Cytoplasmic gamma-actin contributes to a compensatory remodeling response in dystrophin-deficient muscle

Abstract

Dystrophin mechanically links the costameric cytoskeleton and sarcolemma, yet dystrophin-deficient muscle exhibits abnormalities in cell signaling, gene expression, and contractile function that are not clearly understood. We generated new antibodies specific for cytoplasmic gamma-actin and confirmed that gamma-actin most predominantly localized to the sarcolemma and in a faint reticular lattice within normal muscle cells. However, we observed that gamma-actin levels were increased 10-fold at the sarcolemma and within the cytoplasm of striated muscle cells from dystrophin-deficient mdx mice. Transgenic overexpression of the dystrophin homologue utrophin, or functional dystrophin constructs in mdx muscle, restored gamma-actin to normal levels, whereas gamma-actin remained elevated in mdx muscle expressing nonfunctional dystrophin constructs. We conclude that increased cytoplasmic gamma-actin in dystrophin-deficient muscle may be a compensatory response to fortify the weakened costameric lattice through recruitment of parallel mechanical linkages. However, the presence of excessive myoplasmic gamma-actin may also contribute to altered cell signaling or gene expression in dystrophin-deficient muscle.

Fig. 1.

Retention of costameric actin on peeled sarcolemma from transgenic mdx mice. (A) The domain structure of dystrophin expressed in wild-type mice, the transgenic proteins expressed on the mdx background, and the phenotype of each transgenic line. The shaded regions identify the actin filament binding (ABD) or glycoprotein complex binding (CRCT) domains. H1–H4, hinge modules 1–4; SR1–SR24, spectrin repeats 1–24. (B) Confocal images of peeled sarcolemma stained with Alexa Fluor 568-phalloidin. (Scale bar: 20 μm.)

Fig. 2.

Characterization of γ-actin-specific antibodies. (A) Gels equally loaded with purified sarcomeric α-actin, platelet actin (a 4:1 mixture of β- and γ-isoforms), and brain γ-actin were either stained with Coomassie blue (CB) or transferred to nitrocellulose and Western-blotted with mAbs specific for α- or β-actin. (B) Western blots were stained with affinity-purified pAbs raised against the unique amino-terminal peptide sequence of cytoplasmic γ-actin or mAbs from mice immunized with both purified brain γ-actin and the amino-terminal sequence of cytoplasmic γ-actin. Where indicated, 10 μg/ml of either the amino-terminal γ-actin or β-actin peptide was preincubated with the γ-actin antibodies. (C) Purified platelet β/γ-actin was resolved by 2D electrophoresis and transferred to poly(vinylidene difluoride), and the blot was serially probed with γ-actin pAbs, γ-actin mAbs, and finally β-actin mAbs. (D) Ten-micrometer transverse and longitudinal cryosections of control tibialis anterior muscle stained with the γ-actin mAb. (Scale bar: 50 μm.)

Fig. 3.

γ-Actin levels in mdx versus control muscle. (A) Western blots of control and mdx skeletal muscle homogenates and high-speed pellets or supernatants were stained with γ-actin pAb 7577 or mAb sarcomeric α-actin. (B) γ-Actin immunoreactivity in homogenates from control (Con) and mdx muscle resolved by 2D electrophoresis. (C) γ-Actin immunoreactivity in supernatants from control and mdx skeletal muscle before (Sup.) and after (Void) application to DNase I matrix and in the SDS eluate from the DNase I matrix.

Fig. 4.

Developmental time course and localization of γ-actin in control (Con) and mdx muscle. (A) Western blots of DNase I-enriched supernatants from skeletal muscle of different age control and mdx mice were stained for γ- and α-actin. (B) Western blots of KCl-washed microsomes from control and mdx skeletal muscle stained for γ-actin and Na⁺/K⁺-ATPase immunoreactivity. (C) Transverse cryosections of control and mdx tibialis anterior muscle stained for γ-actin (green) and laminin (red). (Scale bar: 50 μm.)

Fig. 5.

Tissue specificity of elevated γ-actin in mdx mice and restoration to normal levels by transgene expression. (A) Western blots of DNase I-enriched supernatants from skeletal muscle of control (Con), mdx, and transgenic mdx mice expressing utrophin or dystrophin constructs depicted in Fig. 1 were stained for γ- and α-actin. (B) γ-Actin immunoreactivity in SDS extracts from organs of control and mdx mice. (C) Western blots of DNase I-enriched supernatants of cardiac muscle from control, mdx, and Fiona transgenic mdx mice stained for γ- and α-actin.