Investigation of muscular dystrophy and the possible therapeutic effect of FHL1

Skeletal muscle mass is maintained by a balance between two opposing processes, namely muscle growth (hypertrophy) and muscle degeneration, that act to either increase or decrease muscle mass respectively. Any shift in this balance can have dramatic effects on muscle mass. Once such example is the debilitating muscle wasting which occurs secondary to prolonged myofibre degeneration in the inherited neuromuscular disorder Duchenne muscular dystrophy (DMD). DMD is caused by a mutation in the dystrophin gene, located on the X chromosome, which leads to complete absence of dystrophin protein expression at the sarcolemmal membrane of myofibres, resulting in progressive muscle degeneration and wasting. To date no cure or long-term effective treatment is available for DMD. The four-and-a-half LIM (FHL) domain protein FHL1 regulates skeletal muscle mass and promotes hypertrophy via regulation of NFATc1 transcriptional activity. FHL1 could therefore serve as a novel therapeutic target for muscle wasting in human myopathies such as DMD. This thesis describes the phenotypic characterisation of FHL1 transgenic mice overexpressing HA-tagged FHL1 specifically in skeletal muscle. FHL1 promotes skeletal muscle hypertrophy and increased muscle mass, which is sustained into adult life and is associated with increased numbers of Pax-7 positive satellite cells. To determine whether FHL1 expression has therapeutic benefit for DMD, FHL1 transgenic mice were crossed with a mdx mouse model of human DMD, to generate FHL1-Tg/mdx mice. Transgenic expression of FHL1 normalises the muscle histological features of young and adult mdx mice, as FHL1-Tg/mdx mice exhibit improved muscle membrane integrity, reduced skeletal muscle degeneration and muscle inflammation relative to mdx mice. Several studies have reported that activation of the calcineurin/NFATc1 pathway leads to increased sarcolemmal localisation of the dystrophin homolog, utrophin A, and can improve the mdx clinicopathological phenotype. To determine whether FHL1 improves mdx skeletal muscle membrane integrity and reduces susceptibility to contraction-induced muscle damage via upregulating utrophin A, the level and localisation of utrophin A in FHL1-Tg/mdx skeletal muscle was systematically analysed. Transgenic expression of FHL1 in mdx muscle promotes the upregulation of utrophin A mRNA and protein levels in skeletal muscle, associated with enhanced sarcolemmal localisation of utrophin A and reassembly of dystrophin-associated-proteins, β-dystroglycan and syntrophin, in mature and regenerating myofibres. This indicates that localisation of utrophin A to the sarcolemma in FHL1-Tg/mdx muscle is sustained in adult myofibres and utrophin A is able to functionally compensate for the lack of dystrophin. To determine the molecular mechanism by which FHL1 enhances the sarcolemmal localisation of utrophin A in mdx muscle, the activity of the calcineurin/NFATc1 pathway was systematically analysed. In C2C12 cells, FHL1 enhances the transactivation of the utrophin A promoter via the calcineurin/NFATc1 pathway. In FHL1-Tg/mdx muscle, NFATc1 and FHL1 localised to the nucleus in mature and regenerating fibres exhibiting sarcolemmal utrophin A upregulation. In conclusion, this study has identified that overexpression of FHL1 in skeletal muscle enhances utrophin A localisation at the sarcolemma and this may be mediated via the calcineurin/NFATc1 pathway, to a level sufficient to improve muscle membrane integrity, reduce susceptibility to contraction induced muscle damage and prevent the progression of muscular dystrophy in mdx muscle. Therefore, this thesis identifies FHL1 as a novel regulator of utrophin A and the findings in this study may lead the way to new therapies for DMD and other diseases associated with muscle wasting