Myofilament lengths of cat skeletal muscle: theoretical considerations and functional implications.

The cat is the primary model for neuromuscular research. However, sarcomere geometry, in particular thin-myofilament lengths of cat skeletal muscles, is not known, thus preventing adequate muscle modeling on the sarcomere level. The purpose of this study was to determine thin-myofilament lengths in cat skeletal muscle. It was found that average thin-myofilament lengths of cat tibialis anterior muscles (1.12 microns) were larger than the average values reported for frog (approximately 0.95 microns), rat (1.09 microns), and rabbit muscles (1.09 microns) and were smaller than the values reported for monkey (1.16 microns) and human skeletal muscles (1.27 microns). According to the cross-bridge theory of muscular contraction, this result implies that the range of sarcomere length on the ascending limb of the force-length relation for cat muscle is between those of frog, rat, and rabbit on the one side and monkey and human on the other side. It is speculated that the differences in thin-myofilament lengths of different animals are related to the functional demands of these muscles in everyday movement tasks. Isolated experimental observations appear to support this speculation.     

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.     

Structural states in the Z band of skeletal muscle correlate with states of active and passive tension

In skeletal muscle Z bands, the ends of the thin contractile filaments interdigitate in a tetragonal array of axial filaments held together by periodically cross-connecting Z filaments. Changes in these two sets of filaments are responsible for two distinct structural states observed in cross section, the small-square and basketweave forms. We have examined Z bands and A bands in relaxed, tetanized, stretched, and stretched and tetanized rat soleus muscles by electron microscopy and optical diffraction. In relaxed muscle, the A-band spacing decreases with increasing load and sarcomere length, but the Z lattice remains in the small-square form and the Z spacing changes only slightly. In tetanized muscle at sarcomere lengths up to 2.7 micron, the Z lattice assumes the basketweave form and the Z spacing is increased. The increased Z spacing is not the result of sarcomere shortening. Further, passive tension is not sufficient to cause this change in the Z lattice; active tension is necessary.     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.     

THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.     

Capillary tortuosity in skeletal muscles of mammals depends on muscle contraction

Capillary orientation (anisotropy) was compared in hindlimb muscles of mammals of different size and/or different aerobic capacity (dog, goat, pony, and calf). All muscles were fixed by vascular perfusion at sarcomere lengths ranging from 1.5 to 2.7 micron. The ratios of capillary counts per fiber cross-sectional area on two sets of sections (0 and 90 degrees) to the muscle fiber axis were used to estimate capillary anisotropy and the coefficient c(K,0) relating 1) capillary counts on transverse sections (a commonly used parameter to assess muscle capillarity) and 2) capillary length per volume of fiber (i.e., capillary length density). Capillary orientation parallel to the muscle fiber axis decreased substantially with muscle fiber shortening. In muscles fixed at sarcomere lengths of 2.69 microns (dog vastus intermedius) and 1.52 microns (dog gastrocnemius), capillary tortuosity and branching added 7 and 64%, respectively, to capillary length density. The data obtained in this study are highly consistent with the previously demonstrated relationship between capillary anisotropy and sarcomere length in extended vs. contracted rat muscles, by use of the same method. Capillary anisotropy in mammalian locomotory muscles is curvilinearly related to sarcomere length. No systematic difference was found in capillary tortuosity with either body size, athletic ability, or aerobic capacity. Capillary tortuosity is a consequence of fiber shortening rather than an indicator of the O2 requirements of the tissue.     

Sarcomere length-joint angle relationships of seven frog hindlimb muscles.

The sarcomere length-joint angle relationship was measured in 7 different muscle-joint complexes (n = 43 muscles) of the frog hindlimb (Rana pipiens). Muscles studied included the cruralis, iliacus internus, gastrocnemius, gluteus magnus, gracilis major, semimembranosus and the semitendinosus. Muscle-joint complexes were mounted in a jig and submerged in chilled Ringer's solution. Joints were rotated throughout their range of motion, while sarcomere length was measured by laser diffraction. Muscles were then formalin fixed and architectural properties determined by microdissection of individual muscle fibers. Sarcomere length change per degree of joint rotation (dLs/d theta) ranged from a low of 3.7 nm/degree for the cruralis muscle acting at the knee to a high of 12.5 nm/degree for the semitendinosus muscle acting at the hip. Values for dLs/d theta were significantly different between all muscles (p < 0.001), and dLs/d theta values for muscles acting at the hip were significantly greater than those for muscles acting at the knee (p < 0.005). dLs/d theta was negatively correlated with fiber length, suggesting a balance between fiber length and moment arm in most muscle-joint systems. However, many exceptions to this generalization were noted. These data suggest that different muscle-joint systems are 'designed' for differential contribution of muscle force production to the joint torque profile. The low variability of these data also suggests that sarcomere number is tightly regulated in these muscle-joint systems but not simply as a result of the total in vivo muscle excursion.     

Z band dynamics as a function of sarcomere length and the contractile state of muscle

The Z band in skeletal muscle has two distinct structural states--a relaxed (small square or ss) form and a maximally activated (basket weave or bw) form. We have examined by electron microscopy and optical diffraction Z lattice forms and dimensions and A band spacings in relaxed, tetanized, stretched, and stretched-and-tetanized rat soleus muscle. We have tested the independent contributions of passive load, active tension, and sarcomere length to Z band state. As the A band spacing decreased with increasing load and increasing sarcomere length in the untetanized muscles, the Z lattice remained in the ss form and the Z spacing changed only slightly. Computer-enhanced images from digitized electron micrographs showed that the ss Z lattice resisted deformation regardless of load or method of stretching. In contrast, when the muscle was tetanized at sarcomere lengths of up to 2.7 microns, the Z lattice assumed the bw form and the Z spacing was increased by 20%. Regardless of lattice form, Z spacing did not vary significantly with sarcomere length. Images from freeze-substituted preparations showed both lattice forms comparable to those in images from glutaraldehyde-fixed muscles. Thus, Z band state appears to be a function of the presence (or absence) of active tension. Our previous three-dimensional model is compatible with these observations and with the sub-structures revealed by computer-enhanced images of both lattice forms.     

Strain-induced reorientation of an intramuscular connective tissue network: implications for passive muscle elasticity

The most abundant intramuscular connective tissue component, the perimysium, of bovine M. sternomandibularis muscle was shown to be a crossed-ply arrangement of crimped collagen fibres which reorientate and decrimp on changing muscle fibre sarcomere length. Reorientation of perimysial strands was observed by light microscopy and identification of these strands as collagen fibres was confirmed by high-angle X-ray diffraction. Mean collagen fibre direction with respect to the muscle fibres ranged from approximately 80 degrees at sarcomere length = 1.1 micron to approximately 20 degrees at 3.9 microns. This behaviour was well described by a model of a crimped planar network surrounding a muscle fibre bundle of constant volume but varying length. Modelling of the mechanical properties of the perimysium at different sarcomere lengths produced a load-sarcomere length curve which was in good agreement with the passive elastic properties of the muscle, especially at long sarcomere lengths. It is concluded that the role of the perimysial collagen network is to prevent over-stretching of the muscle fibre bundles.     

Sarcomere length organization as a design for cooperative function amongst all lumbar spine muscles

The functional design of spine muscles in part dictates their role in moving, loading, and stabilizing the lumbar spine. There have been numerous studies that have examined the isolated properties of these individual muscles. Understanding how these muscles interact and work together, necessary for the prediction of muscle function, spine loading, and stability, is lacking. The objective of this study was to measure sarcomere lengths of lumbar muscles in a neutral cadaveric position and predict the sarcomere operating ranges of these muscles throughout full ranges of spine movements. Sarcomere lengths of seven lumbar muscles in each of seven cadaveric donors were measured using laser diffraction. Using published anatomical coordinate data, superior muscle attachment sites were rotated about each intervertebral joint and the total change in muscle length was used to predict sarcomere length operating ranges. The extensor muscles had short sarcomere lengths in a neutral spine posture and there were no statistically significant differences between extensor muscles. The quadratus lumborum was the only muscle with sarcomere lengths that were optimal for force production in a neutral spine position, and the psoas muscles had the longest lengths in this position. During modeled flexion the extensor, quadratus lumborum, and intertransversarii muscles lengthened so that all muscles operated in the approximate same location on the descending limb of the force-length relationship. The intrinsic properties of lumbar muscles are designed to complement each other. The extensor muscles are all designed to produce maximum force in a mid-flexed posture, and all muscles are designed to operate at similar locations of the force-length relationship at full spine flexion.     

Energetics of activation in frog skeletal muscle at sarcomere lengths beyond myofilament overlap.

Experiments were designed to gain information about the effects of extremely long sarcomere lengths (greater than 3.8 microns) on muscle activation. The amount of energy liberated in an isometric twitch by muscles stretched to sarcomere lengths where myofilament overlap is vanishingly small (greater than 3.6 microns) is thought to be an indirect measure of the Ca2+ cycled during contraction. The effects of altering sarcomere length from 3.8 to 4.3 microns on the amount of Ca2+ cycled was measured using twitch energy liberation as an indicator of the Ca2+ cycled. Twitch energy liberation decreased by approximately 20% over this sarcomere length region, suggesting that the amount of Ca2+ released by a single action potential is not altered dramatically when a muscle is stretched to extreme lengths.     

Interaction between series compliance and sarcomere kinetics determines internal sarcomere shortening during fixed-end contraction

The interaction between contractile force and in-series compliance was investigated for the intact skeletal muscle-tendon unit (MTU) of Rana pipiens semitendinosus muscles during fixed-end contraction. It was hypothesized that internal sarcomere shortening is a function of the length-force characteristics of contractile and series elastic components. The MTUs (n=18) were dissected, and, while submerged in Ringer's solution, muscles were activated at nine muscle lengths (-2 to +6 mm relative to optimal length in 1 mm intervals), while measuring muscle force and sarcomere length (SL) by laser diffraction. The MTU was clamped either at the bone (n=6), or at the proximal and distal ends of the aponeuroses (n=6). Muscle fibers were also trimmed along with aponeuroses down to 5-20 fibers and identical measurements were performed (n=6). The magnitude of shortening decreased as MTU length increased. The magnitude of shortening ranged from -0.08 to 0.3 microm, and there was no significant difference between delta SL as a function of clamp location. When aponeuroses were trimmed, sarcomere shortening was not observed at L(0) and longer. These results suggest that the aponeurosis is the major contributor to in-series compliance. Results also support our hypothesis but there also appear to be other factors affecting internal sarcomere shortening. The functional consequence of internal sarcomere shortening as a function of sarcomere length was to skew the muscle length-tension relationship to longer sarcomere lengths.     

Sarcomere length dependence of the force-velocity relation in single frog muscle fibers.

The force-velocity relation of single frog fibers was measured at sarcomere lengths of 2.15, 2.65, and 3.15 microns. Sarcomere length was obtained on-line with a system that measures the distance between two markers attached to the surface of the fiber, approximately 800 microns apart. Maximal shortening velocity, determined by extrapolating the Hill equation, was similar at the three sarcomere lengths: 6.5, 6.0, and 5.7 microns/s at sarcomere lengths of 2.15, 2.65, and 3.15 microns, respectively. For loads not close to zero the shortening velocity decreased with increasing sarcomere length. This was the case when force was expressed as a percentage of the maximal force at optimal fiber length or as a percentage of the sarcomere-isometric force at the respective sarcomere lengths. The force-velocity relation was discontinuous around zero velocity: load clamps above the level that kept sarcomeres isometric resulted in stretch that was much slower than when the load was decreased below isometric by a similar amount. We fitted the force-velocity relation for slow shortening (less than 600 nm/s) and for slow stretch (less than 200 nm/s) with linear regression lines. At a sarcomere length of 2.15 microns the slopes of these lines was 8.6 times higher for shortening than for stretch. At 2.65 and 3.15 microns the values were 21.8 and 14.1, respectively. At a sarcomere length of 2.15 microm, the velocity of stretch abruptly increased at loads that were 160-170% of the sarcomere isometric load, i.e., the muscle yielded. However, at a sarcomere length of 2.65 and 3.15 microm yield was absent at such loads. Even the highest loads tested (260%) resulted in only slow stretch.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optical Diffraction Studies of Muscle Fibers

A new technique to monitor light diffraction patterns electrically is applied to frog semitendinosus muscle fibers at various levels of stretch. The intensity of the diffraction lines, sarcomere length change, and the length-dispersion (line width) were calculated by fast analogue circuits and displayed in real time. A heliumneon laser (wavelength 6328 Å) was used as a light source. It was found that the intensity of the first-order diffraction line drops significantly (30-50%) at an optimal sarcomere length of 2.8 μm on isometric tetanic stimulation. Such stimulation produced contraction of half-sarcomeres by about 22 nm presumably by stretching inactive elements such as tendons. The dispersion of the sarcomere lengths is extremely small, and it is proportional to the sarcomere length (less than 4%). The dispersion increases on stimulation. These changes on isometric tetanic stimulation were dependent on sarcomere length. No vibration or oscillation in the averaged length of the sarcomeres was found during isometric tetanus within a resolution of 3 nm; however, our observation of increased length dispersion of the sarcomeres together with detection of the averaged shortening of the sarcomere lengths suggests the presence of asynchronous cyclic motions between thick and thin filaments. An alternative explanation is simply an increase of the length dispersion of sarcomeres without cyclic motions.     

Muscle anatomy is a primary determinant of muscle relaxation dynamics in the lobster (<Emphasis Type="Italic">Panulirus interruptus</Emphasis>) stomatogastric system

We stained sarcomere thin filaments with fluorescently labeled phalloidin, measured sarcomere and muscle length, and calculated sarcomere number in pyloric and gastric mill muscles. A wide range of sarcomere lengths (3.25–12.29 μm), muscle lengths (5.9–21.1 mm), and sarcomere numbers (648–3,036) were observed. Sarcomere number differences occurred both because of changes in sarcomere length and muscle length, and sarcomere and muscle length varied independently. This independence, the wide range of sarcomere numbers present, and the muscles being all ‘slow’, graded muscles allowed us to use these data to test Huxley and Neidergerke’s (1954) hypothesis that muscle dynamics depend on sarcomere number. The time constants of exponential fits to contraction relaxations were used to measure muscle dynamics, and comparison of theoretical predictions and experimental results quantitatively confirm the predicted dependence. The differing dynamics of the various pyloric muscles are likely functionally important, and the dependence of muscle dynamics on sarcomere number implies that sarcomere number is likely closely regulated in these muscles. The stomatogastric system may thus be an excellent model system for studying the mechanisms regulating muscle sarcomere number.     

Muscle and sarcomere lengths in the hind limb of the rabbit (Oryctolagus cuniculus) during a galloping stride

Rabbits were filmed galloping, and the length changes of the principal hind limb muscles were determined. Sarcomere lengths were measured in carcasses set by rigor mortis in four of the positions adopted during a stride. These sarcomere lengths were measured by means of a diffraction technique, devised for the purpose, using an ordinary microscope. Expected sarcomere lengths for three of the positions were predicted from that observed in the fourth, together with muscle length changes. A theoretical length-tension curve for rabbit muscle was constructed, using A and I filament lengths, it was shown that when the muscles were active, their sarcomere lengths corresponded to the plateau of the length-tension curve.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Muscle Compliance and the Longitudinal Transmission of Mechanical Impulses

The time required for a mechanical impulse to propagate from one end to the other was measured directly in frog sartorius muscles and in fiber bundles from the semitendinosus muscle. When the fibers were fully activated, the transmission velocity was 170 mm/ms. In resting fibers the transmission time was three to four times greater than in activated fibers. Control experiments indicated that the transmission time across the tendons was negligible. A muscle compliance of 55–80 Å per half sarcomere was estimated from these data. The "measurement time" of the method was calculated to be about 15 µs. This relatively short measurement time makes the method potentially useful for detecting changes in cross-bridge compliance.     

Changes in thick filament length in Limulus striated muscle

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.     

Thin filaments are not of uniform length in rat skeletal muscle

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3- , 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9- d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.