Artificial gravity as a countermeasure to microgravity: a pilot study examining the effects on knee extensor and plantar flexor muscle groups.

The goal of this project was to examine the effects of artificial gravity (AG) on skeletal muscle strength and key anabolic/catabolic markers known to regulate muscle mass. Two groups of subjects were selected for study: 1) a 21 day-bed rest (BR) group (n = 7) and 2) an AG group (n = 8), which was subjected to 21 days of 6 degrees head-down tilt bed rest plus daily 1-h exposures to AG (2.5 G at the feet). Centrifugation was produced using a short-arm centrifuge with the foot plate approximately 220 cm from the center of rotation. The torque-velocity relationships of the knee extensors and plantar flexors of the ankle were determined pre- and posttreatment. Muscle biopsy samples obtained from the vastus lateralis and soleus muscles were used for a series of gene expression analyses (mRNA abundance) of key factors implicated in the anabolic vs. catabolic state of the muscle. Post/pre torque-velocity determinations revealed greater decrements in knee extensor performance in the BR vs. AG group (P < 0.04). The plantar flexors of the AG subjects actually demonstrated a net gain in the torque-velocity relationship, whereas in the BR group, the responses declined (AG vs. BR, P < 0.001). Muscle fiber cross-sectional area decreased by approximately 20% in the BR group, whereas no losses were evident in the AG group. RT-PCR analyses of muscle biopsy specimens demonstrated that markers of growth and cytoskeletal integrity were higher in the AG group, whereas catabolic markers were elevated in the BR group. Importantly, these patterns were seen in both muscles. We conclude that paradigms of AG have the potential to maintain the functional, biochemical, and structural homeostasis of skeletal muscle in the face of chronic unloading.

New perspectives about human laryngeal muscle: single-fiber analyses and interspecies comparisons.

BACKGROUND: In companion studies on canine and rodent laryngeal muscle, we observed that (1) muscle fibers in both the canine and rodent posterior cricoarytenoid (PCA) muscles have a slower myosin heavy-chain (MyHC) isoform profile than those in the thyroarytenoid (TA) muscle; (2) the muscle fiber composition of PCA and TA muscles in canines and rodents is complex given the presence of so-called hybrid fibers (fibers coexpressing various combinations of MyHC isoforms); (3) the types and proportions of hybrid fibers are both muscle specific and, in some cases, region specific; and (4) the MyHC isoform profile of canine laryngeal muscle appears to be slower than that of rodent laryngeal muscle, suggesting the possibility that larger mammals have a slower MyHC isoform profile. OBJECTIVES: Given the findings of these companion studies and the fact that very little is known about the MyHC isoform composition of laryngeal muscle fibers, the primary objectives of this study were to determine (1) the types of MyHC isoforms found in the human PCA and TA muscles, (2) if there were regional differences in MyHC isoform composition, (3) if hybrid fibers commonly occur in human laryngeal muscle, and (4) if the MyHC isoform profile of human laryngeal muscle is slower than that of canine and rodent laryngeal muscle. RESULTS AND CONCLUSIONS: The findings of this study clearly demonstrate that both the PCA and TA muscles in humans express 3 types of MyHC isoforms (ie, slow type I, fast type IIA, and fast type IIX MyHC isoforms). At the single-fiber level, there were distinct regional differences and hybrid fibers were a common occurrence. Finally, the data demonstrate that the PCA and TA muscles of humans have a slower MyHC profile than that found in either canine or rodent laryngeal muscle.

Are hybrid fibers a common motif of canine laryngeal muscles? Single-fiber analyses of myosin heavy-chain isoform composition.

BACKGROUND: The canine lateral cricoarytenoid muscle contains a large proportion of muscle fibers that coexpress various combinations of myosin heavy-chain isoforms (ie, so-called hybrid fibers). OBJECTIVE: To test the hypothesis that hybrid fibers are a common motif throughout laryngeal muscles. DESIGN: The posterior cricoarytenoid, canine cricothyroid, and thyroarytenoid muscles were removed from 5 beagle dogs. The posterior cricoarytenoid and canine cricothyroid muscles were each dissected into horizontal, oblique, and rectus regions. The thyroarytenoid was separated into medial and lateral regions. Approximately 40 single fibers were microdissected from each region ( approximately 1800 total fibers were sampled) and placed into a denaturing sample buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then used to separate the individual myosin heavy-chain isoforms. RESULTS: Each laryngeal muscle contained hybrid fibers; however, the types and proportions of hybrid fibers were clearly muscle specific. Within a given muscle, there were relatively minor regional differences in the types and proportions of hybrid fibers. CONCLUSION: If the myosin heavy-chain isoform composition of a single fiber can be used as a "physiological marker," then the extent of hybridism may reveal the diversity of activity required of a given laryngeal muscle.

Single-fiber myosin heavy-chain isoform composition of rodent laryngeal muscle: modulation by thyroid hormone.

BACKGROUND: Studies have shown that canine laryngeal muscle contains a large number of muscle fibers that coexpress varying combinations of myosin heavy-chain (MyHC) isoforms. Currently, it is not clear whether this phenomenon is unique to canine laryngeal muscle or occurs in all mammals. OBJECTIVES: To examine the single-fiber MyHC isoform composition of rodent laryngeal muscle and to examine the plasticity of single-fiber MyHC isoform composition via manipulation of thyroid state. RESULTS: (1) Findings of single-fiber electrophoretic analyses clearly demonstrate that most fibers in both the posterior cricoarytenoid and thyroarytenoid muscles exhibit MyHC polymorphism. However, the proportions and patterns of polymorphism appear to be muscle specific. (2) Although the fast type IIL isoform was observed in fibers from both muscles, it was always coexpressed in combination with other MyHC isoforms (ie, no pure type IIL fibers were found), and always represented a minor proportion of the total MyHC pool. (3) Altering the thyroid state proved a useful tool for exploring the scope of MyHC isoform expression in these muscles. While the posterior cricoarytenoid muscle seemed more sensitive to the thyroid state, transitions in both muscles were primarily confined to the fast type IIX and IIB MyHC isoforms. CONCLUSION: The findings of this study support the concept that single-fiber MyHC polymorphism occurs commonly in mammalian laryngeal muscle.

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism.

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

MHC polymorphism in rodent plantaris muscle: effects of mechanical overload and hypothyroidism.

In a previous study, it was shown that a combined treatment of hyperthyroidism and hindlimb suspension effectively converted the slow-twitch soleus muscle to a fast-twitch muscle. The objective of this study was to test the hypothesis that hypothyroidism [absence of triiodothyronine (-T(3))] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan muscle was composed of approximately 13 different fiber types as defined by myosin heavy chain (MHC) isoform content. The largest proportion of fibers ( approximately 35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e., type IIX/IIB fibers). In this context, the combined intervention of -T(3) and OV produced a significant reduction in the relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all four MHC protein isoforms. The mechanical consequences of these transitions, however, were modest, producing a 15% decrease in maximal shortening velocity. The findings of this study demonstrate that -T(3) + OV does produce a partial shift toward a slower phenotype; however, the high degree of polymorphism found in the Plan muscle represents a unique design that appears to minimize the functional consequences of these significant MHC transitions.

Novel transitions in MHC isoforms: separate and combined effects of thyroid hormone and mechanical unloading.

Single-fiber (n = 3,818 fibers) electrophoretic analyses were used to delineate the separate and combined effects of hyperthyroidism (T3) and hindlimb suspension (HS) on the myosin heavy chain (MHC) isoform composition (1-, 2-, and 4-wk time points) of the rat soleus muscle. The key findings of this study are as follows. First, T3 and HS both altered the distribution of MHC isoforms at the single-fiber level; however, the populations of fibers produced by these two interventions were clearly different from one another. Second, T3 + HS rapidly converted the soleus into a fast muscle, producing large increases in the relative contents of the fast type IIx and IIb MHC isoforms which were primarily expressed in several populations of hybrid fibers (e.g., types I/IIa/IIx, I/IIx/IIb, I/IIa/IIx/IIb). Finally, T3 + HS produced unique populations of hybrid fibers that did not adhere to the Ileft arrow over right arrow IIaleft arrow over right arrow IIxleft arrow over right arrow IIb sequential scheme of MHC plasticity. Collectively, the findings of this study demonstrate that the intervention of T3 + HS is a powerful model for manipulating and studying MHC isoform plasticity in slow skeletal muscle.

Adaptive gait responses to plantar heel pain.

Neuropathic foot ulcers in people with diabetes result from repetitive stress aggravated by a lack of protective sensation. Protective sensation causes individuals without this impairment to produce alterations in their gait in response to painful stimuli. This study evaluates the adaptive gait responses to pain in individuals with sensate feet. The gaits of 18 such control subjects were studied with a foot switch gait analyzer without painful stimuli. Each then had his or her gait analyzed with three successively larger painful stimuli (2, 3.3, and 4.6 mm beads) placed below the heel. This study showed that subjects compensated for the painful stimuli by reducing the single limb support duration of the affected side at bead sizes of 3.3 and 4.6 mm and by reducing the unaffected side's swing phase and single limb support as a percentage of the gait cycle at the 4.6-mm bead size only. Gait adaptations to painful stimuli may indicate another possible avenue, in addition to pressure redistribution, in the assessment of programs aimed at prevention and treatment of diabetic foot ulcers.

A new concept in laryngeal muscle: multiple myosin isoform types in single muscle fibers of the lateral cricoarytenoid.

This report describes the first known investigation of canine laryngeal muscle in which single fibers were dissected and their myosin heavy chain (MHC) isoform content was analyzed. Both SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot techniques were used. The data from single fiber SDS-PAGE indicate that the lateral cricoarytenoid (LCA) is predominantly a fast muscle composed of the following MHC isoforms: Type I, 16.3%; Type IIA, 71.3%; Type IIX, 10.4%; and Type IIB, 2.0%. The results reveal a phenomenon that, to our knowledge, has not been previously described for laryngeal muscle: the presence of two or more MHC isoforms in a single canine LCA muscle fiber. A large number (41%) of muscle fibers coexpressed two or more MHC isoforms. The three most common patterns of coexpression were Type IIA/IIX (72%), Type IIA/I (16%), and Type IIA/IIX/I (8%). Interestingly, the fast Type IIX MHC isoform was typically present with other isoforms and rarely found by itself in individual fibers. Additional experiments are underway to determine whether other laryngeal muscles exhibit such an unusually high ratio of MHC isoform polymorphism.

The "Space Cycle" Self Powered Human Centrifuge: a proposed countermeasure for prolonged human spaceflight.

The Self Powered Human Centrifuge, or Space Cycle, is a countermeasure to the adverse physiologic effects of prolonged human exposure to spaceflight microgravity. This unique device simultaneously provides exercise, impact loading and gravity analogous acceleration to emulate conditions on Earth. One or two crewmembers pedal themselves about a shaft mounted to the space craft located "above" their heads. This creates a short arm centrifuge with a head-to-toe acceleration orientation. The potential advantages of the Space Cycle include: a) reversal of cephalad fluid shift, minimizing post flight orthostatic intolerance; b) pedaling to maintain muscular and cardiovascular fitness; and c) enhancement of skeletal homeostasis by impact loading with a pedal-crank mounted cam and frame mounted resistive device. Other anticipated advantages include generation of usable electricity, physiologic monitoring and a means of mass measurement. Motion sickness is controlled with restraints and virtual reality headsets. The device is compatible with International Space Station dimensional constraints.

Single-fiber and whole muscle analyses of MHC isoform plasticity: interaction between T3 and unloading.

Previous data suggest that separate interventions of hyperthyroidism (T3) and hindlimb suspension (HS) act on some but not all slow type I fibers in the soleus muscle. This may be due to the presence of "refractory" fibers that are unresponsive to either of these interventions. Alternatively, T3 and HS might act on different populations of slow type I fibers in the soleus muscle. Adult female Sprague-Dawley rats were assigned to 1) control, 2) T3, 3) HS, or 4) T3+HS. Nine animals were assigned to each group. Single-fiber electrophoretic analyses (n = 40 per muscle) of the soleus muscle demonstrated that the HS reduced the percentage of slow type I fibers from approximately 80% (control) to approximately 40% (HS) of the fiber population. Although hyperthyroidism affected a greater percentage of slow type I fibers than HS, a small population (approximately 10% of the slow type I fibers) were unaffected by T3. The combined intervention, in contrast, transformed all slow type I fibers into fibers expressing various combinations of fast myosin heavy chain (MHC) isoforms. These findings demonstrate that the soleus muscle does not contain so-called refractory fibers. They further suggest that the soleus muscle contains different populations of slow type I fibers that vary in their sensitivity to altered physiological conditions.

Determinants of work produced by skeletal muscle: potential limitations of activation and relaxation.

The objective of this study was to estimate the limitations imposed by the kinetics of activation and relaxation on the ability of slow skeletal muscle to produce mechanical work. These estimates were made by the following methods: 1) using the work loop technique and measuring the actual mechanical work (WA) produced by rat soleus muscles (n = 6) at four different frequencies (0.5, 1, 2, and 4 Hz) and seven different amplitudes of length change (1, 2, 3, 4, 5, 6, and 7 mm); 2) determining the force-velocity relationships of the soleus muscles and using this data to quantify the theoretical mechanical work (WT) that could be produced under the work loop conditions described above; and 3) subtracting WA from WT. The difference between WT and WA was interpreted to represent limitations imposed by activation and relaxation. Under certain conditions (high frequency, small strain), factors controlling the kinetics of activation and relaxation reduced the mechanical work of the soleus muscle by approximately 60%. Hence, activation and relaxation collectively represent important factors limiting the production of mechanical work by slow skeletal muscle.

Optimal shortening velocities for in situ power production of rat soleus and plantaris muscles.

Force-velocity (FV) relationships have been used previously to calculate maximal power production and to identify an optimal velocity of shortening (V(opt)-fv) to produce such power in skeletal muscle. The cyclical nature of muscle position during locomotion for muscles such as the soleus and plantaris is such that either constant force or velocity is rarely attained. In the present study, the work loop technique, a technique developed to measure maximal attainable power output from muscles undergoing cyclic length changes, was undertaken to determine whether simulating in vivo function alters the power-velocity relationship of the soleus and plantaris and, in particular, the velocity of shortening that produces maximal power (V(opt)-wl). FV relationships were determined for both soleus (n = 4) and plantaris (n = 4) muscles in situ from adult female Sprague-Dawley rats by measuring shortening velocities during afterloaded isotonic contractions. The velocity that produced maximal power using FV relationships, V(opt)-fv, was 54.6 +/- 0.7 mm/s for the plantaris vs. 20.2 +/- 1.2 mm/s for the soleus. Then, the work loop technique was employed to measure net power from these same muscles at multiple cycling frequencies (1.5 to 4.0 Hz for the soleus; 4.0 to 8.0 Hz for the plantaris). Multiple power-velocity curves were generated (one at each cycle frequency) by varying the strain (1-8 mm). Thus, at each cycle frequency, V(opt)-wl could be identified. For both the plantaris and soleus, V(opt)-wl at each cycle frequency was not different from their respective V(opt)-fv value. Thus both fast and slow skeletal muscles have inherent optimal shortening velocities, identifiable with FV relationships, that dictate their respective maximal attainable mechanical power production using the work loop technique.

Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency.

This study tested for the hypothesis that mechanical loading, not stimulation frequency per se, plays a key role in determining the plasticity of myosin heavy chain (MHC) protein isoform expression in muscle undergoing resistance training. Female Sprague-Dawley rats were randomly assigned to resistance-training programs that employed active 1) shortening (n = 7) or 2) lengthening contractions (n = 8). The medial gastrocnemius (MG) muscles in each group trained under loading conditions that approximated 90-95% of maximum isometric tetanic tension but were stimulated at frequencies of 100 and approximately 25 Hz, respectively. Lengthening and shortening contractions were produced by using a Cambridge ergometer system. The MG muscles trained every other day, performing a total of 16 training session. Both training programs produced significant (P < 0.01) and similar reductions in the fast type IIB MHC protein isoform in the white MG muscle, reducing its relative content to approximately 50% of the total MHC protein isoform pool. These changes were accompanied by increases in the relative content of the fast type IIX MHC protein isoform that were of similar magnitude for both groups. The results of this study clearly demonstrate that stimulation frequency does not play a key role in modulating MHC isoform alterations that result from high-resistance training.

Are soft echoes really soft? Intravascular ultrasound assessment of mechanical properties in human atherosclerotic tissue.

To examine the accuracy of intravascular ultrasound (IVUS) in assessing the biophysical properties of atherosclerotic plaque, 33 human iliac arteries were imaged with a 25 MHz IVUS transducer and classified into four groups on the basis of IVUS appearance: minimally diseased arterial wall, bright echogenic plaque with acoustic shadowing, bright echogenic plaque without shadowing, and hypoechogenic plaque (so-called "soft echoes"). The hardness of each plaque was assessed with an ultrasensitive compression ergonometer. The radial static stress-strain relations fit well (r > 0.98) to exponential curves, providing a compression stiffness constant (K) defined as the coefficient of the exponential power. K for bright echogenic plaque with shadowing was significantly greater than that of the other tissues. However, K among minimally diseased entire arterial wall, hypoechogenic plaque, and bright echogenic plaque without shadowing was not significantly different, but these tissues are not physically soft compared with adipose tissue. Therefore, tissue characterization by IVUS distinguishes calcified from noncalcified plaque and accurately predicts its biomechanical hardness. However, soft echoes, although less firm than calcium, do not necessarily correspond to soft tissue.

Prediction of normal values for lactate threshold estimated by gas exchange in men and women.

Lactate threshold (LT) is an index of exercise capacity and can be estimated from the gas exchange consequences of a metabolic acidosis (LT(GE)). In recent years, it has emerged as a diagnostic tool in the evaluation of subjects with exercise limitation. The purpose of this study was to develop LT(GE) prediction equations on a relatively large sample of adults and to cross-validate each equation. A total of 204 healthy, sedentary, nonsmoking subjects (103 men and 101 women), aged 20-70 years, underwent graded exercise testing on a cycle ergometer. The V-slope technique was used to detect LTGE as the oxygen uptake (VO2) at the breakpoint of the carbon dioxide output versus VO2 relationship. Multiple linear regression was used to develop 12 equations with combinations of the following predictor variables: age, height, body mass, and fat-free mass. Eight of the equations are gender-specific and four are generalized with gender as a dummy variable. The equations were cross-validated using the predicted residual sum of squares (PRESS) method. The results demonstrate that the equations had relatively high multiple correlations (0.577-0.863) and low standard errors of the estimate (0.123-0.228 1 x min(-1)). The PRESS method demonstrated that the equations are generalizable, i.e., can be used in future studies without a significant loss of accuracy. Since we tested only healthy, sedentary subjects, our equations can be used to predict the lower limit of normal for a given subject. Using individual data for healthy and diseased subjects from the literature, we found that our gender-specific equations rarely miscategorized subjects unless they were obese and mass was a predictor variable. We conclude that our equations provide accurate predictions of normal values for LT(GE) and that they are generalizable to other subject populations.

Microgravity-induced transformations of myosin isoforms and contractile properties of skeletal muscle.

This study examined the effects of microgravity (14 days) on 1) the contractile properties of the soleus (Sol), an antigravity skeletal muscle; and 2) the myosin heavy chain (MHC) protein and mRNA isoform content of the Sol, vastus intermedius (VI), plantaris (Plan), and tibialis anterior (TA) muscles. The force-velocity relationships of the flight Sol muscles had a significant reduction in maximal isometric tension (-37%) and a corresponding increase in maximal shortening velocity (+20%). Additionally, the force-frequency relationship of the flight Sol muscles was shifted to the right of the ground-based control group. Microgravity had the greatest effect on muscle fiber composition in the Sol muscle, with a reduction in slow muscle fibers and a corresponding increase in muscle fibers categorized as hybrid fibers. The estimated absolute MHC isoform content was altered to the greatest extent in the Sol and VI muscles, with significant decreases and elevations in the slow type I and fast type IIX MHC protein isoforms, respectively. Consistent with the protein data, both the flight Sol and VI muscles exhibited significant elevations in the fast type IIX MHC mRNA isoform. In contrast, however, the flight Plan and TA groups had significant increases in the fast type IIB MHC mRNA isoform content without corresponding changes at the protein level. The results of this study suggest that spaceflight of even short duration produces important changes in the contractile properties of antigravity skeletal muscle. These changes are mediated by alterations in MHC phenotype and reductions in muscle mass. In some instances, the alterations in MHC mRNA isoform content seemed to be uncoupled from those occurring at the protein level. This apparent uncoupling between mRNA and protein expression demonstrates that the effects of microgravity must be better understood at the transcriptional, translational, and post-translational levels.

Alterations in phenotypic and contractile properties of the rat diaphragm: influence of hypothyroidism.

This study examined the influence of experimental hypothyroidism on myosin isoform distribution and contractile function of the costal diaphragm. Adult female Sprague-Dawley rats were randomly assigned to control (n = 12) or hypothyroid groups (n = 13) over a 6-wk treatment period. In comparison to the control group, in the hypothyroid group the relative distribution of type I myosin heavy chain (MHC) was increased 35% (P < 0.05), whereas type IIb MHC decreased 63% (P < 0.05). Similarly, Ca(2+)-activated myosin adenosinetriphosphatase activity (nmol Pi.mg-1.min-1) in the hypothyroid group was reduced 30% compared with the control group (P < 0.05). Furthermore, significant reductions in diaphragmatic maximal tetanic specific tension (Po; N.cm-2; -21%) and maximal shortening velocity (Vmax; muscle length/s; -25%) were observed in the hypothyroid group. These data provide the first evidence that hypothyroid produces a fast-(type IIb) to-slow (type I) shift in costal diaphragmatic MHC isoform profile that is highly correlated to the observed decrease in Vmax. Finally, the present findings indicate that hypothyroidism does not alter myofibrillar content or noncontractile elements of the diaphragm, thereby suggesting an alternative mechanism(s) to explain the reduction in specific Po.

Influence of mechanical loading on myosin heavy-chain protein and mRNA isoform expression.

The overall objective of the studies reported herein was to examine the effects of high-resistance training on myosin heavy-chain (MHC) protein and mRNA isoform expression. The findings from these studies can be summarized as follows: 1) there was a substantial increase in the fast type IIX MHC protein isoform content of the trained red and white medial gastrocnemius muscles, but this did not occur until after the eighth training session (i.e., 16 days); 2) single-fiber analyses demonstrated that many so-called fast type IIB fibers contained small amounts of the fast type IIX MHC protein isoform and that the high-resistance training program altered the bias of fast type IIB-type IIX MHC protein isoform distribution in these fibers but did not increase the number of fibers that could be categorized as exclusively fast type IIX fibers; 3) the high-resistance training program produced a rapid (i.e., after two training sessions) elevation in the fast type IIX MHC mRNA isoform and a corresponding repression of the fast type IIB MHC mRNA isoform; and 4) the dose-response study revealed that as few as 10 contractions (40 s) per training session were capable of elevating the expression of the fast type IIX MHC mRNA isoform by approximately 250%. These collective findings demonstrate that high-resistance training is a powerful modulator of MHC protein isoforms and that pretranslational mechanisms are very sensitive to even small amounts of high-resistance training.