Functional muscle analysis of the Tcap knockout mouse

Abstract

Autosomal recessive limb-girdle muscular dystrophy type 2G (LGMD2G) is an adult-onset myopathy characterized by distal lower limb weakness, calf hypertrophy and progressive decline in ambulation. The disease is caused by mutations in Tcap, a z-disc protein of skeletal muscle, although the precise mechanisms resulting in clinical symptoms are unknown. To provide a model for preclinical trials and for mechanistic studies, we generated knockout (KO) mice carrying a null mutation in the Tcap gene. Here we present the first report of a Tcap KO mouse model for LGMD2G and the results of an investigation into the effects of Tcap deficiency on skeletal muscle function in 4- and 12-month-old mice. Muscle histology of Tcap-null mice revealed abnormal myofiber size variation with central nucleation, similar to findings in the muscles of LGMD2G patients. An analysis of a Tcap binding protein, myostatin, showed that deletion of Tcap was accompanied by increased protein levels of myostatin. Our Tcap-null mice exhibited a decline in the ability to maintain balance on a rotating rod, relative to wild-type controls. No differences were detected in force or fatigue assays of isolated extensor digitorum longus (EDL) and soleus (SOL) muscles. Finally, a mechanical investigation of EDL and SOL indicated an increase in muscle stiffness in KO animals. We are the first to establish a viable KO mouse model of Tcap deficiency and our model mice demonstrate a dystrophic phenotype comparable to humans with LGMD2G.

Figure 1.

Targeted disruption of the Tcap gene locus and establishment of mouse lines expressing normal and mutant Tcap. (A) Schematic diagram illustrating the targeting construct, the WT Tcap allele, and the predicted recombinant allele with exons 1 and 2 replaced by a lacZ/Neo cassette. The 15.4-kb targeting construct included the plasmid vector, the long 5′ and short 3′ homology arms, a lacZ reporter gene and neomycin-resistance (Neo) cassette. Homologous recombination resulted in replacement of exons 1 and 2 of the Tcap gene locus with a DNA fragment containing a lacZ/Neo gene. (B) PCR primers (LAN1/A2) external and internal (Lac2/Lac3) to the targeting construct (as shown in A) were used to characterize the targeted locus in ES cells. The 3′ homology arm of the targeting construct was amplified by high-fidelity PCR from genomic DNA of ES cells using the primers LAN1 and A2. LAN1 anneals to the recombinant allele inside the Neo cassette and A2 anneals 3′ to the SA, outside the region used to create the targeting construct. LAN1/A2 and Lac2/3 primer sets amplify fragments within the KO allele measuring 2.9 and 1.5 kb, respectively; WT1/A2 amplifies a 2.8 kb fragment within the WT allele. (C) Detection of the recombinant gene using PCR primers (shown in A) of mouse tail DNA from each of the founder lines. Neg, negative control; Pos, positive control, W, wild-type; H, heterozygote; K, knockout.

Figure 2.

Immunohistochemistry and western blot of WT and Tcap KO mouse hindlimb muscles. (A) Staining pattern in longitudinal thin muscle sections reveals thin bands (top panel), consistent with Tcap's z-disc localization, whereas thick bands are observed with myosin staining (bottom panel). Fluorescence imaging was accomplished using primary monoclonal antibodies against myosin heavy chain (MF-20) and full-length Tcap. Bar = 50 µm. (B) Representative western immunoblot of WT and Tcap KO muscle lysates. Gastrocnemius lysate from 2-month-old mice was loaded at 20, 40 and 60 µg per lane (top panel). Anti-GAPDH antibody was used as a loading control (bottom panel).

Figure 3.

Tcap KO mouse muscles reveal atrophic scattered fibers with central nucleation. Isopentane-frozen gastrocnemius muscles from WT (A–C) and Tcap KO (D–F) mice were stained with hematoxylin and eosin (A, D) or NADH (B, E), showing scattered atrophic fibers in the Tcap KO mice, with coarse basophilic inclusions in some small fibers and an increase in the number of centrally nucleated fibers (D, inset). Toluidine blue staining of Epon-embedded quadriceps muscle also reveals atrophic fibers in Tcap KO mice (C, F). Scale Bar = 100 µm in A and D, and 200 µm in B, C, E and F.

Figure 4.

Tcap KO mouse muscles display ultrastructural abnormalities. Electron microscopy of Tcap KO muscles revealed prominent mitochondria in atrophic fibers (arrowhead) (A–C) whereas contractile filaments are appropriately organized (D). Tubular aggregates (arrow) were also present in numerous muscle fibers in the Tcap KO muscles (E, F). Scale bar = 500 nm.

Figure 5.

Myostatin protein expression is increased in Tcap KO mouse muscles. Immunoblots were performed on muscle lysates from WT and Tcap KO gastrocnemius muscles probed with an anti-myostatin antibody. (A) To evaluate the myostatin antibody, recombinant purified human myostatin protein was subjected to both non-reducing (−) and reducing (+) conditions as indicated. One hundred and twenty-five, 250 and 500 ng purified myostatin protein were loaded in each condition. Bands at ∼26 and ∼13 kDa were clearly seen, indicating the sensitivity of the antibody to detect intact and cleaved C-terminal dimer myostatin proteins, respectively. (B) Both the 45-kDa precursor and 26-kDa processed myostatin bands were observed, but only in Tcap KO muscle lysates and not in WT muscles. (C) To assess the sensitivity of myostatin antibody in skeletal muscle tissue, female rats (n = 3) were suspended by their tails (HLU) for four weeks, SOL muscles were subsequently removed and muscle lysates were probed with anti-myostatin antibody. Intensity of myostatin bands (∼26 kDa) increased relative to GAPDH bands (∼36 kDa) during HLU compared with non-HLU controls. Densitometry analysis (D) of MSTN bands demonstrated significantly greater intensity (P < 0.05) relative to GAPDH bands in the HLU group compared with non-HLU controls. Equal protein loading of lanes was confirmed by intensity of GAPDH bands.

Figure 6.

Tcap and myostatin associate in mouse skeletal muscle tissue. HLU was used to increase myostatin expression in skeletal muscle. Adult female mice (n = 2) were suspended by their tails (HLU) for 2 weeks. (A) Three independent thin sections of SOL muscles were stained with primary antibodies directed against either Tcap or myostatin and conjugated to red or green fluorescent secondary antibodies, respectively, as indicated. In merged images (second column from the right) the relative amount of color overlap was determined with computer software. The mean (±SD) percent overlap between Tcap and myostatin was 20.5 ± 0.9%, suggesting a biological association between the two proteins. The far-right column shows detail of the area indicated by the box. (B) Representative line-scan analysis of fluorescent intensity. Points along the line drawn on the image connecting two points, labeled ‘a’ and ‘b’, were sampled to generate an intensity profile of two fluorescent colors, red (Tcap) and green (myostatin). Peak intensity for both Tcap and myostatin line scans correlate with the regular repeating z-disc striations observed in longitudinal thin sections of muscle.

Figure 7.

Tcap KO mice exhibit a decline in motor performance by 4 months of age. The ability to maintain balance on a rotating rod was compared between WT and Tcap KO mice by measuring the time (in seconds) to fall from a Rotor Rod. Performance was assessed longitudinally, and compared with WT controls, Tcap KO mice (open circles) exhibited impaired performance relative to controls at 2 months (n = 32, 62±6 versus 77 ± 7 s), 3 months (n = 32, 65 ± 6 versus 72 ± 12 s) and 4 months (n = 30, 56 ± 6 versus 71 ± 7), respectively. Data indicate mean ± SE.

Figure 8.

Stress profiles of EDL and SOL muscles. Absence of the Tcap protein was not associated with a change in the stress frequency (A), fatigue (B) or recovery (C) profiles of EDL or SOL muscles. Solid black: WT, dashed gray: Tcap KO. n = 7–9 muscles per group.

Figure 9.

Passive stiffness of EDL and SOL muscles. (A) Representative EDL passive stretch profiles for the two stress-relaxation protocols. (B, C) Independent of muscle type, absence of Tcap was associated with increased series elastic stiffness and series modulus of elasticity for the 5%, 7 s passive stretch protocol but not the 15%, 20 s passive stretch condition. Independent of genotype, series elastic stiffness and modulus of elasticity were greater in EDL (13.8 ± 0.6 g/mm and 2.2 ± 0.1 MPa, respectively) than SOL (11.4 ± 1.2 g/mm and 1.5 ± 0.2 MPa, respectively; P < 0.05) muscles in the 5%, 7 s passive stretch condition, but not the 15%, 20 s condition (data not shown). (D, E) SOL parallel elastic stiffness and modulus of elasticity were greater in the KO muscles compared with WT muscles stretched 5% for 7 s, but not different when stretched 15% for 20 s. Solid black: WT, dashed gray: Tcap KO; * different from WT; n = 7–9 muscles per group (P < 0.05).