Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)

Abstract

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Copyright 2009 Elsevier Inc. All rights reserved.

Figures

Figure 1

A) Schematic of a structural organization of skeletal muscle and sarcomere (bottom). B) Schematic of the human nebulin sequence. Nebulin has a highly modular structure, with in the central region (M9–M162) seven modular repeats arranged into twenty-two super-repeats. The four NEM2 patients studied here harbor mutations in exon 45 (one patient) that is part of super-repeat 7, and exon 55 (three patients) that is part of super-repeat 9.

Figure 2

A) SDS-PAGE (for typical example gel result of a control and NM patient, see left panel; NM patient ID: 174-1) revealed that nebulin protein levels (normalized to MHC) in nebulin-based NM muscle are reduced to ~20% of the nebulin levels found in control muscle (middle panel). Actin protein levels in nebulin-based NM muscle are reduced to ~80% of controls (right panel). B) Expression analysis of proteins regulating sarcomeric force generation (left panel shows Western blot result for a control and NM patient; NM patient ID: 4–5). Total protein levels of tropomyosin, relative to actin, were slightly increased in NM. Total protein levels of troponin C, I, and T, relative to actin, were on average reduced but this reduction was not statistically significant. In light grey bar the nebulin/actin ratio is shown, illustrating the predominant decrease in nebulin protein level. Note that NM muscle showed a shift towards slow isoforms of the troponins (see text for details). C) Specialized SDS-PAGE to separate MHC isoforms in muscle from the four NM patients and four control subjects. Note the strong predominance of myosin heavy chain slow in NM muscle, whereas control muscle expresses slow, 2A, and 2X isoforms. NM patient IDs, from left to right: 76-1, 4-5, 4-4, 174-1.

Figure 3

Myofibrils from controls and nebulin-based NM patients stained for actin. A) Actin staining with phalloidin shows broad and homogenous staining in control myofibrils, whereas actin staining intensity in NM myofibrils gradually decreases from Z-disk towards the middle of the sarcomere. Analysis of phalloidin line scan intensities (B) revealed significantly reduced average thin filament (TF) lengths in NM myofibrils (C).

Figure 4

Force–Ca2+ characteristics of skinned muscle from NM and control muscle (note that the results from NM muscle fibers were compared to those from control muscle fibers expressing solely MHC slow). A) Typical chart recording showing the force response to incremental Ca2+ concentrations in a NM (174-1) fiber preparation B) Left panel: the force generated in response to incubation with incremental increase of [Ca2+]; note the rightward shift of the force–Ca2+ relationship in NM vs control muscle. Middle panel, the Ca2+ concentration needed for 50% of maximal force generation was significantly higher (i.e., lower pCa50) in NM vs control muscle, whereas no difference was found in the Hill coefficient (nH, right panel).

Figure 5

Tension cost of NM and control fibers. A) Example of a maximally activated NM (174-1) fiber bundle (pCa 4.5) with developed force at bottom and [ATP] at the top. The slope of the [ATP] vs time trace was divided by fiber volume (in mm3) to determine ATP consumption rate. B) ATP consumption rate was normalized to tension to determine the tension cost. Tension cost is significantly higher in NM fibers.

Figure 6

Ktr measurements of NM and control fibers. A) Example of ktr measurement at pCa 4.5 with superimposed the results of a NM (174-1) and a control fiber. B) Ktr is significantly lower in NM compared to control fibers.

Figure 7

Schematic of the mechanisms underlying muscle weakness in NEM2 patients. In NEM2 patients, force is depressed by (1) decreased and non-uniform thin filament lengths to reduce the amount on thin-thick filament overlap in a sarcomere length dependent manner (for details see Ottenheijm et al. [9]); (2) altered cross bridge cycling kinetics to reduce the fraction of force generating cross bridges; (3) a decrease of myofilament calcium sensitivity to reduce submaximal force generation.