Functional characterization of extraocular muscle in transgenic mouse models

Abstract

Extraocular muscles (EOMs) are among the fastest and most fatigue resistant skeletal muscles; they are categorized as a separate group of muscles or ‘allotype’ since they represent a unique group of highly specialized muscles anatomically and physiologically different from other skeletal muscles. The distinct origin and innervation of EOMs are probably responsible for their different gene and protein expression. This also applies for the excitation–contraction coupling (ECC) and the calcium handling in EOMs. The main goal of this thesis is to establish methods to study the ECC in mouse EOMs and apply these new techniques on different mouse models. Calcium is an ubiquitous second messenger mediating numerous physiological processes, including muscle contraction and neuronal excitability. Ca2+ is stored in the ER/SR and is released into the cytoplasm via the opening of intracellular inositol trisphosphate receptor and ryanodine receptor calcium channels. Whereas in skeletal muscle, isoform 1 of the RYR is the main channel mediating calcium release from the SR leading to muscle contraction, the function of ubiquitously expressed ryanodine receptor 3 (RyR3) is far from clear. The previous finding of our group, that RyR3 is highly expressed in EOMs versus limb muscles (Sekulic-Jablanovic et al., 2015) is the basis of the first paper entitled “Extraocular muscle function is impaired in ryr3−/− mice”. By using the RyR3 KO mouse we were able to show that RyR3 is an essential factor for normal vision, which is the first real functional role of this channel. In detail, the loss of RyR3 reduced the peak force and altered the twitch kinetic of isolated EOMs. Additionally, we found altered calcium transient kinetic in isolated single EOM fibers. Mutations in the RYR1 gene are associated with neuromuscular disorders and patients with recessive mutations are severely affected and characteristically display ptosis and/or ophthalmoplegia. Previously we constructed a compound heterozygous RyR1p.Q1970fsX16+p.A4329D (DKI) mutant mice based on a patient biopsy (Klein et al., 2012) and characterized the skeletal muscles (Elbaz et al., 2019b; a). In order to gain insight into the mechanism leading to extraocular muscle involvement, we investigated the biochemical, structural and physiological properties of EOMs from this mouse model. We also investigated the properties of EOMS of heterozygous single mutation carriers as well as mice carrying the homozygous mutation. These studies constitute the second paper entitled “Molecular basis of impaired extraocular muscle function in a mouse model of congenital myopathy due to compound heterozygous RYR1 mutations”. In this paper we were able to show a significant reduction in the ex vivo force of the DKI mouse, while the other lines do not show any changes. These findings were constant with a reduction in the peak calcium transients and in several ECC involved proteins. Interestingly, we also found a tremendous reduction in the specific MyHC-EO isoform. To sum it up, our results shows that the combination of a reduced content of ryanodine and dihydropyridine receptors, changes in the calcium release units and reduced MyHC-EO leads to impaired vision. The methods and findings in this thesis will be the start of further investigation into EOM specific disorders or neuromuscular disorders which where EOMs are spared or heavy affected.