Effects of Acute Muscle Contraction on Titin Stiffness-Related Contractile Properties
in Rat Fast-Twitch Skeletal Muscle

Jiayu Shi
Graduate School of Integrated Arts and Sciences
Hiroshima UniversityMarch 2022

In a half sarcomere of a striated muscle, the filament known as titin runs from the Z-disk to the Mband. Passive force is the force of a muscle that is stretched but not activated. Most of the passive
force is generated by titin. The contribution of titin to active force has been long thought to benegligible, because titin stiffness is much lower than that of the acto-myosin. However, recent studies
have suggested the more important role of titin in active force than previously thought. In cardiac
muscles, it has been shown that acute exercise resulted in an increase in passive force. Based on thesefindings, it might be expected that the changes in titin stiffness would affect active force. However,no studies have investigated whether titin stiffness can undergo changes with acute muscle
contractions in skeletal muscles. The aim of this study was to elucidate acute muscle contraction on
titin stiffness-related contractile properties in rat fast-twitch muscle. To this end, four separatedexperiments (1-4) were performed in this study.
In the experiment 1, the effects of isometric contraction (ISC) on passive force were examined.
Intact gastrocnemius muscles of the rats were electrically stimulated in situ until the force was reduced
to ∼50% of the initial force. Immediately after cessation of the stimulation, the superficial regions ofthe muscles were dissected and subjected to skinned fiber analysis. The ISC resulted in a decrease inpassive force. Protein kinase Cα-treatment increased the passive force in stimulated fibers to restinglevels. The ISC had no effect on the maximum Ca2+-activated force (max Ca2+ force) at a sarcomere
length (SL) of 2.4-μm. Stretching the SL to 3.0 μm led to the augmentation of the max Ca2+ force. Theextent of the increase was smaller in rested than in stimulated fibers.
In the experiment 2, the effects of ISC on length-dependent activation (LDA), residual force
enhancement (RFE), and passive force enhancement (PFE) were examined. The electrical stimulationand the skinned fiber preparation were performed in a manner similar to those in the experiment 1.

The ISC led to a decrease in myofibrillar (my-) Ca2+ sensitivity at 2.6-μm SL. Although a stretchof SL from 2.6 to 3.0 μm increased my-Ca2+ sensitivity in both rested and stimulated fibers, theextent of the increase was higher in the stimulated than in the rested fibers. To evaluate RFE, thefibers were stretched before activation (the force developed by the activation was referred to as
M1 and the passive force after M1 was called P1) and during activation (the force was referred toas M2 and the passive force after M2 was called P2). The ISC decreased M1 and M2 in bothrested and stimulated fibers. A relative difference (the ratio of M2 to M1) between M1 and M2
did not differ between rested and stimulated fibers. P1, but not P2, decreased in stimulated fibers.
In both rested and stimulated fibers, P2 was greater than P1. However, the degree of the difference
differed between rested and stimulated fibers. Absolute (P2 minus P1) and relative (the ratio ofP2 to P1) differences were greater in stimulated than in rested fibers.
In the experiment 3, the effects of eccentric contraction (ECC) on passive force were examined.

Intact GAS muscles were electrically stimulated in situ to subject the muscles to 200 repeated
ECCs. The skinned fiber preparation was performed in a manner similar to that in experiment 1.
ECCs brought about a decrease in the max Ca2+ force at 2.4 μm SL. A stretch of the SL to 3.0 μm
led to the augmentation of the max Ca2+ force in both rested and stimulated fibers. The degree ofthe augmentation in stimulated fibers resembled that in rested fibers. The ECC resulted in an
increase in the titin-based passive force. Protein kinase A-treatment reduced the passive force instimulated fibers to the resting levels.
In the experiment 4, the effects of ECC on LDA, RFE, and PFE were examined. The electricalstimulation and the skinned fiber preparation were performed in a manner similar to those inexperiment 3. ECC tended to decrease my- Ca2+ sensitivity at 2.6-μm SL. Although a stretch ofSL from 2.6 to 3.0 μm increased my-Ca2+ sensitivity in both rested and stimulated fibers, theextent of the increase was higher in the stimulated than in the rested fibers. M1 and M2 weredecreased in stimulated fibers. An absolute difference or a relative difference between M1 andM2 did not differ between the rested and stimulated fibers. P1 and P2 were increased in stimulatedfibers. An absolute difference between P2 and P1 was larger in the stimulated than in the restedfibers.
The results obtained from this study indicate that ISC suppresses the passive force, whereas ECCraises it. The decreased passive force may contribute to muscle fatigue. The altered passive force isascribable, at least in part, to a reduction in phosphorylation levels by protein kinase Cα and protein
kinase A for ISC and ECC, respectively. Both contraction modes are capable of potentiating LDA andPFE, but not RFE. The potentiated LDA and PFE arguably help produce greater force compared tothat without the potentiation. It is suggested that some of titin stiffness-based contractile properties
may function to resist muscle fatigue in the muscles of the exercising body.