Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

Abstract

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations. Electronic supplementary material The online version of this article (10.1007/s00401-019-02034-8) contains supplementary material, which is available to authorized users.

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“…To investigate this specific point, we used immunofluorescence to label acetyl histone H3 (Lys9/Lys14) (AcH3). This histone modification is well documented as a marker of actively transcribed regions of DNA, and nuclear abundance/fluorescence intensity of this marker is positively correlated with global nuclear transcription levels [22,31]. The mean fluorescence intensity of AcH3 within individual myonuclei was significantly lower in XLMTM and AAVLow dogs than in healthy, AAVMid and AAVHigh animals (Fig.…”

“…For the measurement of nuclear coordinates, fibres were mounted at a fixed sarcomere length of ≈2.20 µm. This was a prerequisite for exact determination of nuclear spatial organization as it allowed accurate comparisons between myofibres [22,[31][32][33]. At RT, arrays were fixed in 4% PFA for 10 min, washed × 3 in phosphate buffered saline (PBS), further permeabilized in 0.1% triton-X100/PBS for 10 min, and subsequently subjected to actin staining (Rhodamineconjugated Phalloidin at 1:100 in PBS, Molecular Probes, R415) and nuclear staining (DAPI at 1:1000 in PBS, Molecular Probes, D3571).…”

“…Other skeletal muscle diseases with altered nuclear spacing notably include those caused by mutations in genes encoding nuclear envelope proteins [21,36], dynamin 2 (centronuclear myopathy) [12] and skeletal muscle actin/ nebulin (nemaline myopathy) [31]. In light of these various disorders, it has been hypothesized that optimal muscle fibre performance requires proper myonuclear number and positioning, MND volume and synthetic activity [42].…”

“…For the measurement of nuclear coordinates, fibres were mounted at a fixed sarcomere length of ≈2.20 µm. This was a prerequisite for exact determination of nuclear spatial organization as it allowed accurate comparisons between myofibres [ 22 , 31 – 33 ].…”

“…We also sought to investigate whether the presence of centrally located nuclei might directly interfere with muscle contraction, since these nuclei are buried between the contractile filaments. We isolated membrane-permeabilized muscle fibres and ran a series of functional and morphological measures including an evaluation of the 3D spatial arrangement of myonuclei in relation to myofibre size, using our image analysis algorithm applied to confocal images [22,31]. We then correlated these results with a marker of global nuclear transcriptional output, muscle architecture at the ultrastructural level, and measurements of intrinsic contractile force generation.…”