B-lymphocytes from Malignant Hyperthermia-susceptible Patients Have an Increased Sensitivity to Skeletal Muscle Ryanodine Receptor Activators

Abstract

Malignant hyperthemia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1␤ after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1␤ from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1␤ production. Malignant hyperthermia (MH)1 is a pharmacogenetic disease triggered by volatile anesthetics and the depolarizing muscle relaxant succinylcholine in predisposed individuals (1-4). The clinical signs of an impending MH reaction are highly variable and are caused by a hypermetabolic state with muscle rigidity, metabolic acidosis, rhabdomyolysis, tachycardia, and/or an increase in body temperature (5). In some individuals MH reactions appear to be triggered by physical exercise or emotional stress. The latter observation has led to the suggestion that MH, heat stroke, and exercise-induced rhabdomyolysis might have a common denominator (2, 6, 7). The underlying causes of MH are abnormalities in the skeletal muscle calcium metabolism (8, 9) and molecular genetic studies have mapped the primary locus of MH to chromosome 19q, the gene encoding the ryanodine receptor calcium release channel (RYR1) (2, 4, 10). Approximately 50% of MH families have mutations in the RYR1 gene, and mutations have been reported in other loci (for recent reviews, see Refs. 11 and 12).The ryanodine receptors are large tetrameric oligomers that function as intracellular calcium release channels. Three different isoforms have been identified at the molecular level: type 1 (RYR1), which is preferentially expressed in skeletal muscle; type 2, which is in the heart and cerebellum; and type 3, which is in the central nervous system as well as in a variety of other tissues (13-16). RYR1 can be pharmacologically activated by a number of compounds, among which are caffeine, halothane, thymol, 4-chloro-m-cresol, E218, bastadin, polylysine, and calcium (17-21). Activation causes the channel to open and thus to a transient calcium flow from the sarcoplasmic reticulum,...

Gain of function of the immune system caused by a ryanodine receptor 1 mutation

“…Recently it was shown that RyR1s are also expressed in other cell types, including neurons, smooth muscle cells and immune cells, specifically in Blymphocytes and dendritic cells (DC) (Sei et al, 1999;Girard et al, 2001;Bracci et al, 2007;Uemura et al, 2007;O'Connell et al, 2002). DCs are the most potent antigen presenting cells connecting innate and adaptive immunity.…”

“…They are located in peripheral tissues where they continuously sample the environment for the presence of foreign antigens; when these are encountered DCs process them and then migrate to secondary lymphoid organs where they present the processed antigens in conjunction with major histocompatibility complex II (MHCII) molecules to T-lymphocytes and initiate a specific immune response (Banchereau et al, 2000). Studies on the role of RyR1 in immune cells in vitro has established that in B-lymphocytes its activation is coupled to cytokine release (Girard et al, 2001) whereas in DCs it leads to enhanced maturation, release of proinflammatory cytokines and enhanced ability to prime T-cells (Bracci et al, 2007).…”

Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes

“…To examine the function of RYR1 channel variants in EBV-immortalized lymphoblastoid cells, we recorded extracellular acidification, as a measure of the energy metabolism of activated cells, with a sensitive pHmetric biosensor. RYR1 is expressed in human B lymphocytes where it functions as a CaP [Sei et al, 1999], and CaP 2+ P homeostasis is altered in B cells of MHS individuals [Sei et al, 2002;Girard et al, 2001]. We demonstrate that the increase in the acidification rate of immortalized lymphoblastoid cells in response to 4-CmC is mainly due to the activation of the RYR1 calcium channel; therefore this assay can be used to analyze the activity of RYR1 in this cellular system.…”

“…Previous studies have also demonstrated that human B-lymphocytes express RYR1 [Sei et al, 1999] and could be used as a model to test the effect of sequence variants on RYR1 function [Sei et al, 2002[Sei et al, , 1999Girard et al, 2001]. We therefore applied the proton release assay in EBV-immortalized lymphoblastoid cells from patients carrying RYR1 variants.…”

“…Various methods have been developed to characterize the function of RYR1 variants: analysis of calcium release in human primary myotubes [Wehner et al, 2004[Wehner et al, , 2002Girard et al, 2002;Brinkmeier et al, 1999] and in immortalized B-lymphocytes from patients or after expression by transfection in various cell types [Yang et al, 2003;Girard et al, 2001;Censier et al, 1998;Tong et al, 1997], and determination of the channel openings in a ryanodine binding assay [Sambuughin et al, 2001]. Moreover, metabolic tests in vivo [Bina et al, 2006;Anetseder et al, 2003Anetseder et al, , 2002Textor et al, 2003] and in vitro [Klingler et al, 2002] have been developed to distinguish between MHS and MHN muscles.…”