Figure 8. MstnPP aggregates and protofibrils (PF) are cytotoxic to C2C12 mouse myoblasts. C2C12 cytotoxicity assay using the WST-1 reagent where the difference in absorbance at 450 and 630 nm directly correlates to cell density after incubation with increasing concentrations of MstnPP soluble aggregates (SA, 5–25 mM), protofibrils (PF, 5–25 mM), 25 mM MstnPP dimer and 25 mM PF/F (fibrils). Concentrations are expressed as monomer equivalents. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Error bars represent the standard error of the mean for triplicate samples from two independent experiments. Statistical significance was calculated using a paired Student’s t-test where ** P,0.01 and * P,0.05. doi:10.1371/journal.pone.0009170.g008 production of misfolded proteins overwhelms the UPR, protein aggregation may result in the generation of cytotoxic amyloid protofibrils [41,42] and mature amyloid fibrils, contributing to amyloid diseases, including sIBM [32]. After refolding of MstnPP, misfolded yet soluble aggregates that form amyloid protofibrils and fibrils at a lowered pH can be observed. Although elevated temperature has been used to produce mature MstnPP fibrils quickly in this study, a physiological temperature allows protofibril and mature fibril formation over an extended time period; by comparison, fibril formation is expected to occur over many years in vivo. sIBM is characterised by amyloid aggregation of the Ab protein and severe muscle atrophy [30,33]. Previous studies have shown that MstnPP associates with Ab fibrils in diseased cells [31] and that ER stress results in upregulation of MstnPP in a cultured human sIBM-model [45]. Increased activity of the myostatin growth factor following MstnPP upregulation may contribute to atrophy in sIBM, however another study showed MstnPP accumulated with Ab/AbPP within aggresomes following ER stress by proteasome inhibition in CHMFs [46]. This accumula- tion in aggresomes may prevent processing of MstnPP and decrease levels of circulating growth factor, as mature myostatin growth factor is absent in immunoblots of CHMFs transiently expressing AbPP [46]. The in vitro formation of MstnPP aggregates and amyloid species suggests a similar phenomenon could occur in vivo having a negative effect on cell viability and/or assisting in Ab fibrillogen- esis. The cytotoxicity exhibited by MstnPP aggregates and protofibrils in C2C12 cells supports a mechanism in which aggregation by MstnPP in sIBM contributes to muscle degener- ation. This may occur independently of atrophy resulting from increased myostatin growth factor signalling. Even though MstnPP aggregates were localised to the cytoplasm, aggresomes and nuclear regions of sIBM tissue in previous studies [31,46], an absence of extracellular MstnPP was not shown. Since MstnPP is secreted, possibly for local inhibition of muscle growth [14], post- Myostatin Amyloid Formation secretion aggregation or secretion of aggregated/misfolded MstnPP may contribute to muscle fiber atrophy in sIBM via a cytotoxic mechanism that may involve alterations to endo- and exocytosis [65], alterations to Ca2+ homeostasis and the generation of reactive oxygen species [41]. The presence of pore-like structures under TEM suggests that the cytotoxic mechanism may include permeabilization of extracellular and/or intracellular membranes [43]. The results presented here show that human MstnPP is able to form cytotoxic amyloid-like aggregates and amyloid fibrils in vitro, raising the possibility that amyloid formation by MstnPP in vivo may contribute to the pathogenesis of sIBM. Materials and Methods MstnPP b-Aggregation Propensity by In Silico Analysis MstnPP amino acids 21–375 were used in b-aggregation propensity prediction algorithms. Tango [52] and Waltz [55] output was reproduced from www.tango.embl.de; PASTA output was taken from www.protein.cribi.unipd.it/pasta [54]. Default settings were used for all programs. Expression of Recombinant MstnPP Full-length human myostatin cDNA was used as a template for PCR cloning with primers encompassing cDNA corresponding to human MstnPP amino acid 21 (C-terminal to the signal peptidase cleavage site) to the C-terminal amino acid 375. PCR fragments were cloned into a modified pET vector via BamHI and XhoI restriction sites. Positive colonies were selected by colony PCR. The pET-MstnPP plasmid was isolated, sequenced and used to transform E. coli BL21(DE3) cells for protein expression. Transformed BL21 cells containing pET-MstnPP were grown in a starter culture of 20 mL LB in the presence of 100 mg/mL ampicillin (LB amp) at 37uC overnight. The starter culture was used to inoculate 2 L LB amp and cells were grown at 25uC to an OD600 of 0.8. Expression was induced by the addition of 0.1 mM IPTG and cells were incubated overnight (16–18 hours). Cells were collected by centrifugation at 4,0006g for 30 min at 4uC and stored at 220uC. Inclusion Body Isolation and Solubilisation and Refolding of Recombinant MstnPP The frozen cell pellet from each 2 L culture was thawed at room temperature and resuspended in a final volume of 70 mL 20 mM Tris-HCl pH 8.5. Cells were lysed by two passages through a French Press at 5,000 psi and the inclusion body pellet was collected by centrifugation at 30,0006g at 4uC for 30 min. The inclusion bodies were washed with two 20 mL volumes of wash buffer (50 mM Tris HCl pH 8.5, 0.5 M NaCl, 10% glycerol, 0.5% Triton-X 100) and two washes with the same volume of milliQ H2O. Pellets were collected after each wash by centrifu- gation at 30,0006g for 10 min at 4uC. Solubilisation and refolding were carried out using methods published previously for the zebra- fish myostatin precursor protein [8] with some modifications. Each inclusion body pellet from 2 L of cell culture was solubilised in 8 mL 6 M guanidine hydrochloride (GndHCl), 50 mM Tris-HCl pH 8.5, 1 mM EDTA, 0.1 mM DTT, at room temperature, with shaking for 5 hours. Insoluble proteins and cell debris were removed by centrifugation at 17,0006g, 4uC for 30 min. The supernatant was acidified to pH ,5.0 by adding a few drops of concentrated HCl and dialysed overnight against 6 M GndHCl, 50 mM MES pH 5.0, 1 mM EDTA to remove DTT. After dialysis, the sample was centrifuged again as above. The solubilised protein (8 mL) was rapidly diluted in 150 mL of freshly PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9170