DOI: 10.5061/dryad.xd2547dhh
title: Rapid unloading experiments from Ttn112-158 muscles

description: Evidence suggests that the giant muscle protein, titin, functions as a
 tunable spring in active muscle. However, the mechanisms for increasing
 titin stiffness with activation are not well understood. Previous studies
 have suggested that during muscle activation, titin binds to actin which
 engages the PEVK region of titin thereby increasing titin stiffness. In
 this study, we investigated the role of PEVK titin in active muscle
 stiffness during rapid unloading. We measured elastic recoil of active and
 passive soleus muscles from Ttn112-158 mice characterized by a 75%
 deletion of PEVK titin and increased passive stiffness. We hypothesized
 that activated Ttn112-158 muscles are stiffer than wild-type muscles as a
 result of the increased stiffness of PEVK titin. Using a servomotor force
 lever, we compared the stress-strain relationships of elastic elements in
 active and passive muscles during rapid unloading and quantified the
 change in stiffness upon activation. The results show that the elastic
 modulus of Ttn112-158 muscles increased with activation. However, elastic
 elements developed force at 7% longer lengths and exhibited 50% lower
 active stiffness in Ttn112-158 soleus muscles than wild-type muscles.
 Thus, despite having a shorter, stiffer PEVK segment, during rapid
 unloading, Ttn112-158 soleus muscles exhibited reduced active stiffness
 compared to wild-type soleus muscles. These results are consistent with
 the idea that PEVK titin contributes to active muscle stiffness, however,
 the reduction in active stiffness of Ttn112-158 muscles suggests that
 other mechanisms compensate for the increased PEVK stiffness.
description: <strong>Animals</strong>
 Ttn<sup>112-158</sup> mice were obtained from the
 Granzier laboratory at the University of Arizona, and a breeding colony
 was established at the Claremont Colleges. The
 Ttn<sup>112-158</sup> deletion corresponds to a deletion of 47
 exons which encode 1586 amino acids in the PEVK region of titin and
 represents ~75% of the wild-type PEVK sequence (Brynnel et al., 2018).
 Soleus muscles were extracted from Ttn<sup>112-158</sup> mice
 euthanized with an overdose of isoflurane confirmed by cervical
 dislocation. The Institutional Animal Care and Use Committee at the
 Claremont Colleges approved the experimental protocol (IACUC Protocol
 #019-004) and use of these animals. <strong>Whole
 Muscle Experiments</strong> Whole muscle ex vivo
 experiments were conducted on 17 soleus muscles from age-matched
 Ttn<sup>112-158</sup> mice of both sexes (age = 98.8 ± 9.5
 days, 10 females, 7 males). The experimental setup was described in
 previous publications (Hessel et al., 2021; Monroy et al., 2017). In
 brief, the soleus muscles were dissected and tied off securely at the
 muscle-tendon junction to minimize the contribution of extramuscular
 connective tissues to the experiments. The distal end of each muscle was
 attached to an inflexible hook and the proximal end was attached to a dual
 servomotor muscle lever (Aurora Scientific, Inc., Series 300B, Aurora, ON,
 Canada). All experiments were conducted at a constant temperature
 (21–23°C) in a Krebs–Henseleit bath (in mM: 137 NaCl, 5 KCl, 1
 NaH<sub>2</sub>PO<sub>4</sub>, 24
 NaHCO<sub>3</sub>, 2 CaCl<sub>2</sub>, 1 MgSO4,
 and 11 dextrose, pH 7.4) buffered with 95% O<sub>2</sub> and
 5% CO<sub>2</sub>. Muscles were stimulated using an electrical
 field generated between two platinum electrodes connected to an Aurora
 Scientific 701C bi-phase stimulator. At this temperature, maximum
 isometric tetanic force remains stable for several hours and within 10% of
 the maximum isometric stress at a normal body temperature of 37°C (James
 et al., 2015). A custom LabVIEW (National Instruments Corp., Austin, TX,
 United States) program was used to control the muscle lever and record
 force, length, and time at a sampling rate of 4 kHz. At
 the start of each experiment, a muscle was stretched to its optimal length
 (L<sub>0</sub>), defined as the length at which maximum
 isometric twitch force is produced. Muscles were stimulated at 70–80 Hz
 for 800–1,000 ms to measure maximum isometric tetanic force (Monroy et
 al., 2017). Maximum isometric stress (P<sub>0</sub>,
 Ncm<sup>−2</sup>) was determined by dividing maximum isometric
 tetanic force by the physiological cross-sectional area (PCSA), calculated
 using standard methods (Hakim et al., 2013; Lieber and Ward, 2011; Monroy
 et al., 2017). Briefly, to determine PCSA, muscle mass was multiplied by
 the cosine of the pennation angle (8.5°; Burkholder et al., 1994), and
 divided by the product of muscle fiber length (L<sub>f</sub>)
 and the density of mammalian skeletal muscle ( 1.06 g
 cm<sup>−3</sup>; Sacks and Roy, 1982). Brynnel et al., (2018)
 reported that fiber and muscle lengths (L<sub>f)</sub> were ~
 13.5% longer in Ttn<sup>112-158</sup> muscles. However, the
 fiber length to muscle length ratio was not different from wild-type
 muscles. The maximum isometric tetanic force was measured periodically
 throughout an experiment, and a muscle was removed from the analysis if
 force dropped by more than 10%. Of the 17 muscles used in these
 experiments, two muscles were removed from the analysis due to a decrease
 in maximum force. <strong>Elastic
 properties</strong> A series of rapid unloading
 tests was used to measure elastic recoil of active and passive soleus
 muscles from TtnD1<sup>12-158</sup> mice (Fig. 1). The initial
 stress and change in stress were matched for active and passive trials
 (Fig. 1A, B). Muscles were stimulated isometrically at optimal length to
 an initial stress followed by a series of 5–8 step decreases in load that
 varied from 5–90% of initial stress. Muscles were allowed to recover for ~
 4–5 minutes between each trial. The order of the tests was somewhat random
 although the greatest step decrease in load was often conducted at the end
 because muscles needed longer to recover from these trials. Among muscle
 preparations, the initial stress ranged from 6.7–15.1
 Ncm<sup>-2</sup> which corresponded to 40–70% maximum
 isometric tetanic stress (P<sub>0</sub>). Maximum isometric
 tetanic stress ranged from 15–28 N/cm<sup>-2</sup>. The
 duration of stimulation was varied to achieve the desired initial stress
 in activated muscles. Muscles were stretched passively
 (1.02–1.07 L<sub>0</sub>) to a length at which the initial
 steady-state passive stress equaled the initial active stress. Due to some
 variability in the initial stress, the actual unloading steps varied
 slightly between active and passive trials. In wild-type muscles, the
 initial stresses were more variable (2.9–2.1
 Ncm<sup>-2</sup>), but most (11 out of 14) fell within the
 same range of stresses as the current study (Monroy et al., 2017).
 Wild-type muscles were stretched to 1.09–1.17 L<sub>0</sub>
 until the passive force reached 10–40% P<sub>0</sub> (Monroy
 et al., 2017). For each test, the initial rapid recoil
 distance (mm, L/L<sub>0</sub>) was measured (Fig. 1C, D). When
 the load is reduced rapidly, active muscles shorten biphasically, with an
 initial rapid change in length due to recoil of elastic elements and a
 later slow phase due to cross-bridge cycling (Jewell and Wilkie, 1958;
 Lappin et al., 2006; Monroy et al., 2007; Monroy et al., 2017; Wilkie,
 1956). We measured the distance shortened from the onset of unloading to
 the intersection of the lines that best fit the initial and slow phases of
 shortening (Fig 1C, Lappin et al., 2006; Monroy et al., 2017). In passive
 muscles, we measured the distance shortened to the steady-state length
 following initial oscillations (Fig. 1D). Data collected from each muscle
 and state were used to model the stress-strain relationships.
description: Analyzed data are .xlsx files.