Granzier, H. L. (2015). Reply to Tskhovrebova et al.: Titin's IA junction does not control thick filament length. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1173.
Granzier, H., Anderson, B. R., Bogomolovas, J., Labeit, S., & Granzier, H. L. (2010). The effects of PKCalpha phosphorylation on the extensibility of titin's PEVK element. Journal of structural biology, 170(2).
Post-translational modifications, along with isoform splicing, of titin determine the passive tension development of stretched sarcomeres. It was recently shown that PKCalpha phosphorylates two highly-conserved residues (S26 and S170) of the PEVK region in cardiac titin, resulting in passive tension increase. To determine how each phosphorylated residue affects myocardial stiffness, we generated three recombinant mutant PEVK fragments (S26A, S170A and S170A/S26A), each flanked by Ig domains. Single-molecule force spectroscopy shows that PKCalpha decreases the PEVK persistence length (from 0.99 to 0.68 nm); the majority of this decrease is attributable to phosphorylation of S26. Before PKCalpha, all three mutant PEVK fragments showed at least 40% decrease in persistence length compared to wildtype. Furthermore, Ig domain unfolding force measurements indicate that PEVK's flanking Ig domains are relatively unstable compared to other titin Ig domains. We conclude that phosphorylation of S26 is the primary mechanism through which PKCalpha modulates cardiac stiffness.
Winter, J. M., Joureau, B., Lee, E. J., Kiss, B., Yuen, M., Gupta, V. A., Pappas, C. T., Gregorio, C. C., Stienen, G. J., Edvardson, S., Wallgren-Pettersson, C., Lehtokari, V. L., Pelin, K., Malfatti, E., Romero, N. B., Engelen, B. G., Voermans, N. C., Donkervoort, S., Bönnemann, C. G., , Clarke, N. F., et al. (2016). Mutation-specific effects on thin filament length in thin filament myopathy. Annals of neurology, 79(6), 959-69.
Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation.
Granzier, H. L., Hutchinson, K. R., Tonino, P., Methawasin, M., Li, F. W., Slater, R. E., Bull, M. M., Saripalli, C., Pappas, C. T., Gregorio, C. C., & Smith, J. E. (2014). Deleting titin's I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function. Proceedings of the National Academy of Sciences of the United States of America, 111(40), 14589-94.
Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (∼ 40-fold higher) but low, relative to that of the myosin-based thick filament (∼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.
Granzier, H., Fukushima, H., Chung, C. S., & Granzier, H. L. (2010). Titin-isoform dependence of titin-actin interaction and its regulation by S100A1/Ca2+ in skinned myocardium. Journal of biomedicine & biotechnology, 2010.
Titin, also known as connectin, is a large filamentous protein that greatly contributes to passive myocardial stiffness. In vitro evidence suggests that one of titin's spring elements, the PEVK, interacts with actin and that this adds a viscous component to passive stiffness. Differential splicing of titin gives rise to the stiff N2B and more compliant N2BA isoforms. Here we studied the titin-isoform dependence of titin-actin interaction and studied the bovine left atrium (BLA) that expresses mainly N2BA titin, and the bovine left ventricle (BLV) that expresses a mixture of both N2B and N2BA isoforms. For comparison we also studied mouse left ventricular (MLV) myocardium which expresses predominately N2B titin. Using the actin-severing protein gelsolin, we obtained evidence that titin-actin interaction contributes significantly to passive myocardial stiffness in all tissue types, but most in MLV, least in BLA, and an intermediate level in BLV. We also studied whether titin-actin interaction is regulated by S100A1/calcium and found that calcium alone or S100A1 alone did not alter passive stiffness, but that combined they significantly lowered stiffness. We propose that titin-actin interaction is a "viscous break" that is on during diastole and off during systole.
