Calcium homeostasis and role of ryanodine receptor type 1 (RyR1) in immune cells

Ryanodine receptors are intracellular Ca2+ release channels located in the membrane of the Endoplasmatic/Sarcoplasmatic Reticulum. Ryanodine receptor 1 isoform is preferentially expressed in skeletal muscle where it is responsible for release of Ca2+ from the SR, an event that leads to muscle contraction. Point mutations in the gene encoding ryanodine receptor 1 have been linked to disease such as Malignant Hyperthermia, Central core disease and Multi-minicore disease.
Malignant Hyperthermia is a pharmacogenetic disorder with autosomal dominant inheritance and abnormal Ca2+ homeostasis in skeletal muscle in response to triggering agents. In susceptible individuals, a malignant hyperthermia crisis may be triggered by commonly used halogenated anaesthetics (halothane, isoflurane) or muscle relaxants (succhinylcholine). The main symptoms are hypermetabolism and muscle rigidity. Without treatment, death would occur in more than 80% of cases. Although a genetic-chip based diagnostic approach is under development, the invasive in vitro contracture test remains the “gold standard” to diagnose this disorder.
Central core disease is a slowly progressive myopathy characterized by muscle weakness and hypotonia. Central core disease is characterized histologically by the presence of central cores running along longitudinal axis of the muscle fiber.
Multi-minicore disease disease is a more severe, rare, autosomal recessive myopathy characterized histologically by the presence of multi-minicores in only a small number of sarcomeres. So far, no effective therapy has been developed to treat muscle weakness in central core disease and multi-minicore disease patients and their diagnosis is difficult on the basis of clinical findings alone. Histological examination of muscle tissue in these diseases is essential.
Recent data has shown that ryanodine receptor 1 is also expressed in some areas of the central nervous system as well as in cells of the immune system, specifically B-lymphocytes and dendritic cells.
The first part of my thesis focuses on the role of the ryanodine receptor 1 in dendritic cell. We first show that both immature and mature in vitro derived dendritic cells as well as circulating plasmocytoid cells express the ryanodine receptor 1 Ca2+ release channel within the endoplasmatic reticulum. Pharmacological activation of the ryanodine receptor 1 leads to the rapid release of Ca2+ from intracellular stores, and in the presence of sub-optimal concentrations of microbial stimuli, provides synergistic signals resulting in dendritic cell maturation and stimulation of T cell function. Furthermore, we were interested in unravelling more direct roles of this receptor in dendritic cells function. Interestingly, ryanodine receptor 1 activation in dendritic cells causes a very rapid increase in surface expression of major histocompatibility complex II molecules. In order to dissect the physiological route of ryanodine receptor 1 activation in vivo we hypothesized that a possible functional partner of ryanodine receptor 1 in dendritic cells could be, an L-type Ca2+ channel. We were able to show that human dendritic cells express the cardiac isoform of the L-type Ca2+ channel, which acts as a ryanodine receptor 1 functional partner on the plasma membrane of dendritic cells. We show that depolarization of dendritic cells by the addition of potassium chloride activates L-type Ca2+ channels initiating Ca2+ influx and activation of Ca2+ release via ryanodine receptor 1 and that this process could be prevented by nifedipine or ryanodine. Physiologically potassium could be released from dying cells within an inflamed tissue or from T- cells into immunological synapse during dendritic cell T-cell engagement and these events could be possible routes for activation of L-type Ca2+ channel- ryanodine receptor 1 signalling in dendritic cells in vivo. Thus, in vivo, activation of the ryanodine receptor 1 signalling cascade may be important during the early stages of infection, providing the immune system with rapid mechanisms to initiate an early response, facilitating the presentation of antigens to T cells.
While continuing our investigation on Ca2+ homeostasis in dendritic cells we noticed that spontaneous Ca2+ oscillations occur in immature dendritic cells but not in dendritic cells stimulated to undergo maturation with lipopolysaccharide or other toll like-receptor agonists. We investigated the mechanism and role of spontaneous Ca2+ oscillations in immature dendritic cells and found that they are mediated by the inositol-1,4,5-trisphosphate receptor since they were blocked by pre-treatment of cells with the inositol-1,4,5-trisphosphate receptor antagonist Xestospongin C and 2-Aminoethoxydiphenyl borate. A component of the Ca2+ signal is also due to influx from the extracellular environment. As to the biological function of these high frequency oscillations, our results indicate that they are associated with the translocation of a Ca2+ dependent transcription factor (nuclear factor of activated T-cells) into the nucleus of immature dendritic cells. In fact, once the Ca2+ oscillations are blocked with the 2-aminoethoxydiphenyl borate or by treating cells with lipopolysaccharide, nuclear factor of activated T-cells remains cytoplasmic.
The results from the first part of my thesis provide novel insights into the physiology of dendritic cells, role of ryanodine receptor 1 signaling and Ca2+ as an important second messenger in these cells.
The second aim of my thesis deals with functional properties of ryanodine receptor 1 carrying mutations linked to neuromuscular disorders, which is important from diagnostic point of view but also to understand the basic pathophysiological mechanism leading to these different diseases.
Since functional ryanodine receptors 1 are expressed in B-lymphocytes we investigated Ca2+ homeostasis in B-lymphocytes transformed with Epstein Barr virus from patients carrying the mutation linked to malignant hyperthermia and healthy donors.
In the first study from the Swiss population we investigated four novel mutations found in malignant hyperthermia susceptible pedigrees: (p.D544Y, p.R2336H, p.E2404K and p.D2730G). We found that the resting Ca2+ levels were significantly higher in cells from all four mutations bearing individuals compared to controls. These four mutations were also found to significantly affect either 4-chloro-m-cresol or caffeine dose response curves suggesting higher sensitivity of ryanodine receptor 1 to pharmacological activation in patients carrying these mutations.
In the second study we examined patients from the Swedish population carrying five different novel mutations (p.E1058K, p.R1679H, p.H382N, p.K1393R and p.R2508G). The first 4 patients had serious malignant hyperthermia clinical reactions and thereafter have tested by the in vitro contracture test and classified as malignant hyperthermia susceptible; the patient with the fifth mutation, p.Arg2508Gly, had been diagnosed as a central core disease. In this study as well functional studies were performed on Epstein Barr virus transformed B-lymphocytes from patients carrying mutations and healthy donors. Our results from the Swedish population suggest that ryanodine receptor 1 mutations also lead to abnormal Ca2+ homeostasis. Results from these and other studies support the use of Epstein Barr virus transformed -B-lymphocytes as an alternative, non-invasive, protocol for the diagnosis and the functional proof that a mutation in the ryanodine receptor causes alterations in Ca2+ homeostasis. This is a pre-requisite for the molecular diagnosis of malignant hyperthermia. These results also provide new concepts for the treatment of muscular pathologies involving mutations in ryanodine receptor 1.