Slawomir Wojcik

Endoplasmic reticulum stress induces myostatin precursor protein and NF-κB in cultured human muscle fibers: Relevance to inclusion body myositis

Abstract Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-κB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-κB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-κB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.

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

Increased amyloid-β precursor protein (AβPP) and amyloid-β (Aβ) accumulation appear to be upstream steps in the pathogenesis of sporadic inclusion-body myositis (s-IBM). BACE1, participating in Aβ production is also increased in s-IBM muscle fibers. Nogo-B and Nogo-A belong to a family of integral membrane reticulons, and Nogo-B binding to BACE1 blocks BACE1 access to AβPP, decreasing Aβ production. We studied Nogo-B and Nogo-A in s-IBM muscle and in our IBM muscle culture models, based on AβPP-overexpression or ER-stress-induction in cultured human muscle fibers (CHMFs). We report that: (1) in biopsied s-IBM fibers, Nogo-B is increased, accumulates in aggregates, is immuno-co-localized with BACE1, and binds to BACE1; Nogo-A is undetectable. (2) In CHMFs, (a) AβPP overexpression increases Nogo-B, Nogo-A, and BACE1, (b) ER stress increases BACE1 but decreases Nogo-B and Nogo-A, (c) Nogo-B and Nogo-A associate with BACE1. Accordingly, two novel mechanisms, AβPP overexpression and ER stress, are involved in Nogo-B and Nogo-A expression in human muscle. We propose that in s-IBM muscle the Nogo-B increase may represent an attempt by muscle fiber to decrease Aβ production. However, the increase of Nogo-B seems insufficient because Aβ continues to accumulate and the disease progresses. We propose that manipulations, which increase Nogo-B in s-IBM muscle might offer a new therapeutic opportunity.

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

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-β (Aβ); (2) by immunoblots, both myostatin and myostatin precursor are increased; and (3) by immunoprecipitation, myostatin precursor complexes with Aβ. Our study suggests that myostatin/myostatin precursor, either alone, or bound to Aβ, 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

Amyloid-beta precursor protein (AbetaPP) and its fragment amyloid-beta (Abeta) are increased in s-IBM muscle fibers and appear to play an important role in the pathogenic cascade. alphaB-Crystallin (alphaBC) was shown immunohistochemically to be accumulated in s-IBM muscle fibers, but the stressor(s) influencing alphaBC accumulation was not identified. We now demonstrate, using our experimental IBM model based on genetic overexpression of AbetaPP into cultured normal human muscle fibers, that: (1) AbetaPP overexpression increased alphaBC 3.7-fold (p=0.025); (2) additional inhibition of proteasome with epoxomicin increased alphaBC 7-fold (p=0.002); and (3) alphaBC physically associated with AbetaPP and Abeta oligomers. We also show that in biopsied s-IBM muscle fibers, alphaBC was similarly increased 3-fold (p=0.025) and physically associated with AbetaPP and Abeta oligomers. We propose that increased AbetaPP is a stressor increasing alphaBC expression in s-IBM muscle fibers. Determining the consequences of alphaBC association with Abeta oligomers could have clinical therapeutic relevance.

In inclusion-body myositis muscle fibers Parkinson-associated DJ-1 is increased and oxidized

Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. The muscle-fiber molecular phenotype exhibits similarities to both Alzheimer-disease (AD) and Parkinson-disease (PD) brains, including accumulations of amyloid-beta, phosphorylated tau, alpha-synuclein, and parkin, as well as evidence of oxidative stress and mitochondrial abnormalities. Early-onset autosomal-recessive PD can be caused by mutations in the DJ-1 gene, leading to its inactivation. DJ-1 has antioxidative and mitochondrial-protective properties. In AD and PD brains, DJ-1 is increased and oxidized. We studied DJ-1 in 17 s-IBM and 18 disease-control and normal muscle biopsies by: (1) immunoblots of muscle homogenates and mitochondrial fractions; (2) real-time PCR; (3) oxyblots evaluating DJ-1 oxidation; (4) light- and electron-microscopic immunocytochemistry. Compared to controls, in s-IBM muscle fibers DJ-1 was: (a) increased in the soluble fraction, monomer 2-fold (P = 0.01), and dimer 2.8-fold (P = 0.004); (b) increased in the mitochondrial fraction; (c) highly oxidized; and (d) aggregated in about 15% of the abnormal muscle fibers. DJ-1 mRNA was increased 3.5-fold (P = 0.034). Accordingly, DJ-1 might play a role in human muscle disease, and thus not be limited to human CNS degenerations. In s-IBM muscle fibers, DJ-1 could be protecting these fibers against oxidative stress, including protection of mitochondria.

Myostatin precursor protein is increased and associates with amyloid-β precursor protein in inclusion-body myositis culture model

Myostatin, also called growth and differentiation factor-8, is a member of the transforming growth factor-b superfamily [1,2]. Myostatin is a secreted protein considered a negative regulator of muscle growth during development and of muscle mass during adulthood [1]. In mouse models, knocking out the myostatin gene, overexpressing proteins neutralizing myostatin, or natural mutations of the myostatin gene cause increased muscle mass [1]. In cattle, naturally occurring myostatin gene mutations leading to inactive protein cause ‘double-muscle cattle’ [2]. Recently reported was a child in whom a homozygous myostatin gene mutation that results in reduced production of myostatin protein, was associated with increased muscle bulk and strength [3]. Conversely, mature myostatin protein has been reported increased in muscle tissue of patients with HIV-associated muscle wasting [4], and increased myostatin-precursor protein (MstnPP) mRNA reported in muscle wasting associated with osteoarthritis [5]. Within muscle fibres, myostatin is synthesized as a MstnPP [6,7]. MstnPP and its mRNA, and mature myostatin, are predominantly expressed in skeletal muscle tissue (reviewed in [1]). MstnPP, a 375-amino-acid protein translated from a 3.1 kb mRNA [4], consists of three structural domains: a signal sequence; an N-terminal 28 kDa propeptide, also referred to as latency associated peptide [7]; and a C-terminal 12 kDa mature myostatin peptide [6,7]. Intracellular processing of myostatin from MstnPP has been proposed to occur through furin [6,8]. We recently showed in biopsied sporadic inclusion-body myositis (s-IBM) muscle fibres that both MstnPP and myostatin dimer were significantly increased, and MstnPP was physically associated with amyloid-b precursor protein (AbPP) [9]. Moreover, by lightand electron-microscopic immunocytochemistry, MstnPP/myostatin colocalized with amyloid-b (Ab)/AbPP [9]. s-IBM is severely progressive, the most common muscle disease of older persons, and there is no successful treatment [10]. Histological hallmarks of s-IBM include: (1) vacuolar degeneration and atrophy of muscle fibres, accompanied by intramuscle-fibre accumulations of ubiquitinated protein aggregates, including Ab/AbPP; (2) muscle-fibre atrophy; and (3) mononuclear lymphocytic inflammation [10–12]. Recently demonstrated in s-IBM muscle fibres were inhibition of 26S proteasome activity and presence of aggresomes [13]. Accumulation of AbPP/Ab appears to be an early upstream step in the s-IBM pathogenesis, because: (i) abnormal accumulation of AbPP epitopes appears to precede other abnormalities in IBM muscle fibres [11]; and (ii) several aspects of the s-IBM phenotype, including Ab accumulation, proteasome inhibition and aggresome formation, were produced in cultured normal human muscle fibres (CHMFs) after long-term overexpression of AbPP in them [13–15]. The latter provides a useful IBM human-muscle tissue-culture model. The aim of the present study was to utilize this model to investigate possible mechanisms responsible for increased MstnPP and myostatin within the biopsied s-IBM muscle fibres. We cultured human muscle fibres from satellite cells obtained from six normal diagnostic muscle biopsies, as described [13–15] and referenced therein. Into well-differentiated 3-week-old cultured muscle fibres we transferred a 3 kb human AbPP-cDNA encoding 751-AbPP using a replication-deficient adenovirus vector at 0.3 ¥ 10 pfu/ml culture medium, as detailed previously [14,15]. In addition, we treated some of the cultures with 1 mm epoxomicin (Biomol Research Laboratories, Plymouth Meeting, PA, USA) [13], an irreversible proteasome inhibitor [16]. Four days after AbPP gene transfer and 24 h after epoxomicin treatment, control and AbPP-overexpressing CHMFs (AbPP+ CHMFs) were processed for lightand electron-microscopic immunocytochemistry, immunoblotting, combined immunoprecipitation/immunoblotting, and reverse transcriptase polymerase chain reaction (RT-PCR), as described [13–15,17]. For all the myostatin studies, we used an anti-myostatin rabbit polyclonal antibody (Chemicon,