Study of elasticity of titin at the level of the single molecule

Titin is the third myofilament of striated muscle. It spans the half sarcomere and gives rise to passive muscle stiffness upon sarcomere stretch. Because passive muscle stiffness plays important physiological roles (for example, it determines the filling behavior of the heart), extensibility and elasticity of a whole titin molecule have been intensively studied in the past using cardiac myocyte and skeletal muscle fiber mechanics.; In my doctoral work, various spring elements (serially-linked Ig domains, PEVK segment and N2B-Us) that comprise titin extensible I-band region were stretched using force-mode atomic force microscopy (AFM) that is capable of characterizing the mechanical properties of the single molecule in the nano-scale range. AFM results were analyzed with the entropic elasticity model and results indicated that the elasticity of each element differs markedly, thus they are mechanically distinct.; Using mechanical parameters established in the present work, the extensibility of three spring elements and force-extension relation of cardiac titin upon sarcomeric stretch were calculated and compared with experimentally determined values. The calculation closely simulated the complex extension of the three elements of titin in the sarcomere as well as the unique passive force-sarcomere length relation of cardiac myocytes. Thus, in vivo behavior of cardiac titin can be demonstrated with serially linked and mechanically distinct springs using parameters obtained from AFM studies.; The modulation of titin's elasticity by physiological levels of calcium was also studied using AFM, fluorescence and cellular mechanics. Calcium-induced mechanical and conformational changes were observed in a recombinant PEVK fragment that contains an E(glutamate)-rich motif, but it was absent from fragments without the motif. Mutagenesis in the E-rich motif identified 4 amino acids that are involved in conferring calcium sensitivity. The calcium-PEVK interaction was also effective in vivo myocyte mechanics in which titin-based passive force was shown to be increased by calcium. Taken together, the E-rich motif used in this study regulates titin-calcium interaction, which results in titin-based passive force in myocytes to be calcium-dependent. Thus, calcium in muscle may play an important role in regulating muscle contraction actively and passively.