Hendrikus L Granzier

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.

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.

During postnatal development, major changes in mechanical properties of skeletal muscle occur. We investigated passive properties of skeletal muscle in mice and rabbits that varied in age from 1 day to approximately 1 year. Neonatal skeletal muscle expressed large titin isoforms directly after birth, followed by a gradual switch toward progressively smaller isoforms that required weeks-to-months to be completed. This suggests an extremely high plasticity of titin splicing during skeletal muscle development. Titin exon microarray analysis showed increased expression of a large group of exons in neonatal muscle, when compared to adult muscle transcripts, with the majority of upregulated exons coding for the elastic proline-glutamate-valine-lysine (PEVK) region of titin. Protein analysis supported expression of a significantly larger PEVK segment in neonatal muscle. In line with these findings, we found >50% lower titin-based passive stiffness of neonatal muscle when compared to adult muscle. Inhibiting 3,5,3'-tri-iodo-L-thyronine and 3,5,3',5'-tetra-iodo-L-thyronine secretion did not alter isoform switching, suggesting no major role for thyroid hormones in regulating differential titin splicing during postnatal development. In summary, our work shows that stiffening of skeletal muscle during postnatal development occurs through a decrease in titin isoform size, due mainly to a marked restructuring of the PEVK region of titin.

Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5-1.0) than mice (N2BA:N2B ratio ~0.2). To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL) to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1-400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL "overshoot" at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.