Decreased force enhancement in skeletal muscle sarcomeres with a deletion in titin

Movement involves the coordination of dynamic muscle contractions produced by proteins within sarcomeres. For nearly 60 years, contractile force has been understood as a Ca²⁺-dependent cyclical formation of cross-bridges between thick (myosin) and thin (actin) filaments in the sarcomere (Huxley and Simmons, 1971) (Fig. 1). Titin, a viscoelastic spring in the sarcomere, was discovered decades after the cross-bridge model was developed (Maruyama, 1976). Thus, the cross-bridge theory does not account for this force-bearing protein in the sarcomere. Based on the cross-bridge theory, active, steady-state, isometric force must be proportional to the number of cross-bridges that can form (given by the sarcomere length for the plateau and descending limb regions of the force–length relationship), with maximal force produced at optimal filament overlap (Gordon et al., 1966). However, active muscle force greatly exceeds this predicted maximum force following stretching (Abbot and Aubert, 1952). Stretch-induced residual force enhancement has been observed at all structural levels, from entire muscles down to single sarcomere preparations (Leonard et al., 2010; Rassier, 2012). Therefore, an unidentified mechanism generates enhanced force in sarcomeres that persists in the isometric steady-state following active stretch. Previous investigations sought to describe this residual force enhancement within the framework of the cross-bridge theory. However, this property persists beyond filament overlap, where cross-bridges cannot contribute to force (Leonard and Herzog, 2010). Evidence suggests that titin becomes stiffer during muscle contraction, enhancing force during and following active stretch (Herzog and Leonard, 2002; Leonard and Herzog, 2010; Nishikawa et al., 2012; Powers et al., 2014).

A role for titin in active muscle force production has long been speculated (Edman et al., 1982; Noble, 1992) but remains controversial. Titin's function within the sarcomere was traditionally considered structural, but its dynamic nature has since been demonstrated with widespread structural, mechanical and signaling roles (Granzier and Labeit, 2004; Labeit et al., 2003; Leonard and Herzog, 2010; Nishikawa et al., 2012). Titin molecules (six per half thick filament) anchor the thin filaments at the Z-disc (Maruyama, 1976), running the full length of the thick filament to the M-line (Gregorio et al., 1999) to form a titin network that maintains the structural integrity of each sarcomere (Fig. 1). Structurally, titin's I-band consists of springs in series that resist sarcomere stretch. Upon stretch, the proximal immunoglobulin (Ig) domains elongate under low force, followed by stretching of the PEVK (Proline-Glutamate-Valine-Lysine) region, which contributes the majority of titin-based force (Fig. 1) (Granzier and Labeit, 2004). Located between the proximal Ig domain and the PEVK region, the N2A segment can also elongate during long sarcomere stretches and is a ‘hot spot’ for titin-based signaling (Granzier and Labeit, 2004). The distal Ig segment, connecting titin's I- and A-bands, is the most rigid of titin's springs. Transient increases in titin-based stiffness (15–20%) are observed when Ca²⁺ ions bind to the Ig and PEVK segments of titin (DuVall et al., 2013; Labeit et al., 2003). However, observations of up to 200% increase in titin stiffness in actively stretched rabbit and mouse sarcomeres provide evidence of an additional mechanism for modulation of titin-based force (Leonard and Herzog, 2010; Powers et al., 2014). This mechanistically elusive increase in titin-based stiffness in actively stretched sarcomeres is referred to as ‘titin force enhancement’ henceforth.

Titin force enhancement occurs in a force-dependent manner, decreasing in magnitude with reduced contractile force preceding stretch (Leonard and Herzog, 2010). Initiation of titin force enhancement requires strong cross-bridge formation (Herzog and Leonard, 2002; Joumaa et al., 2008; Powers et al., 2014), suggesting that the mechanisms underlying titin force enhancement and cross-bridge cycling are intimately connected in an active sarcomere. Distinct from Ca²⁺-dependent cross-bridges, titin force enhancement persists following Ca²⁺ deactivation (Joumaa et al., 2008) and cannot be attributed solely to the effects of Ca²⁺ (Powers et al., 2014). Therefore, it appears that titin's spring properties are modulated in actively stretched sarcomeres by an unknown mechanism that occurs in the presence of Ca²⁺ when cross-bridges cycle and contractile force is generated. It has been suggested that titin force enhancement could occur if titin binds to the thin filament to shorten and stiffen its available spring length during active sarcomere stretch (Herzog, 2014; Leonard and Herzog, 2010; Nishikawa et al., 2012; Powers et al., 2014). Conceptually, titin binding to the thin filaments provides a mechanism that can account for large, rapid and reversible force enhancement in actively stretched sarcomeres (Schappacher-Tilp et al., 2015). Therefore, if this hypothetical titin binding could be prevented, actively stretched sarcomeres would be more compliant (Fig. 2).

A spontaneous titin mutation presents the opportunity for this focused investigation of the mechanism underlying titin force enhancement. A titin mutation that occurs in muscular dystrophy with myositis (mdm) mice results in a predicted 83 amino acid deletion in the N2A and PEVK regions of the titin protein (Fig. 2) (Garvey et al., 2002). Muscles from mdm mice are actively more compliant (Monroy et al., 2012; Taylor-Burt et al., 2015), possibly owing to the deletion in titin's I-band region (Fig. 2). This suggests that modulation of titin stiffness in active sarcomeres by the proposed titin–thin filament interaction may be affected by the mdm mutation. We took this opportunity to investigate the mechanism of titin force enhancement by comparing the contractile properties of normal (WT) with mutant (mdm) myofibrils (composed of intact sarcomeres in series) during active and passive stretch to long sarcomere lengths beyond filament overlap. We hypothesized that titin force enhancement would be deficient if a critical region involved in titin–thin filament binding was deleted in the mdm mutation.

A single myofibril preparation was used for mechanical testing of mdm (muscular dystrophy with myositis, with a deletion in N2A–PEVK titin; Garvey et al., 2002) and WT mouse psoas sarcomeres (Fig. 2). For a thorough description of the methods employed in single myofibril experiments, see Powers et al. (2014). Briefly, experiments were conducted using an inverted light microscope (Axiovert 200M, Zeiss, Germany) equipped with a ×100 oil immersion objective (numerical aperture 1.3) and a ×2.5 Optovar (Immersol™, 518F, Zeiss). Force was calculated using custom-built nanofabricated silicon nitride nanolevers with a stiffness of 21 or 68 pN nm⁻¹ (Fig. 3).

Mdm mice were acquired from a colony established at Northern Arizona University Animal Care Facility (Flagstaff, AZ, USA) using breeding pairs of B6C3Fe a-/a-mdm mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Homozygous recessive Ttn^mdm (mdm) mice were confirmed by genotyping (Lopez et al., 2008). The Institutional Animal Care and Use Committee at Northern Arizona University approved the use of these animals. Age matched C57-BL/6 mice from the University of Calgary were used as a healthy comparison. Ethics approval for the study was granted by the Life and Environmental Sciences Animal Ethics Committee of the University of Calgary.

Strips of mouse psoas muscle were extracted from euthanized normal (WT) and mutant (mdm) animals (male and female, 28–40 days old, mean±s.e.m. 33±3.4 days). Muscles were tied to wooden sticks to preserve the in situ length, placed in a rigor-glycerol solution (−20°C, pH 7.0) with protease inhibitors (Complete^®, Roche Diagnostics, Montreal, QB, Canada) and stored at −20°C for 15–22 days (Leonard and Herzog, 2010). Muscle strips were homogenized in a rigor solution at 4°C and tested at 20°C (Leonard and Herzog, 2010). For experiments, chemically and mechanically isolated mdm and WT myofibrils were glued to one nanolever arm at one end and attached to a stiff glass needle (motor) at the other end (Fig. 3). Myofibril lengths were manually adjusted to an initial average sarcomere length of 2.5 μm. Average sarcomere length was determined by dividing the total length of the myofibril by the number of sarcomeres. Myofibrils were stretched by a piezoelectric motor controlled by custom-written LabView software at a rate of 0.1 μm per sarcomere per second to limit velocity-dependent increases in force. Displacement of the attached nanolever relative to the unattached reference arm was measured using a custom-written MATLAB program. Force was calculated from the measured displacement and lever stiffness and expressed in units of stress (nN μm⁻²) by normalizing the calculated force to the cross-sectional area of each myofibril. Myosin and actin content in WT and mdm psoas was determined using 12% SDS-PAGE (Laemmli, 1970).

Individual myofibrils were suspended in a relaxing solution (pCa 8.0) for passive stretch. For active experiments, the bath was flushed with a Ca²⁺-rich activation solution (pCa 3.5) prior to stretch. Myofibrils were stretched from an average initial sarcomere length of 2.5 µm to an average final length of 6.0 µm. At a sarcomere length of 4.0 µm, the mouse sarcomere is beyond myofilament overlap (Powers et al., 2014); thus, titin-based force within the intact sarcomere was considered at average sarcomere lengths exceeding 4.0 µm (Fig. 4).

A repeated measures two-way analysis of variance and simple effect test were used to determine statistical differences between stresses by genotype and experimental condition. To compare individual slopes by condition, Kruskal–Wallis non-parametric analyses and Mann–Whitney U with Bonferroni corrections were used. t-tests were used to compare independent groups. Significance was determined by an alpha level of 0.01.

Isolated myofibrils from mdm and WT were indistinguishable with no difference in diameter (Fig. 3; P=0.98; mean±s.e.m. mdm: 1.28±0.06 µm, N=19; WT: 1.27±0.05 µm, N=18) or contractile protein content, expressed as the ratio of myosin to actin content (myosin/actin) in mdm (1.39±0.09) and WT (1.41±0.09) psoas. Single isolated mdm and WT mouse psoas myofibrils were stretched actively (with Ca²⁺) or passively (without Ca²⁺) from an average sarcomere length of 2.5 µm to far beyond thick and thin filament overlap (6.0 µm) to observe titin-based stress in the absence of cross-bridges. Passive stress did not differ between mdm (N=10) and WT (N=9) myofibrils for the duration of stretch (P>0.01; Fig. 4). Active stress was lower in mdm (N=8) compared with wt myofibrils (N=7) at all sarcomere lengths (P<0.01; Fig. 4).

Individual slopes, indicative of titin stiffness at lengths beyond filament overlap, were compared by group and genotype for active WT (mean±s.d., 69.3±9.5 nN μm⁻³, R²=0.99, N=7), passive WT (21.9±5.9 nN μm⁻³, R²=0.99, N=9), active mdm (21.1±7.5 nN μm⁻³, R²=0.99, N=7) and passive mdm (22.2±11.1 nN μm⁻³, R²=0.99, N=10) myofibrils. Titin stiffness was three-times greater at the end of stretch (average sarcomere length, 6.0 µm) in active compared with passive WT myofibrils (Fig. 4). The slopes of active and passively stretched mdm myofibrils did not differ, indicating no change in titin stiffness from passive to activated mdm myofibrils (Fig. 4).

There was no difference in stress between passively stretched WT and mdm myofibrils, suggesting that the passive mechanical properties of mdm titin are not affected by the mdm deletion in titin (Fig. 4). Previous studies report no detectable difference in the molecular weight of mdm (predicted to be missing only 83 of over 30,000 amino acids) and WT titin (Huebsch et al., 2005). While we recognize that additional amino acids to those predicted based on the mutated titin gene may be deleted from mdm titin (Buck et al., 2014), the modest amino acid deletion in mdm titin (Fig. 2) does not alter titin's spring properties in a passive intact psoas sarcomere. Previous observations of increased passive stress in whole-muscle preparations from mdm mice (Huebsch et al., 2005; Lopez et al., 2008; Monroy et al., 2012; Taylor-Burt et al., 2015) are likely due to large endomysial and perimysial connective tissue deposits measured in mdm muscles (Lopez et al., 2008). With negligible connective tissue in the myofibril preparation, increased passive force was not observed in the present study and, thus, is unlikely to be the result of inherent changes to the passive stiffness of mdm titin.

The mechanism of titin force enhancement is of primary interest in this study. We observed a 3- to 4-fold increase in titin stiffness in actively compared with passively stretched WT mouse sarcomeres (4.0–6.0 μm), which was not observed in mdm sarcomeres (Fig. 4). This observation suggests that the mechanism of titin force enhancement is inhibited by the mdm mutation. In a passively stretched sarcomere, proximal Ig domains elongate, bearing little force until recruitment of the PEVK segment, which contributes the majority of titin-based force in the sarcomere (Granzier and Labeit, 2004). PEVK (Labeit et al., 2003) and Ig domains (DuVall et al., 2013) become measurably stiffer when Ca²⁺ ions are released from the sarcoplasmic reticulum during activation. Additional observations demonstrate Ca²⁺-dependent PEVK interactions with the thin filament (Kellermayer and Granzier, 1996) as well as interactions between tropomyosin, actin filaments and titin (Raynaud et al., 2004). The results from this and previous studies demonstrate a distinct modulation of titin stiffness when passive sarcomeres are activated and cross-bridges are allowed to cycle and generate force (Leonard and Herzog, 2010; Powers et al., 2014). The mechanism of titin force enhancement stiffens titin and, consequently, stiffens the actively stretched sarcomere as well. It was initially suggested that titin force enhancement may occur when titin binds to the thin filament, becoming shorter and stiffer in actively stretched sarcomeres (Leonard and Herzog, 2010). A binding location was later predicted at N2A, limiting titin's extendible spring length to include only its stiffest PEVK and distal Ig segments (Nishikawa et al., 2012). A mathematical model predicting N2A binding to the thin filament underestimated experimental values of titin force enhancement (Powers et al., 2014; Schappacher-Tilp et al., 2015). Thus, PEVK must become stiffer (beyond the effects of Ca²⁺ and beyond the effects of N2A binding) in an actively stretched sarcomere to explain experimental observations of titin force enhancement. Nishikawa et al. (2012) previously suggested that PEVK may become stiffer when it is wound around the thin filament with translation and rotation of cross-bridges (Nishikawa et al., 2012). It is also possible that PEVK binds directly to the thin filament at one or many sites along its length to become stiffer (Schappacher-Tilp et al., 2015) or to a different protein in the sarcomere such as tropomyosin (Raynaud et al., 2004). Whether by binding or winding or an alternative mechanism, we acknowledge that there are many ways in which titin may become stiffer in an activated sarcomere. Nevertheless, these and previous findings in mdm suggest that PEVK and N2A play a critical role in the mechanism of titin force enhancement.

Some considerations are worth noting regarding the effect of the mdm deletion on the structure and function of the titin protein. Protein conformation is dependent on the interactions of the individual amino acids within a protein. Even a small amino acid deletion can lead to modifications in protein conformation that subsequently alter its function (Akhter et al., 2014). In healthy muscle, length-specific titin isoforms that vary in stiffness are routinely generated by selective amino acid deletion during post-translational processing (Granzier and Labeit, 2004). Local conditions can also initiate post-translational processing of titin to generate length-specific titin isoforms that suit the altered mechanical needs of the muscle (Granzier and Labeit, 2004). While the mdm mutation is predicted to remove 74 amino acids from distal N2A and 9 amino acids from proximal PEVK (Fig. 2), the effect of the mdm mutation on post-translational processing is unknown. Subtle deletions in titin have previously been shown to elicit variant splicing during post-translational processing, resulting in additional, unpredicted deletion of amino acids along the length of the protein (Buck et al., 2014). Therefore, we acknowledge the possibility that additional amino acids to those in N2A and PEVK may be missing from mdm titin with further consequences to the function of the protein. In the absence of a comprehensive amino acid sequence of mdm titin, and with no measurable change in the molecular weight of mdm titin (Garvey et al., 2002; Huebsch et al., 2005), we assume a reasonably accurate prediction primarily affecting the N2A and PEVK segments of mdm titin (Fig. 2). However, whether the amino acids deleted in mdm titin directly or indirectly affect the mechanism of titin force enhancement is not clear. Determining the amino acid sequence of the mutant titin is an important focus of future investigation as the amino acids deleted in mdm may prove critical to the function of titin in an active sarcomere.

Considering the mechanism of titin force enhancement, one must acknowledge that mdm sarcomeres generated considerably less active stress than wt myofibrils within and beyond filament overlap (Fig. 4). With no increase in mdm stiffness during active stretch, we speculate that the titin force enhancement mechanism does not occur in mdm sarcomeres and that contractile dysfunction plays a role in the loss of titin force enhancement in mdm. Titin force enhancement is abolished with chemical inhibition of cross-bridges (Powers et al., 2014) and the magnitude of titin force enhancement has been shown to be dependent on contractile force (Leonard et al., 2010). These previous observations along with the present observation that active force and titin force enhancement are both affected in mdm sarcomeres leads us to the intriguing question: is there an inherent dysfunction in cross-bridge function that eliminates titin force enhancement in mdm or does the mdm deletion in titin alter the active mechanics of the sarcomeres?

Mdm sarcomeres generated lower active force within the region of filament overlap than normal WT sarcomeres. Gel analyses confirmed that myosin/actin were not differentially degraded in mdm. Thus, at the same sarcomere length and presumably similar overlap of thick and thin filaments, mdm myofibrils should generate comparable forces to WT myofibrils during active stretch unless there is inherent contractile protein malfunction. While it is reasonable to suspect that actin and myosin may have been affected by secondary effects of the mdm mutation, there is currently no evidence to suggest that structural changes to the contractile proteins may render mdm sarcomeres functionally deficient. Mdm sarcomeres do not differ in size or striation and appear physically unaltered (Fig. 3). Nevertheless, decreased active force has been observed in various preparations in mdm (Lopez et al., 2008; Monroy et al., 2012; Taylor-Burt et al., 2015) and down to the basic contractile unit of the muscle. This suggests that active mdm sarcomeres are deficient in their ability to generate force. Characterization of the mdm mutation by Lopez et al. (2008) showed an increase in the slow isoform of myosin heavy chain with mdm disease progression. However, whether (and how) a switch in myosin heavy chain isoforms results in contractile dysfunction in mdm is unclear. The N2A protein signaling complex is also disturbed in mdm, affecting various signaling pathways and the levels of numerous signaling molecules in the mdm sarcomere (Witt et al., 2004). However, how perturbed signaling may contribute to the contractile dysfunction observed in mdm remains speculative.

Our current hypothesis is that when the cross-bridges cycle and generate force, titin binds to the thin filament, shortening its extendable spring length to become stiffer in actively stretched sarcomeres (Herzog and Leonard, 2002; Leonard and Herzog, 2010; Nishikawa et al., 2012; Powers et al., 2014). How cross-bridge cycling alters the environment in the sarcomere to allow for titin binding is not understood. We previously demonstrated that movement of the regulatory protein troponin C does not initiate titin force enhancement (Powers et al., 2014). However, it is possible that movement of (or interaction with) the regulatory protein tropomyosin exposes potential titin binding site(s) on the thin filaments. We acknowledge the possibility that titin force enhancement may occur by a number of mechanisms. Nevertheless, with more than 300% titin force enhancement in WT myofibrils compared with 30% in mdm myofibrils at 6.0 µm, we demonstrate that following the mdm mutation in the titin gene, the titin force enhancement mechanism is lost.

Although various observations indicate a mechanism by which titin becomes up to four times stiffer in an actively stretched sarcomere (Leonard and Herzog, 2010; Powers et al., 2014), there is currently no direct evidence that modulation of titin force is elicited by titin binding to the thin filament. Since early investigations of residual force enhancement, the ‘recruitment of a passive element’ has been suggested as an explanation for the observations of increased force following active stretch (Edman et al., 1978). Subsequent studies have explored the possible recruitment of titin, the primary passive element in the sarcomere. Still, the details of titin recruitment remain vague and subject to speculation. With previous research demonstrating some affinity of titin for actin (Bianco et al., 2007; Kellermayer and Granzier, 1996) and the ability to reproduce experimental observations of sarcomere force enhancement by modeling titin–thin filament binding (Powers et al., 2014; Schappacher-Tilp et al., 2015), we remain in support of the idea that titin binds to the thin filament in activated skeletal muscle sarcomeres (Fig. 5), offering a rapid and reversible increase in stiffness. How this mechanism occurs and how it influences whole-muscle function and, subsequently, animal locomotion, are important topics for future investigations. In the case of the mdm mouse, movement is severely compromised, with animals exhibiting an abnormal gait (Huebsch et al., 2005; Taylor-Burt et al., 2015) while actively stretched sarcomeres are three times more compliant than wt sarcomeres (Fig. 4). Whether the compliance of actively stretched mdm sarcomeres directly contributes to deficiencies in mdm mouse locomotion cannot be deduced from the results of the present study. The ability to scale mechanical observations from subcellular, to whole-muscle and in vivo observations, where force enhancement is also present (Herzog and Leonard, 2002), is an important consideration for the design of future experiments investigating titin force enhancement. Whether titin is primarily (or partially) responsible for in vivo residual force enhancement observations remains an interesting consideration.

While titin has traditionally been viewed as a contributor strictly to passively stretched sarcomeres, it is now evident that its mechanical role extends to actively stretched sarcomeres. A major stiffening of titin has been observed in actively stretched intact sarcomeres of rabbit (Leonard and Herzog, 2010) and mouse (Powers et al., 2014). In this study, we have used a titin mutation model (mdm) to investigate how an amino acid deletion affects the elusive mechanism of titin force enhancement in skeletal muscle sarcomeres. The results suggest that the mechanism of titin force enhancement does not occur in mdm sarcomeres. Actively stretched mdm sarcomeres generate much lower force than healthy WT controls, suggesting that contractile force and titin force enhancement are intimately connected. Decreased active force in mdm has been observed previously in whole muscle (Monroy et al., 2012; Taylor-Burt et al., 2015) and fiber bundles (Lopez et al., 2008), and now in myofibrils, introducing the possibility that titin plays a critical role in active force that translates across the muscle hierarchy. Decreased active stress prior to stretch (2.5 µm) suggests that titin force enhancement is initiated immediately upon activation and is coupled to the sarcomere's ability to generate contractile force.

Titin can no longer be viewed as a passive structural spring in the sarcomere, but should be considered a dynamic adaptable spring, adjusting its stiffness to suit the temporary and extended mechanical demands of the sarcomere. However titin force enhancement may occur, this mechanism stiffens the structural network of the sarcomere for enduring the high forces of cross-bridges and stretch during dynamic locomotion. Titin-based force enhancement is essentially absent in mdm sarcomeres where amino acids in N2A and PEVK titin are deleted, indicating these specific regions along titin are paramount in increasing titin stiffness in an active sarcomere. With this spontaneous titin mutation (mdm), nature has provided us with a stepping-stone to explore the long-elusive mechanism of residual force enhancement and to further establish the role of titin in an active sarcomere. It is time to reconsider traditional views of skeletal muscle contraction as an exclusively cross-bridge-based mechanism and embrace the role of titin in the active sarcomere.

The authors acknowledge Geoffrey Power, Scott Sibole and Azim Jinha for contributing to the final version of this paper. Rene Fuqua and Uzma Tahir assisted in mdm tissue collection. The nanofabricated cantilevers were built by Tim Leonard at the NanoScale Facility at Cornell University (Ithaca, NY, USA), supported by the National Science Foundation. Statistical assistance was provided by Professor Tak Fung, University of Calgary.