Quadriceps myopathy caused by skeletal muscle-specific ablation of β(cyto)-actin

Abstract

Quadriceps myopathy (QM) is a rare form of muscle disease characterized by pathological changes predominately localized to the quadriceps. Although numerous inheritance patterns have been implicated in QM, several QM patients harbor deletions in dystrophin. Two defined deletions predicted loss of functional spectrin-like repeats 17 and 18. Spectrin-like repeat 17 participates in actin-filament binding, and thus we hypothesized that disruption of a dystrophin-cytoplasmic actin interaction might be one of the mechanisms underlying QM. To test this hypothesis, we generated mice deficient for β(cyto)-actin in skeletal muscles (Actb-msKO). Actb-msKO mice presented with a progressive increase in the proportion of centrally nucleated fibers in the quadriceps, an approximately 50% decrease in dystrophin protein expression without alteration in transcript levels, deficits in repeated maximal treadmill tests, and heightened sensitivity to eccentric contractions. Collectively, these results suggest that perturbing a dystrophin-β(cyto)-actin linkage decreases dystrophin stability, which results in a QM, and implicates β(cyto)-actin as a possible candidate gene in QM pathology.

Fig. 1.

Skeletal muscle-specific ablation of β_cyto-actin. (A) Representative western blots of actin isoform expression from actin-rich eluates of skeletal muscle from control and Actb-msKO mice. (B) Quantification of actin isoform expression from quadriceps extracts from three control (WT) and three Actb-msKO mice at 3 months of age. α_st-actin served as a loading control. Actb-msKO skeletal muscle showed a 59% decrease in β_cyto-actin expression. Error bars represent s.e.m. (C) Cryosections of 10 μm from control and Actb-msKO quadriceps were stained with DAPI (blue), β_cyto-actin (green) and laminin (red). Control skeletal muscle showed sarcolemmal staining, which was absent in Actb-msKO skeletal muscle. Endomyosial capillaries (arrowheads) showed strong β_cyto-actin immunoreactivity, probably explaining the remaining β_cyto-actin signal in actin-rich elutes of Actb-msKO muscle. Scale bar: 50 μm.

Fig. 2.

Quadriceps myopathy in Actb-msKO mice. (A–F) Representative 10 μm sections stained with hematoxylin and eosin, and respective quantification of the proportion of centrally nucleated fibers from control and Actb-msKO tibialis anterior (A,B), triceps (C,D) and quadriceps (E,F) muscles at 1, 3, 6 and 12 months of age (n=3 mice per genotype per timepoint). *P≤0.05. Error bars represent s.e.m. Scale bar: 100 μm.

Fig. 3.

β_cyto-actin colocalizes with dystrophin and is necessary for dystrophin stability. (A) Cryosections of 10 μm from control quadriceps were stained with β_cyto-actin (green) and dystrophin (red). (B) Higher magnification image of box from A. Dystrophin and β_cyto-actin colocalize at the sarcolemma. (C) Representative western blots of dystrophin (Dys), β-dystroglycan (DG), utrophin(Utr) and α-sarcoglycan (SG) from SDS-extracted skeletal muscle. Post-transfer Coomassie Brilliant Blue (CBB)-stained gel shows equivalent loading. The most prominent band represents myosin heavy chain. (D) Quantification of dystrophin, β-dystroglycan, utrophin and α-sarcoglycan expression from skeletal muscle of 3- and 12-month-old Actb-msKO mice. Dystrophin expression was decreased approximately 50%, β-dystroglycan expression was decreased approximately 15–20%, utrophin levels were increased 6–13%, and α-sarcoglycan expression was decreased approximately 28% compared with control skeletal muscle. Error bars represent s.e.m. (E) Ethidium bromide-stained agarose gel showing quantitative RT-PCR products of dystrophin and GAPDH. (F) Quantification of dystrophin transcript normalized to GAPDH. Ablation of β_cyto-actin did not alter dystrophin transcript levels. (G) Cryosections of 10 μm from control and Actb-msKO quadriceps were stained with dystrophin (green) and DAPI (blue). Ablation of β_cyto-actin led to decreased dystrophin staining at the sarcolemma. (H) Longitudinal sections of 20 μm from control and Actb-msKO quadriceps were stained with dystrophin (green) and DAPI (blue). Costamere organization is preserved in Actb-msKO skeletal muscle.

Fig. 4.

Ablation of β_cyto-actin does not alter cage behavior or force-generating capacity. (A–C) Quantification of 24-hour cage behavior in control (n=6) and Actb-msKO (n=7) mice indicated there was no significant decrease in the number of jumps (A), ambulatory distance (B), or the number of vertical counts (C) in Actb-msKO mice at 3–4 months of age. (D–F) Whole body tension (D), twitch force (E) and specific force (F) were not altered by ablation of β_cyto-actin. Error bars represent s.e.m.

Fig. 5.

Actb-msKO skeletal muscle shows increased susceptibility to stressful stimuli. (A) Repeated maximal treadmill tests show a significant reduction in running time on the second trial in Actb-msKO mice. (B) Quantification of serum CK activity following treadmill test. Actb-msKO did not exhibit elevated sarcolemmal permeability. (C) Percentage force drop following five eccentric contractions on isolated extensor digitorum longus muscles. At 3 and 12 months of age, Actb-msKO muscle was significantly more susceptible to eccentric-contraction-induced force loss. (D) Quantification of force production from an in vivo eccentric contraction protocol testing the anterior leg compartment of control and Actb-msKO mice indicates a statistically significant column factor exists as determined by Two-way ANOVA. (E) Contraction number when force drop percentage was within 2% of final force drop percentage. Actb-msKO mice reached their final force drop percentage significantly faster than control mice. *P≤0.05 compared with control values, as determined by t-test.