Matteo 2015 Abstract MiPschool London 2015

From Bioblast
Mitochondrial and nuclear IP3-mediated Ca2+ signaling in core myopathies.


Matteo S, Jenny Sharpe J, Muntoni F, Duchen MR, Szabadkai G (2015)

Event: MiPschool London 2015

Disruption of cellular Ca2+ homeostasis underlies most muscle pathologies. It is particularly apparent in core myopathies such as Central Core Disease (CCD) and Multi Minicore Disease (MmD), caused by mutations in the ryanodine receptor one (RyR1) Ca2+ release channel [1]. Several lines of evidence indicate that depolarizing stimuli induce two independent cellular Ca2+ signals in muscle fibers: a fast signal associated with excitation-contraction coupling (ECC) and a slow signal that is preferentially formed in the nucleus and regulates gene expression. While the role of RyR1 mutations in the disruption of ECC has been extensively studied, the role of the nuclear Ca2+ dependent pathways remains completely unexplored in these diseases [2]. Recently, a signalling complex in which IP3 produced by plasma membrane receptors directly binds to nuclear IP3R, bypassing sarcoplasmic reticulum (SR) calcium signaling machinery and directly regulating transcriptional events, has been described in rat cardiomyocytes [3].

To examine whether in skeletal muscle IP3R mediated local nuclear Ca2+ signalling controls gene expression leading to maladaptive pathological phenotype, we characterized the nuclear and cytosolic RyR1 and IP3R mediated Ca2+ signal generating system in a cellular model based on C2C12 mouse myotubes, using single live cell imaging. Our data suggest that loss of function of RyR1 induced by siRNA transfection (condition that mimics certain CCD phenotypes) increases cytosolic and nuclear IP3-mediated calcium responses; moreover western blot analysis showed that RyR1 silencing induces up-regulation of IP3R.

In addition, we collected Ca2+ imaging data from CCD affected primary myotubes isolated from clinical biopsies. Preliminary data suggest that in these patients (mutation G2060C in RyR1 domain 2 and mutation N4575T in RyR1 domain 3) nuclear IP3 mediated Ca2+ signals are greater than cytosolic one. Since in cell lines the ECC machinery is not exclusively mediated by physical interaction of RyR and DHPR even after depolarizing stimulation, we decided to generate a set of data directly from FDB mouse fibers injected by electroporation with short-hairpin RNA to silence RyR1. Efficiency of RyR1 silencing in fibers has been successfully tested by measuring caffeine mediated Ca2+ signals using fura-2 and imaging experiments are currently ongoing. Finally, to identify the genes altered by RyR1 loss of function we have performed microarray analysis from EDL (fast twitch) and soleus (slow twitch) muscles of RyRI4859T pathologically mutant expressing animals, an established knock-in mouse model of CDD. Here we show that, in both cases, a large set of mitochondrial genes are the most significantly deregulated gene group. In particular, the most affected genes are those of co-regulators involved in regulating mitochondrial biogenesis (PGC-1 family of co-regulators or transcription factors Nrf-1, Nrf-2, TFAM and the nuclear receptor ERR family and PPARs): this is an essential outcome since a common feature of central and multi-mini cores is the loss of mitochondria from the central axes of the myofibers [1].

β€’ Keywords: Central core disease, Multi minicore disease, Excitation-contraction coupling

β€’ O2k-Network Lab: UK London Duchen MR


Organism: Mouse  Tissue;cell: Skeletal muscle, Other cell lines  Preparation: Intact cells 

Regulation: Calcium 

Abstract continued

Our principal future aim is to measure mitochondrial Ca2+ signaling and quantify gene expression patterns in fully differentiated myofibers from human CCD affected tissues to determine (i) whether deregulation of mitochondrial gene expression or mitochondrial Ca2+ overload can lead to mitochondrial dysfunction and degradation in core myopathies and (ii) whether re-establishment of normal mitochondrial Ca2+ homeostasis and metabolism through genetic or pharmacological treatment could improve the disease phenotype.


1-Dept Biomedical Sc, Univ Padua, Italy. - [email protected]

2-Dept Cell Developm Biol, Univ College London, UK

3-Dubowitz Neuromuscular Center MRC Center Neuromusc Dis, Univ College London, Inst Child Health, UK


  1. Jungbluth H, Sewry CA, Muntoni F (2011) Core myopathies. Semin Pediatr Neurol 18:239-49.
  2. Casas M, Figueroa R, Jorquera G, Escobar M, MolgΓ³ J, Jaimovich E (2010) IP3-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers. J Gen Physiol 136:455–67.
  3. Ibarra C, Vicencio JM, Estrada M, Lin Y, Rocco P, Rebellato P, Munoz JP, Garcia-Prieto J, Quest A, Chiong M, Davidson SM, Bulatovic I, Grinnemo KH, Larsson O, Szabadkai G, UhlΓ©n P, Jaimovich E, Lavandero S (2013) Local control of nuclear calcium signaling in cardiac myocytes by perinuclear microdomains of sarcolemmal insulin-like growth factor 1 receptors novelty and significance. Circ Res 112:236-45.
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