Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency

Abstract

Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin's N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.

Figures

Figure 1.

(A) Schematic of a skeletal muscle sarcomere. (B) Schematic of the human nebulin sequence. Nebulin has a highly modular structure, within the central region (M9-M162) seven modular repeats arranged into twenty-two super-repeats. Exon 55 codes for 35 amino acids that are part of repeat 5 and 6 (super-repeat 9).

Figure 2.

(A) Western blotting studies with antibodies against nebulin’s N-terminus (Neb-N), C-terminus (Neb-C) and serine-rich domain (Neb-Sr). Nebulin expression is reduced in NM-NEB muscle, with the most severe reduction at the N-terminal end. Bottom: titin and myosin loading controls. (B and C) SDS–PAGE revealed approximately 8-fold reduced nebulin expression (normalized to myosin) in NM-NEB muscle.

Figure 3.

(A) Left: Force–sarcomere length relation of control fibers expressing slow and fast isoforms of myosin heavy chain. Right: SDS–PAGE of muscle homogenates from controls reveal expression of slow and fast (2A, and 2X) myosin heavy chain isoforms. NM-NEB homogenates contain only slow isoforms of myosin heavy chain. The majority of single fibers isolated from control biopsies expressed only a single myosin heavy chain isoform. (B) Left: The force–sarcomere length relation of control fibers has a characteristic force plateau followed by a descending limb. The force–sarcomere length relation of NM-NEB fibers is shifted leftward compared with control fibers, and the force plateau is absent. Right: Plotting stress (force normalized to fiber CSA) versus sarcomere length illustrates >65% reduction in stress generating capacity in NM fibers. (C) The force–sarcomere length relation of NebKO muscle is also shifted to the left (as found for NM fibers in B). Maximal stress is more than 50% reduced in NebKO compared with wt muscle. Data are based on ∼50 fibers per group.

Figure 4.

(A) Myofibrils from controls and NM-NEB patients stained for α-actinin and tropomodulin (Tmod). Left: Note the tropomodulin doublet in the middle of the sarcomere in control myofibrils, but diffuse staining in NM-NEB myofibrils. Middle: overlay of line scan intensity profile of α-actinin and tropomodulin. Right: The distance between tropomodulin staining (measured across the Z-disk, and indicated as Tmod-α-actinin-Tmod) is significantly reduced in NM-NEB myofibrils. (B) Alpha-actinin and tropomodulin staining of NebKO myofibrils is similar to the staining pattern of NM-NEB myofibrils in (A). (C) Left: Actin staining with phalloidin shows broad and homogenous staining in control myofibrils, whereas actin staining intensity in NM-NEB myofibrils gradually decreases from Z-disk towards the middle of the sarcomere. Middle and right: Analysis of phalloidin line scan intensities revealed significantly reduced average thin filament (TF) lengths in NM-NEB myofibrils. (D) Actin staining of NebKO myofibrils shows high resemblance with staining of NM myofibrils shown in (C). Data are based on ∼15 fibers per group.

Figure 5.

(A) Electron micrograph of control and NM-NEB myofibrils. Note that the H-zone (thin filament devoid zone) is only visible in control sarcomeres. (B) Similar findings were obtained in NebKO myofibrils. (C) Control and NM-NEB myofibrils labeled with phalloidin-biotin, followed by streptavidin-nanogold and silver enhancement (Bars: 0.5 µm). Bottom: Schematic showing how the distance between silver grains and the Z-disk was defined. (D) Histograms of obtained distances for control and NM-NEB myofibrils. Results suggest that in NM-NEB myofibrils thin filaments vary in length between ∼0.4 and ∼1.3 µm. (E) Predicted force–sarcomere length relations for control and NM-NEB muscle, assuming that thin filament length in NM-NEB muscle varies between ∼0.4 and 1.3 µm, and is uniform in control muscle at ∼1.3 µm. Note the leftward shift of the predicted force–sarcomere length relation and the absence of a force plateau in NM-NEB muscle. (F) The predicted force–sarcomere length relation of wt and NebKO muscle (based on previously published phalloidin labeling patterns) (6). The shift of the NebKO muscle is similar to that of NM-NEB fibers.