Abstract
Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein.
Highlights
Tropomyosin (TM) is a component of the muscle sarcomeric thin filament where it plays a central role in the calciumdependent regulation of striated muscle contraction
We aimed to investigate the effects of five different b-TM mutations on myofibril organisation and to examine the localisation of mutant b-TMs within the cytoskeleton and myofilament
We examined the accumulation of p62, a polyubiquitinatebinding protein, in human and C2C12 myoblasts, transfected with WT- and mutant-b-TMEGFP constructs
Summary
Tropomyosin (TM) is a component of the muscle sarcomeric thin filament where it plays a central role in the calciumdependent regulation of striated muscle contraction. In tropomyosin a heptad repeat motif forms a parallel a-helical coiled-coil structure, which is required for the correct formation of dimers, as well as for the interaction with proteins along the length of actin thin filaments [2,3,4]. There are three major tropomyosin isoforms: aTM, b-TM and c-TM, encoded by the TPM1, TPM2 and TPM3 genes respectively. Beta-tropomyosin (b-TM) is mainly expressed in slow, type 1 and to some extent in fast muscle fibres and cardiac muscle [5]. The a-TM muscle isoform is predominantly expressed in cardiac muscle and fast type 2, muscle fibres, whereas c-TM is predominantly expressed in slow muscle fibres and in the heart [2]. Each gene uses alternative promoters, alternative splicing and differential RNA processing to introduce multiple striated muscle, smooth muscle and cytoskeletal transcripts [2,6]
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