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Medicine

Functional Characterization of Endogenously Expressed Human RYR1 Variants

Published: June 9th, 2021

DOI:

10.3791/62196

1Department of Biomedicine, Basel University Hospital, 2Department of Life Sciences, University of Ferrara, 3Department of Anesthesia, Basel University Hospital

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.

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 heterologo....

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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 mo.......

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[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.......

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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 .......

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The work described in this manuscript was supported by grants from the Swiss National Science Foundation (SNF) and the Swiss Muscle Foundation.

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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

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