A nonlinear model of passive muscle viscosity.

The material properties of passive skeletal muscle are critical to proper function and are frequently a target for therapeutic and interventional strategies. Investigations into the passive viscoelasticity of muscle have primarily focused on characterizing the elastic behavior, largely neglecting the viscous component. However, viscosity is a sizeable contributor to muscle stress and extensibility during passive stretch and thus there is a need for characterization of the viscous as well as the elastic components of muscle viscoelasticity. Single mouse muscle fibers were subjected to incremental stress relaxation tests to characterize the dependence of passive muscle stress on time, strain and strain rate. A model was then developed to describe fiber viscoelasticity incorporating the observed nonlinearities. The results of this model were compared with two commonly used linear viscoelastic models in their ability to represent fiber stress relaxation and strain rate sensitivity. The viscous component of mouse muscle fiber stress was not linear as is typically assumed, but rather a more complex function of time, strain and strain rate. The model developed here, which incorporates these nonlinearities, was better able to represent the stress relaxation behavior of fibers under the conditions tested than commonly used models with linear viscosity. It presents a new tool to investigate the changes in muscle viscous stresses with age, injury and disuse.

Intersarcomere dynamics of single muscle fibers during fixed-end tetani.

The contraction dynamics of end and center regions of single fibers have been measured during fixed-end tetani. Experimental control and data acquisition are provided by a digital system that can acquire diffraction data as fast as every 260 microseconds for 300-700 ms. Tension records are simultaneously displayed on a storage oscilloscope. Resting sarcomere length variation between the end and center regions was analogous to that of Gordon et al. (1966). During the rapid rise in force (less than 45 ms), the end regions contract almost twice as fast as the center regions. During the slow rise in force, the velocity of contraction of the end regions was 3.8 times the velocity of stretch of the center regions. In addition, factors that affected the rate and extent of the slow rise in tension also affected the rate and extent of end shortening. In 58% of the cases studied, the amount of shortening observed in the end region was enough to explain the extent of the slow rise in tension. These data support the explanation of creep first proposed by A. V. Hill (1953) and used by Gordon et al. (1966) to justify their use of the back-extrapolation technique in measuring the isometric force-generating capability of a single fiber. These data also indicate that the laser diffraction technique may provide an effective, noninvasive method for studying sarcomere dynamics during creep and related phenomena.

The mechanical strength of side-to-side tendon repair with mismatched tendon size and shape.

Tendon transfers frequently require coaptation of two mismatched tendons. In this cadaver study, ultimate load, stiffness, and Young's modulus were measured in tendon-to-tendon attachments with mismatched donor and recipient tendons, using pronator teres (PT) to extensor carpi radialis brevis (ECRB) and flexor carpi ulnaris (FCU) to extensor digitorum communis (EDC). FCU-to-EDC attachments failed at higher loads than PT-to-ECRB attachments, but they had similar modulus and stiffness values. Ultimate tensile strength of the tendon attachments exceeded the maximum predicted contraction force of any of the transferred muscles, with safety factors of four-fold for the FCU-to-EDC and two-fold for the PT-to-ECRB transfers. This implies that size and shape mismatches should not be contraindications to tendon attachment in transfers. The strength safety factors suggest that postoperative immobilization of these transfers is unnecessary.

Cloning and characterization of the S1 domain of four myosin isoforms from functionally divergent fiber types in adult Rana pipiens skeletal muscle.

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.

Myosin isoforms in anuran skeletal muscle: their influence on contractile properties and in vivo muscle function.

Functional studies on isolated single anuran skeletal muscle cells represent classic experiments from which much of our understanding of muscle contraction mechanisms have been derived. Because of their superb mechanical stability when isolated, single anuran fibers provide a uniquely powerful model system that can be exploited to understand the relationship between myosin heavy chain (MHC) and myosin light chain (MLC) composition and muscle fiber function. In this review, we summarize historic and recent studies of MHC and MLC expression patterns in the fiber types of anuran species. We extend the traditional classification scheme, using data from recent reports in which frog MHCs have been cloned, to reveal the molecular basis of frog muscle fiber types. The influence of MHC and MLC isoforms on contractile kinetics of single intact fibers is reviewed. In addition, we discuss more subtle questions such as variability of myosin coexpression along a single cell, and its potential influence on contractile function. The frog jump is used as a model system to elucidate principles of muscular system design, including the role of MHC isoforms on in vivo muscle function. Sequence information is used from cloned frog MHCs to understand the role of specific regions of the myosin motor domain in regulating contractile function and the evolutionary origins of fast and slow amphibian MHCs. Finally, we offer promising future possibilities that combine molecular methods (such as recombinant gene transfer) with single cell contractile measurements to address questions regarding myosin structure/function and gene regulation.

Muscle damage is not a function of muscle force but active muscle strain.

Contractile properties of rabbit tibialis anterior muscles were measured after eccentric contraction to investigate the mechanism of muscle injury. In the first experiment, two groups of muscles were strained 25% of the muscle fiber length at identical rates. However, because the timing of the imposed length change relative to muscle activation was different, the groups experienced dramatically different muscle forces. Because muscle maximum tetanic tension and other contractile parameters measured after 30 min of cyclic activity with either strain timing pattern were identical (P > 0.4), we concluded that muscle damage was equivalent despite very different imposed forces. This result was supported by a second experiment in which the same protocol was performed at one-half the strain (12.5% muscle fiber length). Again, there was no difference in maximum tetanic tension after cyclic 12.5% strain with either strain timing. Data from both experiments were analyzed by two-way analysis of variance, which revealed a highly significant effect of strain magnitude (P < 0.001) but no significant effect of stretch timing (P > 0.7). We interpret these data to signify that it is not high force per se that causes muscle damage after eccentric contraction but the magnitude of the active strain (i.e., strain during active lengthening). This conclusion was supported by morphometric analysis showing equivalent area fractions of damaged muscle fibers that were observed throughout the muscle cross section. The active strain hypothesis is described in terms of the interaction between the myofibrillar cytoskeleton, the sarcomere, and the sarcolemma.

Muscle damage induced by eccentric contractions of 25% strain.

Contractile and morphological properties were measured in the rabbit tibialis anterior muscle 1 h after isometric contraction (IC), passive stretch (PS), or eccentric contraction (EC). Maximal tetanic tension (Po) was reduced after 30 min of PS (P less than 0.001), IC (P less than 0.001), or EC (P less than 0.0001). However, the magnitude of the force deficit was a function of the treatment method. After 30 min of cyclic PS, Po decreased by 13%, whereas after IC or EC, Po decreased by 31 and 69%, respectively. The time course of tension decline in the various groups suggested that the EC-induced injury occurred during the first few minutes of treatment. Although the morphology of samples from the PS and IC groups appeared normal, eccentrically exercised muscles exhibited portions of abnormally large fibers (diam greater than or equal to 110 microns) when viewed in cross section. Examination of 231 such fibers from 6 muscles revealed that all enlarged fibers were exclusively of the fast-twitch glycolytic fiber type. Although no ultrastructural abnormalities were observed in any of the muscles from the IC or PS groups, a significant portion of the fibers in the EC group displayed various degrees of disorganization of the sarcomeric band pattern. Taken together, these studies highlight the importance of fiber oxidative capacity in EC-induced injury, which may be related to the damage mechanism.

Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction.

The time course of loss of the 55,000-Da intermediate filament protein desmin was measured in rabbit muscles subjected to cyclic eccentric contraction. Rabbit extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were examined 5 or 15 min after eccentric exercise and 1 h or 1 day after 30 min of an eccentric exercise protocol (n = 16 rabbits). The earliest change noted was a significant loss of desmin labeling in 2.5 +/- 0.63% of the rabbit EDL muscle fibers (P < 0.005) 5 min after initiation of eccentric exercise. Some loss of TA fiber desmin was also apparent at this time period (0.24 +/- 0.19%), but the magnitude was not significantly different from zero (P > 0.2). Fifteen minutes after initiation of exercise, desmin loss was more pronounced, increasing to 7.4 +/- 1.4 and 4.6 +/- 1.0% in the EDL and TA, respectively (P < 0.005). Finally, 1 day after 30 min of eccentric exercise, the percentage of fibers without desmin staining rose to 23.4 +/- 3.7 and 7.7 +/- 2.4% in the EDL and TA, respectively (P < 0.001). Loss of desmin staining occurred in the absence of contractile or metabolic protein disruption. Increased staining intensity of the intrasarcomeric cytoskeletal protein titin and an inability to exclude plasma fibronectin were also observed in most but not all fibers that had lost desmin staining. Desmin disruption thus represents a very early structural manifestation of muscle injury during eccentric contraction. Cytoskeletal disruption may predispose the contractile apparatus to previously reported structural damage.

Myosin and actin filament lengths in diaphragms from emphysematous hamsters.

In vitro studies of the diaphragm from emphysematous animals have, in some instances, shown an alteration in its sarcomere length-tension relationship and a decreased maximal specific tension. To our knowledge, it has never been determined whether such functional changes may be indicative of ultrastructural adaptations, e.g., changes in filament lengths and thus cross-bridge number. To address this, we compared filament lengths in diaphragms from hamsters in which emphysema was induced by endotracheal instillation of elastase (E) 5 mo before the hamsters were killed with those from control hamsters (C; saline instillation). Diaphragms were then fixed by vascular perfusion with buffered glutaraldehyde in situ at airway pressures set to approximate the physiological range of lung volumes from residual volume (RV) to total lung capacity (TLC). Ultrathin sections (50-70 nm) were taken parallel to the muscle fiber axis and examined by electron microscopy (x33,000). Sarcomere and filament length measurements were calibrated using an actin periodicity of 39 nm and an M-band width of 86 nm to correct for dimensional changes during preparation. Emphysema increased the change in lung volume from -20 to +25 cmH2O airway pressure (from RV to TLC) by approximately 88%, and the displacement volume of excised lung at 0 cmH2O airway pressure was increased by approximately 138% on average. Neither myosin (C = 1.592 +/- 0.027; E = 1.572 +/- 0.035 micron; P = 0.72) nor actin (C = 1.210 +/- 0.035; E = 1.221 +/- 0.014 micron; P = 0.76) filament lengths were affected by emphysema. Thus, filament length changes do not underlie the diaphragm functional adaptations observed previously in emphysema.

Contractile and cellular remodeling in rabbit skeletal muscle after cyclic eccentric contractions.

The time course of muscle contractile and cellular properties was studied in rabbit ankle flexor muscles after injury produced by eccentric exercise. Cyclic eccentric exercise was produced by increasing the tibiotarsal angle of the rabbit while activating the peroneal nerve by use of transcutaneous electrodes. Muscle properties were measured 1, 2, 3, 7, 14, and 28 days after exercise to define the time course of muscle changes after injury. A control group receiving only isometric contraction was used to study the effect of cyclic activation itself. The magnitude of the torque decline after 1 day was the same with use of isometric or eccentric exercise, but eccentric exercise resulted in a further decrease in torque after 2 days, at which time isometrically exercised muscles had fully recovered. The most prominent morphological changes in the injured muscle fibers were the loss of antibody staining for the desmin cytoskeletal protein and deposition of intracellular fibronectin, even when the injured muscle fibers retained their normal complement of contractile and enzymatic proteins. The presence of fibronectin inside the myofibers indicated a loss of cellular integrity. Invasion by inflammatory cells was apparent on the basis of localization of embryonic myosin. Thus eccentric exercise initiates a series of events that results in disruption of the cytoskeletal network and an inflammatory response that could be the mechanism for further deterioration of the contractile response.

Statistical significance and statistical power in hypothesis testing.

Experimental design requires estimation of the sample size required to produce a meaningful conclusion. Often, experimental results are performed with sample sizes which are inappropriate to adequately support the conclusions made. In this paper, two factors which are involved in sample size estimation are detailed--namely type I (alpha) and type II (beta) error. Type I error can be considered a "false positive" result while type II error can be considered a "false negative" result. Obviously, both types of error should be avoided. The choice of values for alpha and beta is based on an investigator's understanding of the experimental system, not on arbitrary statistical rules. Examples relating to the choice of alpha and beta are presented, along with a series of suggestions for use in experimental design.

Torque history of electrically stimulated human quadriceps: implications for stimulation therapy.

The time course of knee extension torque was measured in human quadriceps muscles during 30 min of transcutaneous neuromuscular electrical stimulation (NMES). Ninety subjects were divided into six experimental groups (n = 15 per group), which received stimulation at one of the following frequency/duty cycle combinations: 10 Hz/50%, 30 Hz/50%, 50 Hz/50%, 10 Hz/70%, 30 Hz/70%, and 50 Hz/70%. Two-way analysis of variance revealed that the magnitude of the relative torque decrease (the percentage of decrease in torque relative to the initial value) was significantly different between frequencies (p < 0.005) and duty cycles (p < 0.02), with no significant interaction (p > 0.6). Increasing either frequency or duty cycle caused a greater decrease in torque. In spite of this result, there was no significant difference between groups in the total activity (torque-time integral) achieved during the 30 min treatment session. The magnitude of this activity corresponded to only about 7-14 maximum voluntary contractions. Finally, the average torque during the treatment session was significantly different among groups (p < 0.001), being greatest for the 50 Hz/50% group and least for the 10 Hz/70% group. Taken together, these data suggest that a smaller number of longer duration contractions produces the greatest muscle tension. They also suggest that the absolute torque levels achieved with NMES are relatively low compared with voluntary muscular activity.

Recovery of the dog quadriceps after 10 weeks of immobilization followed by 4 weeks of remobilization.

Skeletal muscle fiber areas were measured in three heads of the dog quadriceps after 10 weeks of immobilization followed by 4 weeks of remobilization. Two-way analysis of variance demonstrated a significant decrease in both type 1 (p less than 0.005) and type 2 (p less than 0.001) fiber area. However, there was no significant difference among the three heads of the quadriceps (p less than 0.2). Although muscle fiber areas had not returned to control levels following remobilization, the area fraction of perimysial and epimysial connective tissue was not significantly different from control values (p greater than 0.15). These data suggest that although the degree of muscle atrophy following 10 weeks of immobilization is severe and muscle specific, following 4 weeks of remobilization, muscles uniformly recover to about 70% of control values.

Equal effectiveness of electrical and volitional strength training for quadriceps femoris muscles after anterior cruciate ligament surgery.

Neuromuscular electrical stimulation and voluntary muscle contraction are two exercise modes widely used in rehabilitation to strengthen skeletal muscle. Since there is no debate as to which mode is most effective, we compared electrical stimulation with voluntary contraction performed at matched intensities following reconstructive surgery of the anterior cruciate ligament. Forty men and women, aged 15-44, were randomly assigned to either an electrical stimulation or a voluntary contraction group. None of the subjects had a previous history of neuromuscular injury. The subjects received treatment for 30 minutes a day, 5 days a week, for 4 weeks. Knee extension torque was monitored during treatment to try to match the absolute muscular tensions (quantified as "activity") achieved during therapy. To match the activity of the subjects in the electrical stimulation group, who were treated at the highest stimulation intensity they could tolerate, the subjects in the voluntary contraction group were paced at progressively increasing intensities corresponding to 15, 25, 35, and 45% of the injured limb's maximum voluntary torque during weeks 1, 2, 3, and 4, respectively. We found no significant difference between the groups in terms of maximum voluntary knee extension torque throughout the study period. In addition, 1 year after surgery, there was still no significant difference between groups with regard to knee extension torque (p > 0.4). These data suggest that neuromuscular electrical stimulation and voluntary muscle contraction treatments, when performed at the same intensity, are equally effective in strengthening skeletal muscle that has been weakened by surgical repair of the anterior cruciate ligament.

Synovial fluid nutrient delivery in the diathrial joint: an analysis of rabbit knee ligaments.

The role of synovial fluid in providing nutrition to rabbit knee ligaments and menisci was evaluated by intraarticular injection of a labeled collagen precursor, tritiated proline. Incorporation of this substrate as tritiated hydroxyproline was measured in collateral and cruciate ligaments and menisci. The injectate volume (0.35 ml) did not appreciably change the overall joint pressure as measured by a wick catheter; therefore, no alteration of synovial membrane diffusion characteristics resulted. The concentration of the injected proline (0.52 mg%) was well below that normally present in serum (2.65 mg%). Therefore, incorporation of this substrate was not driven by a concentration gradient and represented normal uptake of synovial fluid and physiological incorporation of label as measured by the presence of tritiated hydroxyproline. Autoradiography was performed on all ligaments and menisci, and demonstrated concentration of the isotope and its metabolite (tritiated proline and tritiated hydroxyproline, respectively) in and around fibroblasts. This study indicates that rabbit knee ligaments and menisci can derive nutrition from a synovial fluid source.

Local compression patterns beneath pneumatic tourniquets applied to arms and thighs of human cadavera.

Distributions of tissue fluid pressure were examined beneath a standard pneumatic tourniquet in six upper extremities and six lower extremities of fresh human cadavera, disarticulated at the shoulder and hip, respectively. A standard 8-cm-wide tourniquet cuff was applied at mid-humerus or mid-femur position. Tissue fluid pressures were measured by 100-cm-long slit catheters inserted parallel to the bone at four tissue depths: subcutaneous, subfascial, mid-muscle, and adjacent to bone. All arms and thighs were studied at the following cuff pressures: 100, 150, 200, 250, 300, 400, and 500 mm Hg. Tissue fluid pressure was always maximal in subcutaneous tissue at mid-cuff. Transmission of cuff pressures to deeper tissues was significantly less (p less than 0.01) in the thighs with a girth of 40-52 cm than in the arms with a girth of 22-33 cm. At the four tissue depths studied, tissue fluid pressures fell steeply in a longitudinal direction near the cuff edge to levels near zero at points 1-2 cm outside each cuff edge. Our results suggest that wider cuffs are required on thighs than on arms to provide a bloodless field during limb surgery and to minimize underlying tissue injury associated with high cuff pressures. Our recommendation for wider tourniquet cuffs than those presently used during orthopaedic surgery is contrary to recent prevailing knowledge.

Long-term measurement of muscle function in the dog hindlimb using a new apparatus.

The objective of this study was to develop an apparatus for reliable, reproducible, and minimally invasive measurements of long-term, myoneural function. Twenty conditioned dogs were anesthetized and placed supine with one hindlimb secured in a boot apparatus. The hindpaw was attached to a force transducer that was connected to a recorder for continuous monitoring of torque. Muscles within the anterolateral compartment were stimulated by percutaneous electrodes over the peroneal nerve near the fibular head. This elicited isometric dorsiflexion of the hindpaw. Twitch and tetanic torques correlated positively with dog weight whereas other skeletal-muscle function parameters (time to peak tension, one-half relaxation time, and endurance) were independent of dog weight. Muscle function results were consistent with an overall compartmental composition of 30% Type I and 70% Type II fibers. Repetitive testing of twitch and tetanic torques in the dog legs yielded coefficients of variance of 3-4% (intraday) and 7% (interday). Thus, about one-half of the interday variability may be accounted for by diet, exercise, and other physiological conditions that change daily. The apparatus was also used to detect myoneural degeneration following tourniquet ischemia. The results indicate that this procedure for evaluating muscle function yields reliable and quantitative results noninvasively, and thus allows long-term testing of muscle function in normal and diseased hindlimbs of dogs.

Kappa Delta Award paper. Tissue fluid pressures: from basic research tools to clinical applications.

The two basic research tools developed to measure tissue fluid pressure (wick catheter) and osmotic pressure (colloid osmometer) have undergone extensive validation and refinement over the past 20 years. Using these techniques, basic science investigations were undertaken of edema in Amazon reptiles, pressure-volume relations in animals and plants, adaptive physiology of Antarctic penguins and fishes, edema in spawning salmon, tissue fluid balance in humans under normal conditions and during simulated weightlessness, and orthostatic adaptation in a mammal with high and variable blood pressures--the giraffe. Following and sometimes paralleling this basic research have been several clinical applications related to use of our colloid osmometer and wick technique. Applications of the osmometer have included insights into (a) reduced osmotic pressure of sickle-cell hemoglobin with deoxygenation and (b) reduced swelling pressure of human nucleus pulposus with hydration or certain enzymes. Clinical uses of the wick technique have included (a) improvement of diagnosis and treatment of acute and chronic compartment syndromes, (b) elucidation of tissue pressure thresholds for neuromuscular dysfunction, and (c) development of a better tourniquet design for orthopaedics. This article demonstrates that basic research tools open up areas of basic, applied, and clinical research.

Growth hormone secretagogue increases muscle strength during remobilization after canine hindlimb immobilization.

Twenty-two beagles were divided into two equal groups, and the right hindlimb of each animal was immobilized at 105 degrees of knee flexion by external fixation. After 10 weeks of fixation, the device was removed, allowing free mobility for the following 5 weeks. Each day throughout the 15 weeks, one group received a growth hormone secretagogue (treatment) at a dose of 5 mg/kg, and the other received a lactose placebo (control). At weeks 0, 10, and 15, strength as indicated by maximum isometric extension torque was measured in the right hindlimb, biopsies of the vastus lateralis muscle were taken, and the dogs were weighed. Weekly blood samples were analyzed for insulin-like growth factor-1, blood urea nitrogen, and creatine phosphokinase. Between weeks 0 and 10, tetanic torque declined by about 60% (p < 0.001) in both groups, with no significant difference between the groups (p > 0.7). Between weeks 10 and 15, tetanic torque in the treated group increased by 0.81 Nm; this was significantly greater than the increase of 0.25 Nm in the placebo group (p < 0.05). The diameters of slow (type-1) and fast (type-2) fibers measured from the vastus lateralis muscle followed the same trend. At all time points, fiber diameter correlated strongly with torque; this argues against nonmuscular causes such as nerve injury for strength loss. The mean levels of insulin-like growth factor-1 increased 100% by week 4 in the treated group and remained elevated by about 60% throughout the experiment. Levels of insulin-like growth factor-1 in the placebo group decreased 30% within week 1 and remained depressed throughout the experiment. Our interpretation of these data suggests that the growth hormone secretagogue elevated levels of serum insulin-like growth factor-1, which in turn increased the size and strength of the quadriceps muscle during remobilization. These data may ultimately have therapeutic application to humans during rehabilitation after prolonged inactivity.