The expression of WT and mutant β-TMs-_EGFP in human myotubes.

<p>WT and mutant β-TMs were transfected in human myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) WT-β-TM was expressed in human myotubes and incorporated well into endogenous sarcomeric thin filaments as visualised with phalloidin (arrows). (B) The E41K-β-TM mutant induced diffuse perinuclear aggregates in myotubes (arrows). (C) The K49del-β-TM_EGFP mutant produced cytoplasmic rod-shaped filamentous actin in human myotubes. (D) The cells transfected with G53ins-β-TM mutant differentiated into myotubes, showed the integration of the mutant TM into sarcomeric structures and developed the cross-striated myofibrils in an advanced and developed state (arrow). (D’) The G53ins mutant produced diffuse cytoplasmic labeling at the far end of the transfected myotubes (arrow)_. (E) The transfection of the E122K-β-TM_EGFP construct generally resulted in the appearance of cytoplasmic rod-like structures located at the periphery of the transfected myotubes (arrow). (F) The cells transfected with N202K-β-TM_EGFP differentiated into myotubes, showed an accumulation of mutant N202K-β-TM, co-localisation with polymerised actin (arrows). Cells were imaged using a Zeiss Axio Observer microscope. Scale bar  = 10 µm.</p

Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

<div><p>Mutations in <i>TPM2</i> 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-TM_EGFP mutant showed perinuclear aggregates. The G53ins-β-TM_EGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TM_EGFP and E122K-β-TM_EGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TM_EGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TM_EGFP 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.</p></div

The expression of E122K and N202K-β-TM_EGFP and empty EGFP in C2C12.

<p>The cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM_EGFP mutant was transfected in (A) myoblasts and (B) myotubes. (A) The E122K-β-TM mutant was mislocalised and produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short arrows). The E122K mutant induced the perinuclear aggregates in differentiated C2C12 (B and B”; long arrows). C2C12 myoblasts transfected with N202K-β-TM_EGFP appeared cytopathic, with a thickened, ruffled cell surface (C–C”; long open arrows). Aggregates in the peripheral (D–D”; short closed arrows) and perinuclear (D–D”; long open arrows) areas of differentiated cells were detected. The transfection of C2C12 myoblasts with empty EGFP vector resulted in the formation of well-organised stress fibres (E–E”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

The expression of E202K-β-TM_EGFP and empty EGFP constructs in human cells.

<p>The E202K-β-TM_EGFP mutant was transfected in human (A–B) myoblasts and (C) myotubes. (D) Empty EGFP construct was transfected in myoblasts. Cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The transfection of human myoblasts with mutant N202K-β-TM induced the diffuse cytoplasmic labeling of stress fibres (A–A’; long arrows) and small phalloidin-labeled aggregates in the cytoplasm (A’ and A”; short arrows). Mutant N202K-β-TM formed clouds around the nucleus (B and B”; short arrows), in addition to a well-defined organised filamentous structure of stress fibres (B–B”; long arrows). In many N202K-β-TM_EGFP-transfected myotubes, more marked changes in actin structures were observed (C–C”). A large accumulation of mutant N202K-β-TM with the co-localisation of polymerised actin appeared, suggesting the disruption of endogenous actin filaments (C–C”; short arrows). Human myoblasts transfected with empty EGFP vector formed well-organised filamentous structures (D–D”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

The expression of E122K-β-TM_EGFP in human cells.

<p>The E122K-β-TM_EGFP mutant was transfected in human (A–C) myoblasts and (D) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM_EGFP mutant formed aggregates of endogenous actin in human myoblasts (A–A”; inset (B–B”)). Moreover, the E122K-β-TM_EGFP incorporated in clouds with an unorganised filamentous structure (C–C”). E122K-β-TM_EGFP formed intranuclear rods in human myoblasts that were only detectable with phalloidin labeling (C–C”; short arrows). It also integrated well with stress fibres (A–A” and C–C”; long arrows). The transfection of the E122K-β-TM_EGFP construct generally resulted in the less well-defined phalloidin labeling of actin filaments at the far end of the transfected myotubes with the appearance of small rod-like structures located at the periphery (D–D”; inset, short arrows). The rod-shaped structures did not label with phalloidin, indicating that they were not accessible to phalloidin or were not composed of filamentous actin. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

The expression of K49del-β-TM_EGFP in human cells.

<p>(A–D) The K49del-β-TM_EGFP mutant was transfected in human myoblasts and (E) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The K49del-β-TM co-localised with cytoplasmic and nuclear aggregates of endogenous actin, detectable with phalloidin labeling (A–C”; short arrows). It produced clouds around the nucleus (A and D) but was also incorporated into stress fibres and filamentous lamellipodia (A and A”, B and B”, and D and D”; long arrows). In addition, it induced thickened filamentous structures of endogenous actin (A’–A”; arrow heads). The K49del-β-TM produced small, long rod-shaped intranuclear structures. A subset of aggregates labeled with phalloidin but was not detectably composed of EGFP-tagged mutant K49del-β-TM (D’–D”; short arrows). Frequently, cytoplasmic thickened filamentous structures with no co-localisation of F-actin were found in myoblasts transfected with K49del-β-TM (D and D”; arrow heads). The K49del-β-TM_EGFP mutant produced rod-shaped filamentous actin, cytoplasmic aggregates and cloud patterns in human myotubes (E). The rod-shaped structures were labeled with phalloidin, indicating co-localisation with F-actin (E–E”; short arrows). Cytoplasmic thickened filamentous structures were also observed (E and E”; arrow heads). The K49del-β-TM_EGFP mutant was also incorporated into filamentous actin (E; long arrow). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

Untagged β-TM constructs form abnormal aggregates in human myoblasts.

<p>Human myoblasts transfected with untagged WT-, E41K-, K49del- and G53ins-β-TM constructs and labeled with phalloidin (green) and β-TM (red) and DAPI (blue) to highlight cell nuclei. Stress fibres appeared well aligned in human myoblasts transfected with WT-β-TM (A). Abnormal aggregates were observed in human myoblasts transfected with untagged E41K-, K49del- and G53ins-β-TM constructs (B–D). Intranuclear rod-shaped aggregates labeled by phalloidin are detected in human myoblasts transfected with the untagged K49del-β-TM construct, demonstrating that intranuclear aggregation is an inherent property of K49del mutation and does not result from EGFP-tagging (C; arrows).</p

The expression of wild-type-β-TM_EGFP and E41K-β-TM_EGFP in C2C12.

<p>(A–B) Wild-type β-TM_EGFP and (C–D) E41K-β-TM_EGFP mutant were transfected in (A and C) the myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. WT-β-TM expressed in C2C12 (A) myoblasts or (B) myotubes incorporated well into C2C12 stress fibres (A) and endogenous filamentous actin (B; long arrow) as visualised with phalloidin. The E41K-β-TM mutant appeared to be diffused in transfected myoblasts and intense EGFP aggregates co-localised with endogenous actin in the peripheral area of the cells (C–C”; short open arrows). The E41K-β-TM mutant formed perinuclear aggregates in myotubes that did not show phalloidin labeling (D–D’’; long open arrows). C2C12 cells appeared as fused myoblasts rather than differentiated myotubes in six-day differentiated cultures. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

The expression of K49del and G53ins β-TM-EGFP in C2C12.

<p>The K49del β-TM-EGFP mutant was transfected in (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) The K49del-β-TM mutant was mislocalised and showed the diffuse labeling of phalloidin in transfected myoblasts. It induced nuclear and cytoplasmic aggregates (A–A’; long open arrows). The K49del-β-TM mutant produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short open arrows), in addition to perinuclear aggregates in differentiated C2C12 (B and B”; short closed arrows). (B) The K49del-β-TM mutant showed some integration with actin filaments of differentiated C2C12 (B–B”; long arrows). The G53ins mutant appeared mislocalised, showed the diffuse labeling of phalloidin in transfected myoblasts (C–C”) and formed cytoplasmic aggregates (C–C”; long open arrows). Intense aggregates in the peripheral area of both myoblasts (C–C”; short arrows) and differentiated cells (D–D”; short arrows) were observed. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar  = 10 µm.</p

Incorporation of β-TM mutants into cytoskeleton and sarcomeric filaments in cultured human myoblasts and differentiated myotubes.

<p>(A) Western blot images from a single experiment showing levels of β-TM_EGFP within the insoluble (Insol) and soluble (Sol) protein pools in myoblasts (Myo) and in six-day differentiated cultures (D6). Bands intensity was quantified through densitometric analysis. The mean data from triplicate experiments is shown in (B).</p