Myostatin is increased and complexes with amyloid-β within sporadic inclusion-body myositis muscle fibers

Abstract

Myostatin is a negative regulator of muscle mass and strength. Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease of older persons and is characterized by pronounced muscle wasting. s-IBM is of unknown etiology and pathogenesis, and it lacks definitive treatment. We have now demonstrated in samples from 12 s-IBM biopsies that: (1) by light and electron microscopic immunocytochemistry, myostatin/myostatin precursor is accumulated within muscle fibers and co-localized with amyloid-beta (Abeta); (2) by immunoblots, both myostatin and myostatin precursor are increased; and (3) by immunoprecipitation, myostatin precursor complexes with Abeta. Our study suggests that myostatin/myostatin precursor, either alone, or bound to Abeta, may play a novel role in the pathogenesis of s-IBM.

AβPP-overexpression and proteasome inhibition increase αB-crystallin in cultured human muscle: Relevance to inclusion-body myositis

“…To evaluate if in s-IBM muscle fibers αBC physically associates with AβPP and/or Aβ, a combined immunoprecipitation-immunoblot technique was performed, essentially as above and as described [4,[23][24][25]. In brief, 100 μg of total muscle protein from s-IBM and control muscle biopsies were individually immunoprecipitated in precipitation buffer containing 5 μg of anti-αBC antibody, The immunoprecipitates were electrophoresed: a) on 12% NuPAGE gel in MES-SDS NuPAGE buffer to visualize Aβ oligomers, and b) on 4-12% NuPAGE gel in MOPS-SDS NuPAGE buffer to visualize AβPP.…”

NOGO is increased and binds to BACE1 in sporadic inclusion-body myositis and in AβPP-overexpressing cultured human muscle fibers

“…Double-immunoXuorescence utilized goat polyclonal antibody against Nogo-A/B in combination with one of the following: (a) mouse monoclonal antibody against BACE1, or (b) rabbit polyclonal antibody against caveolin-1. To block non-speciWc binding of an antibody to Fc receptors, all sections were preincubated with 10% normal goat or donkey serum [15,26,37,40]. Omission of the primary antibody or its replacement with non-immune sera or irrelevant antibody was the control for staining speciWcity, and these always produced negative results.…”

“…Immunoblots of s-IBM and control muscle biopsies were performed as previously described [15,26,37,40]. In brief, 20 m-thick frozen muscle sections were collected at ¡25°C and rapidly homogenized on ice in RIPA buVer and protease inhibitor cocktail (Roche Diagnostic GmBH, Mannheim, Germany).…”

“…Single-and double-labeled gold immuno-electron-microscopy were performed on 10-m unWxed frozen sections of s-IBM biopsies adhered to the bottom of 35-mm Petri dishes, as described [3,15,26,37,40]. Two diVerent species-speciWc secondary antibodies, conjugated to either 12 or 6 nm gold particles (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), were used in this study.…”

“…ImmunoXuorescence was performed on 10-m-thick transverse sections of fresh-frozen muscle biopsies of all the diseases and controls, as described [3,15,26,37,40], using the same antibodies that were used for the immunoblots. Double-immunoXuorescence utilized goat polyclonal antibody against Nogo-A/B in combination with one of the following: (a) mouse monoclonal antibody against BACE1, or (b) rabbit polyclonal antibody against caveolin-1.…”