Cytoplasmic γ-actin expression in diverse animal models of muscular dystrophy

Abstract

We recently showed that cytoplasmic γ-actin (γ cyto -actin) is dramatically elevated in striated muscle of dystrophin-deficient mdx mice. Here we demonstrate that γ cyto -actin is markedly increased in golden retriever muscular dystrophy (GRMD), which better recapitulates the dystrophinopathy phenotype in humans. γ cyto -Actin was also elevated in muscle from α-sarcoglycan null mice, but not in several other dystrophic animal models, including mice deficient in β-sarcoglycan, α-dystrobrevin, laminin-2, or α7 integrin. Muscle from mice lacking dystrophin and utrophin also expressed elevated γ cyto -actin, which was not restored to normal by transgenic overexpression of α7 integrin. However, γ cyto -actin was further elevated in skeletal muscle from GRMD animals treated with the glucocorticoid prednisone at doses shown to improve the dystrophic phenotype and muscle function. These data suggest that elevated γ cyto -actin is part of a compensatory cytoskeletal remodeling program that may partially stabilize dystrophic muscle in some cases where the dystrophinglycoprotein complex is compromised.

Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle

“…Of note, γ cyto -actin staining was markedly brighter in TA and soleus muscles from mdx and mdx /mTR mice (Figure 8), consistent with increased γ cyto -actin levels in dystrophic muscles (Hanft et al. , 2006, 2007). Both increased γ cyto -actin and correspondingly increased Tmod3 levels were confirmed by Western blotting (Supplemental Figure S4).…”

“…Dystrophic muscles exhibit dramatic up-regulation of γ cyto -actin, presumably as a compensatory fortification response to sarcolemmal fragility ( Hanft et al. , 2006 , 2007 ). On the basis of these observations, we speculated that depletion of sarcomeric Tmods from dystrophic muscle would result in incorporation of excess γ cyto -actin into the longer thin filaments, as observed in myofibrils from γ cyto -actin–overexpressing muscle ( Jaeger et al.…”

Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

“…For muscle fractionation experiments, TA muscles were dissected in icecold relaxing buffer, relaxed for 24 h at 4°C, and homogenized with a Tissumizer (Teledyne Tekmar) in 10 vol of ice-cold rigor buffer (100 mM KCl, 2 mM MgCl 2 , 10 mM K 3 PO 4 , 1 mM EDTA, and 1 mM DTT, pH 7.0) supplemented with protease inhibitor cocktail (1:1,000; Sigma-Aldrich), extrasarcomeric  cyto -actin has been thought to play a stabilizing and/or force-transmitting role at the sarcolemma Renley et al, 1998;Rybakova et al, 2000;Hanft et al, 2006Hanft et al, , 2007. However, experiments involving targeted genetic perturbation of  cyto -actin expression in skeletal muscle have produced inconsistent results.…”

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms

“…Mitochondrial associated membranes (MAMs) are a functionally important interface between mitochondria and the sarco-/endo-plasmic reticulum involved in mitochondrial respiration, Ca 2+ crosstalk, cell death signaling, lipid metabolism, and mitochondrial dynamics [30–34]. Therefore, we biochemically isolated MAM, mitochondrial, and endoplasmic reticulum fractions from wildtype mouse liver [35] and performed western blot analysis to identify the fractions containing β cyto - and γ cyto -actin immunoreactivity using previously established antibodies specific to each isoform (Figure 4) [24,12,36,37]. As expected, both γ cyto - and β cyto -actin were present in the cytosolic fraction (verified by tubulin immunoreactivity), weakly present in the crude mitochondrial fraction (identified by cytochrome C), and most enriched in the isolated MAM fraction verified by the presence of FACL4, a lipid enzyme localized to the MAM [38,39].…”

“…Samples were transferred onto PVDF membrane for 1h at 100v. Antibodies to ATP5A (ab14748; Abcam, Milton, Cambridge, UK), BiP (C50B12; Cell Signaling Technology, Danvers, MA, USA), β cyto -Actin AC-15 (A5441; Sigma-Aldrich, St. Louis, MO, USA), γ cyto -Actin (mAb 2–4; as published in [37]), Cytochrome C (ab13575; Abcam, Milton, Cambridge, UK), calsequestrin (3516; Abcam, Milton, Cambridge, UK), Fis1 (HPA017430; Sigma-Aldrich, St. Louis, MO, USA), OPA1 (612606; BD Biosciences, San Jose, CA, USA), MFN2 (D2D10; Cell Signaling Technology, Danvers, MA, USA), FACL4 (ab155282; Abcam, Milton, Cambridge, UK), desmin (D1033; Sigma-Aldrich, St. Louis, MO, USA), GAPDH (Sigma-Aldrich, St. Louis, MO, USA), IP3R-3 (610312; BD Biosciences), IRE1α (14C10; Cell Signaling Technology, Danvers, MA, USA), PDI (C81H6; Cell Signaling Technology, Danvers, MA, USA), PERK (D11A8; Cell Signaling Technology, Danvers, MA, USA), SDHA (ab14715; Abcam, Milton, Cambridge, UK), SERCA1 (ab2819; Abcam, Milton, Cambridge, UK), sAmk1 (ARP42566-T100; Aviva Systems Biology, San Diego, CA, USA), Tubulin B512 (T6074; Sigma-Aldrich, St. Louis, MO, USA), and UQCR (ab110252; Abcam, Milton, Cambridge, UK), were used at the recommendation concentrations. Membranes were imaged on a Licor Odyssey (Lincoln, NE, USA).…”