The Effects of Titin Degradation on Passive Stiffness Properties of Skinned Rabbit Psoas Fibers during Osmotic Compression

Abstract

Muscle is disproportionately stiff over short stretch distances. This effect is largely calcium-sensitive, and can be eliminated in actively contracting skinned fibers by inhibiting crossbridge formation with pharmacological agents such as 2,3-butanedione monoxime (BDM). However, elevated short range stiffness can be induced in the absence of calcium in relaxed skinned muscle fibers by osmotic compression, an effect which is not attenuated by BDM. This study aimed to determine if titin, the primary source of tension and stiffness in relaxed muscle, could account for the elevated short range stiffness in osmotically compressed, relaxed skinned fibers. Accordingly, skinned rabbit psoas fibers (n=9) were held at 2.8 μm sarcomere length and osmotically compressed using 7.5% dextran-T-500 in calcium-free relaxing solution. Muscles were stretched by 2% (∼0.057 μm/sarcomere) over 0.66 s, and returned to initial length over another 0.66 s. Dextran treatment resulted in a 5.6 ± 0.6 fold increase in short range stiffness, with an apparent length limit of 4.5±0.2 nm per sarcomere (Phase 1). Beyond this limit (Phase 2), stiffness was not different (100.4±3.0%) than dextran-free stiffness. To selectively degrade titin, fibers were held in relaxing solution containing both dextran and trypsin (0.25 μg/mL) until mechanical failure, with periodic application of the stretch protocol. In the stretch immediately preceding failure, Phase 2 stiffness was 86±3% lower than initial values, while Phase 1 stiffness only decreased by 31±3%. Additionally, tension at peak stretch increased 12.5±2.1 μN (33±3%) upon osmotic compression, matching the 12.6±1.1 μN intercept of the Phase 2 regression line on the Δtension versus Δlength graph. Thus, short range stiffness appears additive to Phase 2, titin-based stiffness, and is best accounted for as a separate entity acting in parallel to titin.