Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca2+ from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP3) receptors (InsP3Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP3R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion–induced calcium entry, whereas its NH2 terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells

Functional effects of mutations in the skeletal muscle ryanodine receptor type 1 (RYR1) linked to malignant hyperthermia and central core disease

Malignant hyperthermia (MH) is a pharmacogenetic disorder with autosomal dominant inheritance. In susceptible individuals, a MH 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 will occur in more than 80% of cases. Although a genetic-chip based diagnostic approach is under development, the invasive in vitro contracture test (IVCT) remains the “gold standard” to diagnose the disorder. Central core disease (CCD) is a slowly progressive myopathy characterised by muscle weakness and hypotonia; affected individuals show delayed motor development and remain physically compromised. Multi-minicore (MmD) disease is a more severe, rare, autosomal recessive myopathy characterised 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 CCD and MmD patients and their diagnosis is difficulton the basis of clinical findings alone and a histological examinationof muscle tissue is essential. MmD, CCD and MH are thought to result from a defect in the components involved in excitation-contraction mechanisms and all three diseases are linked to point mutations in the gene encoding the sarcoplasmic reticulum ryanodine receptor calcium release channel (RYR1). The aim of the thesis is to increase our knowledge of the underlying mechanisms which lead to the three different pathologies from mutations in the same gene, namely the ryanodine receptor type 1. Cultured skeletal muscle cells as well as immortalized B-lymphocyte cell lines were used to assay the underlying functional effects of RYR1 mutations, both cell types having the advantage of naturally expressing the ryanodine receptor type 1. The first element of my thesis reports our investigations of the functional characteristics of the ryanodine receptor in cells carrying the following RYR1 mutations: (i) V2168M mutation linked with Malignant hyperthermia; (ii) 2 substitutions, I4898T and R4893W and 1 deletion R4214-F4216 associated with central core disease and (iii) 3 substitutions P3527S, V4849I and R999H associated with CCD/MmD mixed phenotypes. The second aim of my thesis deals with the downstream effects of Ca2+ dysregulation, in particular, the possible role of the ryanodine receptor in the immune system. For this purpose, we have established whether RYR1 mutations influence the release of two cytokines: interleukin-1β and interleukin-6 and if so whether the latter effect may influence the clinical symptoms of MH, CCD or MmD. In the long run, this work may help to develop a non-invasive approach for the diagnosis of MH susceptibility as well as new concepts for the treatment of these muscular pathologies

Inverse regulation of the cytosolic Ca²⁺ buffer parvalbumin and mitochondrial volume in muscle cells via SIRT1/PGC-1α axis.

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca²⁺-signaling components. Changes in Ca²⁺ signaling and associated Ca²⁺-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca²⁺-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV⁻/⁻) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV⁻/⁻ mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca²⁺](c) resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis

The Contractile Phenotype of Skeletal Muscle in TRPV1 Knockout Mice Is Gender-Specific and Exercise-Dependent

International audienceThe transient receptor potential vanilloid 1 (TRPV1) belongs to the transient receptor potential superfamily of sensory receptors. TRPV1 is a non-selective cation channel permeable to Ca 2+ that is capable of detecting noxious heat temperature and acidosis. In skeletal muscles, TRPV1 operates as a reticular Ca 2+-leak channel and several TRPV1 mutations have been associated with two muscle disorders: malignant hyperthermia (MH) and exertional heat stroke (EHS). Although TRPV1 −/− mice have been available since the 2000s, TRPV1's role in muscle physiology has not been thoroughly studied. Therefore, the focus of this work was to characterize the contractile phenotype of skeletal muscles of TRPV1-deficient mice at rest and after four weeks of exercise. As MS and EHS have a higher incidence in men than in women, we also investigated sex-related phenotype differences. Our results indicated that, without exercise, TRPV1 −/− mice improved in vivo muscle strength with an impairment of skeletal muscle in vitro twitch features, i.e., delayed contraction and relaxation. Additionally, exercise appeared detrimental to TRPV1 −/− slow-twitch muscles, especially in female animals

Inverse Regulation of the Cytosolic Ca2+ Buffer Parvalbumin and Mitochondrial Volume in Muscle Cells via SIRT1/PGC-1α Axis

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca(2+)-signaling components. Changes in Ca(2+) signaling and associated Ca(2+)-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca(2+)-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca(2+)](c) resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis

Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease

In this study we report for the first time the functional properties of human myotubes isolated from patients harboring the native RYR1 I4898T and R4893W mutations linked to central core disease. We examined two aspects of myotube physiology, namely excitation-contraction and excitation-secretion coupling. Our results show that upon activation of the ryanodine receptor (RYR), myotubes release interleukin-6 (IL-6); this was dependent on de novo protein synthesis and could be blocked by dantrolene and cyclosporine. Myotubes from the two patients affected by central core disease showed a 4-fold increase in the release of the inflammatory cytokine IL-6, compared with cells derived from control or malignant hyperthermia susceptible individuals. All tested myotubes released calcium from intracellular stores upon stimulation via surface membrane depolarization or direct RYR activation by 4-chloro-m-cresol. The functional impact on calcium release of RYR1 mutations linked to central core disease or malignant hyperthermia is different: human myotubes carrying the malignant hyperthermia-linked RYR1 mutation V2168M had a shift in their sensitivity to the RYR agonist 4-chloro-m-cresol to lower concentrations, whereas human myotubes harboring C-terminal mutations linked to central core disease exhibited reduced [Ca2+]i increase in response to 4-chloro-m-cresol, caffeine, and KCl. Taken together, these results suggest that abnormal release of calcium via mutated RYR enhances the production of the inflammatory cytokine IL-6, which may in turn affect signaling pathways responsible for the trophic status of muscle fibers

Volumetric analysis of mitochondrial content in WT and PV-transfected C2C12 cells.

<p>A–C: Confocal images of WT C2C12 cell loaded with Mito Tracker Red (A), Calcein-AM (B), DAPI (C): x–y image Z-stack projection (large image), x–z (bottom) and y-z (right) image. Scale bar is 40 µm. These images were transferred to Imaris 3D to calculate relative mitochondrial volume. D–F: Bar histographs of relative mitochondrial volume (D), cytoplasmic volume (E) and relative nuclear volume (F) in C2C12 WT (light bars) and PV-clones (dark bars) either in control medium (left bars) or incubated for 48 h with Br-A23187 (1 µM; right bars). D) The relative mitochondrial volume was increased in WT C2C12 after ionophore treatment (*; p<0.05). The mitochondrial volume in PV-clones, both in control medium or after 48 h ionophore treatment was lower than in untreated WT C2C12 cells; * p<0.05 vs. WT; n>30 cells). The decrease in mitochondrial volume in PV-clones after ionophore treatment was not significant (n.s.) E) No differences in cytoplasmic volumes were detected. F) The nuclear volume in ionophore-treated WT C2C12 was slightly larger than in untreated WT cells (*; p<0.05).</p

Effect of mild ionophore treatment on PV expression levels in PV-transfected C2C12 cells.

<p>A) Detection of mRNA for PV and GAPDH (for normalization) in control (WT) and PV-transfected myotubes by RT-PCR. No signal was detected in WT myotubes before and after ionophore treatment. B) Ionophore treatment did not significantly affect PV mRNA levels (striped bars) of PV-clones at 24–72 h after treatment in comparison to untreated cells (black bars). C) Protein expression levels of PV and GAPDH in WT and PV-clones subjected to Ca²⁺ ionophore treatment (1 µM Br-A23187 for 48 h (+) or untreated cells (−) determined by Western blot analysis. D) Semi-quantitative analysis of PV Western blot signals in C2C12 PV-clones exposed to Br-A23187 for 24–72 h (striped bars) in comparison to untreated cells (black bars). A significant increase in PV expression levels was observed at 48 and 72 h (*; p<0.05; n = 8 values at each time point). E–F: Relative GFP protein expression in GFP-positive C2C12 clones in total protein extracts. Cells were treated for 48 h with the differentiation medium alone (black bar) or with 1 µM Br-A23187 (striped bar). A representative Western blot from clone PV1 is shown in E. For the normalization, GAPDH and/or the Ponceau Red-staining of the nitrocellulose membrane were used. Values were from 2 or 3 clones (PV1–3) and ≥2 independent experiments/clone. G: Stably-transfected C2C12 cells differentiated to myotubes for 6 days were fixed and immunostained for PV expression. PV in multinucleated myotubes is homogenously expressed throughout the cytoplasm.</p

Quantitative analysis of the kinetics of intramitochondrial membrane potential (ΔΨ_m) and mitochondrial [Ca²⁺]_m changes in C2C12 WT and PV-clones after KCl-induced depolarization.

<p>Results are from at least 3 independent myotube preparations and number of cells analyzed ranged from 6–16 per genotype (WT and PV-clones) and conditions (100^# and 300^# mM KCl; *p<0.05, **p<0.01).</p>#<p>The concentrations represent values of the added solutions to the perfusion chamber. The actual concentration sensed by the cells is assumed to be at least 2-fold lower.</p