Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties

Abstract

Nebulin (NEB) is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy.

Fig. 1.

Characterization of isometric stress production in wild-type (WT) and nebulin (NEB) knockout (KO) mice at postnatal days 1 and 7 (P1 and P7, respectively; n = 8 WT at P1, 8 NEB-KO at P1, 10 WT at P7, and 4 NEB-KO at P7) and its relationship to muscle ultrastructure. A: Fisher's protected least-significant difference (PLSD) test showed that the NEB-KO gastrocnemius muscle (GAS) generated significantly less stress than the WT GAS at P1. The differences were even more pronounced by P7, indicating that the WT GAS exhibited both extrinsic and intrinsic postnatal enhancement of contractile function, whereas the NEB-KO GAS exhibited postnatal deterioration, indicating rapidly progressive myopathy. *P < 0.05 compared with WT; **P < 0.0001 compared with WT. B: transmission electron microscopy on tibialis anterior muscle confirmed that the WT muscle maintains normal sarcomere structure during postnatal development, whereas the NEB-KO muscle exhibits progressive deterioration. Deterioration in NEB-KO muscle is distinguished by wavy, fragmented, and disrupted Z-disks. P6, postnatal day 6. Scale bar = 500 nm.

Fig. 2.

Relative quantification of sarcolipin RNA transcript levels in WT and NEB-KO tibialis anterior muscle tissue from P1 to P11 (n = 3 WT and 3 NEB-KO at each time point). All data are shown as the fold expression of sarcolipin compared with WT muscle at P1. Sarcolipin upregulation in NEB-KO muscle increased with age, whereas sarcolipin transcript levels decreased monotonically in WT muscle. The y-axis uses a logarithmic scale for clarity.

Fig. 3.

Myosin heavy chain isoform distributions in the WT and NEB-KO GAS at P1 and P7 (n = 4 WT at P1, 4 NEB-KO at P1, 4 WT at P7, and 4 NEB-KO at P7). A: sample silver-stained SDS-PAGE gel with bands ordered by increasing electrophoretic mobilities. After band intensities were quantified using densitometry, Student's t-test found no significant differences in embryonic (Emb), neonatal (Neo), type 1, and type 2b myosin isoform levels at both P1 (B) and P7 (C). Type 2a and 2x isoforms were not detectable in either genotype at either postnatal day.

Fig. 4.

Responses of the WT and NEB-KO GAS at P1 to cyclic isometric activation (n = 6 WT and 6 NEB-KO). A: isometric stress achieved during a series of 10 isometric tetani (Iso1–Iso10) spaced 2 min apart. The NEB-KO GAS generated less stress than the WT GAS throughout the isometric exercise bout, although the difference became most pronounced beyond Iso6. B: change in isometric stress production across the isometric exercise bout expressed as an absolute change in stress. C: change in isometric stress production across the isometric exercise bout expressed as a percentage of Iso1 stress. Student's t-test indicated that the NEB-KO GAS was more vulnerable than the WT GAS to stress decline during cyclic activation. *P < 0.05 compared with WT; **P < 0.01 compared with WT.

Fig. 5.

Length-tension curves of the WT and NEB-KO GAS at P1 (n = 8 WT and 8 NEB-KO). Mechanical function was expressed as maximum isometric stress (A) or a fraction of peak tension (B). Note the leftward shift and narrowing of the length-tension curve of the NEB-KO GAS relative to the WT GAS, which are consistent with shorter thin filament length in NEB-KO muscle. Despite the difference in thin filament length, “neutral” sarcomere lengths in both the WT GAS (n = 4) and NEB-KO GAS (n = 4) were identical (inset).

Fig. 6.

Sarcomere length in the WT and NEB-KO GAS at P1 either at slack length or stretched by +1 fiber length (n = 4 WT slack, 4 WT stretched, 4 NEB-KO slack, and 4 NEB-KO stretched). Fisher's PLSD test revealed no significant difference in sarcomere length at either slack or stretched conditions, indicating that deletion of NEB does not interfere with the ability of the GAS to distribute tensile strains among its sarcomeres.

Fig. 7.

Parabolic regression analysis of the length-tension properties of the WT and NEB-KO GAS at P1 (n = 8 WT and 8 NEB-KO). A and B: representative length-tension curves from tests of an individual WT GAS (A) and NEB-KO GAS (B) with parabolic regression applied to the data. The regression equations in A and B were y = −1.98x² − 0.16x + 0.95 (R² = 0.97) and y = −1.28x² − 0.80x + 0.84 (R² = 0.99), respectively. All regression curves were required to meet the goodness-of-fit criterion of R² > 0.9, or else they were excluded from this study and further analysis. C: optimum relative muscle length (L_opt) of the GAS. D: Full-width at half-maximum (FWHM) of the length-tension curves generated for the GAS. Student's t-test revealed lower L_opt and reduced FWHM in the NEB-KO GAS, both of which are qualitatively consistent with shorter thin filament length in NEB-KO muscle. *P < 0.05 compared with WT; **P < 0.01 compared with WT.

Fig. 8.

Passive length-tension curves of the WT and NEB-KO GAS at P1 (n = 8 WT and 8 NEB-KO). Student's t-test found no significant differences in passive tensile stress at any muscle length, although there was a trend toward lower passive stresses in the NEB-KO GAS.