Rapid unloading experiments from Ttn112-158 muscles

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.Analyzed data are .xlsx files.Animals Ttn112-158 mice were obtained from the Granzier laboratory at the University of Arizona, and a breeding colony was established at the Claremont Colleges. The Ttn112-158 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 Ttn112-158 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. Whole Muscle Experiments Whole muscle ex vivo experiments were conducted on 17 soleus muscles from age-matched Ttn112-158 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 NaH2PO4, 24 NaHCO3, 2 CaCl2, 1 MgSO4, and 11 dextrose, pH 7.4) buffered with 95% O2 and 5% CO2. 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 (L0), 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 (P0, Ncm−2) 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 (Lf) and the density of mammalian skeletal muscle ( 1.06 g cm−3; Sacks and Roy, 1982). Brynnel et al., (2018) reported that fiber and muscle lengths (Lf) were ~ 13.5% longer in Ttn112-158 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. Elastic properties A series of rapid unloading tests was used to measure elastic recoil of active and passive soleus muscles from TtnD112-158 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-2 which corresponded to 40–70% maximum isometric tetanic stress (P0). Maximum isometric tetanic stress ranged from 15–28 N/cm-2. The duration of stimulation was varied to achieve the desired initial stress in activated muscles. Muscles were stretched passively (1.02–1.07 L0) 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-2), 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 L0 until the passive force reached 10–40% P0 (Monroy et al., 2017). For each test, the initial rapid recoil distance (mm, L/L0) 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