Individual Sarcomere Lengths in Whole Muscle Fibers and Optimal Fiber Length Computation

Estimation of muscle fiber optimum length is typically accomplished using either laser diffraction or by counting the number of sarcomeres in a portion of the muscle fiber, measuring the distance that encompasses those sarcomeres and dividing by the number of sarcomeres to obtain an average sarcomere length. If the sarcomeres are not uniformly distributed, either of these techniques could produce errors when estimating optimum lengths. The purposes of this study were: to describe new software that automatically analyzes digital images of skeletal muscle fibers to measure individual sarcomere lengths; and to use this software to measure individual sarcomere lengths along complete muscle fibers to examine the influence of computing whole muscle fiber properties from portions of the fiber. Six complete muscle fibers were imaged using a digital camera attached to a microscope. The images were then processed to achieve the best resolution possible, individual sarcomeres along the image were detected, and each individual sarcomere length was measured. The software accuracy was compared with that of manual measurement and was found to be as accurate. In addition, the time to measure individual sarcomere lengths was greatly reduced using the software compared with manual measurement. The arrangement of individual sarcomere lengths demonstrated long‐range correlations, which indicates problems in assuming only a portion of a fiber can be used to determine whole fiber properties. This study has provided evidence on the number of sarcomeres which must be analyzed to infer the properties of whole muscles. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Light diffraction patterns and sarcomere length variation in striated muscle fibers ofLimulus

Summary Light diffraction patterns produced byLimulus striated muscle fibers were examined. Segments of fibers were glycerinated, fixed or bathed in relaxing solution. Profiles of the intensity of a diffracted order vs. the angle of incidence of the laser beam often exhibited narrow peaks with the fiber at rest length. The incident angles at which the intensity of left and right orders is greatest are used to calculate the sarcomere length, supporting the notion that regions of the fiber are organised into Bragg reflecting planes. These profiles developed subpeaks and broadened upon stretch of the fiber. The broad angle scan profiles are suggested to result from a decrease in the regular packing of myofibrils as the fiber is lengthened. The angular width of the subpeaks is used to estimate the thickness of clusters of myofibrils. The variation in sarcomere length along the fiber, as determined by the 0th to 1st diffraction order spacing, was dependent upon the fiber preparation. Glycerinated fibers and those bathed in relaxing solution showed more variation than fixed fibers. The variation of sarcomere length is compared to the variation in thick filament lengths inLimulus reported by Dewey et al. (1982) lengths inLimulus reported by Dewey et al. (1982). These results are compared to those obtained from frog fiber segments.     

Heterogeneity of fiber and sarcomere length in the human masseter muscle.

This study deals with regional differences in the architectural design of the human masseter muscle. For a number of defined muscle regions the three-dimensional coordinates of origin and insertion points, and the lengths of the muscle fibers and the sarcomeres were determined in the closed jaw position. Measurements were made from cadavers and the data were used as input for a model predicting sarcomere length at other mandibular positions. At a closed jaw average muscle fiber length of the muscle regions ranged between 19.0-30.3 mm. The fibers appeared to be considerably longer (35%) anteriorly than posteriorly in the muscle, and deeply situated fibers were on average 5% shorter than superficially situated ones. Average sarcomere length of the regions ranged between 2.27-2.55 microns, indicating that at a closed jaw position sarcomeres are at suboptimum length and have different positions on the length-tension curve. In the deep layer of the muscle sarcomeres were significantly shorter (6%) than in the superficial layer. Within the superficial layer sarcomere lengths did not differ significantly, but in the deep layer sarcomeres were shorter (8%) posteriorly than anteriorly in the muscle. The model shows that jaw displacement had a different effect on sarcomere length in the muscle regions. When the jaw was rotated about a transverse axis (open/close rotation) sarcomere excursions were relatively small in the posterior muscle regions and large in the anterior regions. The reverse was true when the jaw was rotated contralaterally about a vertical axis. It is concluded that, due to heterogeneity in fiber and sarcomere lengths, the distribution of maximal isometric tension across the muscle at full effort is not uniform.     

Heterogeneity of fiber and sarcomere length in the human masseter muscle

This study deals with regional differences in the architectural design of the human masseter muscle. For a number of defined muscle regions the three-dimensional corrdinates of origin and insertion points, and the lengths of the muscle fibers and the sarcomeres were determined in the closed jaw position. Measurements were made from cadavers and the data were used as input for a model predicting sarcomere length at other mandibular positions. At a closed jaw average muscle fiber length of the muscle regions ranged between 19.0–30.3 mm. The fibers appeared to be considerably longer (35%) anteriorly than posteriorly in the muscle, and deeply situated fibers were on average 5% shorter than superficially situated ones. Average sarcomere length of the regions ranged between 2.27–2.55 μm, indicating that at a closed jaw position sarcomeres are at suboptimum length and have different positions on the length-tension curve. In the deep layer of the muscle sarcomeres were significantly shorter (6%) than in the superficial layer. Within the superficial layer sarcomere lengths did not differ significantly, but in the deep layer sarcomeres were shorter (8%) posteriorly than anteriorly in the muscle. The model shows that jaw displacement had a different effect on sarcomere length in the muscle regions. When the jaw was rotated about a transverse axis (open/close rotation) sarcomere excursions were relatively small in the posterior muscle regions and large in the anterior regions. The reverse was true when the jaw was rotated contralaterally about a vertical axis. It is concluded that, due to heterogeneity in fiber and sarcomere lengths, the distribution of maximal isometric tension across the muscle at full effort is not uniform.     

Inhibition of force production in compressed skinned muscle fibers of the frog

Calcium-activated force development in skinned frog muscle fibers is inhibited by osmotically compressing the fiber, probably owing to a decrease in spacing between the myofilaments. This inhibition depends upon sarcomere length in that fibers at long lengths must be compressed further than those at short lengths to achieve the same degree of inhibition. As a result, this length dependency of inhibition tends to compensate for the reduction of force due solely to the decrease in interfilament spacing which occurs with stretch in intact fibers.     

Non-uniform distribution of strain during stretch of relaxed skeletal muscle fibers from rat soleus muscle

Tension and regional average sarcomere length (L_s) behavior were examined during repeated stretches of single, permeabilized, relaxed muscle fibers isolated from the soleus muscles of rats. We tested the hypothesis that during stretches of single permeabilized fibers, the global fiber strain is distributed non-uniformly along the length of a relaxed fiber in a repeatable pattern. Each fiber was subjected to eight constant-velocity stretch and release cycles with a strain of 32% and strain rate of 54% s⁻¹. Stretch-release cycles were separated by a 4.5 min interval. Throughout each stretch-release cycle, sarcomere lengths were measured using a laser diffraction technique in which 20 contiguous sectors along the entire length of a fiber segment were scanned within 2 ms. The results revealed that: (1) the imposed length change was not distributed uniformly along the fiber, (2) the first stretch-release cycle differed from subsequent cycles in passive tension and in the distribution of global fiber strain, and (3) a characteristic “signature” for the L_s response emerged after cycle 3. The findings support the conclusions that longitudinal heterogeneity exists in the passive stiffness of individual muscle fibers and that preconditioning of fibers with stretch-release cycles produces a stable pattern of sarcomere strains.     

The physiological cross-sectional area of motor units in the cat tibialis anterior

The physiological cross-sectional area (CSA) of a motor unit (MU), taken as the sum of fiber areas measured on a single section through the approximate midlength of the MU, has been compared with the physiological CSA more strictly defined as the sum of the maximal areas to be found anywhere along the length of each of the MU fibers. The CSA at intervals along the fiber length was measured in fibers selected from four glycogen-depleted, isolated MUs in the cat tibialis anterior (TA), and profiles of the summed areas made. In one MU, measurements were also taken on all the MU's fibers at less frequent intervals. The profiles demonstrate that the summed CSA based on each fiber's maximum CSA may exceed that derived from observation on any single section by as much as 20%. As a consequence, values that have been reported for specific tension (force per unit area) of MUs in the TA and probably other muscles may have been overestimated, especially for those MUs of fast type. Estimates were also made of the share of the MU's total force transmitted directly to the tendons of origin and insertion via endings of the blunt musculotendinous type as distinct from tapering intrafascicular endings acting through in-series connective tissue and non-MU fibers. In two MUs of slow type in which most fibers ran from tendon to tendon, “partial tapering” extending over 1 cm of the fiber length accounted for a third of the total physiological CSA, and indicated yet another mode for relay of the MU's force to the tendon. © 1993 Wiley-Liss, Inc.     

Contractile properties of bundles of fiber segments from skeletal muscles

Our purpose was to determine whether contractile properties of bundles of skeletal muscle fiber segments were significantly different from those of bundles of intact fibers. In frog muscles, the only difference between the contractile properties of fiber segments and intact fibers was a lower maximum velocity of shortening (Vo) for the fiber segments. In mammalian muscles, the contraction time (TPT), relaxation time (RT1/2), and maximum tetanus tension (Po) of bundles of fiber segments were not different from those of intact fibers, but the rate of tension development (dP/dt), twitch-to-tetanus ratio (Pt/Po) and Vo were lower. The lower dP/dt and Pt/Po resulted from increased compliance due to damaged sarcomeres near cut ends. Within 4-9 mm of a cut end, membrane potentials were less than control values, and sarcomeres lengthened during a fixed-end contraction. after the length of fiber segments was corrected for the exact portion that was not shortening, the Vo of fiber segments was not different from that of intact fibers. We conclude that valid estimates of contractile properties can be obtained from bundles of skeletal muscle fiber segments.     

Enzymatic heterogeneity of the capillary bed of rat skeletal muscles

This study of the capillaries in rat skeletal muscle involved the use of a histochemical method that allows one to distinguish between arterial and venous portions of capillaries. Under controlled staining conditions, the arterial portion of the capillary bed reacts positively for alkaline phosphatase (AP) activity, and the venous portion is positive for dipeptidylpeptidase IV (DPP IV) activity. A short transitional capillary segment is positive for the activity of both enzymes. Capillaries of the normal soleus muscle and the red and white portions of the sternomastoid muscle have been quantitatively analyzed. Quantitative data demonstrated differences in capillary dimensions among the muscles studied. Capillaries of the white part of the sternomastoid were the longest, and they had the shortest DPP IV-positive segment (8% of the total capillary length). Capillaries of the soleus muscle were the shortest, and they also had short DPP IV-positive segments (16%). In contrast, the DPP IV-positive segments of the red part of the sternomastoid occupied 60% of the total capillary length. Survey cross sections reveal a mosaic distribution of patches of capillaries stained for AP and DPP IV activity. This study reveals that within given bundles of muscle fibers, the capillaries that run parallel to the muscle fibers are aligned relative to one another in such a manner that their arterial and venous segments are in register.     

Lens structure in MIP-deficient mice

In this study we used correlative light, scanning, and transmission (freeze-etch) electron microscopy to characterize lens structure in normal mice and compare it with that in mice deficient in the major intrinsic protein (MIP) of fiber cells. Grossly, wild-type lenses were transparent and had typical Y sutures at all of the ages examined. These lenses had fibers of uniform shape (hexagonal in cross section) arranged in ordered concentric growth shells and radial cell columns. In addition, these fibers had normal opposite end curvature and lateral interdigitations regularly arrayed along their length. Ultrastructural evaluation of these fibers revealed anterior and posterior end segments characterized by square array membrane on low-amplitude wavy fiber membrane. Approximately 13% of the equatorial or mid segments of these same fibers were specialized as gap junctions (GJs). In contrast, heterozygote lenses, while initially transparent at birth, were translucent by 3 weeks of age, except for a peripheral transparent region that contained fibers in the early stages of elongation. This degradation in clarity was correlated with abnormal fiber structure. Specifically, although the mid segment of these fibers was essentially normal, their end segments lacked normal opposite end curvature, were larger than normal, and had a distinct non-hexagonal shape. As a result, these fibers failed to form typical Y sutures. Furthermore, the nuclear fibers of heterozygote lenses were even larger and lacked any semblance of an ordered packing arrangement. Grossly, homozygote lenses were opaque at all ages examined, except for a peripheral transparent region that contained fibers in the early stages of elongation. All fibers from homozygote lenses lacked opposite end curvature, and thus failed to form any sutures. Also, these fibers were essentially devoid of interlocking devices, and only 7% of their mid segment was specialized as GJs. The results of this study suggest that MIP has essential roles in the establishment and maintenance of uniform fiber structure, and the organization of fibers, and as such is essential for lens function.     

Fiber type composition of the human female trapezius muscle: Enzyme-histochemical characteristics

The anatomy of the human trapezius muscle is complex, with an extensive origin and fibers running in different directions. The muscle is commonly divided into three different muscle portions according to the fiber direction: the descending, transverse, and ascending portions. In a previous study in males, the structure of the muscle differed between different portions with respect to the enzyme-histochemical fibertype profile. The lower regions of the descending portion and the transverse and the ascending portions had a predominance of type I fibers. The type II fibers were more frequent in the upper regions of the descending portion, and the cross-sectional fiber area in this region of the muscle was smaller. In this study, we have investigated the trapezius muscle in females and compared the results with those from males. The different portions of the female muscle had a relatively even fiber-type composition. However, there tended to be fewer type I fibers and more type IIB fibers in the descending portion of the muscle, and the fibers of the lower regions of the descending portion were somewhat larger. The fiber-type distribution pattern was similar to that of the male trapezius muscle, but the mean cross-sectional area of the fibers in the female muscle was considerably smaller. Thus, our conclusion is that the trapezius muscle of females has a similar activity pattern as that of males. The significantly smaller cross-sectional fiber area, however, may indicate a lower functional capacity which may be of importance in the development of neck and shoulder dysfunction in females.     

Regional differences in fiber characteristics in the rat temporalis muscle

The behavioral differences in muscle use are related to the fiber type composition of the muscles among other variables. The aim of this study was to examine the degree of heterogeneity in the fiber type composition in the rat temporalis muscle. The temporalis muscle was taken from 10‐week‐old Wistar strain male rats (n = 5). Fiber types were classified by immunohistochemical staining according to their myosin heavy chain content. The anterior temporalis revealed an obvious regional difference of the fiber type distribution, whereas the posterior temporalis was homogeneous. The deep anterior temporalis showed a predominant proportion of type IIA fibers and was the only muscle portion displaying slow type fibers (< 10%). The other two muscle portions, the superficial anterior and posterior temporalis, did not differ significantly from each other and contained mainly type IIB fibers. Moreover, the deep anterior temporalis was the only muscle portion showing slow type fibers (< 10%). In the deep portion, type IIX fibers revealed the largest cross‐sectional area (1943.1 ± 613.7 µm²), which was significantly (P < 0.01) larger than those of type IIA and I + IIA fibers. The cross‐sectional area of type IIB fibers was the largest in the remaining two muscle portions and was significantly (P < 0.01) larger than that of type IIX fibers. In conclusion, temporalis muscle in rats showed an obvious heterogeneity of fiber type composition and fiber cross‐sectional area, which suggests multiple functions of this muscle.     

Relationship between Sarcomere Length and Active Force in Rabbit Papillary Muscle1

Isometric peak twitch force (stimulation frequency 0.5/s; 29.5–30.5°C) was correlated with sarcomere length in isolated papillary muscles of the rabbit. Sarcomere length was measured from photographic recordings (1.5 ms exposure time) performed at rest between contractions and at the time of isometric peak twitch force. The sarcomere length at rest was found to be relatively uniform throughout the preparation and to be linearly related to the overall muscle length within the range L_max- 0.85 L_max. The distribution of sarcomere lengths increased considerably as the muscle went from rest to activity. Studies of surface markers showed different degrees of shortening (or elongation) of individual segments along the length of the preparation. The mean resting sarcomere length at L_max (the optimum muscle length for force production) was 2.44±0.01 μm (grand mean ± S.E., 7 muscles). The mean active sarcomere length at L_max was 2.29 ± 0.04 /μm. Active force declined steeply as the muscle length was reduced below L_max. At a resting sarcomere length of 2.0 μm, active force was approximately 1/3 of the maximum. The observed differences between the length-tension relationships in myocardium (twitch responses) and skeletal muscle (tetanic contractions) are discussed on the basis of a length dependency of the activation process in cardiac muscle.     

Functional heterogeneity in a multipinnate muscle

Many mammalian muscles have a complex internal architecture. This type of structure could allow a single muscle to produce a variety of force vectors through selective regional contractions. This hypothesis was tested electromyographically in the multipinnate pig masseter by recording simultaneously from several intramuscular sites. It was found that the activity in different portions of the masseter varied systematically during the various phases of mastication. Anatomical correlates of the differential activity included fasciculus orientation and length, sarcomere length in specific jaw positions, and histochemical fiber type. The usual assumptions made about muscles for biomechanical analysis, such as uniform contraction and constant line of action, are inappropriate for complex muscles such as the pig masseter.     

Heterogeneity of mean sarcomere length in different fibres: effects on length range of active force production in rat muscle

Isometric active length-force characteristics of rat semimembranosus lateralis muscles [rats,n = 6, body mass = 292 (SD 9) g] were recorded. Muscles were photographed during the force plateaus of the tetani to determine lengths of proximal, intermediate and distal fibres. From the mean number of sarcomeres in series in those fibres, mean sarcomere length at different muscle lengths was calculated. The heterogeneity of mean sarcomere lengths for each muscle was quantified according to the coefficient of variation of sarcomere lengths at muscle optimal length. Absolute muscle length changes were equal to fibre length changes. At all muscle lengths, mean sarcomere lengths in the distal fibres were significantly greater than those in proximal and intermediate fibres. The heterogeneity of mean sarcomere lengths augmented the length range of active force production between muscle optimal length and active slack length by about 38% to 43%. We concluded that in a muscle with a low degree of pennation, the heterogeneity of mean sarcomere lengths should be considered as a substantial contributor to the length range over which active force can be produced. In our experiment, the length ranges of active force production between optimal and slack length showed considerable differences (range 18.7–24.9 mm). The coefficient of correlation between length ranges and mean number of sarcomeres in series in various muscle regions was extremely low (r = –0.14) and not significant. The coefficient of correlation between length ranges and heterogeneity was high (r = 0.88) and significant. These data would suggest that muscles with a similar number of sarcomeres in series can exhibit quite different functional characteristics.     

Myocardial sarcomere dynamics during isometric contraction.

1. Sarcomere lengths were measured during rest and throughout the time course of isometric contractions in thin, isolated rat papillary muscles using light diffraction techniques. 2. Shortening of the sarcomere length occurred upon contraction at all muscle lengths, averaging 7% at optimal length and more at shorter lengths. Relative to the narrow range of sarcomere lengths spanning the length--tension curve, this degree of shortening was considerable. 3. Local changes of sarcomere length were quantitatively paralleled by local changes of tissue segment length, the latter demarcated by microspheres lodged within the muscle tissue. At all but the shortest muscle lengths, sarcomere shortening was fully accounted for by equivalent lengthening of the non-striated regions near the clamped ends of the preparation. 4. It seems likely that these regions near the clamped ends of the preparation. 4. It seems constitute the source of the large series elasticity characteristic of isolated papillary muscle preparations such as this.     

Finite element model of intramuscular pressure during isometric contraction of skeletal muscle

The measurement of in vivo intramuscular pressure (IMP) has recently become practical and IMP appears well correlated with muscle tension. A numerical model of skeletal muscle was developed to examine the mechanisms producing IMP. Unipennate muscle is modelled as a two-dimensional material continuum that is incompressible and nonlinearly anisotropic. The finite element technique is used to calculate IMP and muscle stress during passive stretch and during isometric contraction. A novel element models the contractile portion of muscle, incorporating sarcomere length-force and velocity-force relations. A range of unipennate muscle geometries can be modelled. The model was configured to simulate the rabbit tibialis anterior muscle over a range of lengths. Simulated IMP and stress results were validated against animal experimentation data. The simulation agreed well with the experimental data over the range of 0.8-1.1 of the optimal length. Severe pressure gradients were produced near the musculo-tendinous junctions while IMP was more uniform in the central muscle belly. IMP and muscle stress in relaxed (unstimulated) muscle increased nonlinearly with muscle length. IMP and stress in isometrically contracting muscle showed a local maximum at optimal length and were reduced at shorter lengths. At muscle lengths longer than optimal, stress and IMP increased predominately due to tension in the passive elastic structures.     

A-band length, striation spacing and tension change on stretch of active muscle.

1. Single muscle fibres of the frog were stretched during tetanus and filmed down the interference microscope. 2. Sarcomere lengths and A- and I-band widths were measured from cine films taken of the experimental sequences. 3. When a fibre was stretched during a tetanus, its A-band widths remained constant while its I-band widths changed according to the extent of the stretch. This behaviour is identical with that of a fibre stretched at rest. 4. Sarcomere lengths remained uniform along the fibre during a tetanus plus stretch, increasing as the I-band widths increased when the fibre was stretched. 5. At sarcomere lengths longer than LPmax (the length corresponding to greatest isometric tetanus tension) where the isometric tension diminishes as the fibre is lengthened, if the stretch was imposed during a tetanus the tension was higher after the stretch than before, showing a plateau which lasted for the duration of the tetanus. 6. This effect increased with longer sarcomere lengths and was not due to increased resting tension.