Modelling myopathies in zebrafish

Abstract

Muscle diseases, or myopathies, are a group of rare congenital diseases that severely incapacitate the patients and in some cases are fatal. Most of these diseases result from mutations in genes that code for proteins of the sarcomere, the contractile unit of the muscle. In this thesis I studied two groups of myopathies, nemaline and desmin related myopathies. Although markedly distinct myopathies, the general pathological and clinical features are similar, as both nemaline and desmin myopathy patients suffer from muscle weakness and atrophy, and present disrupted muscle fibres with protein aggregates. The aggregates are however different in protein content and shape: nemaline myopathy aggregates are usually rod shaped and electron dense when visualised by electron microscopy, and often contain actin and α-actinin, while desmin myopathy aggregates are irregularly shaped and contain desmin, HSPB5, filamin C, and dystrophin. In some severe cases of nemaline myopathy patients also present with foetal akinesia, a disorder characterised by a reduction or loss of foetal movement, which is fatal in utero or shortly after birth. Nemaline myopathies result from mutations in proteins of the actin-rich thin filaments, but can also result from mutations in a non-structural protein, BTB and kelch domain-containing protein 13 (KBTBD13). KBTBD13 is a member of the BTB and kelch domain-containing family composed of more than 70 members, shown to be involved in ubiquitination of proteins. Here I describe the identification of a member of this family, kelch-like family member 40 (KLHL40), as a foetal akinesia and nemaline myopathy-causing gene. Using a zebrafish knockdown model I confirmed that mutations in KLHL40 disrupt muscle structure and function, consistent with the patients’ pathology. Additionally, I identified three other members of the BTB and kelch domain-containing family to be expressed in the skeletal muscle, klhl31, klhl41a, and klhl40b, and characterised the role of klhl31 in muscle formation and function. Using a knockdown approach I identified klhl31 as a potential nemaline myopathy with akinesia causing gene and proposed a model for how mutations in this family of proteins cause nemaline myopathy. To study the pathobiology of desmin myopathy I generated three zebrafish models expressing desminL345P, desminI451M, and desminS460I. Expression of mutant desmin resulted in the formation of aggregates in the skeletal muscle and presence of mitochondria in the myofibrils, recapitulating the patients’ pathology. Expression of desminS460I resulted in a more severe pathology than in the other transgenic lines, with clear disruption of the muscle fibres, formation of aggregates and Z-disc thickening, and also impaired swimming behaviour, suggesting the pathological features and muscle function may be related. This is the first model for desmin myopathy showing impaired muscle function in vivo. Here I show the generation of zebrafish models for KLHL40 nemaline myopathy with foetal akinesia and three models for desmin myopathy. I also found that loss of klhl31 results in a pathology that resembles nemaline myopathy with foetal akinesia, suggesting that mutations in KLHL31 may also result in this myopathy. These animal models can be used to further understand disease mechanisms and ultimately used to test for compounds that may improve the function of the muscle.