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.