The Viscoelastic Properties of Passive Eye Muscle in Primates. I: Static Forces and Step Responses

The viscoelastic properties of passive eye muscles are prime determinants of the deficits observed following eye muscle paralysis, the root cause of several types of strabismus. Our limited knowledge about such properties is hindering the ability of eye plant models to assist in formulating a patient's diagnosis and prognosis. To investigate these properties we conducted an extensive in vivo study of the mechanics of passive eye muscles in deeply anesthetized and paralyzed monkeys. We describe here the static length-tension relationship and the transient forces elicited by small step-like elongations. We found that the static force increases nonlinearly with length, as previously shown. As expected, an elongation step induces a fast rise in force, followed by a prolonged decay. The time course of the decay is however considerably more complex than previously thought, indicating the presence of several relaxation processes, with time constants ranging from 1 ms to at least 40 s. The mechanical properties of passive eye muscles are thus similar to those of many other biological passive tissues. Eye plant models, which for lack of data had to rely on (erroneous) assumptions, will have to be updated to incorporate these properties

A Mathematical Model of Muscle Containing Heterogeneous Half-Sarcomeres Exhibits Residual Force Enhancement

A skeletal muscle fiber that is stimulated to contract and then stretched from L1 to L2 produces more force after the initial transient decays than if it is stimulated at L2. This behavior has been well studied experimentally, and is known as residual force enhancement. The underlying mechanism remains controversial. We hypothesized that residual force enhancement could reflect mechanical interactions between heterogeneous half-sarcomeres. To test this hypothesis, we subjected a computational model of interacting heterogeneous half-sarcomeres to the same activation and stretch protocols that produce residual force enhancement in real preparations. Following a transient period of elevated force associated with active stretching, the model predicted a slowly decaying force enhancement lasting >30 seconds after stretch. Enhancement was on the order of 13% above isometric tension at the post-stretch muscle length, which agrees well with experimental measurements. Force enhancement in the model was proportional to stretch magnitude but did not depend strongly on the velocity of stretch, also in agreement with experiments. Even small variability in the strength of half-sarcomeres (2.1% standard deviation, normally distributed) was sufficient to produce a 5% force enhancement over isometric tension. Analysis of the model suggests that heterogeneity in half-sarcomeres leads to residual force enhancement by storing strain energy introduced during active stretch in distributions of bound cross-bridges. Complex interactions between the heterogeneous half-sarcomeres then dissipate this stored energy at a rate much slower than isolated cross-bridges would cycle. Given the variations in half-sarcomere length that have been observed in real muscle preparations and the stochastic variability inherent in all biological systems, half-sarcomere heterogeneity cannot be excluded as a contributing source of residual force enhancement

Making Muscle Elastic: The Structural Basis of Myomesin Stretching

The muscle M-band protein myomesin comprises a 36-nm long filament made of repetitive immunoglobulin–helix modules that can stretch to 2.5-fold this length, demonstrating substantial molecular elasticity

Conformation-regulated mechanosensory control via titin domains in cardiac muscle

The giant filamentous protein titin is ideally positioned in the muscle sarcomere to sense mechanical stimuli and transform them into biochemical signals, such as those triggering cardiac hypertrophy. In this review, we ponder the evidence for signaling hotspots along the titin filament involved in mechanosensory control mechanisms. On the way, we distinguish between stress and strain as triggers of mechanical signaling events at the cardiac sarcomere. Whereas the Z-disk and M-band regions of titin may be prominently involved in sensing mechanical stress, signaling hotspots within the elastic I-band titin segment may respond primarily to mechanical strain. Common to both stress and strain sensor elements is their regulation by conformational changes in protein domains

Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension

Expression and purification of large nebulin fragments and their interaction with actin.

cDNA clones encoding mouse skeletal muscle nebulin were expressed in Escherichia coli as thioredoxin fusion proteins and purified in the presence of 6 M urea. These fragments, called 7a and 8c, contain 28 and 19 of the weakly repeating approximately 35-residue nebulin modules, respectively. The nebulin fragments are soluble at extremely high pH, but aggregate when dialyzed to neutral pH, as assayed by centrifugation at 16,000 x g. However, when mixed with varying amounts of G-actin at pH 12 and then dialyzed to neutral pH, the nebulin fragments are solubilized in a concentration-dependent manner, remaining in the supernatant along with the monomeric actin. These results show that interaction with G-actin allows the separation of insoluble nebulin aggregates from soluble actin-nebulin complexes by centrifugation. We used this property to assay the incorporation of nebulin fragments into preformed actin filaments. Varying amounts of aggregated nebulin were mixed with a constant amount of F-actin at pH 7.0. The nebulin aggregates were pelleted by centrifugation at 5200 x g, whereas the actin filaments, including incorporated nebulin fragments, remained in the supernatant. Using this assay, we found that nebulin fragments 7a and 8c bound to actin filaments with high affinity. Immunofluorescence and electron microscopy of the actin-nebulin complexes verified that the nebulin fragments were reorganized from punctate aggregates to a filamentous form upon interaction with F-actin. In addition, we found that fragment 7a binds to F-actin with a stoichiometry of one nebulin module per actin monomer, the same stoichiometry we found in vivo. In contrast, 8c binds to F-actin with a stoichiometry of one module per two actin monomers. These data indicate that 7a can be incorporated into actin filaments to the same extent found in vivo, and suggest that shorter fragments may not bind actin filaments in the same way as the native nebulin molecule

New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly

N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-1B). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly