Titin force enhancement following active stretch of skinned skeletal muscle fibres

Abstract

In actively stretched skeletal muscle sarcomeres, titin-based force is enhanced, increasing the stiffness of active sarcomeres. Titin force enhancement in sarcomeres is vastly reduced in mdm, a genetic mutation with a deletion in titin. Whether loss of titin force enhancement is associated with compensatory mechanisms at higher structural levels of organization, such as single fibres or entire muscles, is unclear. The aim of this study was to determine whether mechanical deficiencies in titin force enhancement are also observed at the fibre level, and whether mechanisms compensate for the loss of titin force enhancement. Single skinned fibres from control and mutant mice were stretched actively and passively beyond filament overlap to observe titin-based force. Mutant fibres generated lower contractile stress (force divided by cross-sectional area) than control fibres. Titin force enhancement was observed in control fibres stretched beyond filament overlap, but was overshadowed in mutant fibres by an abundance of collagen and high variability in mechanics. However, titin force enhancement could be measured in all control fibres and most mutant fibres following short stretches, accounting for ∼25% of the total stress following active stretch. Our results show that the partial loss of titin force enhancement in myofibrils is not preserved in all mutant fibres and this mutation likely affects fibres differentially within a muscle. An increase in collagen helps to reestablish total force at long sarcomere lengths with the loss in titin force enhancement in some mutant fibres, increasing the overall strength of mutant fibres.

Optimal length, calcium sensitivity, and twitch characteristics of skeletal muscles from mdm mice with a deletion in N2A titin

“…A decrease in series elastic stiffness by increased tendon length would be consistent with these results (Bahler et al, 1967;Mayfield et al, 2016). However, mdm muscle preparations in this study had little to no tendon, but do show an increase in endomysial and perimysial connective tissue (Lopez et al, 2008;Powers et al, 2017), and have altered elastic properties that are consistent with greater compliance of active muscle (Monroy et al, 2017). However, internal aponeuroses or internal extracellular matrix components of muscles are not mechanistically (based on Hill-type muscle models) in-series with active fibres, but in-parallel, and thus will have no effect on force transmission.…”

“…Selectively degrading titin reduces force almost linearly (Horowits et al, 1986;Li et al, 2018) and leads to positional instability of thick filaments (Horowits, 1992;Horowits and Podolsky, 1988). Therefore, it is possible that decreased stiffness of titin in mdm muscles, as measured by the slope of the stress versus strain curve during long stretches (Powers et al, 2016(Powers et al, , 2017, decreases sarcomere stability at shorter sarcomere lengths. However, at longer sarcomere lengths, titin bears more force (Linke et al, 1998;Trombitás et al, 1998), which may help to stabilize the sarcomeres.…”

“…Skinned fibers were prepared from mdm and wild-type muscles using standard techniques (Joumaa and Herzog, 2014). Muscles were stored in a relaxing solution [in mmol l −1 : potassium propionate (170), magnesium acetate (2.5), MOPS (20), K 2 EGTA (5) and ATP (2.5), pH 7.0] for 12 h at 4°C, then in a relaxingglycerol (50:50) solution at −20°C for 4 to 6 weeks (Powers et al, 2017). To limit protein degradation, all solutions contained one tablet of protease inhibitor (Complete, Roche Diagnostics, Mannheim, Germany) per 100 ml of solution.…”

Effects of a titin mutation on negative work during stretch-shortening cycles in skeletal muscles

“…Differences in work loop characteristics between WT and mdm muscles mdm is a recessive mutation that results in a 779 bp deletion and a predicted ∼83 amino acid deletion at the border between the N2A and PEVK regions of titin (Garvey et al, 2002). Previous studies demonstrate that mdm muscles exhibit increased passive tension compared with WT muscles (Lopez et al, 2008;Monroy et al, 2017;Powers et al, 2017) due to an increase in collagen content (Powers et al, 2017). No increase in passive stress was observed in myofibrils from mdm psoas (Powers et al, 2016).…”

“…We also found that the maximum stress during the stretch phase of active work loops in mdm muscles decreased in proportion to the reduction in P 0 , suggesting that cross bridges also contribute to the reduction of negative work in mdm muscles. Although no direct evidence suggests that cross-bridge function per se is impaired in mdm muscles, some fibers have weak striation patterns at 30-50 days of age, which could contribute to reduced isometric stress (Powers et al, 2017). The actin and myosin content of single mdm myofibrils appears normal (Powers et al, 2016), and gene expression studies show little or no change in expression of thick or thin filament proteins, or proteins involved in calcium cycling (Witt et al, 2004).…”