Characterization of the excitation - contraction coupling in extraocular muscles

Excitation-contraction coupling (ECC) is the physiological mechanism whereby an electrical signal detected by the dihydropyridine receptor, is translated into an increase in [Ca2+], by activating ryanodine receptors. Mutations in RYR1, the gene encoding the ryanodine receptor 1, are the underlying cause of several congenital myopathies including Central core disease, Multiminicore disease, some forms of Centronuclear myopathy and Congenital fiber type disproportion. Patients with recessive but not dominant RYR1 mutations show a significant reduction of ryanodine receptor protein in muscle biopsies as well as ophthalmoplegia or involvement of the extraocular muscles (EOM). This specific involvement indicates that this group of muscles may express different proteins involved in excitation-contraction coupling compared to limb muscles. The focus of this thesis is the characterization of the excitation-contraction coupling toolkit of human EOM. The main goal was to identify differences or similarities with other skeletal muscles in the context of the previously mentioned diseases which affect skeletal muscles. My results indicate that the transcripts of the main genes involved in skeletal excitation-contraction coupling are downregulated, while at the same time, we report increased expression of the ryanodine receptor 3, cardiac calsequestrin and alfa 1 subunit of the cardiac dihydropiridine receptor. In addition, the finding of increase in excitation-coupled calcium entry in the EOM compared to leg muscles (LM) completes the picture of the EOM muscles as a specific muscle group with a unique mode of calcium handling and their selective involvement in neuromuscular disorders. Facial weakness and ptosis have also been described in patients with mutations in RYR1. Having this in mind, we were interested in investigating the relation between the facial muscle orbicularis oculi (OO), EOM and LM and the excitation-contraction coupling toolkit in human biopsies and myotubes derived from individuals which do not have any known neuromuscular disorder. According to our results, OO show more similarities to leg muscles than to EOM. In addition, we found high expression levels of dystrophin and utrophin and this is significant from the perspective of Duchenne muscular dystrophy (DMD). In fact in this condition EOM are spared from pathology and the same is true in mdx (dystrophin deficient) mouse models. In mdx mice it is believed that utrophin compensates for the lack of dystrophin. Our findings that UTRN is expressed at higher level in OO compared to LM in normal conditions strongly support this theory of a compensatory effect by utrophin when dystrophin is missing. Further investigations in my thesis focus on two isoforms of the ryanodine receptor, namely RyR1 and RyR3. Ryanodine receptor 1 plays a crucial role in the process of excitation-contraction coupling in skeletal muscle. According to our study on normal human EOM, the expression of this receptor is decreased compared to its expression levels in human leg muscles, however the expression level of RyR3 is significantly increased. Because of these latter results, we reasoned that the reported behavioral impairment reported in RYR3 KO mice, may actually be due to alterations of EOM function. Our preliminary data show that in fact RYR3 KO mice exhibit visual impairment as measured using their optokinetic reflex. We are currently investigating the role of RyR3 in EOM calcium homeostasis. Taken all together, this thesis shows that different involvement of EOM and OO in neuromuscular disorders is due to their different excitation contraction coupling toolkit component. Furthermore, EOMs exhibit characteristics that deserve further attention as further investigations may lead to the discovery of protective mechanisms in neuromuscular disorders with potential therapeutic benefit.

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