Background-The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results-SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (PϽ0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (nϭ7), intermediate in those with pseudonormal filling (nϭ6), and lowest in the group with restrictive filling (nϭ7). Mechanical measurements on LV muscle strips dissected from these hearts (nϭ8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O 2 consumption (nϭ10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Conclusions-Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.

Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: Ϸ28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calciumdependent molecular spring that adapts to the physiological state of the cell.T itin comprises a multifunctional myofilament system in muscle, with a single molecule spanning the half-sarcomere (1-5). A large portion of the molecule functions as a molecular spring that, when extended, develops force. This force underlies the passive muscle force, which maintains the structural integrity of the contracting sarcomere and influences the filling behavior of the heart (3). Physiological force levels are largely determined by extension of the proline-glutamate-valine-lysine (PEVK) rich segment (6, 7). PEVK-like sequences are found in many titin-like proteins among evolutionary divergent organisms (8-11). The PEVK region of the human titin gene contains 114 exons; most code for conserved Ϸ28-residue PEVK repeats and 10 are more complex and encode E-rich motifs (12, 13). Here, we studied recombinant proteins that contain both PEVK repeats and glutamate (E)-rich motifs. Previous work suggests that the PEVK segment binds calcium with high affinity, raising the possibility that the extensibility of the PEVK segment may be calcium regulated (14). Thus, an important goal was to examine the effect of calcium on the mechanical behavior of the PEVK segment. MethodsProteins. We engineered only PEVK sequences (''naked'' PEVK fragment) or PEVK sequences flanked by Ig-like domains (PEVK-Ig fusion fragment). Fragments were cloned, expressed, and purified by using routine methods (12, 15). All fragments were His-tagged at their amino terminus and, except when intended for structural analysis, had two Cys residues at the carboxyl terminus for covalent attachment to gold-coated substrates. Purified proteins were dialyzed into AB buffer (in mM; 25 Mops, pH 7.4͞150 KCl͞1 EGTA͞1 DTT), quick frozen, and stored at Ϫ80°C. For additional details, see Supporting Text, which is published as supporting information on the PNAS web site.Single Molecule Mechanics. Molecules were stretched by using an atomic force microscope specialized for stretching molecules (15, 16). Proteins (Ϸ100 g͞ml) were allowed to bind for Ϸ10 min to gold-coated microscope slides (precleaned glass slides and freshly cleaved mica were also...