Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts
Circulation Research 95:7 (2004) 708-716
Abstract:
In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (≈3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from ≈30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by ≈10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.Multiple sources of passive stress relaxation in muscle fibres
Physics in Medicine and Biology 49:16 (2004) 3613-3627
Abstract:
The forces developed during stretch of nonactivated muscle consist of velocity-sensitive (viscous/viscoelastic) and velocity-insensitive (elastic) components. At the myofibrillar level, the elastic-force component has been described in terms of the entropic-spring properties of the giant protein titin, but entropic elasticity cannot account for viscoelastic properties, such as stress relaxation. Here we examine the contribution of titin to passive stress relaxation of isolated rat-cardiac myofibrils depleted of actin by gelsolin treatment. Monte Carlo simulations show that, up to ∼5 s after a stretch, the time course of stress relaxation can be described assuming unfolding of 1-2 immunoglobulin domains per titin molecule. For extended periods of stress relaxation, the simulations failed to correctly describe the myofibril data, suggesting that in situ, titin-Ig domains may be more stable than predicted in earlier single-molecule atomic-force-microscopy studies. The reasons behind this finding remain unknown; simply assuming a reduced unfolding probability of domains - an effect found here by AFM force spectroscopy on titin-Ig domains in the presence of a chaperone, alpha-B-crystallin - did not help correctly simulate the time course of stress relaxation. We conclude that myofibrillar stress relaxation likely has multiple sources. Evidence is provided that in intact myofibrils, an initial, rapid phase of stress relaxation results from viscous resistance due to the presence of actin filaments. © 2004 IOP Publishing Ltd.Association of the Chaperone αβ-crystallin with Titin in Heart Muscle
Journal of Biological Chemistry 279:9 (2004) 7917-7924
Abstract:
αβ-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. αβ-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of αβ-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. αβ-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, αβ-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between αβ-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by αβ-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with αβ-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.The elasticity of single titin molecules using a two-bead optical tweezers assay
Biophysical Journal 87:2 (2004) 1112-1135