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Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity UC Irvine ICTS Publications Title Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity Permalink https://escholarship.org/uc/item/5jb1c92t Journal Journal of Applied Physiology, 75(6) ISSN 8750-7587 1522-1601 Authors Haddad, F. Herrick, R. E Adams, G. R et al. Publication Date 1993-12-01 DOI 10.1152/jappl.1993.75.6.2471 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity FADIA HADDAD, ROBERT E. HERRICK, GREGORY R. ADAMS, AND KENNETH M. BALDWIN Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92717 HADDAD, F ADIA, ROBERT E. HERRICK, GREGORY R. ADAMS, which of the faster MHCs are upregulated in response to AND KENNETH M. BALDWIN. Myosin heavy chain expression in conditions of zero gravity. rodent skeletal muscle: effects of exposure to zero gravity. J. Appl. In a previous study, we reported that 14 days of rodent Physiol. 75(6): 2471- 2477, 1993.-This study ascertained the hindlimb suspension decreased the relative level of ex­ effects of 9 days of zero gravity on the relative (percentage of pression of the type I MHC while increasing the relative total) and calculated absolute (mg/muscle) content of isomyo­ content of both the type Ila and IIb MHC isoforms in the sin expressed in both antigravity and locomotor skeletal mus­ antigravity vastus intermedius (VI) as studied at both cle of ground control (CON) and flight-exposed (FL) rats. Re­ the protein and mRNA levels of analyses (5). Isomyosin sults showed that although there were no differences in body changes in the fast-twitch plantaris were similar to those weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) in the VI but were of lesser magnitude (5). but not in the vastus lateralis (VL) or the tibialis anterior. Both To expand on these observations, a study was under­ total muscle protein and myofibril protein content were not taken, as part of the integrated National Aeronautics and different between the muscle regions examined in the FL and Space Administration (NASA) Space Life Sciences 1 CON groups. In the VI, there were trends for reductions in the mission, to ascertain whether exposure to zero gravity relative content of type I and Ila myosin heavy chains (MHCs) induces the same type of adaptational response in iso­ that were offset by increases in the relative content of both type myosin expression as that reported in the ground-based Ilb and possibly type Ilx MHC protein (P > 0.05). mRNA levels models (5, 11, 13). Herein, we report that 9 days of zero­ were consistent with this pattern (P < 0.05). The same pattern gravity exposure induced an altered pattern suggesting a held true for the red region of the VL as examined at both the reduction in both the relative and absolute content of protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute con­ type I and Ila MHC protein that was partially offset by tent of both type I and Ila MHCs that were offset by calculated small net increases in type llb and possibly type Ilx increases in type Ilb MHC in both VI and red VL. Collectively, MHC content in the high-oxidative red VI and vastus these findings suggest that there are both absolute and relative lateralis (VL) skeletal muscles. However, no changes oc­ changes occurring in MHC expression in the "red" regions of curred in either the fast low-oxidative VL or in the non­ antigravity skeletal muscle during exposure to zero gravity that weight-bearing tibialis anterior. could affect muscle function. METHODS myofibril protein; fast mysoin; slow myosin; type I isomyosin; type Ila isomyosin; type IIb isomyosin; antigravity skeletal Experimental design. The rodents used in this study muscle were part of the integrated Space Life Sciences 1 mission research project, which was a 9-day mission flown aboard the space shuttle Columbia in early June 1991. Approxi­ PREVIOUS STUDIES have shown that the chronic elimina­ mately 30 days before launch, young male Sprague-Daw­ tion of weight-bearing activity induces both fiber atrophy ley rats (Taconic Farms, Germantown, NY), weighing and a transformation in the pattern of isomyosin expres­ ~80 g, were randomly assigned to either a flight (FL; n = sion in rodent skeletal muscle (1, 5, 8, 10, 13). Available 10) or a control (CON; n = 10) group. Each of these two evidence, based on 1) immunohistochemical analyses of groups was subdivided into an additional two groups (n = myosin heavy chains (MHCs) in single fibers (8, 10) and 5 each) and designated for tissue processing as recovery 2) electrophoretic analyses of native isomyosins in whole (R+0) or recovery plus mission length (R+9). Before muscles (1, 5, 13, 15), suggests that slow myosin (type I) launch the animals were housed in groups of five at the expression is downregulated and that of the faster myo­ Kennedy Space Center vivarium and provided ad libitum sin isoforms is upregulated in muscles used extensively with water and a Purina-type food bar diet modified for for posture and/or locomotion when rodents are exposed dispension in the shuttle cages. Approximately 33 h be­ to either the ground-based model of hindlimb suspension fore launch, the FL animals were transferred to the Ani­ (5, 13, 15) or a zero-gravity environment (1, 8, 10). The mal Experimental Modules (AEMs}, which is a facility magnitude of these isoform shifts is dependent on both designed for grouped animal housing that can be accom­ the type of muscle affected (1, 8, 10) and the duration of modated in the middeck of the shuttle. The AEMs were unloading (1, 11, 13). However, it remains uncertain placed aboard the shuttle ~29 h before launch. CON 0161-7567 /93 $2.00 Copyright© 1993 the American Physiological Society 2471 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2472 ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN animals were maintained in grouped housing in the Ken­ are taken into consideration, it is possible to estimate the nedy Space Center vivarium in cages of similar size as the absolute content of a given MHC (mg/muscle) in a partic­ AEMs. The logistics of the 9-day mission and subsequent ular muscle by postflight period were identical to those described in a muscle wt (g) X myofibril yield (mg/g) companion paper (2). Tissue preparation. Both at recovery (R+0) and recov­ X %myosin in myofibril pool (0.45) ery plus mission length (R+9), FL and CON animals (1) X relative % of MHC isoform in myosin pool were killed by decapitation, and the muscles were rapidly removed along with other organs by a dissecting team = MHC isoform (mg)/muscle type coordinated by the NASA Ames Research Center. For this particular experiment, we were provided with both This approach was used in the present study to ascertain the impact of altered muscle weight on the average the right and left vastus complex and tibialis anterior. The right vastus complex was rapidly dissected and sepa­ amount of MHCs maintained in FL and CON muscles. RNA isolation and slot-blot analysis. Total RNA was rated into the VI, the red VL, and the white VL, and these muscles were immediately frozen in liquid nitrogen and isolated from frozen muscle samples according to the RNAzol method (Tel-Test), which is based on the proce­ stored at -70°C until used for MHC mRNA analysis. Then the right tibialis anterior was quickly frozen. Next, dure published by Chomczynski and Sacchi (3). In our the same muscles on the left side were processed by first hands, this technique is favored over the cesium chloride ultracentrifugation technique because it provides greater removing connective tissue and fascia, weighing them, and then storing them in glycerol cooled to -20°C. The yields ofundegraded RNA, free of DNA and proteins, on the basis of analyses using agarose gel-ethidium bromide muscles on the left side were used for protein analyses stain as well as on the basis of the ratio of optical density and electrophoresis of the MHC. Myofibril isolation and gel electrophoresis. The muscle at 260 nm to the optical density at 280 nm ( ~2.0). samples were first rinsed free of glycerol and then homog­ MHC mRNA-specific oligonucleotides, 20 bases in enized in 250 mM sucrose, 100 mM KCl, and 5 mM length, were purchased from Chemgenes (Waltham, MA). These oligo probes are complementary to the 3' EDTA (pH 6.8). An aliquot was used for total protein determination (6). Myofibrils were then prepared and pu­ untranslated regions of MHC mRNA and are highly spe­ cific to the type I, Ila, and IIb MHCs (7). The probes were rified by a detergent washing technique as described pre­ viously (13, 14). After protein measurements (6), myofi­ 5' end labeled with P to a specific activity of 4-5 X 10 1 1 brils were stored at a protein concentration of 1 mg/ml in counts• min- • pmo1- by the T4-polynucleotide kinase a storage buffer consisting of 50% glycerol, 100 mM reaction (9). Labeled oligonucleotides were separated from unlabeled nucleotides and from unincorporated [-y- Na P 0 , 5 mM ethylene glycol-bis(/3-aminoethyl ether)­ 4 2 7 32P] ATP by urea polyacrylamide gel electrophoresis. The N,N,N,N-tetraacetic acid, and 2 mM 2-mercaptoeth­ anol (pH 8.8). MHCs were separated using sodium do­ probes were tested for specificity by using Northern blot decyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis with both positive and negative controls for each according to a modification of the method of Danielli­ probe. Betto et al. (4), as described in detail previously (5). After For slot-blot analysis; total RNA (1-2 µg) was blotted staining and destaining, the gels were scanned densito­ onto a Nytran nylon membrane (Schleicher & Schuell, metrically at 630 nm in a Zeineh soft-laser densitometer Keene, NH) by using a standard slot-blot procedure. The (Biomedical Instruments, Fullerton, CA) interfaced with filters were subsequently fixed with ultraviolet irradia­ an IBM PC computer equipped with software to perform tion. Prehybridization, hybridization, and washing pro­ peak-area integration analysis. The areas of the peaks cedures for the blots were carried out according to the corresponding to each band were summated, and then manufacturer's recommendations. After the final wash, the area for each peak was expressed as a percentage of · the membranes were autoradiographed with an intensi­ the total area. In this way, MHC isoform was expressed fying screen at -70°C for 1-4 days. After hybridization as a percentage of the total myosin or total MHC present with the oligo probe, the membrane was washed with 1 % in the gel. In the system used for the MHC separation of SDS at 100°C for 15 min to completely remove the probe, these samples, we were not capable of consistently sepa­ rinsed with RNase free water, prehybridized as above, rating the so-called type Ilx MHC from the type Ila and rehybridized with a P end-labeled oligo(dT) probe, which hybridizes to poly(A) RNA (total mRNA). mRNA MHC, as the two proteins comigrate closely together; thus, what we refer to as the type Ila MHC band is in levels were quantified by densitometric scanning of the reality a combined type IIa-IIx MHC complex (see DIS­ autoradiogram, and each specific band was normalized CUSSION). to its corresponding. oligo( dT) signal. The quantified In previous studies, we reported that myofibrils can be amount of each MHC mRNA in a given muscle for each quantitatively extracted from skeletal muscle and puri­ of the experimental groups was then normalized to the fied with relatively little protein loss during the proce­ level found in the combined two CON groups, which were dure (13-15). Also, we have provided evidence that the not different from one another. These control values myosin content relative to the other proteins making up were set at 100%, and the experimental values were ex­ the myofibril pool remains relatively constant at ~41- pressed as a percentage of the normal control levels. 45% of the subcellular fraction in normal, atrophying, Statistical analysis. The data are presented as means ± and hypertrophying skeletal muscle (1, 13-15). When SE. All data were analyzed using a one-way analysis of variance with a post hoc Newman-Keul's test being per- these factors as well as the data reported in Tables 1-3 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2473 formed when F ratios were significant. MHC relative TABLE 2. Total muscle and myofibril protein content of content values were arcsine transferred before the statis­ control and flight animals tical analysis was performed. Because MHC mRNA and R+O R + 9 protein content did not differ between the two CON groups, both the protein and mRNA data bases of the Control Flight Control Flight two CON groups were combined into one group, and they Muscle protein were used for comparisons with the two FL groups. The VI 200±19 215±18 211±16 221±17 0.05 level of confidence was selected for statistical signifi­ RVL 322±18 316±30 300±25 301±22 cance. WVL 297±21 301±23 317±17 309±16 Myofibril protein RESULTS VI 95±6 87±10 100±10 96±15 Body weight, muscle weight, and protein content. Body RVL 140±12 141±13 100±18 154±15 weights were not different between FL and CON groups 164±27 WVL 156±27 171±35 100± 21 for the time points investigated (Table 1). The 9-day Values are means± SE in mg/g; n = 5 animals each in R + 0andR + flight induced a significant reduction in muscle weight in 9 groups. RVL, red VL; WVL. white VL. only the VI (Table 1); this difference was nearly identical to that reported in a companion study on this same mus­ muscles appears to be the increased relative expression cle (2). Although there was a small nonsignificant reduc­ of the type Ilb MHC coupled with the trend for a de­ tion in the weight of the VL, this response was less than crease in the relative content type I MHC. The impact on that reported in a companion project on this mission (2). the type Ila MHC pool appears to be different in these We have no insight as to why the weight differential for two red muscles, i.e., possibly increasing in the VI and the VL was less in this study except that the animals in possibly decreasing in the red VL (see DISCUSSION) . No this study were group housed and consequently their in­ changes were observed in either the white VL or tibialis herent interaction with one another may have had an anterior (Table 3). The 9-day recovery period appeared impact on movement activity involving the faster muscle to reverse the isomyosin shift in the red VL but not in the groups. Both total protein and myofibril protein content VI (Table 3). This differential response is surprising in were not different among the muscle types for either time view of the observation that the VL was not as responsive point (Table 2). This finding suggests that the 9-day mis­ as the VI to the atrophying effects of zero gravity (Ta­ sion produced a uniform reduction in the protein pool ble 1). across the muscle to account for the difference in VI In an attempt to estimate the degree that zero gravity weight. However, we noticed that muscles composed pre­ affects the absolute content of a given MHC in different dominantly of fast fibers had higher total and myofibril types of skeletal muscle (see METHODS), we calculated protein content than was previously observed (1). There for the VI that there is on average 1.72 and 1.40 mg/mus­ are two possibilities causing this discrepancy. First, the cle of type I and Ila-Ilx MHC, respectively, in the CON present study involved a different rat strain than that group. In the VI of the FL group there is on average 1.04, used previously (1). Second, these particular muscles 1.13, and 0.05 mg/muscle of type I, Ila-Ilx, and Ub MHC, could have had a lower than normal water content. respectively. Thus, on an absolute basis, there appears to MHC protein analyses. Figure 1 depicts representative be a net reduction in both the type I and Ila-Ilx MHC MHC gels for the different muscles of FL and CON pool by 40 and 19%, respectively, whereas trace amounts groups for the R+0 time point. The CON VI expresses a of the type Ilb MHC begin to appear in this particular similar proportion of type I and Ila MHC. The red VL muscle as a result of zero-gravity exposure. With the use expresses a bias toward the type Ila MHC, whereas the of this same approach for the red VL (and the muscle white VL and tibialis anterior are biased toward the ex­ masses reported in Table 1), the following was observed. pression of a high relative content of the type llb MHC. In the CON group, 6.4, 37.7, and 19.8 mg/muscle of type As presented in Table 3, there was a small but distinct I, type Ila-Ilx, and Ilb MHC were expressed, whereas in shift in the pattern of MHC distribution in both the VI the FL group this changed to 3.7, 31.3, and 26.4 for type I, and the red VL. The common response emerging in both Ila-Ilx, and lib MHC protein, respectively. Thus, in the red VL, the pattern of response closely resembles that TABLE 1. Body weight and wet muscle weights of control observed for the VI in that there is also a net reduction in and flight animals the type I and Ila-II MHC pool by 42 and 17%, respec­ R + O R + 9 tively, whereas that of the type Ilb MHC pool is in­ creased by 33%. Therefore, although the absolute reduc­ Control Flight Control Flight tion in muscle mass is relatively small in the VL (Table BW,g 320± 16 333 ± 29 352± 10 344±11 1), there is, nevertheless, a distinct net loss in both type I Vl,mg 73±3* 77± 3* 57±2t 86± 2 and Ila MHC protein that is largely offset by the increase VL,mg 1,014±33 968±33 1,139±18 1,083±35 in type Ilb MHC protein content. Therefore, despite the TA,mg 550±17 545±18 534±16 571±2 lack of dramatic change in the mass of the VL, there Values are means± SE; n = 5 rats each in recovery group (R + O) appears to be some dramatic transformation occurring in and in recovery plus mission length (9 days) group (R + 9). BW, body some of the fibers in this muscle in response to both zero weight; VI, vastus intermedius; VL, vastus lateralis; TA, tibialis ante­ gravity and weight-bearing recovery (Tables 3 and 4). rior. Significantly different (P < 0.05) from: * all other values in same MHC mRNA. The relative signals for isomyosin group; t R + 9 control group. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2474 ZERO GRAVITY EFFECTS ON MYOSIN H EAVY CHAIN RECOVERY+ 0 IIa - --> , , ·•• Fl • Ii :nu I I--- > C C F C F C S OL VI RED WHITE I Ia ---> ~· --·- I --- >-11111111.- C F C C C F WHITE VI SOL TA FIG. L Gel pattern of myosin heavy chains (MHCs) of soleus (SOL), vastus intermedius (VI), red vastus lateralis (VL) (RED), white VL (WHITE), and tibialis anterior (TA) of control (C) and flight (F) groups at recovery (R+0). A: VI and red VL. B: TA and white VL. Note greater appearance of type Ilb MHC in VI and red VL of F group. TABLE 4. Myosin heavy chain mRNA expression in TABLE 3. Myosin heavy chain protein expression in rodent skeletal muscles in response to spaceflight muscles of control and flight animals Myosin Heavy Chain Type Myosin Heavy Chain Type Ila lib Ila-llx Ilb 100±6 100±15 VI Control 53.4±2.4 46.6±2.3 VI Control 100±8 68±7* 22±4* 204±30* R + 0 46.8± 4.2 51.1± 4.2 2.1 ± 1.3* R+0 45.6± 2.7 50.9± 2.3 3.4±0.9* R+9 108± 4 127± 14* 64±11 R+9 100± 5 100±2 100±13 RVL Control 9.7± 1.7 61.1± 1.8 29.2± 1.3 RVL Control 42.9± 3.5* R +0 98±11 38±3* 220±25* R + 0 6.3± 1.5 50.7± 2.5* 73±5* 84±8 R + 9 5.8±1.0 60.7±1.7t 33.5±2.2t R+9 92±5 WVL 100±11 100±14 100±6 WVL Control 7.4±0 .8 92.6±0.8 Control R +0 91±14 90± 16 91±6 R+0 7.0± 0.4 93.0±0.4 90±7 R+9 7.1±0.7 93.0±0.7 R + 9 119±5 128± 17 TA Control 27.6±0.9 71.3±1.7 TA Control 100±5 100±8 100±9 R +o 28.9±1.2 71.3±1.2 R+0 113±4 118±8 91±6 109±6 91±12 98± 10 R + 9 28.4±0.9 72.0± 1.1 R + 9 Values are means ± SE expressed as percentage of total myosin Values are means ± SE expressed as percentage relative to control value; n = 10 control rats and 5 rats each in R + 0 and R + 9 groups. pool; n = 10 control rats and 5 rats each in R + 0 and R + 9 groups. * Significantly different (P < 0.05) from a ll other values in same group. Statistical analyses were performed on arcsin -transformed values to correct for binomial distribution of proportions. Significantly different (P < 0.05) from: • control value; t R + 0 value. ther e is a net loss in t he absolute amount of type I MHC protein in the red VL (due to atrophy), whereas the corresponding mRNA pool is relatively unaffected (Ta­ mRNA expression in the various muscle types are de­ ble 4). This suggests t hat protein degradation of type I picted in Fig. 2. Generally, the mRNA signals for the MHC may be the dominating factor affecting the relative various muscles parallel, in relative intensity, the pattern amount of this MHC isoform in the red VL. In the white presented for MH C protein expression shown in Fig. l. VL and tibialis anterior, the mRNA dat a are consistent Also, in t hose muscles responsive to MHC protein with t h e pattern of response noted for the MHC protein changes (VI and red VL) as a result of zero-gravity expo ­ data (Table 4, Fig. 4), which further suggests that these sure, a similar pattern of response occurred for the particular muscles were insensitive to zero-gravity expo­ mRNA data with the following exceptions. First, in the sure. VI, the reduction in the type Ila mRNA signal appears to be opposite of t he response seen for type Ila protein ex­ DISCUSSION pression (Fig. 3). Second, the relative reduction in type I MHC protein for t he red VL appears to exceed the re­ The key findin gs of this study indicat e that, after as duction seen for its corresponding mRNA signal (Fig. 3). litt le as 9 days in zero gravity, both atrophy and subtle This is furth er illustrated (see above) by the fact that alterations in MH C expression, as examined at both the Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2475 mRNA and prot ein levels, occur in rodent skeletal mus­ cles known to be involved in both antigravity and loco­ 100 motor function . Also, because both the t otal and myofi­ D Protein bril protein content were not different between CON and FL groups, the net loss in muscle protein that occurred in  mRNA ., muscles such as the VI appeared to be uniform across 40 c:,, cellular fractions. .. .c;; The findings reported herein provide further evidence ;!. t hat either t he lowering of skeletal muscle force produc ­ tion and/or the altering of the act ivity patterns of the -20 muscle during exposure to zero gravity induces a selec­ -40 tive net loss of bot h t he slow type I MHC and the faster -60 type Ila MHC in muscles rout inely used for most weight­ -80 bearing activities. Our calculations (see RESULTS) sug­ Type I Type IIA Type lib gest that the fibers expressing an abundance of type I MHC may be more responsive t h an fibers expressing the type Ila-Ilx MHC, which is consistent with previous re­ ports that used histological approaches to demonstrate a greater degree of atrophy of slow fibe rs (10) . Moreover, the data also suggest a unique process occurring to redi­ rect the pattern of MHC expressed in t hese muscles and favoring an increase in expression of t he fast type Ilb MHC. As a result, in a muscle expressing a relatively ., c:,, ,: MHC mRNA Type: !!! ;!. 0 11B IIA -20 -40 -60 -80 VI Type I Type IIA Type llb FIG. 3. P ercent change relative to normal control level of expres­ sion of MHC mRNA and protein in VI (top) and red VL (bottom). Normal control values were set as 100%. Data were plotted from Tables 3 and 4. high content of slow fibe r s such as the VI, which nor- mally expresses an abundance of the type I MHC (Table RVL 3), the fast myosins could become t h e functionally domi ­ nant isoform(s) expressed after extended exposure to microgravity. Furthermore, t he changes reported in the present study appear to be a resu lt of a t rue atrophy process, since the body weights (and presumably the muscle weights before flight) of the two groups did not differ before or after t he spaceflight. Thus , the atrophy WVL process associated with spaceflight appears to be highly specific to particula r subsets of muscle fibers, i.e., those expressing either type I or type Ila MHC in regions of muscles used extensively for antigravity and locomotor function. Also, it would appear t hat , despite the atrophy process occurring in fibers expressing these MHCs, there is a concurrent process occurring to expand the type Ilb MH C pool in some fibers, which appears to be contr olled TA in part by pretranslational processes leading to enhanced t ype Ilb MHC protein expression (Table 4, Fig. 3). F The findings reported herein at both the protein and mRNA level for the VI and red VL are str ikingly similar to t hose observed in a previous flight experiment involv­ FIG. 2. Slot blots of mRNA e xt racted from different muscles of F ing native myosin analysis (1) and to those observed in and C groups for R+O time point. Note greater signal for type IIb mRNA in VI and VL. RVL, red VL; WVL, white VL. ground-based studies using the model of hindlimb sus- Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2476 ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN discrepancy in MHC mRNA and protein is that the type Ila MHC protein pool may be decreasing while that of D Protein the type Ilx MHC pool is increasing with an overall net increase occurring in the relative size of the type Ila-Ilx  mRNA 60 MHC protein band. To resolve this issue one must sepa­ rate the type Ila-Ilx MHC band into its type Ila and Ilx Gl 0) ,: components. In an attempt to resolve this issue, we are in .t::. "' the process of refining our electrophoretic techniques to improve separation of the type Ila-Ilx MHC band. In preliminary experiments we estimated in the CON VI -20 samples that 75 ± 3% of the type Ila-Ilx MHC band was -40 type Ila MHC and 25 ± 3% represented type Ilx MHC. In the FL samples, the type Ila MHV component accounted -60 for only 61 ± 3% compared with 39% for type Ilx MHC. -80 Thus, based on these distributions, it would indeed ap­ Type I Type IIA Type llb pear that there was both an absolute and relative reduc­ tion in the amount of type Ila MHC that was expressed in the VI of the FL animals, which is consistent with the mRNA data. We suspect that the same pattern occurred 120 in the red VL. In conclusion, the present study provides further evi­ dence that zero gravity exerts a significant impact on both the quantity and type of MHCs expressed in mus­ cles routinely used for both antigravity and locomotor activity. The findings further suggest that molecular <I> 0) events occurring at both the pretranslational and post­ .t::. translational levels of regulating MHC gene expression "' (.) are likely involved in these muscle adaptations to condi­ ;!. tions of unloading. Finally, it appears that future experi­ -20 ments involving exposure to zero gravity need to be con­ ducted for longer durations to obtain a better under­ -40 standing of the transformation in the steady-state -60 pattern of isomyosin expression in both locomotor and -80 antigravity skeletal muscle. Type I Type IIA Type llb The authors thank the research support team organized by the FIG. 4. Percent change relative to normal control level of expres­ NASA Ames Research Center for coordinating this research project. sion of MHC mRNA and protein in white VL (top) and TA (bottom). This research was supported by NASA Grant NAG-2-555. Normal control values were set at 100%. Data were plotted from Tables Address reprint requests to K. M. Baldwin. 3 and 4. Received 29 March 1993; accepted in final form 9 July 1993. pension to study MHC mRNA expression (5, 13). For REFERENCES example, Thomason et al. (13) reported a decrease in type I MHC synthesis in the soleus after 7 days of limb 1. BALDWIN, K. M., R. E. HERRICK, E. ILYINA-KAKUEVA, AND V. s. unweighting even though mRNA levels were not reduced. 0RGANOV. Effects of zero gravity on myofibril content and isomyo­ In the present study, we observed in the red VL, but not sin distribution in rodent skeletal muscle. FASEB J . 4: 79- 83, 1990. in the VI, that the relative reduction in type I MHC pro­ 2. BALDWIN, K. M., R. E. HERRICK, AND S. A. MCCUE. Substrate oxidation capacity in skeletal muscle: Effects of exposure to zero tein exceeded that of mRNA (Fig. 3), which further sug­ gravity. J. Appl. Physiol. 75: 2466-2470, 1993. gests that posttranslational events, possibly associated 3. CttOMCZYNSKI, P ., AND N. SACCHI. Single step method of RNA with protein degradation processes (12), are likely con­ isolation by acid guanidinium thiocyanate-phenol-chloroform ex• tributing to the reduction in net protein accumulation traction. Anal. Biochem. 162: 156-159, 1987. relative to the changes occurring in the mRNA pool. 4. DANIELLI-BETTO, D ., E. ZERBATO, AND R. BETTO. Type I, Ila, and IIb myosin heavy chain electrophoretic analysis of rat muscle Also, Diffee et al. (5) reported a differential pattern in fibers. Biochem. Biophys. Res. Commun. 138: 981-987, 1986. type Ila MHC mRNA expression compared with the rela­ 5 . DIFFEE, G. M., F . HADDAD, R. E. HERRICK, AND K. M. BALDWIN. tive expression of type Ila MHC protein in slow muscles Control of myosin heavy chain expression: interaction of hypothy­ of rats suspended for 14 days. In that study the type Ila roidism and hindlimb suspension. Am. J . Physiol. 261 (Cell Physiol. MHC mRNA signal was depressed in suspended rats, 30): C1099-Cll06, 1991. 6. GORNALL, A.G. , C. J. BARDAWILL, AND M. M. DAVID. Determina­ whereas the (supposed) relative content of type Ila MHC tion of serum protein by means of the biuret method. J. Biol. Chem. protein appeared to have been increased (5). This was 177: 751-756, 1949. the identical pattern seen for these isoforms in the VI 7 . GUSTAFSON, T. A., B. E. MARKHAM, AND E. MORKIN. Effects of from the present study (Fig. 3). thyroid hormone on alpha-actin and myosin heavy chain gene ex­ One possible explanation to account for this particular pression in cardiac and skeletal muscles of rat: measurement of Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZRRO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2477 !ism and /3-myosin heavy-chain rnRNA in unweighted soleus mus­ mRNA content using synthetic oligonucleotide probes. Circ. Res. 59: 194-201, 1986. cle. Am. J. Physiol. 257 (Regulatory Integrative Comp. Physiol. 26): 8. JIANG, B., Y. 0HIRA, R. R. ROY, Q. NGUYEN, E. I. lLYINA-KA­ R300-R305, 1989. KUEVA, V. OGANOV, AND V. R. EDGERTON. Adaptation of fibers in 12. THOMASON, D. B., AND F. W. BOOTH. Atrophy of the soleus muscle fast-twitch muscles of rats to spaceflight and hindlimb suspenRion. by hindlirnb unweighting. J. Appl. Physio/. 68: 1-12, 1990. J. Appl. Physiol. 73, Suppl.: 588-658, 1992. 13. THOMASON, D. B., R. E. HERRICK, D. 8URDYKA, AND K. M. BALD­ WIN. Time course of soleus muscle myosin expression during hind­ 9. MAXAM, A. M., AND W. GILBERT. Sequencing end labelled DNA with base specific chemical cleavages. Metlwds Enzymol. 65: 499- limb suspension and recovery. J. Appl. Physiol. 63: 130-137, 1987. 560, 1980. 14. TSIKA, R. w., R. E. HERRICK, AND K. M . BALDWIN. Time-course 10. 0HIRA, Y., B. JIANG, R.R. ROY, V. 0GANOV, E. ILYINA-KAKUEVA, adaptations in rat skeletal muscle isomyosins during compensa­ J. F. MARINI, AND V. R. EDGERTON. Rat soleus muscle fiber re­ tory growth and recovery. J. Appl. Physiol. 63: 2111-2121, 1987. sponses to 14 days of spaceflight and hindlirnb suspension. J. Appl. 15. TS!KA, R. w., R. E. HERRICK, AND K. M. BALDWIN. Effect of ana­ bolic steroids on overloaded and overloaded suspended skeletal Physiot. 73, Suppl.: 51S-57S, 1992. muscle. J. Appl. Physiol. 63: 3128-2133, 1987. 11. THOMASON, D. B., R. B. BIGGS, AND F. W. BOOTH. Protein metabo- Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Physiology Unpaywall

Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.
Journal of Applied Physiology – Dec 1, 1993
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UC Irvine ICTS Publications Title Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity Permalink https://escholarship.org/uc/item/5jb1c92t Journal Journal of Applied Physiology, 75(6) ISSN 8750-7587 1522-1601 Authors Haddad, F. Herrick, R. E Adams, G. R et al. Publication Date 1993-12-01 DOI 10.1152/jappl.1993.75.6.2471 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity FADIA HADDAD, ROBERT E. HERRICK, GREGORY R. ADAMS, AND KENNETH M. BALDWIN Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92717 HADDAD, F ADIA, ROBERT E. HERRICK, GREGORY R. ADAMS, which of the faster MHCs are upregulated in response to AND KENNETH M. BALDWIN. Myosin heavy chain expression in conditions of zero gravity. rodent skeletal muscle: effects of exposure to zero gravity. J. Appl. In a previous study, we reported that 14 days of rodent Physiol. 75(6): 2471- 2477, 1993.-This study ascertained the hindlimb suspension decreased the relative level of ex­ effects of 9 days of zero gravity on the relative (percentage of pression of the type I MHC while increasing the relative total) and calculated absolute (mg/muscle) content of isomyo­ content of both the type Ila and IIb MHC isoforms in the sin expressed in both antigravity and locomotor skeletal mus­ antigravity vastus intermedius (VI) as studied at both cle of ground control (CON) and flight-exposed (FL) rats. Re­ the protein and mRNA levels of analyses (5). Isomyosin sults showed that although there were no differences in body changes in the fast-twitch plantaris were similar to those weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) in the VI but were of lesser magnitude (5). but not in the vastus lateralis (VL) or the tibialis anterior. Both To expand on these observations, a study was under­ total muscle protein and myofibril protein content were not taken, as part of the integrated National Aeronautics and different between the muscle regions examined in the FL and Space Administration (NASA) Space Life Sciences 1 CON groups. In the VI, there were trends for reductions in the mission, to ascertain whether exposure to zero gravity relative content of type I and Ila myosin heavy chains (MHCs) induces the same type of adaptational response in iso­ that were offset by increases in the relative content of both type myosin expression as that reported in the ground-based Ilb and possibly type Ilx MHC protein (P > 0.05). mRNA levels models (5, 11, 13). Herein, we report that 9 days of zero­ were consistent with this pattern (P < 0.05). The same pattern gravity exposure induced an altered pattern suggesting a held true for the red region of the VL as examined at both the reduction in both the relative and absolute content of protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute con­ type I and Ila MHC protein that was partially offset by tent of both type I and Ila MHCs that were offset by calculated small net increases in type llb and possibly type Ilx increases in type Ilb MHC in both VI and red VL. Collectively, MHC content in the high-oxidative red VI and vastus these findings suggest that there are both absolute and relative lateralis (VL) skeletal muscles. However, no changes oc­ changes occurring in MHC expression in the "red" regions of curred in either the fast low-oxidative VL or in the non­ antigravity skeletal muscle during exposure to zero gravity that weight-bearing tibialis anterior. could affect muscle function. METHODS myofibril protein; fast mysoin; slow myosin; type I isomyosin; type Ila isomyosin; type IIb isomyosin; antigravity skeletal Experimental design. The rodents used in this study muscle were part of the integrated Space Life Sciences 1 mission research project, which was a 9-day mission flown aboard the space shuttle Columbia in early June 1991. Approxi­ PREVIOUS STUDIES have shown that the chronic elimina­ mately 30 days before launch, young male Sprague-Daw­ tion of weight-bearing activity induces both fiber atrophy ley rats (Taconic Farms, Germantown, NY), weighing and a transformation in the pattern of isomyosin expres­ ~80 g, were randomly assigned to either a flight (FL; n = sion in rodent skeletal muscle (1, 5, 8, 10, 13). Available 10) or a control (CON; n = 10) group. Each of these two evidence, based on 1) immunohistochemical analyses of groups was subdivided into an additional two groups (n = myosin heavy chains (MHCs) in single fibers (8, 10) and 5 each) and designated for tissue processing as recovery 2) electrophoretic analyses of native isomyosins in whole (R+0) or recovery plus mission length (R+9). Before muscles (1, 5, 13, 15), suggests that slow myosin (type I) launch the animals were housed in groups of five at the expression is downregulated and that of the faster myo­ Kennedy Space Center vivarium and provided ad libitum sin isoforms is upregulated in muscles used extensively with water and a Purina-type food bar diet modified for for posture and/or locomotion when rodents are exposed dispension in the shuttle cages. Approximately 33 h be­ to either the ground-based model of hindlimb suspension fore launch, the FL animals were transferred to the Ani­ (5, 13, 15) or a zero-gravity environment (1, 8, 10). The mal Experimental Modules (AEMs}, which is a facility magnitude of these isoform shifts is dependent on both designed for grouped animal housing that can be accom­ the type of muscle affected (1, 8, 10) and the duration of modated in the middeck of the shuttle. The AEMs were unloading (1, 11, 13). However, it remains uncertain placed aboard the shuttle ~29 h before launch. CON 0161-7567 /93 $2.00 Copyright© 1993 the American Physiological Society 2471 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2472 ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN animals were maintained in grouped housing in the Ken­ are taken into consideration, it is possible to estimate the nedy Space Center vivarium in cages of similar size as the absolute content of a given MHC (mg/muscle) in a partic­ AEMs. The logistics of the 9-day mission and subsequent ular muscle by postflight period were identical to those described in a muscle wt (g) X myofibril yield (mg/g) companion paper (2). Tissue preparation. Both at recovery (R+0) and recov­ X %myosin in myofibril pool (0.45) ery plus mission length (R+9), FL and CON animals (1) X relative % of MHC isoform in myosin pool were killed by decapitation, and the muscles were rapidly removed along with other organs by a dissecting team = MHC isoform (mg)/muscle type coordinated by the NASA Ames Research Center. For this particular experiment, we were provided with both This approach was used in the present study to ascertain the impact of altered muscle weight on the average the right and left vastus complex and tibialis anterior. The right vastus complex was rapidly dissected and sepa­ amount of MHCs maintained in FL and CON muscles. RNA isolation and slot-blot analysis. Total RNA was rated into the VI, the red VL, and the white VL, and these muscles were immediately frozen in liquid nitrogen and isolated from frozen muscle samples according to the RNAzol method (Tel-Test), which is based on the proce­ stored at -70°C until used for MHC mRNA analysis. Then the right tibialis anterior was quickly frozen. Next, dure published by Chomczynski and Sacchi (3). In our the same muscles on the left side were processed by first hands, this technique is favored over the cesium chloride ultracentrifugation technique because it provides greater removing connective tissue and fascia, weighing them, and then storing them in glycerol cooled to -20°C. The yields ofundegraded RNA, free of DNA and proteins, on the basis of analyses using agarose gel-ethidium bromide muscles on the left side were used for protein analyses stain as well as on the basis of the ratio of optical density and electrophoresis of the MHC. Myofibril isolation and gel electrophoresis. The muscle at 260 nm to the optical density at 280 nm ( ~2.0). samples were first rinsed free of glycerol and then homog­ MHC mRNA-specific oligonucleotides, 20 bases in enized in 250 mM sucrose, 100 mM KCl, and 5 mM length, were purchased from Chemgenes (Waltham, MA). These oligo probes are complementary to the 3' EDTA (pH 6.8). An aliquot was used for total protein determination (6). Myofibrils were then prepared and pu­ untranslated regions of MHC mRNA and are highly spe­ cific to the type I, Ila, and IIb MHCs (7). The probes were rified by a detergent washing technique as described pre­ viously (13, 14). After protein measurements (6), myofi­ 5' end labeled with P to a specific activity of 4-5 X 10 1 1 brils were stored at a protein concentration of 1 mg/ml in counts• min- • pmo1- by the T4-polynucleotide kinase a storage buffer consisting of 50% glycerol, 100 mM reaction (9). Labeled oligonucleotides were separated from unlabeled nucleotides and from unincorporated [-y- Na P 0 , 5 mM ethylene glycol-bis(/3-aminoethyl ether)­ 4 2 7 32P] ATP by urea polyacrylamide gel electrophoresis. The N,N,N,N-tetraacetic acid, and 2 mM 2-mercaptoeth­ anol (pH 8.8). MHCs were separated using sodium do­ probes were tested for specificity by using Northern blot decyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis with both positive and negative controls for each according to a modification of the method of Danielli­ probe. Betto et al. (4), as described in detail previously (5). After For slot-blot analysis; total RNA (1-2 µg) was blotted staining and destaining, the gels were scanned densito­ onto a Nytran nylon membrane (Schleicher & Schuell, metrically at 630 nm in a Zeineh soft-laser densitometer Keene, NH) by using a standard slot-blot procedure. The (Biomedical Instruments, Fullerton, CA) interfaced with filters were subsequently fixed with ultraviolet irradia­ an IBM PC computer equipped with software to perform tion. Prehybridization, hybridization, and washing pro­ peak-area integration analysis. The areas of the peaks cedures for the blots were carried out according to the corresponding to each band were summated, and then manufacturer's recommendations. After the final wash, the area for each peak was expressed as a percentage of · the membranes were autoradiographed with an intensi­ the total area. In this way, MHC isoform was expressed fying screen at -70°C for 1-4 days. After hybridization as a percentage of the total myosin or total MHC present with the oligo probe, the membrane was washed with 1 % in the gel. In the system used for the MHC separation of SDS at 100°C for 15 min to completely remove the probe, these samples, we were not capable of consistently sepa­ rinsed with RNase free water, prehybridized as above, rating the so-called type Ilx MHC from the type Ila and rehybridized with a P end-labeled oligo(dT) probe, which hybridizes to poly(A) RNA (total mRNA). mRNA MHC, as the two proteins comigrate closely together; thus, what we refer to as the type Ila MHC band is in levels were quantified by densitometric scanning of the reality a combined type IIa-IIx MHC complex (see DIS­ autoradiogram, and each specific band was normalized CUSSION). to its corresponding. oligo( dT) signal. The quantified In previous studies, we reported that myofibrils can be amount of each MHC mRNA in a given muscle for each quantitatively extracted from skeletal muscle and puri­ of the experimental groups was then normalized to the fied with relatively little protein loss during the proce­ level found in the combined two CON groups, which were dure (13-15). Also, we have provided evidence that the not different from one another. These control values myosin content relative to the other proteins making up were set at 100%, and the experimental values were ex­ the myofibril pool remains relatively constant at ~41- pressed as a percentage of the normal control levels. 45% of the subcellular fraction in normal, atrophying, Statistical analysis. The data are presented as means ± and hypertrophying skeletal muscle (1, 13-15). When SE. All data were analyzed using a one-way analysis of variance with a post hoc Newman-Keul's test being per- these factors as well as the data reported in Tables 1-3 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2473 formed when F ratios were significant. MHC relative TABLE 2. Total muscle and myofibril protein content of content values were arcsine transferred before the statis­ control and flight animals tical analysis was performed. Because MHC mRNA and R+O R + 9 protein content did not differ between the two CON groups, both the protein and mRNA data bases of the Control Flight Control Flight two CON groups were combined into one group, and they Muscle protein were used for comparisons with the two FL groups. The VI 200±19 215±18 211±16 221±17 0.05 level of confidence was selected for statistical signifi­ RVL 322±18 316±30 300±25 301±22 cance. WVL 297±21 301±23 317±17 309±16 Myofibril protein RESULTS VI 95±6 87±10 100±10 96±15 Body weight, muscle weight, and protein content. Body RVL 140±12 141±13 100±18 154±15 weights were not different between FL and CON groups 164±27 WVL 156±27 171±35 100± 21 for the time points investigated (Table 1). The 9-day Values are means± SE in mg/g; n = 5 animals each in R + 0andR + flight induced a significant reduction in muscle weight in 9 groups. RVL, red VL; WVL. white VL. only the VI (Table 1); this difference was nearly identical to that reported in a companion study on this same mus­ muscles appears to be the increased relative expression cle (2). Although there was a small nonsignificant reduc­ of the type Ilb MHC coupled with the trend for a de­ tion in the weight of the VL, this response was less than crease in the relative content type I MHC. The impact on that reported in a companion project on this mission (2). the type Ila MHC pool appears to be different in these We have no insight as to why the weight differential for two red muscles, i.e., possibly increasing in the VI and the VL was less in this study except that the animals in possibly decreasing in the red VL (see DISCUSSION) . No this study were group housed and consequently their in­ changes were observed in either the white VL or tibialis herent interaction with one another may have had an anterior (Table 3). The 9-day recovery period appeared impact on movement activity involving the faster muscle to reverse the isomyosin shift in the red VL but not in the groups. Both total protein and myofibril protein content VI (Table 3). This differential response is surprising in were not different among the muscle types for either time view of the observation that the VL was not as responsive point (Table 2). This finding suggests that the 9-day mis­ as the VI to the atrophying effects of zero gravity (Ta­ sion produced a uniform reduction in the protein pool ble 1). across the muscle to account for the difference in VI In an attempt to estimate the degree that zero gravity weight. However, we noticed that muscles composed pre­ affects the absolute content of a given MHC in different dominantly of fast fibers had higher total and myofibril types of skeletal muscle (see METHODS), we calculated protein content than was previously observed (1). There for the VI that there is on average 1.72 and 1.40 mg/mus­ are two possibilities causing this discrepancy. First, the cle of type I and Ila-Ilx MHC, respectively, in the CON present study involved a different rat strain than that group. In the VI of the FL group there is on average 1.04, used previously (1). Second, these particular muscles 1.13, and 0.05 mg/muscle of type I, Ila-Ilx, and Ub MHC, could have had a lower than normal water content. respectively. Thus, on an absolute basis, there appears to MHC protein analyses. Figure 1 depicts representative be a net reduction in both the type I and Ila-Ilx MHC MHC gels for the different muscles of FL and CON pool by 40 and 19%, respectively, whereas trace amounts groups for the R+0 time point. The CON VI expresses a of the type Ilb MHC begin to appear in this particular similar proportion of type I and Ila MHC. The red VL muscle as a result of zero-gravity exposure. With the use expresses a bias toward the type Ila MHC, whereas the of this same approach for the red VL (and the muscle white VL and tibialis anterior are biased toward the ex­ masses reported in Table 1), the following was observed. pression of a high relative content of the type llb MHC. In the CON group, 6.4, 37.7, and 19.8 mg/muscle of type As presented in Table 3, there was a small but distinct I, type Ila-Ilx, and Ilb MHC were expressed, whereas in shift in the pattern of MHC distribution in both the VI the FL group this changed to 3.7, 31.3, and 26.4 for type I, and the red VL. The common response emerging in both Ila-Ilx, and lib MHC protein, respectively. Thus, in the red VL, the pattern of response closely resembles that TABLE 1. Body weight and wet muscle weights of control observed for the VI in that there is also a net reduction in and flight animals the type I and Ila-II MHC pool by 42 and 17%, respec­ R + O R + 9 tively, whereas that of the type Ilb MHC pool is in­ creased by 33%. Therefore, although the absolute reduc­ Control Flight Control Flight tion in muscle mass is relatively small in the VL (Table BW,g 320± 16 333 ± 29 352± 10 344±11 1), there is, nevertheless, a distinct net loss in both type I Vl,mg 73±3* 77± 3* 57±2t 86± 2 and Ila MHC protein that is largely offset by the increase VL,mg 1,014±33 968±33 1,139±18 1,083±35 in type Ilb MHC protein content. Therefore, despite the TA,mg 550±17 545±18 534±16 571±2 lack of dramatic change in the mass of the VL, there Values are means± SE; n = 5 rats each in recovery group (R + O) appears to be some dramatic transformation occurring in and in recovery plus mission length (9 days) group (R + 9). BW, body some of the fibers in this muscle in response to both zero weight; VI, vastus intermedius; VL, vastus lateralis; TA, tibialis ante­ gravity and weight-bearing recovery (Tables 3 and 4). rior. Significantly different (P < 0.05) from: * all other values in same MHC mRNA. The relative signals for isomyosin group; t R + 9 control group. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2474 ZERO GRAVITY EFFECTS ON MYOSIN H EAVY CHAIN RECOVERY+ 0 IIa - --> , , ·•• Fl • Ii :nu I I--- > C C F C F C S OL VI RED WHITE I Ia ---> ~· --·- I --- >-11111111.- C F C C C F WHITE VI SOL TA FIG. L Gel pattern of myosin heavy chains (MHCs) of soleus (SOL), vastus intermedius (VI), red vastus lateralis (VL) (RED), white VL (WHITE), and tibialis anterior (TA) of control (C) and flight (F) groups at recovery (R+0). A: VI and red VL. B: TA and white VL. Note greater appearance of type Ilb MHC in VI and red VL of F group. TABLE 4. Myosin heavy chain mRNA expression in TABLE 3. Myosin heavy chain protein expression in rodent skeletal muscles in response to spaceflight muscles of control and flight animals Myosin Heavy Chain Type Myosin Heavy Chain Type Ila lib Ila-llx Ilb 100±6 100±15 VI Control 53.4±2.4 46.6±2.3 VI Control 100±8 68±7* 22±4* 204±30* R + 0 46.8± 4.2 51.1± 4.2 2.1 ± 1.3* R+0 45.6± 2.7 50.9± 2.3 3.4±0.9* R+9 108± 4 127± 14* 64±11 R+9 100± 5 100±2 100±13 RVL Control 9.7± 1.7 61.1± 1.8 29.2± 1.3 RVL Control 42.9± 3.5* R +0 98±11 38±3* 220±25* R + 0 6.3± 1.5 50.7± 2.5* 73±5* 84±8 R + 9 5.8±1.0 60.7±1.7t 33.5±2.2t R+9 92±5 WVL 100±11 100±14 100±6 WVL Control 7.4±0 .8 92.6±0.8 Control R +0 91±14 90± 16 91±6 R+0 7.0± 0.4 93.0±0.4 90±7 R+9 7.1±0.7 93.0±0.7 R + 9 119±5 128± 17 TA Control 27.6±0.9 71.3±1.7 TA Control 100±5 100±8 100±9 R +o 28.9±1.2 71.3±1.2 R+0 113±4 118±8 91±6 109±6 91±12 98± 10 R + 9 28.4±0.9 72.0± 1.1 R + 9 Values are means ± SE expressed as percentage of total myosin Values are means ± SE expressed as percentage relative to control value; n = 10 control rats and 5 rats each in R + 0 and R + 9 groups. pool; n = 10 control rats and 5 rats each in R + 0 and R + 9 groups. * Significantly different (P < 0.05) from a ll other values in same group. Statistical analyses were performed on arcsin -transformed values to correct for binomial distribution of proportions. Significantly different (P < 0.05) from: • control value; t R + 0 value. ther e is a net loss in t he absolute amount of type I MHC protein in the red VL (due to atrophy), whereas the corresponding mRNA pool is relatively unaffected (Ta­ mRNA expression in the various muscle types are de­ ble 4). This suggests t hat protein degradation of type I picted in Fig. 2. Generally, the mRNA signals for the MHC may be the dominating factor affecting the relative various muscles parallel, in relative intensity, the pattern amount of this MHC isoform in the red VL. In the white presented for MH C protein expression shown in Fig. l. VL and tibialis anterior, the mRNA dat a are consistent Also, in t hose muscles responsive to MHC protein with t h e pattern of response noted for the MHC protein changes (VI and red VL) as a result of zero-gravity expo ­ data (Table 4, Fig. 4), which further suggests that these sure, a similar pattern of response occurred for the particular muscles were insensitive to zero-gravity expo­ mRNA data with the following exceptions. First, in the sure. VI, the reduction in the type Ila mRNA signal appears to be opposite of t he response seen for type Ila protein ex­ DISCUSSION pression (Fig. 3). Second, the relative reduction in type I MHC protein for t he red VL appears to exceed the re­ The key findin gs of this study indicat e that, after as duction seen for its corresponding mRNA signal (Fig. 3). litt le as 9 days in zero gravity, both atrophy and subtle This is furth er illustrated (see above) by the fact that alterations in MH C expression, as examined at both the Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2475 mRNA and prot ein levels, occur in rodent skeletal mus­ cles known to be involved in both antigravity and loco­ 100 motor function . Also, because both the t otal and myofi­ D Protein bril protein content were not different between CON and FL groups, the net loss in muscle protein that occurred in  mRNA ., muscles such as the VI appeared to be uniform across 40 c:,, cellular fractions. .. .c;; The findings reported herein provide further evidence ;!. t hat either t he lowering of skeletal muscle force produc ­ tion and/or the altering of the act ivity patterns of the -20 muscle during exposure to zero gravity induces a selec­ -40 tive net loss of bot h t he slow type I MHC and the faster -60 type Ila MHC in muscles rout inely used for most weight­ -80 bearing activities. Our calculations (see RESULTS) sug­ Type I Type IIA Type lib gest that the fibers expressing an abundance of type I MHC may be more responsive t h an fibers expressing the type Ila-Ilx MHC, which is consistent with previous re­ ports that used histological approaches to demonstrate a greater degree of atrophy of slow fibe rs (10) . Moreover, the data also suggest a unique process occurring to redi­ rect the pattern of MHC expressed in t hese muscles and favoring an increase in expression of t he fast type Ilb MHC. As a result, in a muscle expressing a relatively ., c:,, ,: MHC mRNA Type: !!! ;!. 0 11B IIA -20 -40 -60 -80 VI Type I Type IIA Type llb FIG. 3. P ercent change relative to normal control level of expres­ sion of MHC mRNA and protein in VI (top) and red VL (bottom). Normal control values were set as 100%. Data were plotted from Tables 3 and 4. high content of slow fibe r s such as the VI, which nor- mally expresses an abundance of the type I MHC (Table RVL 3), the fast myosins could become t h e functionally domi ­ nant isoform(s) expressed after extended exposure to microgravity. Furthermore, t he changes reported in the present study appear to be a resu lt of a t rue atrophy process, since the body weights (and presumably the muscle weights before flight) of the two groups did not differ before or after t he spaceflight. Thus , the atrophy WVL process associated with spaceflight appears to be highly specific to particula r subsets of muscle fibers, i.e., those expressing either type I or type Ila MHC in regions of muscles used extensively for antigravity and locomotor function. Also, it would appear t hat , despite the atrophy process occurring in fibers expressing these MHCs, there is a concurrent process occurring to expand the type Ilb MH C pool in some fibers, which appears to be contr olled TA in part by pretranslational processes leading to enhanced t ype Ilb MHC protein expression (Table 4, Fig. 3). F The findings reported herein at both the protein and mRNA level for the VI and red VL are str ikingly similar to t hose observed in a previous flight experiment involv­ FIG. 2. Slot blots of mRNA e xt racted from different muscles of F ing native myosin analysis (1) and to those observed in and C groups for R+O time point. Note greater signal for type IIb mRNA in VI and VL. RVL, red VL; WVL, white VL. ground-based studies using the model of hindlimb sus- Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. 2476 ZERO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN discrepancy in MHC mRNA and protein is that the type Ila MHC protein pool may be decreasing while that of D Protein the type Ilx MHC pool is increasing with an overall net increase occurring in the relative size of the type Ila-Ilx  mRNA 60 MHC protein band. To resolve this issue one must sepa­ rate the type Ila-Ilx MHC band into its type Ila and Ilx Gl 0) ,: components. In an attempt to resolve this issue, we are in .t::. "' the process of refining our electrophoretic techniques to improve separation of the type Ila-Ilx MHC band. In preliminary experiments we estimated in the CON VI -20 samples that 75 ± 3% of the type Ila-Ilx MHC band was -40 type Ila MHC and 25 ± 3% represented type Ilx MHC. In the FL samples, the type Ila MHV component accounted -60 for only 61 ± 3% compared with 39% for type Ilx MHC. -80 Thus, based on these distributions, it would indeed ap­ Type I Type IIA Type llb pear that there was both an absolute and relative reduc­ tion in the amount of type Ila MHC that was expressed in the VI of the FL animals, which is consistent with the mRNA data. We suspect that the same pattern occurred 120 in the red VL. In conclusion, the present study provides further evi­ dence that zero gravity exerts a significant impact on both the quantity and type of MHCs expressed in mus­ cles routinely used for both antigravity and locomotor activity. The findings further suggest that molecular <I> 0) events occurring at both the pretranslational and post­ .t::. translational levels of regulating MHC gene expression "' (.) are likely involved in these muscle adaptations to condi­ ;!. tions of unloading. Finally, it appears that future experi­ -20 ments involving exposure to zero gravity need to be con­ ducted for longer durations to obtain a better under­ -40 standing of the transformation in the steady-state -60 pattern of isomyosin expression in both locomotor and -80 antigravity skeletal muscle. Type I Type IIA Type llb The authors thank the research support team organized by the FIG. 4. Percent change relative to normal control level of expres­ NASA Ames Research Center for coordinating this research project. sion of MHC mRNA and protein in white VL (top) and TA (bottom). This research was supported by NASA Grant NAG-2-555. Normal control values were set at 100%. Data were plotted from Tables Address reprint requests to K. M. Baldwin. 3 and 4. Received 29 March 1993; accepted in final form 9 July 1993. pension to study MHC mRNA expression (5, 13). For REFERENCES example, Thomason et al. (13) reported a decrease in type I MHC synthesis in the soleus after 7 days of limb 1. BALDWIN, K. M., R. E. HERRICK, E. ILYINA-KAKUEVA, AND V. s. unweighting even though mRNA levels were not reduced. 0RGANOV. Effects of zero gravity on myofibril content and isomyo­ In the present study, we observed in the red VL, but not sin distribution in rodent skeletal muscle. FASEB J . 4: 79- 83, 1990. in the VI, that the relative reduction in type I MHC pro­ 2. BALDWIN, K. M., R. E. HERRICK, AND S. A. MCCUE. Substrate oxidation capacity in skeletal muscle: Effects of exposure to zero tein exceeded that of mRNA (Fig. 3), which further sug­ gravity. J. Appl. Physiol. 75: 2466-2470, 1993. gests that posttranslational events, possibly associated 3. CttOMCZYNSKI, P ., AND N. SACCHI. Single step method of RNA with protein degradation processes (12), are likely con­ isolation by acid guanidinium thiocyanate-phenol-chloroform ex• tributing to the reduction in net protein accumulation traction. Anal. Biochem. 162: 156-159, 1987. relative to the changes occurring in the mRNA pool. 4. DANIELLI-BETTO, D ., E. ZERBATO, AND R. BETTO. Type I, Ila, and IIb myosin heavy chain electrophoretic analysis of rat muscle Also, Diffee et al. (5) reported a differential pattern in fibers. Biochem. Biophys. Res. Commun. 138: 981-987, 1986. type Ila MHC mRNA expression compared with the rela­ 5 . DIFFEE, G. M., F . HADDAD, R. E. HERRICK, AND K. M. BALDWIN. tive expression of type Ila MHC protein in slow muscles Control of myosin heavy chain expression: interaction of hypothy­ of rats suspended for 14 days. In that study the type Ila roidism and hindlimb suspension. Am. J . Physiol. 261 (Cell Physiol. MHC mRNA signal was depressed in suspended rats, 30): C1099-Cll06, 1991. 6. GORNALL, A.G. , C. J. BARDAWILL, AND M. M. DAVID. Determina­ whereas the (supposed) relative content of type Ila MHC tion of serum protein by means of the biuret method. J. Biol. Chem. protein appeared to have been increased (5). This was 177: 751-756, 1949. the identical pattern seen for these isoforms in the VI 7 . GUSTAFSON, T. A., B. E. MARKHAM, AND E. MORKIN. Effects of from the present study (Fig. 3). thyroid hormone on alpha-actin and myosin heavy chain gene ex­ One possible explanation to account for this particular pression in cardiac and skeletal muscles of rat: measurement of Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved. ZRRO GRAVITY EFFECTS ON MYOSIN HEAVY CHAIN 2477 !ism and /3-myosin heavy-chain rnRNA in unweighted soleus mus­ mRNA content using synthetic oligonucleotide probes. Circ. Res. 59: 194-201, 1986. cle. Am. J. Physiol. 257 (Regulatory Integrative Comp. Physiol. 26): 8. JIANG, B., Y. 0HIRA, R. R. ROY, Q. NGUYEN, E. I. lLYINA-KA­ R300-R305, 1989. KUEVA, V. OGANOV, AND V. R. EDGERTON. Adaptation of fibers in 12. THOMASON, D. B., AND F. W. BOOTH. Atrophy of the soleus muscle fast-twitch muscles of rats to spaceflight and hindlimb suspenRion. by hindlirnb unweighting. J. Appl. Physio/. 68: 1-12, 1990. J. Appl. Physiol. 73, Suppl.: 588-658, 1992. 13. THOMASON, D. B., R. E. HERRICK, D. 8URDYKA, AND K. M. BALD­ WIN. Time course of soleus muscle myosin expression during hind­ 9. MAXAM, A. M., AND W. GILBERT. Sequencing end labelled DNA with base specific chemical cleavages. Metlwds Enzymol. 65: 499- limb suspension and recovery. J. Appl. Physiol. 63: 130-137, 1987. 560, 1980. 14. TSIKA, R. w., R. E. HERRICK, AND K. M . BALDWIN. Time-course 10. 0HIRA, Y., B. JIANG, R.R. ROY, V. 0GANOV, E. ILYINA-KAKUEVA, adaptations in rat skeletal muscle isomyosins during compensa­ J. F. MARINI, AND V. R. EDGERTON. Rat soleus muscle fiber re­ tory growth and recovery. J. Appl. Physiol. 63: 2111-2121, 1987. sponses to 14 days of spaceflight and hindlirnb suspension. J. Appl. 15. TS!KA, R. w., R. E. HERRICK, AND K. M. BALDWIN. Effect of ana­ bolic steroids on overloaded and overloaded suspended skeletal Physiot. 73, Suppl.: 51S-57S, 1992. muscle. J. Appl. Physiol. 63: 3128-2133, 1987. 11. THOMASON, D. B., R. B. BIGGS, AND F. W. BOOTH. Protein metabo- Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (128.200.102.124) on March 15, 2018. Copyright © 1993 American Physiological Society. All rights reserved.

Journal

Journal of Applied Physiology – Unpaywall

Published: Dec 1, 1993

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