Figure 5. Circular dichroism spectra showing the structural transitions that occur in amyloid fibril formation. Correctly folded MstnPP dimer (blue); MstnPP soluble aggregates before acidification (green); prefibrillar aggregates after overnight incubation in pH 5.3 at 60uC (red); mixture of prefibrillar aggregates and fibrils after one week incubation (black). doi:10.1371/journal.pone.0009170.g005 protofibrils and fibrils on the viability of C2C12 mouse myoblasts was investigated by monitoring the absorbance of formazan (Fig. 8) produced after addition of WST-1 (Roche). Only viable cells reduce WST-1 to produce formazan with the ratio of absorbances at 450 and 630 nm correlated to the number of viable cells in the culture. C2C12 mouse myoblasts are the standard model cell-line Myostatin Amyloid Formation used for the analysis of myostatin activity [16,18]. Although human MstnPP is used here, sequence identity between the mouse and human myostatin precursors is 96%; the murine cell-line is therefore suitable for initial studies. 25 mM of soluble aggregates and at least 10 mM of protofibrils decreased cell viability significantly compared to buffer only and correctly folded MstnPP dimer controls, at the same pH. A solution containing a mixture of MstnPP protofibrils and fibrils, as well as lower concentrations of soluble aggregates and protofibrils, had a reduced effect. These results indicate that oligomeric species in the amyloid formation pathway of MstnPP affect the normal functioning of C2C12 myoblasts. Discussion Amyloid formation is a predominant feature of sIBM [30] with the Alzheimer’s disease protein Ab associated with sIBM amyloid structures [31,44]. A role for MstnPP in sIBM was proposed after co- localisation and direct association with Ab was observed in diseased cells [31], and also because ER stress causes an upregulation of myostatin expression [45]. We now show that a human MstnPP misfolded species shares the morphological characteristics of amyloid protofibrils and the ability to bind the amyloid-specific dye thioflavin T. The a-helical secondary structure of these aggregates suggests they are most likely to represent an intermediate oligomeric species that occurs in the transition between native protein and b-sheet-rich protofibrils. When subjected to the mildly denaturing conditions of Figure 6. SDS-PAGE and TEM analysis showing MstnPP resistance to trypsin digestion. A. dimer; and B. soluble aggregates. Major bands are indicated: 1. MstnPP monomer; 2. MstnPP dimer; 3. MstnPP soluble aggregates. C. MstnPP fibrils: 4. prefibrillar aggregates plus fibrils. Samples were analysed in sample buffer +/2 bME where bME concentration is 2 M. D. TEM shows resistance of (i) protofibrils and (ii) amyloid fibrils to trypsin. Trypsin digests were performed with a MstnPP:trypsin ratio of 100:1 (A and B) or 20:1 (C and D) for 18 hours at 37uC. doi:10.1371/journal.pone.0009170.g006 PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9170