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. 2011 Feb 22;6(2):e16651.
doi: 10.1371/journal.pone.0016651.

Uncoordinated transcription and compromised muscle function in the lmna-null mouse model of Emery- Emery-Dreyfuss muscular dystrophy

Viola F Gnocchi  1 , Juergen Scharner, Zhe Huang, Ken Brady, Jaclyn S Lee, Robert B White, Jennifer E Morgan, Yin-Biao Sun, Juliet A Ellis, Peter S Zammit
Affiliations

Uncoordinated transcription and compromised muscle function in the lmna-null mouse model of Emery- Emery-Dreyfuss muscular dystrophy

Viola F Gnocchi et al. PLoS One. .

Abstract

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Myonuclear morphology is abnormal in lmna-null mice.
DAPI staining of representative EDL and soleus myofibres from wild-type (WT) lmna+/+mice show that myonuclei are evenly distributed and have similar shape, size and heterochromatin content (a and d). By contrast, myonuclei in lmna −/− myofibres are unevenly distributed, with variable size and shape, and heterogeneous chromatin content and distribution (b and e). Myofibres isolated from the mdx mouse model of DMD contain myonuclei of a more regular size, shape and heterochromatin organization (c and f). Unlike in lmna −/− myofibres, myonuclei in mdx mice are often located in a chain in the centre of the myofibre, indicative of a recent regenerative event (c and f). Representative TEM images of longitudinal sections of soleus muscle from wild-type lmna+/+ (g) and lmna −/− (h) mice. WT myonuclei (thin red arrows) are regularly shaped, and have an even layer of highly condensed chromatin around the nuclear rim, in addition to centrally located condensations (g). Myonuclei (thin red arrows) from lmna null mice are irregularly shaped and have disorganized chromatin throughout with occasional vacuoles (h - red *). A thick red arrow indicates an abnormally elongated myonucleus. Note connective tissue between myofibres and the disruption of the sarcomeric arrangements near the abnormal myonuclei (red open square). Scale bar for (a–f) is 50 µm and 10 µm for (g and h).
Figure 2
Figure 2. Myonuclei cluster at the myotendinous junctions of lmna−/− myofibres.
DAPI staining at the myotendinous junction from wild-type (WT) lmna+/+ EDL and soleus myofibres show that myonuclei are evenly distributed, with similar shape, size and chromatin organization (a and d). Myonuclei tend to cluster at the myotendinous junction from lmna −/− mice, and are unevenly distributed with varying sizes, shapes and condensed chromatin content (b and e). Myonuclei of the myotendinous junction from mdx mice have an overtly normal morphology, although often in centrally located chains that continue to the end of the myofibre (c and f). Scale bar 50 µm.
Figure 3
Figure 3. Variable transcriptional activity and mRNA processing between myonuclei of lmna-null mice.
Representative images of wild-type (WT) lmna+/+ EDL myofibres, immunostained for acetyl-histone H3 (red) and counter-stained with DAPI (white), with merged images (a–c). Myonuclei in WT myofibres have near uniform acetyl-histone H3 immunostaining, suggesting similar transcriptional activity. By contrast, immunostaining of lmna −/− EDL myofibres showed that myonuclei clearly have varying acetyl-histone H3 levels, indicating heterogeneous transcriptional activity, with some immunostaining at near background levels (d–f - arrows) while others appear hyperacetylated (d–f - arrowhead). WT myonuclei also show virtually homogeneous PABPN1 immunostaining (g–i), while in many myonuclei of lmna −/− EDL, PABPN1 is either reduced, or absent (j–l - arrows). Scale bar 50 µm.
Figure 4
Figure 4. Transcriptional activity and mRNA processing are often impaired in myonuclei at the myotendinous junctions of lmna-null mice.
Representative images of wild-type (WT) lmna+/+ EDL myofibres immunostained for acetyl-histone H3 (red) and counter-stained with DAPI (white), with merged images (a–c). Myonuclei at the myotendinous junction (MTJ) in WT myofibres have near homogeneous acetyl-histone H3 immunostaining, as observed along the myofibre, indicating similar levels of transcriptional activity. By contrast, immunostaining of lmna −/− EDL myofibres revealed that myonuclei at the myotendinous junctions clearly had varying acetyl-histone H3 levels, indicating heterogeneous transcriptional activity, ranging from virtually inactive (background levels – arrow in d–f), to hyperacetylated, myonuclei (d–f). WT myonuclei also exhibit regular and homogeneous PABPN1 immunostaining at the myotendinous junction (g–i). In myonuclei at the myotendinous junctions of lmna −/− EDL however, varying PABPN1 levels are observed, with many being virtually unstained (j–l - arrows), indicating an impairment of mRNA processing and maturation. Scale bar 50 µm.
Figure 5
Figure 5. Myotendinous junction structure is abnormal in lmna−/− mice.
Representative TEM images of longitudinal sections of myotendinous junction (MTJ) from EDL (a–c) and soleus (g–i) myofibres of wild-type (WT) lmna+/+ mice. Myonuclei (red arrows) and sarcomere organisation appear normal. Note the extensive inter-digitations between the myofibre and tendon. TEM images of longitudinal sections of myotendinous junctions from EDL (d–f) and soleus (j–l) myofibres from lmna −/− mice. Myonuclei with abnormal shape, size and chromatin organization are evident (red arrows). There is a lack of inter-digitations, abnormal connective tissue (C in panel d) and fat accumulations (asterix in panel f) and sarcomeric disorganisation (boxed area in panel f). Myotendinous junctions in soleus muscle are particularly badly affected (j–l). The tissue architecture in (l) is so disrupted that the muscle region (M) and the connective tissue region (C) are barely distinguishable. Scale bar in each image equals 10 µm.
Figure 6
Figure 6. Force generation is impaired in EDL and soleus muscles lacking lamin A/C.
Muscle specific force produced by EDL (left panel) and soleus (right panel) from wild-type (WT) lmna+/+, lmna −/− and lmna −/+ mice at 4 (white bars) and 5 (grey bars) weeks of age (a). Generation of isometric tetanic force at optimim length by EDL and soleus from WT (—), lmna −/+ (---) and lmna −/− (····) mice at 4 and 5 weeks of age (b). 3 muscles from 3 mice per genotype per age were analyzed. Values are mean ± SEM from three muscles. An asterisk denotes significance level using Student's t-test. *** p<0.001; * p<0.05; ns p>0.05. Each value is compared with that of age-matched WT. The muscle specific force was also significantly different for soleus muscles from lmna −/− mice between 4 and 5 weeks of age.
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