Introduction:
Small heat-shock proteins (sHSPs) generally participate in cellular protein quality-control mechanisms. They are abundant in cardiomyocytes where they bind under diverse stress conditions preferentially to myofilament proteins. The functional role of this association is unclear.
Hypothesis:
HSP27 and αB-crystallin bind to human cardiac titin spring elements and exert a protective effect on cardiomyocyte passive stiffness under stress conditions.
Methods:
Binding of sHSPs to recombinant human titin constructs was characterized by GST-pulldown assay and confirmed by immunofluorescence staining of human donor and failing (DCM) cardiomyocytes. Sedimentation-velocity centrifugation, photometric and chromatographic methods were used to test for titin aggregation and protection from it by sHSPs. Passive force was measured in isolated human cardiomyocytes or single myofibrils, in search for a possible protective effect of sHSPs on mechanical function.
Results:
HSP27 interacted with distinct domains of the human titin-spring region
in vitro
and in cardiomyocytes, and independent of the presence of actin filaments in the sarcomeres. The binding sites on the elastic titin segment resemble those for αB-crystallin and include proximal Ig-domains, the N2-B and N2-A regions, but not the PEVK-domain.
In-vitro
assays revealed a monomeric organization of these titin-spring elements; however, unfolded N2-A domain (mainly composed of Ig-domains) aggregated in pH-6.6 buffer but not in normal-pH buffer, whereas αB-crystallin protected from this effect. The intrinsically disordered N2-Bus titin domain did not aggregate. Single skinned human cardiomyocytes showed greatly increased passive stiffness when pre-stretched under acidic stress (pH 6.6), but αB-crystallin or HSP27 corrected the stiffening. In failing patient heart tissue, both HSP27 and αB-crystallin frequently associated with the elastic I-band region, in contrast to a cytosolic and Z-disk location of these sHSPs in donor heart.
Conclusions:
In cardiomyocytes sHSPs bind to mechanically active titin domains under stress conditions such as intracellular acidosis (
e.g.
, ischemia), protecting the titin springs from aggregation and helping reverse diastolic stiffening.
Comparative analysis of the molecular mechanism of heat shock proteins from pathogenic and non-pathogenic E.coli
