Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Abstract

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8-11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.

Figure 1.

Targeting of the nebulin gene. (A) Targeting strategy. A restriction map of the relevant genomic region of nebulin is shown at the top, the targeting construct is shown in the middle, and the mutated locus after recombination is shown at the bottom. The grey box indicates exon 1, and black boxes indicate frt sites. DTA, Diphteria toxin A chain; Neo, neomycin resistance gene. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNA from electroporated ES cells was digested with SacI and analyzed by Southern blot analysis with a probe as shown in A. The 8.977- and 6.982-kb bands represent wild-type and targeted alleles, respectively. (C) Southern blot analysis of DNA isolated from postnatal day 1 wild-type, nebulin^+/−, and nebulin^−/− mice. DNA was digested with SacI and analyzed by Southern blot analysis with the probe shown in A. (D) Detection of nebulin protein by Western blot analysis. Protein prepared from postnatal day 1 skeletal muscle of wild-type and nebulin^−/− mice was analyzed with antinebulin M161–165 and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies. (E) RT-PCR using primers for exons 2 and 3, 98–102, and 163 and 164 confirmed that nebulin was successfully knocked out.

Figure 2.

Nebulin is expressed in skeletal and cardiac muscle. X-galactosidase staining of tissue from nebulin^+/− Rosa26 mice. (A) X-galactosidase staining of heart sections reveals nebulin expression mainly in atrial cardiomyocytes and only in a small percentage of ventricular cardiomyocytes. Enlarged images of different areas from the heart are shown (arrows refer to the enlarged areas). (B) X-galactosidase staining of heart sections reveals nebulin expression mainly in atrial cardiomyocytes and only in a small percentage of ventricular cardiomyocytes. (bottom right) LacZ-positive cardiomyocyte. (C and D) X-galactosidase staining was also positive for the liver (C) and aorta (D). Bars (C and D), 1 mm.

Figure 3.

Nebulin^−/− mice are born at a similar weight as their wild-type littermates but die at postnatal day 8–11 at a weight ∼25% of that of their wild-type littermates. (A) Nebulin^−/− (knockout; KO) mice at postnatal days 1 and 8 as compared with wild-type (wt) littermate controls. (B) Typical growth of nebulin^−/− (KO) mice compared with wild-type littermate control mice from postnatal day 1–11 when nebulin^−/− mice die. No statistical differences between wild-type and nebulin^+/− pups were observed. Numbers above the bars for nebulin^−/− mice indicate the number of remaining viable mice. Error bars represent SEM.

Figure 4.

TEM of TA muscle from nebulin^−/− mice at postnatal day 1. TEM of relaxed TA muscle from wild-type (A) and nebulin^−/− mice (B) showed relatively normal sarcomeric organization in nebulin^−/− mice, although some sarcomeres were misaligned. TEM of stretched TA muscle from wild-type (C and E) and nebulin^−/− mice (D and F) revealed myofibrillar misalignment with evidence of myofibril splitting (arrowhead) and fragmentation of Z-lines (arrow) in nebulin^−/− mice. M, M-line; Z, Z-line. Bars, 1 μm.

Figure 5.

TEM of diaphragm muscle. Micrographs of longitudinal sections of diaphragm muscle from wild-type (A, C, and E) and nebulin^−/− mice (B, D, F, G, and H) at embryonic day (E) 18.5 (A and B), postnatal day (PN) 1 (C and D), and postnatal day 9 (E–H). In the noncontracting diaphragm at embryonic day 18.5, nebulin^−/− mice showed normal sarcomeric organization with well-aligned sarcomeres (B) and were comparable with wild-type muscle (A). At postnatal day 1, nebulin^−/− mice exhibited misaligned sarcomeres with moderately wider Z-lines compared with wild type (C and D). By postnatal day 9, myofibril architecture was severely disrupted (F–H) with either thick (arrowheads in F), punctate (arrows in F–H), or dissolving Z-lines (G and H). In addition, M-lines became poorly defined, and abnormal accumulations of mitochondria were present. Bars, 500 nm.

Figure 6.

Immunolabeling with antibodies against various cytoskeletal proteins. Immunostaining of frozen sections from the TA muscle stained for nebulin, actin (phalloidin), α-actinin, tropomyosin, tropomodulin, α-myosin, desmin, and palladin. Bar, 10 μm.

Figure 7.

Thin filament lengths in TA muscle from nebulin^−/− mice are shorter compared with wild-type mice. Myofibril image regions, line scan profiles, and thin filament models for wild-type (A) and nebulin^−/− (B) mice stained for α-actinin (red) and tropomodulin (green) or for wild-type (C) and nebulin^−/− (D) mice stained for α-actinin (red) and phalloidin (green). Myofibrils: the regions used for each line scan are indicated on each image by a semitransparent overlay. Image regions above and below the line scan region were used to calculate background values. Profiles: optimized intensity fits for α-actinin (red) and either tropomodulin or phalloidin (green) closely fit the line scan intensity values (gray and black lines). Thin filament models: individual α-actinin (red) and either tropomodulin or phalloidin (green) model distributions optimized to fit the observed line scan intensities by repeating each profile along the length of the line scan for each Z-line. Thin filament lengths determined by the tropomodulin or phalloidin peaks are indicated below the profiles. (B) Histogram of thin filament lengths measured for wild-type and nebulin^−/− mice. Curves show the Gaussian fit to the data.

Figure 8.

Contractile properties of 1-d-old muscles. (A) Isometric contractile properties of mouse TA muscle (inset) from wild-type (black) and nebulin^−/− mice (blue). Note the decreased force generated over a similar time course. This implicates the force-generating/transmitting system. (B) Stress generated by wild-type (black) and nebulin^−/− (hatched) muscles. Note that the nebulin^−/− muscles generate <50% of the wild-type stress (P < 0.001). (C) Physiological cross-sectional area (PCSA) of wild-type (black) and nebulin^−/− (hatched) muscles. See Materials and methods for the definition of PCSA. The PCSA of the two genotypes is nearly identical, demonstrating that both muscles have the same amount of force-generating material, but the nebulin^−/− muscle is of inferior quality, thus explaining the lower stress (see B). Data is presented as mean ± SEM (error bars) for wild-type (n = 14) and nebulin^−/− (n = 9) muscles.