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First published online 23 April 2008
doi: 10.1242/dev.015818

Development 135, 2043-2053 (2008)
Published by The Company of Biologists 2008
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Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

Francesco J. Conti1, Amanda Felder2, Sue Monkley3, Martin Schwander1, Malcolm R. Wood4, Richard Lieber2, David Critchley3 and Ulrich Müller1,*

1 The Scripps Research Institute, Department of Cell Biology and Institute of Childhood and Neglected Disease, La Jolla, CA 92037, USA.
2 University of California and Veterans Administrative Centers, Department of Orthopaedics and Bioengineering, San Diego, CA 92037, USA.
3 University of Leicester, Department of Biochemistry, Leicester LE1 7RH, UK.
4 The Scripps Research Institute, Microscopy Core Facility, La Jolla, CA 92037, USA.


Figure 1
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  		Fig. 1. Generation of the talin 1 conditional allele. (A) Scheme of the targeting strategy. Homologous recombination of the targeting construct into the Tln1 gene introduced a loxP site (triangle) downstream of coding exon 5 and a floxed pgk-neo cassette upstream of the first coding exon (exon 2). CRE expression induced three recombination events: (1) Type II deletion: removal of the neo cassette (Tln1 fl/fl); (2) Type III deletion: removal of exons 1-4 (not shown); (3) Type I deletion: removal of pgk-neo and exons 1-4 (Tln1-). Primers a, b and c were used for the PCR reactions shown in B. (B) Recombination pattern observed by PCR on DNA extracted from E14.5 and P2 wild-type (WT) and Tln1HSA-CREko mutant (MT) littermates. (Top row) A 707 bp band indicative of the recombined allele (Rec) was observed in samples from Tln1HSA-CREko mice. (Middle row) The presence of floxed (fl) and wild-type (wt) alleles was confirmed. (Lower row) The presence of CRE recombinase was confirmed.

 

Figure 2
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  		Fig. 2. Characterization of Tln1HSA-CREko mice. (A-E) Cross-sections of wild-type and Tln1HSA-CREko mutant muscle were stained with antibodies to talin 1 at P8 (A,B) and 6 months (C,D). Talin 1 expression was detected at the sarcolemma in wild-type (A,C) but not mutant mice (B,D). In the mutants, talin 1 expression remained in cells between skeletal muscle fibers (B,D, arrowheads) that were identified as blood vessels by staining with antibodies to VE-cadherin (E). (F) Western blots with extracts from P2 and P8 gastrocnemius and soleus muscle, respectively. Expression of talin 1 was markedly reduced in Tln1HSA-CREko mice. Residual protein levels were likely to be due to talin 1 expression in endothelial cells. Talin 2 expression was detected in muscle extracts, but no compensatory upregulation was evident. Membranes were probed for tubulin ({alpha}-tub) as a loading control. (G) Analysis of Tln1 and Tln2 transcript levels in gastrocnemius muscle by quantitative real-time PCR. Data are plotted relative to Tln1 mRNA levels (n=3; mean ±s.e.; *P=0.004 at P5; P=0.002 at 6 months). (H) Growth curve of wild-type and Tln1HSA-CREko mice up to 60 weeks of age revealed no differences between the two genotypes. Scale bars: 25 µm in A-D; 50 µm in E.

 

Figure 3
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  		Fig. 3. Skeletal muscle defects in Tln1HSA-CREko mice. (A-P) Muscle sections from wild-type and mutant animals were stained with Hematoxylin and Eosin. Longitudinal sections from gastrocnemius (gast) and diaphragm (diap) muscles at P15 (A-D) and P60 (E-H) revealed no defects in the mutants. At 6 months, defects were observed in gastrocnemius and diaphragm muscles in mutants (J,L-N,P), but not in wild types (I,K,O). Pathological changes included enlarged (J,L, arrows) and bent (M, arrow) muscle fibers, and centrally located nuclei (L,N,P, arrowheads). Abnormal muscle fiber morphology was also evident in proximity to MTJs (compare O with P). (Q-X) Six-month-old mice were injected with Evans Blue Dye (EBD) and analyzed for dye uptake by bright field illumination of whole-mount tissue. Wild-type diaphragm (Q) and gastrocnemius muscle (S) did not show dye uptake, unless the muscle fibers were damaged (U) (damage was occasionally observed in wild-type and mutant mice). No dye was incorporated in mutant fibers (R,T). (V-X) Dye uptake was analyzed in sections using immunofluorescence microscopy. Tendon (t) but not muscle (m) in wild type (V) and mutants (W) had incorporated dye. In control damaged muscle from wild types (X), dye uptake was observed. (Y) At 6 months of age, a mild increase in serum creatine kinase levels was observed in the mutants (n≥4; mean ± s.e.). Scale bars: 250 µm in A-P; 200 µm in V-X.

 

Figure 4
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  		Fig. 4. Talin 1 is not essential for costamere assembly. (A-D) Longitudinal and (E-L) cross-sections of muscle from 6-month-old wild-type and Tln1HSA-CREko mice were stained with antibodies to costameric proteins and ECM glycoproteins and analyzed by immunofluorescence microscopy. The integrin β1-subunit, {alpha}-actinin, vinculin, dystrophin, collagen type IV and laminin were localized normally in the mutants. (M-P) Analysis of muscle by transmission electron microscopy. Sarcomere organization was not affected in Tln1HSA-CREko mice (M,N), but sarcomeres occasionally appeared hypercontracted (O,P). The Z-band was always detectable (O,P, arrows), but the A band (O,P, asterisk) was sometimes not evident in contracted muscle fibers. Scale bars: 200 µm in A-L; 2 µm in M,N; 1 µm in O,P.

 

Figure 5
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  		Fig. 5. Expression and phosphorylation of talin 1-associated proteins. (A-C) Extracts from wild-type and Tln1 mutant gastrocnemius muscle were analyzed by western blotting. No changes were observed in levels of FAK (A), Erk1/2 (B, ERK), β1 integrin and vinculin (C). Phosphorylation of FAK at Tyr397 (A, P-FAK) and of Erk1/2 (B, P-ERK) were unaffected. Membranes were probed with {alpha}-tubulin antibodies as a loading control. (D) Protein expression was quantified by densitometry (n≥3; relative expression levels ±s.e.). No statistically significant difference was observed in expression (P-values: β1 integrin, 0.238; vinculin, 0.146; FAK, 0.223; P-FAK, 0.153; ERK, 0.892; P-ERK, 0.165).

 

Figure 6
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  		Fig. 6. Integrin complexes are maintained at the MTJ. (A,B) MTJs in diaphragm muscle from wild-type and Tln1 mutant mice were analyzed by transmission electron microscopy to reveal the lamina densa. A continuous basement membrane (arrowheads) was present at the MTJ in mutants. (C-N) Localization of collagen IV, laminin, integrin {alpha}7β, vinculin, Ilk and talin 2 at MTJs (arrowheads) was analyzed by immunofluorescence microscopy. All analyzed proteins were present at MTJs in wild-type and mutant animals. t, tendon, m, muscle. Scale bars: 500 nm in A,B; 100 µm in C-F; 200 µm in G-N.

 

Figure 7
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  		Fig. 7. Detachment of myofilaments at the MTJ. Electron micrographs of tibialis (A-E), diaphragm (F-J) and soleus (K,L) muscles collected from 1-month-old (A-C,H,I) and 6-month-old (D-G,J-L) wild-type (WT) and Tln1 mutant (MT) mice. In wild-type mice, myofilaments connected to junctional electron-dense plaques are visible (arrowheads in A,D,F). In 1-month-old Tln1HSA-CREko mice, most MTJs were intact (B), but detachment of myofibers from MTJs was occasionally observed (C). In 6-month-old mutant animals, detachment of the myofibers was observed very frequently (E). Necrotic material was present where myofibers detached (asterisks in C,E,G). The thickness of the junctional plaque was reduced, and gaps were present where myofilaments failed to attach (arrows in G). Degeneration (I) and accumulation (K,L) of mitochondria were observed. Necrotic areas were observed in mutant fibers (J,K, asterisk) next to abnormal myofilaments (K, arrows). Scale bars: 3 µm in A-E,H,I; 1 µm in F,G; 10 µm in J-L.

 

Figure 8
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  		Fig. 8. Impaired force generation by Tln1-deficient muscles. (A) The length, mass and physiological cross-sectional area (PCSA) are similar in the fifth EDL muscle from wild-type and Tln1HSA-CREko mice. (B,C) Sample curves representing a single isometric and eccentric contraction, respectively. (D-H) Isometric and eccentric contractile properties of the fifth EDL muscle isolated from 2- and 7-month-old mice. (D) No significant difference was observed in stress exerted by 2-month-old muscles from wild-type and Tln1HSA-CREko mice in the eccentric contraction protocol. (E) Example of force profile of the first isometric contraction in 7-month-old mice. Tln1-deficient muscles exert reduced isometric stress. (F,G) In an eccentric contraction protocol, muscles from 7-month-old Tln1HSA-CREko mice showed a significant reduction in peak stress exerted in the pre-eccentric (P=0.0276) and post-eccentric (P=0.0358) isometric contractions as compared with wild-type controls (n≥4 per genotype per time point). (H) Even though Tln1-deficient muscles generated less force than controls, the force drop was comparable between genotypes, suggesting enhanced susceptibility to damage in the mutants. All force values were normalized against the PCSA of the muscle (mean ±s.e.).

 

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© The Company of Biologists Ltd 2008