Human unilateral lower limb suspension as a model for spaceflight effects on skeletal muscle

The following is the abstract of the article discussed in the subsequent letter:

	ABSTRACT

Widrick J. J., S. W. Trappe, J. G. Romatowski, D. A. Riley, D. L. Costill, and R. H. Fitts. Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function. J Appl Physiol 93: 354-360, 2002. We used Ca²⁺-activated skinned muscle fibers to test the hypothesis that unilateral lower leg suspension (ULLS) alters cross-bridge mechanisms of muscle contraction. Soleus and gastrocnemius biopsies were obtained from eight subjects before ULLS, immediately after 12 days of ULLS (post-0 h), and after 6 h of reambulation (post-6 h). Post-0 h soleus fibers expressing type I myosin heavy chain (MHC) showed significant reductions in diameter, absolute and specific peak Ca²⁺-activated force, unloaded shortening velocity, and absolute and normalized peak power. Fibers obtained from the gastrocnemius were less affected by ULLS, particularly fibers expressing fast MHC isoforms. Post-6 h soleus fibers produced less absolute and specific peak force than did post-0 h fibers, suggesting that reambulation after ULLS induced cell damage. Like bed rest and spaceflight, ULLS primarily affects soleus over gastrocnemius fibers. However, in contrast to these other models, slow soleus fibers obtained after ULLS showed a decrease in unloaded shortening velocity and a greater reduction in specific force.

	ARTICLE

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To the Editor: Widrick et al. (18) have published data from a 12-day unilateral lower limb suspension (ULLS) study conducted in 1994 using the methods originally published by Berg et al. in 1991 (3). Analyses included the morphology and contractile behavior of skinned skeletal muscle fibers obtained from the soleus and gastrocnemius of the ULLS limb. As evidenced by the title, the primary focus of this work was the evaluation of ULLS as a viable model for spaceflight-induced skeletal muscle adaptation. To this end, the authors made extensive comparisons between the ULLS results and those obtained from a National Aeronautics and Space Administration study that included 17 days of spaceflight and an attendant bed rest study of similar duration. A limitation of this paper is that the extensive comparisons were made in the form of prose presented only in the discussion section. None of the spaceflight data were provided. This significantly hampers the reader's ability to evaluate the authors conclusions. This problem is further exacerbated by the fact that the spaceflight and bed rest data have been parsed into eight separate publications (10-17).

There are a number of perplexing inconsistencies related to the presentation of information in the Widrick et al. ULLS paper (18). The authors state, "The finding that ULLS caused preferential atrophy of the soleus fibers is consistent with our earlier results obtained following a 17-day spaceflight" (14, 17). However, in a paper not cited from the same group and the same spaceflight and bed rest studies, Trappe et al. (13) state that "no statistical differences in single-fiber size (type I and II) were noted in the gastrocnemius or the soleus muscles of the crew members" and that "the bed-rest subjects showed no changes in single-fiber size of the soleus type I muscle fibers." In addition, another research group that collected data in the same 17-day bed rest and spaceflight studies conducted muscle contractile analyses (on the opposite leg) at the same time points as those reported in the various papers published by Dr. Fitts' group (7). These investigators found that there was no change in the maximal voluntary contraction of the triceps surae group during or after bed rest or spaceflight (they did see frequency-dependent declines in electrically evoked contractions). These results would seem to more strongly support the authors' contention in Widrick et al. that ULLS is not a high-fidelity model for spaceflight-induced muscle atrophy. Most pointedly, according to Trappe et al., spaceflight did not cause atrophy whereas ULLS did in the Widrick et al. paper. Why these same authors find fiber atrophy from apparently the same subjects from both bed rest and spaceflight is not clear (13, 14, 17). However, with regard to the present issue, there were very significant differences between the design of the ULLS study and that of the bed rest and spaceflight experiments referred to by Widrick et al. The primary difference is due to the loading state of the muscles studied and then compared by Widrick et al. in the paper in question. As detailed by Trappe et al., both the bed rest and spaceflight studies included a serial testing program (2, 13). Each testing bout lasted 30 min and included isometric, force-velocity, and fatigue tests. In aggregate, these tests included 57 maximal contractions plus a number at lower loads. This program consisted of 3 testing days spaced throughout the unloading period such that the longest span without an appreciable resistance load appears to have been 5 days. In fact, Trappe et al. concluded that "the testing sequence employed during the SF and BR may have served as a resistance training countermeasure to attenuate whole muscle strength loss." In addition, both the bed rest and spaceflight studies included the participation of other investigators who also conducted various types of muscle testing that may also have affected the adaptation of the leg muscle (2). As a further complication, the four crew members from the spaceflight study also participated in additional individualized countermeasure exercise activity, the volume of which was not reported. The bed rest and spaceflight studies used a pre- vs. post-design and did not include nontested controls to elucidate the effects of these intensive testing procedures. It is therefore quite reasonable, as suggested by Trappe et al., to conclude that testing activities may well account for the lack of change in myofiber cross-sectional area or maximal voluntary contraction reported by various investigators after bed rest or spaceflight (7, 13). In fairness to these investigators, the lack of nonexercising spaceflight crew members is something that has plagued all studies in this field. A true spaceflight control group has never, to the best of my knowledge, been made available to researchers.

In contrast to the bed rest and spaceflight studies cited by the authors, the ULLS study detailed in Widrick et al. was not reported as including any muscle loading over the entire course of the ULLS period. Thus it is not clear how these authors could so definitively conclude that the greater drop in fiber-specific tension, peak power, or any other performance or structural measure seen with ULLS was a function of the model rather than the differences in the previous activity history of the muscles in the ULLS, bed rest, and spaceflight studies. The intensive testing protocols of the bed rest and spaceflight studies used for comparison to ULLS in Widrick et al. (18) represent a significant difference in treatment and should preclude the comparison of results between these studies.

A second inaccuracy in the Widrick et al. publication involves the ULLS model itself. The authors suggest that the changes in performance seen in previously published ULLS studies may be a function of the relative immobilization and positioning of the lower limb during ULLS. However, in a number of the papers cited by Widrick et al., including two published before their study (4, 5), the ULLS model had been significantly modified to eliminate both immobilization and muscle shortening (1, 4, 5). As implemented in these and other papers, the modified ULLS model provided for the maintenance of the lower limb in normal anatomical positions when sitting or standing (1, 4, 5, 8, 9). With the use of the methods described in these studies, the target limb is free to move during ambulatory activity but remains unloaded. The results reported in these papers have generally had a high degree of fidelity with the spaceflight data that is available. Therefore the Widrick et al. contention that the performance deficits reported in their ULLS study may reflect model-specific adaptations such as increased susceptibility to damage would not apply to the majority of the functional ULLS data published to date (1, 4, 5, 8, 9). In addition, Widrick et al. suggest that indications of cellular degradation with ULLS, for example central core-like lesions, are distinct to this model and do not occur with bed rest. However, the results of Hikida et al. (6) for a 30-day bed rest study are at odds with this finding.

In summary, the conclusion that ULLS does not represent a high-fidelity model of spaceflight-induced skeletal muscle adaptation is at best premature. The comparisons made by Widrick et al. were not valid because of the incongruence in experimental design and the use of a less than optimal ULLS procedure.

The authors are urged to publish a corrigendum to this paper to provide a perspective on these limitations for future readers of this work.

	REFERENCES

1.   Adams, GR, Hather BM, and Dudley GA. Effect of short-term unweighting on human skeletal muscle strength and size. Aviat Space Environ Med 65: 1116-1121, 1994[Medline].

2.  Arnaud SB, Walker K, and Hargens A, eds. Life and Microgravity Sciences Spacelab Mission: Human Research Pilot Study/Six Month Report. Houston, TX: NASA, 1996. (NASA Tech. Memo. 110395)

3.   Berg, HE, Dudley GA, Haggmark T, Ohlsen H, and Tesch PA. Effects of lower limb unloading on skeletal muscle mass and function in man. J Appl Physiol 70: 1882-1885, 1991[Abstract/Free Full Text].

4.   Dudley, GA, Duvoisin MR, Adams GR, Meyer RA, Belew AH, and Buchanan P. Adaptation to unilateral lower limb suspension in humans. Aviat Space Environ Med 63: 678-683, 1992[Medline].

5.   Hather, BM, Adams GR, Tesch PA, and Dudley GA. Skeletal muscle responses to lower limb suspension in humans. J Appl Physiol 72: 1493-1498, 1992[Abstract/Free Full Text].

6.   Hikida, RS, Gollnick PD, Dudley GA, Convertino VA, and Buchanan P. Structural and metabolic characteristics of human skeletal muscle following 30 days of simulated microgravity. Aviat Space Environ Med 60: 664-670, 1989[Medline].

7.   Narici, MV, Kayser B, Barattini P, and Cerretelli P. Changes in electrically evoked skeletal muscle contractions during 17-day spaceflight and bed rest. Int J Sports Med 18: S290-S292, 1997.

8.   Ploutz-Snyder, LL, Tesch PA, Crittenden DJ, and Dudley GA. Effect of unweighting on skeletal muscle use during exercise. J Appl Physiol 79: 168-175, 1995[Abstract/Free Full Text].

9.   Ploutz-Snyder, LL, Tesch PA, Hather B, and Dudley GA. Vulnerability to dysfunction and muscle injury after unloading. Arch Phys Med Rehabil 77: 773-777, 1996[ISI][Medline].

10.   Riley, DA, Bain JL, Thompson JL, Fitts RH, Widrick JJ, Trappe SW, Trappe TA, and Costill DL. Disproportionate loss of thin filaments in human soleus muscle after 17-day bed rest. Muscle Nerve 21: 1280-1289, 1998[ISI][Medline].

11.   Riley, DA, Bain JL, Thompson JL, Fitts RH, Widrick JJ, Trappe SW, Trappe TA, and Costill DL. Decreased thin filament density and length in human atrophic soleus muscle fibers after spaceflight. J Appl Physiol 88: 567-572, 2000[Abstract/Free Full Text].

12.   Riley, DA, Bain JL, Thompson JL, Fitts RH, Widrick JJ, Trappe SW, Trappe TA, and Costill DL. Thin filament diversity and physiological properties of fast and slow fiber types in astronaut leg muscles. J Appl Physiol 92: 817-825, 2002[Abstract/Free Full Text].

13.   Trappe, SW, Trappe TA, Lee GA, Widrick JJ, Costill DL, and Fitts RH. Comparison of a space shuttle flight (STS-78) and bed rest on human muscle function. J Appl Physiol 91: 57-64, 2001[Abstract/Free Full Text].

14.   Widrick, JJ, Knuth ST, Norenberg KM, Romatowski JG, Bain JL, Riley DA, Karhanek M, Trappe SW, Trappe TA, Costill DL, and Fitts RH. Effect of a 17 day spaceflight on contractile properties of human soleus muscle fibres. J Physiol 516: 915-930, 1999[Abstract/Free Full Text].

15.   Widrick, JJ, Norenberg KM, Romatowski JG, Blaser CA, Karhanek M, Sherwood J, Trappe SW, Trappe TA, Costill DL, and Fitts RH. Force-velocity-power and force-pCa relationships of human soleus fibers after 17 days of bed rest. J Appl Physiol 85: 1949-1956, 1998[Abstract/Free Full Text].

16.   Widrick, JJ, Romatowski JG, Bain JL, Trappe SW, Trappe TA, Thompson JL, Costill DL, Riley DA, and Fitts RH. Effect of 17 days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers. Am J Physiol Cell Physiol 273: C1690-C1699, 1997.

17.   Widrick, JJ, Romatowski JG, Norenberg KM, Knuth ST, Bain JL, Riley DA, Trappe SW, Trappe TA, Costill DL, and Fitts RH. Functional properties of slow and fast gastrocnemius muscle fibers after a 17-day spaceflight. J Appl Physiol 90: 2203-2211, 2001[Abstract/Free Full Text].

18.   Widrick, JJ, Trappe SW, Romatowski JG, Riley DA, Costill DL, and Fitts RH. Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function. J Appl Physiol 93: 354-360, 2002[Abstract/Free Full Text].

Gregory R. Adams Department of Physiology & Biophysics University of California, Irvine Irvine, California 92697-4560 E-mail: gradams{at}uci.edu

	REPLY

To the Editor: Dr. Adams states that our study on the effects of short-term unilateral lower limb suspension (ULLS) contains a number of inconsistencies. In his letter to the editor, Adams appears to have no argument with our data per se but rather with our interpretation of this data, particularly as it pertains to other models of non-weight bearing. The intent of our article was to determine the effects of ULLS on single fiber contractile function and then in the discussion to compare these results to the available spaceflight and bed rest data. It was not our intent to discredit the ULLS model. We recognize that both ULLS and spaceflight produce similar degrees of muscle and fiber atrophy and declines in peak force. We agree with Dr. Adams' statement that the published data have generally found a high degree of fidelity between the effects of ULLS and those of spaceflight on skeletal muscle. However, our results identify a potentially important difference in that spaceflight increased and ULLS decreased the maximal shortening velocity (V_o) of the soleus type I fiber (6, 8). It is true, as pointed out by Dr. Adams, that the subjects in the spaceflight study (STS-78) performed exercise testing and exercise countermeasures (a component of all spaceflight studies), whereas the ULLS subjects did not. It is an open issue as to whether or not the exercise during spaceflight affected the soleus type I fiber V_o. However, rats flown in space or hindlimb unloaded under conditions in which there was no exercise showed an increased soleus V_o and, in the case of hindlimb unloading, an elevated type I fiber V_o (1, 3).

In the second paragraph of his letter, Dr. Adams suggests that our spaceflight data contain inconsistencies as to whether or not short-term spaceflight induces muscle atrophy and losses in force. This is an important issue that needs to be clarified. Collectively, the data from the STS-78 flight demonstrated that significant fiber atrophy did occur, with the soleus affected more than the gastrocnemius (6, 7). We studied skinned muscle fibers obtained from astronauts on this flight and observed reductions in slow-twitch soleus fiber cross-sectional area of 16% (8.3% reduction in fiber diameter) coupled with a 21% reduction in peak Ca²⁺-activated force (6). Gastrocnemius fibers were affected much less, with only the slow fibers showing a 6% reduction in force (7). In the same astronauts, LeBlanc et al. (2), using magnetic resonance imaging, found a 10% decline in the volume of the ankle extensors. These results are clearly in agreement and demonstrate that short-term spaceflight induces significant muscle atrophy and loss of force in isolated single fibers. As Dr. Adams points out, in our paper by Trappe et al. (5) we seem to contradict this conclusion as we reported that group averages for soleus fiber cross-sectional area were not significantly altered by the 17-day flight. In actuality these data were not treated statistically because of the small number of subjects (n = 4), and the statement of no statistical significance was inappropriate because no statistics were performed. In the Widrick et al. paper (6), the pre- and postflight fiber diameters were statistically compared by using a nested ANOVA (fibers nested within subject). Nonetheless, if one evaluates the actual data in the two papers, the results are remarkably similar. In both publications, the astronauts most affected, subjects B and D, showed the same percentage decline in fiber diameter or cross-sectional area.

The finding by Trappe et al. (5) and Narici et al. (4) that peak force during a maximum voluntary contraction of the plantarflexors was unaltered postflight shows that despite the reduced cellular capacity of the soleus type I fibers, the astronauts were able to maintain preflight neuromuscular strength. The failure to detect a reduced maximum voluntary contraction of the calf may have resulted from the fact that the gastrocnemius showed only minimal declines in type I fiber force, and thus the reduced force of the soleus fibers was not sufficient to alter the whole calf muscle strength.

Finally, it is not clear to us how Adams arrived at the conclusion that our paper by Widrick et al. (8) attributed the increased susceptibility to reloading damage to a model-specific adaptation to ULLS. As pointed out in our ULLS paper, reloading damage is a problem after not only ULLS, but also bed rest and spaceflight. Astronauts are known to be susceptible to reloading muscle damage and to experience extreme muscle soreness postflight.

	FOOTNOTES

10.1152/japplphysiol.00412.2002

	REFERENCES

1.   Caiozzo, VJ, Baker MJ, Herrick RE, Tao M, and Baldwin KM. Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow muscle. J Appl Physiol 76: 1764-1773, 1994[Abstract/Free Full Text].

2.   Leblanc, AD, Chen L, Shackelford L, Sinitsyn V, Evans H, Belichenko O, Schenkman B, Kozlovskaya I, Oganov V, Bakulin A, Hedrick T, and Feeback D. Muscle volume, MRI relaxation times (T2), and body composition after spaceflight. J Appl Physiol 89: 2158-2164, 2000[Abstract/Free Full Text].

3.   McDondal, KS, and Fitts RH. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity. J Appl Physiol 74: 2949-2957, 1993[Abstract/Free Full Text].

4.   Narici, MV, Kayser B, Barattini P, and Cerretelli P. Changes in electrically evoked skeletal muscle contractions during 17-day spaceflight and bed rest. Int J Sports Med 18: S290-S292, 1997.

5.   Trappe, SW, Trappe TA, Lee GA, Widrick JJ, Costill DL, and Fitts RH. Comparison of a space shuttle flight (STS-78) and bed rest on human muscle function. J Appl Physiol 91: 57-64, 2001.

6.   Widrick, JJ, Knuth SK, Norenberg KM, Romatowski JG, Bain JLW, Riley DA, Karhanek M, Trappe SW, Trappe TA, Costill DL, and Fitts RH. Effect of a 17 day spaceflight on contractile properties of human soleus fibers. J Physiol 516: 915-930, 1999.

7.   Widrick, JJ, Romatowski JG, Norenberg KM, Knuth ST, Bain JLW, Riley DA, Trappe SW, Trappe TA, Costill DL, and Fitts RH. Functional properties of slow and fast gastrocnemius muscle fibers after a 17-day spaceflight. J Appl Physiol 90: 2203-2211, 2001.

8.   Widrick, JJ, Trappe SW, Romatowski JG, Riley DA, Costill DL, and Fitts RH. Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function. J Appl Physiol 93: 354-360, 2002.

J. J. Widrick, J. G. Romatowski, R. H. Fitts Department of Biology Marquette University Milwaukee, Wisconsin 53201 E-mail: robert.fitts{at}marquette.edu

S. W. Trappe, D. L. Costill The Human Performance Lab Ball State University Muncie, Indiana 47306

D. A. Riley Department of Cellular Biology and Anatomy Medical College of Wisconsin Milwaukee, Wisconsin 53226