Reduced myofibrillar connectivity and increased Z-disk width in nebulin-deficient skeletal muscle

Abstract

A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein nebulin. Here we tested whether nebulin is part of a novel biomechanical linker complex, by using a recently developed nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of nebulin and expressed modules within the Z-disk portion of nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with nebulin. Finally, because nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (approximately 80 nm in soleus and approximately 40 nm in EDL fibers) whereas a small subset had severely increased widths (up to approximately 1 microm) and resembled nemaline rod bodies. In summary, structural studies on a nebulin KO mouse show that in the absence of nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.

Fig. 1.

Sarcomeric structure in EDL and soleus skinned skeletal muscle fibers of WT and nebulin KO mice. (A-D) EDL muscle; (E-H) soleus muscle. WT fibers have a regular structure with well-aligned sarcomeres. In KO fibers, sarcomeres are misaligned and the Z-disk structure is non-uniform, with nemaline bodies (arrows). (D,H) Measurement of longitudinal Z-disk displacement (ΔX, defined as longitudinal displacement of nearby Z-disk in adjacent myofibrils) in skeletal muscles. Note large Z-disk displacement in both EDL and soleus KO fibers. Scale bars: 0.5 μm (A,B,E,F); 2 μm (C,D,G,H). (Each result is from a different mouse. Fibers were stretched 80%.)

Fig. 2.

Z-disk displacement in relation to SL of EDL and soleus muscle from nebulin KO and WT muscles. (A,C) EDL muscle; (B,D) soleus muscle. 10-day-old mice were used for A and B and 3-day-old mice for C and D. Results from KO fibers (black circles) are significantly different from WT fibers (white circles) (P<0.001). Each value represents the mean of ~50 measurements made on a single electron micrograph; ~20 micrographs were obtained from randomly selected fibers from five different mice.

Fig. 3.

The mean Z-disk displacement at SLs >2.9 μm. (A) Soleus muscle, (B) EDL muscle. Values are means ± s.d. for 3-day-old and 10-day-old mice. ANOVA with Tukey's HSD post-hoc tests show statistical significance (S) between all KO and WT groups but not within any of the WT and KO groups, with identical results for 3-day-old and 10-day-old mice. No significant differences exist between EDL and soleus fibers. Results are from ~50 measurements made on a single electron micrograph with ~20 micrographs obtained from randomly selected fibers from five different mice.

Fig. 4.

Immunoelectron microscopy of EDL muscle. (A) WT muscle had gold particles (indicating the presence of desmin) near the Z-disks (black arrowheads), whereas the nebulin KO (B) muscle was largely devoid of gold particles (white arrowheads) in the Z-disk regions between myofibrils. Bar, 500 nm. (C) Particle counts in the Z-disk to Z-disk space (intermyofibrillar area enclosed by drawing lines from edge to edge of adjacent Z-disks; see examples on micrograph). Results are from three WT and three KO muscle (from three WT and three KO mice) with 15 micrographs per muscle and approx. five Z-disk regions per micrograph. We also counted particles in randomly selected areas in the A-band region of the sarcomere. Significant differences exist between WT and KO fibers for Z-disk to Z-disk measurements only. Note that no differences were found in the size of the gold particles: 14.1±3.6 nm (WT) and 16.8±4.7 nm (KO).

Fig. 5.

Knockdown of nebulin in primary cultures of quail myotubes perturbs the association of desmin at the Z-disk. Myotubes were double stained with antibodies generated against α-actinin, as a marker of the Z-disk, and desmin, 4-8 days after siRNA treatment. Treatment with nebulin-specific siRNA results in a significant decrease in assembled nebulin (see supplementary material Fig. S3) with a concomitant decrease in assembled Z-disk-associated desmin. In the siRNA-treated cells, desmin staining at the Z-disk was either undetectable (top two rows) or if detectable, was significantly broader and less intense, than in the controls (insets, bottom two rows). The distribution of α-actinin was not disrupted in the identical myofibrils. Scale bar: 10 μm. (Top two rows: example of the most typical cell type in which desmin Z-disk assembly was absent; bottom two rows: example of less typical cell type in which broad and faint Z-disk labeling of desmin was seen.)

Fig. 6.

Introduction of a recombinant nebulin fragment (M160-170), which contains the desmin binding site, perturbs the Z-disk distribution of desmin in primary cultures of quail myotubes. Myotubes were double stained for desmin and α-actinin, 6 days after transfection of mCherry alone or nebulin M160-170 fused to mCherry. Scale bar: 10 μm.

Fig. 7.

Representative electron micrographs of EDL (A,B) and soleus (C,D) muscle from WT (A,C) and nebulin KO 10-day old mice (B,D). In nebulin KO fibers (B and D) wide and irregular Z-disk inclusions that resembled nemaline bodies (arrows) were commonly present. Scale bars: 2 μm (A,B), and 1 μm (C,D). Fibers were stretched 80% in A and D and 60% in B and C.

Fig. 8.

Analysis of Z-disk widths. Histograms of Z-disk widths in EDL (A,B) and soleus (C,D) fibers. Primary peaks were fit by Gaussians. Gaussian peak fit of WT fibers (broken lines) is superimposed on results of KO fibers. Results are from 82 micrographs from five muscles from five different mice. See text for details.