|dc.description.abstract||Myofibrillogenesis is a complex process, which involves assembly of hundreds of structural proteins in a highly ordered manner to form the contractile structural unit of muscle, the sarcomere. Several myopathic conditions reported in humans are caused due to abnormal myofibrillogenesis owing to mutations in the genes coding for many of these structural proteins. These myopathies have highly variable clinical features and time of onset. Since their aetiology is poorly understood, it becomes imperative to have a model system to study the muscle defects. Present study proposes to employ the Indirect Flight Muscle (IFM) system in Drosophila melanogaster as a model to analyse the development/onset of some of these myopathies and resulting pathophysiology.
We have carried out a systematic study on mutations in two major proteins of the sarcomere, actin and myosin, to understand the pathophysiology associated with the disease conditions and in turn gain insights into the process of myofibrillogenesis. To verify whether the human muscle phenotypes are observed in flies, we analysed the IFM for functional and structural defects categorised by the presence of aberrant sarcomeric structures. An important question that we have addressed is whether mutants of the Drosophila IFM recapitulate human conditions and whether it can serve as a good genetic model to study the developmental mechanisms of the human skeletal myopathies in vivo.
Mutations of the human ACTA1 skeletal actin gene produce seven congenital myopathies – actin myopathy, nemaline rod myopathy, intranuclear rod myopathy, congenital fibre type disproportion, congenital myopathy with core-like areas, cap disease and zebra body myopathy. Four known mutations in Act88F—a Drosophila homologue of ACTA1—occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for muscle phenotypes with nemaline structures. Mutant homozygotes show phenotypes ranging from lack of myofibrils to almost normal sarcomeres at eclosion. Whereas, heterozygotes do allow myofibrillar assembly to certain extent; however, atypical structures are seen adjacent to normal sarcomeres. Aberrant Z disc-like structures and serial Z disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all the four Act88F mutants. The electron-dense structures observed in electron micrographs show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. A possible mechanism for the ‘zebra bodies’ is proposed based on this study. Analysis of IFM at early developmental stages shows that in three of the mutants, there is an abnormal myofibril assembly leading to malformed sarcomeres mirrored in the adult stages. In one of the Act88F mutants, normal myofibrils are seen post-eclosion but the IFM show activity dependant progression of muscle degeneration. All the Act88F mutants produce dominant disruption of muscle structure and function which cannot be rescued even by three copies of the wild type Act88F gene implying that the mutants are strong antimorphs.
Myosin myopathies are a group of human muscle diseases with heterogeneous clinical features and are caused by mutations in the skeletal muscle myosin heavy chain. We identified two chemical mutagen generated flightless mutants, Ifm(2)RU1 and ifm(2)RU2 that map closely to myosin heavy chain gene (Mhc) region. Since there are no structural proteins predicted in the mapped region, it was likely that these two are Mhc mutations. We show that Ifm(2)RU1 and ifm(2)RU2 are indeed Mhc mutations and the molecular aberrations affect amino acid residues present in the myosin rod region. Human muscle myosin heavy chain (MyHC) mutations that cause Laing early onset distal myopathy and myosin storage myopathy occur in this domain of the protein. Even though mutations lie in the same region of myosin rod, Ifm(2)RU1 is semidominant, whereas ifm(2)RU2 is recessive. Both the mutants show IFM defects and the presence of abnormal myofibrils. Mutant myofibrillar structures can be rescued with an additional wild type Mhc gene copy. However, the restored myofibrillar structure is incapable of rescuing the flight ability of mutants. The muscle phenotypes are due to defects in thick filament assembly which manifest from the early stages of sarcomere development. The MHC protein rod region is an α-helix that forms coiled-coils which further self assemble to form thick filaments or aggregates as observed in in vitro conditions. Biophysical and biochemical analyses reveal that the coiled-coil structure of mutant rods is not affected, however the thermodynamic stability is altered in ifm(2)RU2 mutation. Interestingly, rod aggregate size and stability are not affected in mutant rods. The Drosophila MHC mutant rods were studied along with four MHC mutant rods that harbour human rod mutations to compare the molecular consequences. The Drosophila mutations do not hamper the rod structure and assembly. Therefore, the defects may arise due to altered interactions with myosin rod binding proteins. Flightin is an extensively studied myosin rod binding protein. The amount and phosphorylation status of flightin are an extremely sensitive measure of thick filament assembly. Flightin phosphorylation is affected in the mutants suggesting a functional dependence on MHC and it also indicates MHC instability.
In the light of the work done, we have assessed the mutations with respect to their structure-functional implications. The acto-myosin interactions responsible for the defects are also discussed. Formation of unusual myofibrillar structures are analysed with regards to the process of myofibrillogenesis. An understanding of this entire process with the information available from IFM is reviewed in detail.
The work so far has helped in understanding the manifestation of myopathies at tissue/cellular levels with insights into the plausible mechanisms of origin of the disease phenotypes. Myopathic condition may arise due to developmental or functional defects. For therapeutic considerations, the fly provides a simple test to inspect the effects of adding extra copies of the wild type gene. We conclude that the Drosophila IFM provide a good model system for the study of human ACTA1 and MyHC myopathies.||en_US