AB-Crystallin Binds to Titin Ig Domains and Increases Stiffness of Skinned Cardiac Trabeculae

Abnormally stiff or compliant cardiac muscle is commonly observed following acute damage or disease. Cardiac stiffness is primarily modulated by the extracellular matrix protein collagen and intracellularly by the giant sarcomeric protein titin. However, it is not clear, particularly in the absence of fibrosis, how stiffness is altered in disease conditions. Recently, a mutation in the small heat shock protein αB-crystallin (R157H) was shown to cause inherited dilated cardiomyopathy. This abundant protein (3-5% of total soluble protein in the heart) is thought to bind titin and may regulate its stiffness. To test this, we measured the passive stiffness of skinned mouse trabeculae (with endogenous αB-crystallin extracted) by extending the sarcomere length from 2.0 to 2.6 µm in relaxing solution and measuring the resulting tension; addition of 1 mg/ml recombinant WT αB-crystallin significantly increased stiffness (linear Young's Modulus, extracted 31.2 ± 3.3; WT αB-crystallin 55.3 ± 8.7 mN/mm2). Passive stiffness at lower sarcomere lengths was increased more than at longer sarcomere lengths, indicating an increased contribution to force by titin relative to collagen. Interestingly, addition of the R157H mutant produced a significantly weaker effect. We show that αB-crystallin binds to fully folded titin Ig domains in vitro using native mass spectrometry and methyl trosy NMR. Our results indicate that αB-crystallin increases muscle stiffness not simply by stabilizing titin domains that have become unfolded during stretching, but by using a novel mechanism that is dissimilar to its regular function as a holdase chaperone. This work illustrates a further complexity in cardiac muscle regulation that may clarify disease pathogenesis and lead to additional therapeutic pathways.