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 heterologo....

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 i....

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