The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

Myotiliini on lihaksen niin kutsutuissa Z-linjassa sijaitseva proteiini, joka toimii omalta osaltaan lihaksen sarkomeerien rakenteen säilyttäjänä ja organisoijana. Myotiliinimutaatiot aiheuttavat erilaisia lihassairauksia, esimerkiksi Limb-Girdle Muscular Dystrophy ja Myofibrillar Myopathy. Myotiliini sitoutuu itsensä ja erilaisia sarkomeerin proteiinia: alfa-aktiniini, aktiini, filamiini C, FATZ ja ZASP. Väitöskirjassa on kolme töitä. Ensimmäisessä selvitettiin hiiren myotiliini geeni ja promoottori. Toisessa osatyössä kartoitettiin myotiliinin ilmentyminen hiiren ja osaksi myös ihmisen sikiönkehityksen eri vaiheissa. Kolmannessa osatyössä tuotettiin myotiliinin suhteen poistogeeninen hiirimalli ja selvitettiin poiston seuraukset. Työ osoitti ettei myotiliinin suhteen poistogeenisellä hiirellä ole normaali-olosuhteissa poikkeavaa fenotyyppiä, myotiliini ei ole välttämätön/on korvattavissa lihasten sikiönkehityksessä ja toiminnassa.