Small Heat Shock Proteins Prevent Titin Aggregation-Induced Stiffening in Human Myocytes

Abstract

During stretch of myocytes, mechanically active domains of the giant elastic titin molecule become unfolded. Unfolded proteins typically expose previously buried hydrophobic sites and are under risk to aggregate and lose their function. As for titin, it is unclear whether domain unfolding leads to aggregation and functional impairment. Here we consider that small heat shock proteins (sHSPs), which are abundantly expressed in myocytes, could help prevent titin aggregation. Various sHSPs, including HSP27 and αB-crystallin, are known to translocate under stress conditions preferentially to the sarcomeres, especially the elastic I-band region, where the titin springs are located. We find that HSP27 and αB-crystallin share binding sites on the titin springs, associating with immunoglobulin-like domain (Ig) containing regions, but not with the intrinsically disordered PEVK-domain. In sarcomeres, sHSP-binding to titin occurs independent of the presence of actin filaments and is enhanced by stretch. The titin spring elements behave in vitro mainly as monomers. However, unfolded Ig regions aggregate in vitro, preferentially under acidic conditions, whereas αB-crystallin protects against aggregation. Disordered titin spring regions do not aggregate. Single human cardiomyocytes in which titin Ig unfolding is promoted reveal elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppress this stiffening. The two sHSPs do not bind to the sarcomeres of stretched cardiomyocytes expressing very compliant titin (Ig domain-unfolding probability very low), but localize to the I-band region in stretched cardiomyocytes expressing stiff titin (Ig domain-unfolding probability high). In diseased human muscle/heart tissue both sHSPs associate with the titin springs, in contrast to cytosolic/Z-disk locations in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains under stress conditions would stiffen myocytes, but sHSPs translocating to these domains prevent titin aggregation and cell stiffening.