N2A Titin Research Articles

ANKRD1 expression is aberrantly upregulated in the mdm mouse model of muscular dystrophy and induced by stretch through NFκB.

The muscular dystrophy with myositis (mdm) mouse model results in a severe muscular dystrophy due to an 83-amino-acid deletion in the N2A region of titin, an expanded sarcomeric protein that functions as a molecular spring which senses and modulates the response to mechanical forces in cardiac and skeletal muscles. ANKRD1 is one of the muscle ankyrin repeat domain proteins (MARPs) a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. The aberrant over-activation of Nuclear factor Kappa B (NF-κB) and the Ankyrin-repeat domain containing protein 1 (ANKRD1) occurs in several models of progressive muscle disease including Duchenne muscular dystrophy. We hypothesized that mechanical regulation of ANKRD1 is mediated by NF-κB activation in skeletal muscles and that this mechanism is perturbed by small deletion of the stretch-sensing titin N2A region in the mdm mouse. We applied static mechanical stretch of the mdm mouse diaphragm and cyclic mechanical stretch of C2C12 myotubes to examine the interaction between NF-κΒ and ANKRD1 expression utilizing Western blot and qRTPCR. As seen in skeletal muscles of other severe muscular dystrophies, an aberrant increased basal expression of NF-κB and ANKRD1 were observed in the diaphragm muscles of the mdm mice. Our data show that in the mdm diaphragm, basal levels of NF-κB are increased, and pharmacological inhibition of NF-κB does not alter basal levels of ANKRD1. Alternatively, NF-κB inhibition did alter stretch-induced ANKRD1 upregulation. These data show that NF-κB activity is at least partially responsible for the stretch-induced expression of ANKRD1.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles.

Residual force enhancement (RFE) is the increase in steady-state force after active stretch relative to the force during isometric contraction at the same final length. The muscular dystrophy with myositis (mdm) mutation in mice, characterized by a small deletion in N2A titin, has been proposed to prevent N2A titin-actin interactions so that active mdm muscles are more compliant than wild type (WT). This decrease in active muscle stiffness is associated with reduced RFE. We investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from WT and mdm mice. On each fiber bundle, we performed active and passive stretches from an average sarcomere length of 2.6-3.0 µm at a slow rate of 0.04 µms-1, as well as isometric contractions at the initial and final lengths. One-way ANOVA showed that SOL and EDL fiber bundles from mdm mice exhibited significantly lower RFE than WT mice (P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. However, it contradicts the results from a previous study that appeared to show that compensatory mechanisms could restore titin force enhancement in single fibers from mdm psoas. We suggest that RFE measured previously in mdm single fibers was an artifact of the high variability in passive tension found in degenerating fibers, which begins after ∼24 days of age. The results are consistent with the hypothesis that RFE is reduced in mdm skeletal muscles owing to impaired Ca2+-dependent titin-actin interactions resulting from the small deletion in N2A titin.

Titin N2A Domain and Its Interactions at the Sarcomere.

Titin is a giant protein in the sarcomere that plays an essential role in muscle contraction with actin and myosin filaments. However, its utility goes beyond mechanical functions, extending to versatile and complex roles in sarcomere organization and maintenance, passive force, mechanosensing, and signaling. Titin’s multiple functions are in part attributed to its large size and modular structures that interact with a myriad of protein partners. Among titin’s domains, the N2A element is one of titin’s unique segments that contributes to titin’s functions in compliance, contraction, structural stability, and signaling via protein–protein interactions with actin filament, chaperones, stress-sensing proteins, and proteases. Considering the significance of N2A, this review highlights structural conformations of N2A, its predisposition for protein–protein interactions, and its multiple interacting protein partners that allow the modulation of titin’s biological effects. Lastly, the nature of N2A for interactions with chaperones and proteases is included, presenting it as an important node that impacts titin’s structural and functional integrity.

Unraveling the mysteries of the titin-N2A signalosome.

The sarcomeric titin springs and accessory proteins modulate muscle force and mechanical signaling at the N2A signalosome.

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

Titin N2A: More than a signaling node?

Stronczek et al. investigate the structure of titin–N2A and how it affects the binding of signaling proteins.

The N2A region of titin has a unique structural configuration.

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca2+-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.

Solution NMR Structure of Titin N2A Region Ig Domain I83 and Its Interaction with Metal Ions

Titin, the largest single chain protein known so far, has long been known to play a critical role in passive muscle function but recent studies have highlighted titin’s role in active muscle function. One of the key elements in this role is the Ca2+-dependent interaction between titin’s N2A region and the thin filament. An important element in this interaction is I83, the terminal immunoglobulin domain in the N2A region. There is limited structural information about this domain, but experimental evidence suggests that it plays a critical role in the N2A-actin binding interaction. We now report the solution NMR structure of I83 and characterize its dynamics and metal binding properties in detail. Its structure shows interesting relationships to other I-band Ig domains. Metal binding and dynamics data point towards the way the domain is evolutionarily optimized to interact with neighbouring domains. We also identify a calcium binding site on the N-terminal side of I83, which is expected to impact the interdomain interaction with the I82 domain. Together these results provide a first step towards a better understanding of the physiological effects associated with deletion of most of the I83 domain, as occurs in the mdm mouse model, as well as for future investigations of the N2A region.

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.

N2A Titin: Signaling Hub and Mechanical Switch in Skeletal Muscle.

Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin’s functions, muscular dystrophy with myositis (mdm), arose spontaneously in mice as a transposon-like LINE repeat insertion that results in a small deletion in the N2A region of titin. This small deletion profoundly affects hypertrophic signaling and muscle mechanics, thereby providing insights into the function of this specific region and the consequences of its dysfunction. The impact of this mutation is profound, affecting diverse aspects of the phenotype including muscle mechanics, developmental hypertrophy, and thermoregulation. In this review, we explore accumulating evidence that points to the N2A region of titin as a dynamic “switch” that is critical for both mechanical and signaling functions in skeletal muscle. Calcium-dependent binding of N2A titin to actin filaments triggers a cascade of changes in titin that affect mechanical properties such as elastic energy storage and return, as well as hypertrophic signaling. The mdm phenotype also points to the existence of as yet unidentified signaling pathways for muscle hypertrophy and thermoregulation, likely involving titin’s PEVK region as well as the N2A signalosome.

Deleting Titin's C-Terminal PEVK Exons Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal Hypertrophy in Skeletal Muscle.

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the TtnΔ219–225 model (MGI: TtnTM 2.1Mgot). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle (WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.

Titin: A Tunable Spring in Active Muscle.

Muscle has conventionally been viewed as a motor that converts chemical to kinetic energy in series with a passive spring, but new insights emerge when muscle is viewed as a composite material whose elastic elements are tuned by activation. New evidence demonstrates that calcium-dependent binding of N2A titin to actin increases titin stiffness in active skeletal muscles, which explains many long-standing enigmas of muscle physiology.

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region.

Titin is a large filamentous protein that spans half a sarcomere, from Z-disk to M-line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein-protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80-I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81-I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔGunfolding , which is increased in the presence of calcium (pCa 4.3), indicating that Ca2+ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca2+ might influence binding events involving N2A.

Calcium-dependent titin-thin filament interactions in muscle: observations and theory.

Gaps in our understanding of muscle mechanics demonstrate that the current model is incomplete. Increasingly, it appears that a role for titin in active muscle contraction might help to fill these gaps. While such a role for titin is increasingly accepted, the underlying molecular mechanisms remain unclear. The goals of this paper are to review recent studies demonstrating Ca2+-dependent interactions between N2A titin and actin in vitro, to explore theoretical predictions of muscle behavior based on this interaction, and to review experimental data related to the predictions. In a recent study, we demonstrated that Ca2+ increases the association constant between N2A titin and F-actin; that Ca2+ increases rupture forces between N2A titin and F-actin; and that Ca2+ and N2A titin reduce sliding velocity of F-actin and reconstituted thin filaments in motility assays. Preliminary data support a role for Ig83, but other Ig domains in the N2A region may also be involved. Two mechanical consequences are inescapable if N2A titin binds to thin filaments in active muscle sarcomeres: (1) the length of titin's freely extensible I-band should decrease upon muscle activation; and (2) binding between N2A titin and thin filaments should increase titin stiffness in active muscle. Experimental observations demonstrate that these properties characterize wild type muscles, but not muscles from mdm mice with a small deletion in N2A titin, including part of Ig83. Given the new in vitro evidence for Ca2+-dependent binding between N2A titin and actin, it is time for skepticism to give way to further investigation.

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

During isometric contractions, the optimal length of skeletal muscles increases with decreasing activation. The underlying mechanism for this phenomenon is thought to be linked to length dependence of Ca2+ sensitivity. Muscular dystrophy with myositis (mdm), a recessive titin mutation in mice, was used as a tool to study the role of titin in activation dependence of optimal length and length dependence of Ca2+ sensitivity. We measured the shift in optimal length between tetanic and twitch stimulation in mdm and wild-type muscles, and the length dependence of Ca2+ sensitivity at short and long sarcomere lengths in mdm and wild-type fiber bundles. The results indicate that the mdm mutation leads to a loss of activation dependence of optimal length without the expected change in length dependence of Ca2+ sensitivity, demonstrating that these properties are not linked, as previously suggested. Furthermore, mdm muscles produced maximum tetanic stress during sub-optimal filament overlap at lengths similar to twitch contractions in both genotypes, but the difference explains less than half of the observed reduction in active force of mdm muscles. Mdm muscles also exhibited increased electromechanical delay, contraction and relaxation times, and decreased rate of force development in twitch contractions. We conclude that the small deletion in titin associated with mdm in skeletal muscles alters force production, suggesting an important regulatory role for titin in active force production. The molecular mechanisms for titin's role in regulating muscle force production remain to be elucidated.

Severe thermoregulatory deficiencies in mice with a deletion in the titin gene TTN.

Muscular dystrophy with myositis (mdm) mice carry a deletion in the N2A region of the gene for the muscle protein titin (TTN), shiver at low frequency, fail to maintain body temperatures (Tb) at ambient temperatures (Ta) <34°C, and have reduced body mass and active muscle stiffness in vivo compared with wild-type (WT) siblings. Impaired shivering thermogenesis (ST) could be due to the mutated titin protein causing more compliant muscles. We hypothesized that non-shivering thermogenesis (NST) is impaired. To characterize the response to cold exposure, we measured Tb and metabolic rate (MR) of WT and mdm mice at four nominal temperatures: 20, 24, 29 and 34°C. Subsequently, we stimulated NST with noradrenaline. Manipulation of Ta revealed an interaction between genotype and MR: mdm mice had higher MRs at 29°C and lower MRs at 24°C compared with WT mice. NST capacity was lower in mdm mice than in WT mice. Using MR data from a previous study, we compared MR of mdm mice with MR of Perognathus longimembris, a mouse species of similar body mass. Our results indicated low MR and reduced NST of mdm mice. These were more pronounced than differences between mdm and WT mice owing to body mass effects on MR and capacity for NST. Correcting MR using Q10 showed that mdm mice had lower MRs than size-matched P. longimembris, indicating that mutated N2A titin causes severe thermoregulatory defects at all levels. Direct effects of the titin mutation lead to lower shivering frequency. Indirect effects likely lead to a lower capacity for NST and increased thermal conductance through decreased body size.

Calcium increases titin N2A binding to F-actin and regulated thin filaments

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A – F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A – actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A – actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin – actin binding occurs in skeletal muscle sarcomeres in vivo.

Calcium Dependent Interaction Between N2A-Halo and F-Actin: A Single Molecule Study

In the last two decades, interactions between titin's I-band region and actin have been widely investigated for skeletal and cardiac isoforms. Previous studies suggested a calcium-dependent interaction between titin and actin. However, previous in vitro motility studies did not quantify the strength of interaction between titin's N2A region and actin in the presence of calcium. Here, we used AFM single molecule force spectroscopy (SMFS) to probe the interaction strength between a recombinant N2A-HALO construct and actin filaments (F-actin), in the presence (pCa = 4.0) and absence of calcium. The N2A-Halo construct consisted of the N2A region of titin (Ig80-IS-Ig81-Ig82-Ig83, where IS is the insertion sequence) with a C-terminal Halo-tag. The construct was immobilized on an AFM cantilever tip via its N-terminal cysteines. The interaction strength was determined by probing the molecule during multiple approach-retraction cycles at physiological pulling rates (300-1000 nm/sec) over various locations on a substrate functionalized with F-actin stabilized on a lipid bilayer with ATP. Preliminary data show that both the yield and strength of N2A-Halo interactions with F-actin nearly doubled in the presence of Ca2+, with multiple peaks in the rupture force distribution at 50 ± 25 pN, 75 ± 10 pN, and 100 ± 25 pN. The force extension curves also showed different rupture lengths, indicating multiple actin binding sites. No significant interactions occurred between N2A constructs and the charged lipid bi-layer surface in the absence of F-actin. These experiments show that calcium increases the interaction probability and binding strength between N2A-Halo and F-actin and that the binding strength between N2A titin and F-actin is nearly 4 times greater than previously reported for PEVK titin - F-actin interactions. These results support a physiological role for titin in active muscle contraction and force enhancement.

Investigation of heat production deficiencies during cold stress and a nonshivering thermogenesis challenge in mice with a deletion in N2A titin

Homeothermic animals strive to maintain core body temperature across varying ambient temperatures. Studies have demonstrated that mice with the muscular dystrophy with myositis (mdm) mutation shiver at a lower than expected frequency, are heterothermic, and have reduced active muscle stiffness in vivo compared to wildtype mice. Mdm mice are characterized by a deletion in the N2A region of titin that results in a lower body mass, stiffer gait, and reduced lifespan. The underlying defect that leads to a lower shivering frequency is not known but could be caused by muscle properties. The titin protein is known to behave like a spring and contribute to passive muscle stiffness. Recent studies have shown that titin contributes to active muscle stiffness as well. Shivering thermogenesis can be modeled as simple harmonic motion, where the spring constant k represents muscle stiffness. This impairment in heat production could be due to the N2A deletion in the titin protein, leading to a more compliant spring and a lower shivering frequency as explained by simple harmonic motion. An alternative hypothesis is that the ability of mdm mice to use nonshivering thermogenesis and/or uncoupling protein 1 (UCP1), found in brown adipose tissue (BAT) to generate heat during cold stress is impaired. The purpose of this study was to evaluate potential causes for the inability of mdm mice to maintain homeothermy during cold stress. We hypothesize that the inability of mdm mice to thermoregulate properly is due primarily to an impairment in heat production (e.g. shivering thermogenesis and nonshivering thermogenesis). Mice underwent a 4‐day cold stress experiment during which they lived in a mouse cage that used a continuous system for indirect calorimetry. The cages were placed in an incubator so that ambient temperature could be controlled. During each experiment, temperature was controlled to either 34°C, 29°C, 25°C, 19°C. Upon conclusion of the cold stress experiment, a nonshivering thermogenesis challenge was employed by administering 1.5 mg/kg of norepinephrine. As expected, wildtype mice increased their metabolic rate during cold stress and during the nonshivering thermogenesis challenge. RQ values reached 0.7, representing pure fat oxidation and utilization of uncoupling protein 1, present in BAT. Core body temperature for an mdm mouse decreased with decreasing ambient temperature within a range of 28C to 37C. During the nonshivering thermogenesis challenge, both wildtype and mdm mice increased their oxygen consumption. Both mdm and wildtype mice increased their oxygen consumption by 37% during the nonshivering thermogenesis challenge. These results suggest that the failure to maintain homeothermy is due to reduced shivering frequency and not a reduced capacity for nonshivering thermogenesis, pending additional experiments.Support or Funding InformationOur funding sources include NSF IOS‐0742483, NSF IOS‐1025806, NSF IOS‐1456868, and the W.M. Keck Foundation.

Shiver me titin! Elucidating titin's role in shivering thermogenesis.

Shivering frequency scales predictably with body mass and is 10 times higher in a mouse than a moose. The link between shivering frequency and body mass may lie in the tuning of muscle elastic properties. Titin functions as a muscle 'spring', so shivering frequency may be linked to titin's structure. The muscular dystrophy with myositis (mdm) mouse is characterized by a deletion in titin's N2A region. Mice that are homozygous for the mdm mutation have a lower body mass, stiffer gait and reduced lifespan compared with their wild-type and heterozygous siblings. We characterized thermoregulation in these mice by measuring metabolic rate and tremor frequency during shivering. Mutants were heterothermic at ambient temperatures of 20-37°C while wild-type and heterozygous mice were homeothermic. Metabolic rate increased at smaller temperature differentials (i.e. the difference between body and ambient temperatures) in mutants than in non-mutants. The difference between observed tremor frequencies and shivering frequencies predicted by body mass was significantly larger for mutant mice than for wild-type or heterozygous mice, even after accounting for differences in body temperature. Together, the heterothermy in mutants, the increase in metabolic rate at low temperature differentials and the decreased tremor frequency demonstrate the thermoregulatory challenges faced by mice with the mdm mutation. Oscillatory frequency is proportional to the square root of stiffness, and we observed that mutants had lower active muscle stiffness in vitro. The lower tremor frequencies in mutants are consistent with reduced active muscle stiffness and suggest that titin affects the tuning of shivering frequency.