometric fluorescence measurements (excitation at 340 nm and 380 nm, emission at 510 nm) are recorded every second for up to 2 min. A few images are obtained before perfusion in order to obtain a steady baseline, subsequently cell perfusion is initiated, first by flushing cells with a solution of Krebs Ringer's solution containing 100 µM La3+ for 5 seconds. This should not result in a change in florescence and is a control to assure that the cell(s) stick to the coverslip throughout the experiment; subsequently a solution containing the selected agonist concentration is flushed over the cell(s). In the example shown in Figure 3A, the cells were stimulated with 5 mM caffeine for 20 seconds. The arrow shown indicates when the caffeine valve was opened and this results in an immediate increase in the 340/380 nm fluorescent ratio. After 20 seconds, the caffeine valve closes, and cells are flushed with Krebs Ringer's solution containing 100 µM La3+; fluorescence is recorded until the baseline is reached. For each caffeine concentration the ΔF, that is the caffeine induced peak 340/380 nm fluorescence - the initial resting 340/380 nm fluorescence is calculated and used to construct a dose response curve as shown in Figure 3B. Mean ΔF from 5-10 cells is averaged for each caffeine concentration. The amount of calcium in intracellular stores can also be monitored. In this case, cells are rinsed with Krebs Ringer's solution containing 0.5 mM EGTA and a solution of 1 µM thapsigargin, 1 µM ionomycin and 0.5 mM EGTA is added by hand to the cells (not through the microperfusion system as ionomycin sticks to the tubing and cannot be washed off) and the fluorescence is recorded for approximately 4-5 min. To calculate the ΔF, a region of interest (ROI) outlining a cell is obtained and the changes in fluorescence within the ROI are calculated using an imaging software.
In summary, when using EBV immortalized B-cells to measure the sensitivity of the RyR1 to a specific agonist, repeated experiments should be performed on cells from one individual, on different days. For measurements on cell populations, the status of the intracellular calcium stores needs to be assessed and the EC50 to agonist induced calcium release is plotted relative to the total amount of calcium that can be released from the stores. One advantage of using this approach is that the [Ca2+] response of millions of cells is averaged; additionally, no microperfusion system and fluorescent microscopes are necessary. The single cell method allows the use of a smaller number of cells and the on-line visualization of changes in [Ca2+] of selected cells.
Single cell [Ca2+]i measurements in human satellite cell derived myotubes
Glass coverslip grown and differentiated myotubes are loaded with Fura-2, transferred to the perfusion chamber and bathed in Krebs Ringer's solution containing 2 mM Ca2+ as described above for EBV cells. The syringes of the perfusion system are filled with the selected agonist and myotubes are stimulated as described above. Since not all cells on the coverslip are multinucleated myotubes, it is important to select the appropriate cell(s) that will be measured. Small groups of myotubes can be stimulated simultaneously. In the example shown in Figure 4, a single myotube was flushed with a solution containing KCl, different concentrations of 4-chloro-m-cresol and finally caffeine, however, normally dose response curves to agonists are constructed, as indicated in the previous section, in order to compare the agonist sensitivity of cells from different individuals12,15,16. KCl is used as a way to depolarize the plasma membrane. In skeletal, muscle plasma membrane depolarization is sensed by the voltage sensing DHPR which thereby undergoes a conformational change leading to activation and opening of the RyR1. On the other hand, 4-chloro-m-cresol and caffeine are direct pharmacological activators of the RyR1.
Figure 1: Generation of primary human muscle biopsy-derived myoblast cultures. (A) Small fragments of muscle (arrows) are placed in inserts containing 0.5 mL growth medium. After 7-14 days satellite cells can be seen (small arrows) adjacent to the muscle tissue and growing on the bottom of the insert. Image taken through a 10x objective (B) and 20x objective (C). The grey boxes in panel A were used to cover the identity of the patient. Please click here to view a larger version of this figure.
Figure 2: Calcium release experiments in EBV immortalized B lymphocytes. Ratiometric [Ca2+]i measurements in a population of Fura-2 loaded cells in suspension. A) The addition of 300 µM 4-chloro-m-cresol (arrow) causes an immediate increase in the cytoplasmic [Ca2+], which subsequently decays back to resting levels within 500 seconds. In this example the ΔF induced by the addition of 300 µM 4-chloro-m-cresol is 1.7 fluorescence units- 1.4 fluorescence units = 0.3 fluorescence units. B) The addition of the SERCA inhibitor thapsigargin (400 nM, arrow) causes a larger increase in the Fura-2 fluorescence ratio which peaks at 2.4 fluorescence units, and subsequently decays to resting levels at 1.45 fluorescence units. The thapsigargin induced [Ca2+] transient represents the total amount of rapidly releasable calcium in the intracellular stores present in the cell population. The peak transient obtained (2.4-1.45= 0.95 units) is used to calculate the percentage of calcium released by a given concentration of 4-chloro-m-cresol. C) Representative dose response curves correlating the ΔF as a percentage of the total amount of rapidly releasable calcium in intracellular stores. For 300 µM 4-chloro-m-cresol this value is 0.3/0.95x100=31.6%. Each symbol represents the mean± SEM% of 5-10 values from EBV immortalized cells from a control (closed circles, dotted line) and an MHS individual (closed squares, continuous line) carrying a RYR1 mutation. The curves were generated using a sigmoidal dose-response curve function. Panels A and B are adapted from Girard et al.11. Please click here to view a larger version of this figure.
Figure 3: Caffeine-induced Ca2+ release in individual EBV-immortalized B lymphocytes from a control individual. A) Top panels, time lapse images (A) Phase-contrast; (B-E), single-cell [Ca2+]I measurements of fura-2-loaded EBV-immortalized lymphocytes: (B) t=0, (C) t=36 s, (D) t=50 s and (E) t=77 s after the application of 5 mM caffeine. Cells were individually stimulated by the addition of caffeine diluted in Krebs-Ringer solution. Scale bar=10 µm. Bottom panel, representative trace obtained after stimulation of a single cell with 5 mM caffeine (arrow). B) Dose response curves showing the caffeine-dependent change in [Ca2+]i, expressed as change in fluorescence ratio (peak ratio 340/380 nm−resting ratio 340/380 nm). Each point represents the mean ± SEM of the change in fluorescence of 4-15 cells. The curves were generated using a sigmoidal dose-response curve function. Closed squares, dotted line, control cells; closed triangles, continuous line, MHS individual carrying a RYR1 mutation. This figure is an adaptation from Ducreux et al.13. Please click here to view a larger version of this figure.
Figure 4: Calcium release stimulated by KCl, 4-chloro-m-cresol and caffeine in human myotubes from a control individual. Left panels: A) phase contrast (B-H), single cell intracellular Ca2+ measurements of Fura-2-loaded human myotubes. B) resting [Ca2+]i ; C) t=2 s after the application of 150 mM KCl. D) t= 2 s after the application of 150 µM 4-chloro-m-cresol; E) t=2 s after the application of 300 µM 4-chloro-m-cresol; F) t=2 s after the application of 600 µM 4-chloro-m-cresol; G) t=2 s after the application of 10 mM caffeine; H) t=20 s after the application of caffeine. Right panel: Plot of time (s) versus fluorescence ratio (340/380 nm) in the stimulated cell. Myotubes were individually stimulated by addition of the agonist in Krebs-Ringer buffer containing 100 µM La3+, thus the increase in [Ca2+]i represents only release of calcium from intracellular stores. Please click here to view a larger version of this figure.
The protocols described in this paper have been successfully utilized by several laboratories to study the impact of RYR1 mutations on calcium homeostasis. The critical steps of the approaches outlined in this paper deal with sterility, cell culturing skills and techniques and availability of biological material. In principle, the use of EBV-immortalized B lymphocytes is simpler and allows one to generate cell lines containing mutant RyR1 channels. The cells can be frozen and stored in liquid nitrogen for many years and cultures can be re-started at any time. Additionally, one can chose whether to monitor calcium homeostasis in cell populations or at the single cell level. The former method is simpler, does not require a fluorescence microscope and allows the investigator to test cell lines generated from different individuals within a short period of time. The limitation being the velocity of cell growth and the availability of a fully equipped (heated and with magnetic stirrer) spectrofluorometer. As an alternative approach flow cytometry in combination with fluorescent calcium indicators can be used to measure calcium fluxes in EBV-immortalized B lymphocytes; in such a way changes in the intracellular calcium concentration can be determined19. If a fluorescent microscope, perfusion chamber and microperfusion system are available, then single cell imaging has the advantage of being more sensitive and giving more detailed information including cell to cell variability, kinetic analysis and identification of subcellular domains involved in calcium release. The latter approach is technically more challenging and requires more equipment.
There are multiple advantages to using EBV-immortalized B lymphocytes, including that other parameters aside [Ca2+]i homeostasis can be measured. For example 4-chloro-m-cresol induced acidification of B cells has been used to differentiate cells from control individuals from MHS patients20,21. Nevertheless one must keep in mind (i) that B cells do not express many of the proteins involved in skeletal muscle excitation contraction coupling that may indirectly influence calcium release, (ii) they are non-excitable cells thus cannot be activated physiologically by plasma membrane depolarization, and finally a report by Monnier et al. found that a mutation identified in a patient was not expressed in EBV-immortalized B cells because of a cryptic splice site22.
Patient derived primary muscle cell cultures have been used by several groups interested in studying the effect of mutations in different genes encoding proteins involved in calcium homeostasis10,23,24. These cells can be differentiated into multinucleated myotubes, respond to plasma membrane depolarization and can be assessed by electrophysiological means. In addition, they can be immortalized in order to obtain cell lines carrying RYR1 mutations25, though the procedure is far more complex than for B-lymphocytes. While it is also true that the muscle cells are slow growing and the time necessary from taking a biopsy to having a sufficient number of cells can be more than one month, it is also possible to store the small pieces of muscle biopsies in freezing medium in liquid nitrogen in order to obtain myoblasts years later. The long culturing times have the added risk of contamination by bacteria, yeasts or molds and as soon as a sufficiently large number of myoblasts have been obtained, it is important to freeze and store them in liquid nitrogen. Our laboratory has successfully applied the same technique outlined above for myotubes, to study calcium changes in human skin-derived fibroblasts transduced with myoD and differentiated into myotubes26. This was done to study the functional effect of RYR1 mutations when muscle biopsy-derived myoblasts were not available. As for B-lymphocytes, 4-chloro-m-cresol induced acidification of myotubes from patients with RYR1 mutations linked to MHS has been tested successfully27.
In conclusion, the use of biological material from patients to study the genotype-phenotype correlation has many advantages the drawback and can be used successfully to study the effect of mutations in RYR1. When using this approach however, it is important to keep in mind that mutations present in other genes may influence calcium homeostasis; therefore, the use of cells from different families harboring the same mutation as well as from family members not harboring the RYR1 mutation as controls, should be performed.
The authors have nothing to disclose.
The work described in this manuscript was supported by grants from the Swiss National Science Foundation (SNF) and the Swiss Muscle Foundation.
|Blood collection tubes||Sarstedt||172202|
|Bovine serum albumin (BSA)||Sigma-Aldrich||A7906|
|Cascade 125+ CCD camera||Photometrics|
|Cascade 128+ CCD||Photometrics|
|Epidermal Growth Factor (EGF)||Sigma-Aldrich||E9644|
|Foetal calf serum||ThermoFisher Scientific||26140079|
|Fura-2/AM||Invitrogen Life Sciences||F1201|
|Glutamax||Thermo Fisher Scientific||35050061|
|Horse serum||Thermo Fisher Scientific||16050122|
|Microperfusion system||ALA-Scientific||DAD VM 12 valve manifold|
|Origin Software||OriginLab Corp||Software|
|Pennicillin/Streptomycin||Gibco Life Sciences||15140-122|
|Perfusion chamber POC-R||Pecon||000000-1116-079|
|Tissue culture dishes||Falcon||353046|
|Tissue culture flask||Falcon||353107|
|Tissue culture inserts||Falcon||353090|
|Trypsin/EDTA solution||ThermoFisher Scientific||25300054|
|Visiview||Visitron Systems GmbH||Software|
|Zeiss Axiovert S100 TV microscope||Carl Zeiss AG|
|Zeiss glass coverslips||Carl Zeiss AG||0727-016|
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