Functional Characterization of Endogenously Expressed Human RYR1 Variants

Summary

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Abstract

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.

Introduction

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 cDNA^4,5, 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 RyR1s^7,8,9, characterization of cells from patients expressing the RYR1 variant endogenously^10,11,12. 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-cresol^10,11,12,13; (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⁺ stores^12,13,14,15; (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.

Protocol

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

1. 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.
2. Isolate mononuclear cells from whole blood by density gradient centrifugation media (e.g., Ficoll-Hypaque, .077 g/L).
   1. Place 30 mL of sterile blood in a 50 mL conical sterile tube.
   2. 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.
   3. 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.
3. 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.
4. 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.
5. 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.
6. Infect mononuclear cells with Epstein-Barr virus.
   1. Use supernatants from B95.8 cell line cultures (containing 10²-10³ transforming units/mL stocked at -80 °C) as a source of EBV.
   2. 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.
7. 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.
8. 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 .

1. For experiments in cell populations using a spectrofluorometer^11,13,14
   1. 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.
   2. 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.
   3. Measure fluorescence changes (ratio 340/380 nm) using a spectrofluorometer equipped with a magnetic stirrer set at maximal velocity and set to 37 °C.
   4. 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⁺.
   5. Place cells in a 3 mL glass spectrofluorometer cuvette and record the fluorescence ratio (340 nm/380 nm excitation, 510 nm emission).
   6. 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.
   7. 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.
   8. Add 400 nM thapsigargin to cells to calculate the total amount of Ca²⁺ present in intracellular stores. Record the peak Ca²⁺.
   9. 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
2. For experiments on single cells¹³
   1. 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.
   2. 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.
   3. 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.
   4. 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⁺.
   5. 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.
   6. 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.
   7. 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.
   8. Construct a dose response curve of 4-cmc versus change in [Ca²⁺], as described above.

3. Preparation of human myotubes from muscle biopsies^10,12,15

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.

1. Rinse muscle biopsy with sterile PBS to remove excess blood and cut into small fragments of about 0.5-1 mm.
2. 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.
3. 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).
4. Release a sufficient number of cells from the biopsy (after approximately 10-14 days), trypsinize as follows:
   1. 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.
   2. 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).
   3. Change the growth medium the next day to remove the EDTA and subsequently change the medium once per week.
5. 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.
6. 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.
7. 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

1. 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).
2. Transfer glass coverslip to the perfusion chamber and rinse cells with Krebs Ringer's solution containing 2 mM CaCl₂.
3. 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).

Results

[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 th...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
4-chloro-m-cresol	Fluka	24940
Blood collection tubes	Sarstedt	172202
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
caffeine	Merk	102584
Cascade 125+ CCD camera	Photometrics
Cascade 128+ CCD	Photometrics
Creatine	Sigma-Aldrich	C-3630
DMEM	ThermoFisher Scientific	11965092
DMSO	Sigma	41639
EGTA	Fluka	3778
Epidermal Growth Factor (EGF)	Sigma-Aldrich	E9644
Ficoll Paque	Cytiva	17144002
Foetal calf serum	ThermoFisher Scientific	26140079
Fura-2/AM	Invitrogen Life Sciences	F1201
Glutamax	Thermo Fisher Scientific	35050061
HEPES	ThermoFisher Scientific	15630049
Horse serum	Thermo Fisher Scientific	16050122
Insulin	ThermoFisher Scientific	A11382II
Ionomycin	Sigma	I0634
KCl	Sigma-Aldrich	P9333
Laminin	ThermoFisher Scientific	23017015
Lanthanum	Fluka	61490
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
poly-L-lysine	Sigma-Aldrich	P8920
RPMI	ThermoFisher Scientific	21875091
Spectrofluorimeter	Perkin Elmer	LS50
Thapsigargin	Calbiochem	586005
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