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Am J Physiol Cell Physiol 293: C723-C728, 2007. First published May 9, 2007; doi:10.1152/ajpcell.00009.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

The roles of myosin ATPase activity and myosin light chain relative content in the slowing of type IIB fibers with hindlimb unweighting in rats

Sheng Zhong and LaDora V. Thompson

Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, Minnesota

Submitted 5 January 2007 ; accepted in final form 4 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that slowing of shortening velocity generated by type IIB fibers from hindlimb-unweighted (HU) rats resulted from a reduced ATPase activity and/or a reduction in the relative content of myosin light chain 3f isoform content (MLC3f). After 2, 3, and 4 wk of HU, maximal unloaded shortening velocity (Vo) of single permeabilized semimembranosus muscle fibers was determined by the slack test. Subsequently, the myosin heavy chain and the relative content of MLC were determined by SDS-PAGE. The ratio of MLC3f to MLC2f was determined by densitometric analysis. In addition, myofibrils were prepared from permeabilized fibers (soleus and semimembranosus muscles) and assayed for resting myosin ATPase and Ca2+-activated myosin ATPase. After HU, Vo declined by 28–40% and the MLC3f/MLC2f ratio decreased by 32 to 48%. A significant correlation between the relative amount of MLC3f and Vo was found (r = 0.48, P < 0.05). Resting myosin ATPase rates were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.86). Ca2+-activated myosin ATPase activities also were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.13). These data suggest that the slowing of maximal unloaded shortening velocity in type IIB fibers with HU is, at least in part, due to a relative change in the essential light chain composition, a decrease in the relative amount of MLC3f and most likely a concomitant increase in MLC1f. However, this reduction in Vo is independent of myosin ATPase activity.

unloading shortening velocity; myosin light chain 3f

SKELETAL MUSCLE PERFORMANCE is significantly altered following periods of muscle unloading or non-weight-bearing conditions (27, 30, 31). For instance, at the individual skeletal muscle fiber level, cross-sectional area, peak force, and specific force (peak force normalized to cross-sectional area) are decreased after muscle unloading (30, 31). The maximal unloaded shortening velocity is elevated in fibers with myosin heavy chain type I isoform (MHC) and decreased in fibers with MHC type IIB after muscle unloading (16, 17, 30, 31, 36). However, the unloading-induced changes in fibers with MHC type IIA are not consistent (7, 34, 35).

Unloaded shortening velocity (Vo) is directly related to, and dependent on, the specific activity of myosin ATPase (3). ATPase is predominantly determined by the MHC isoform because the MHC contains the catalytic site for ATPase activity (23). Therefore, the muscle unloading-induced changes in velocity may be related to changes in ATPase activity (6). Indeed, the unloading-induced increase in velocity is correlated with an elevation in ATPase activity in MHC type I fibers (17). Currently, the relationship between changes in ATPase and velocity in MHC type IIB fibers following unloading is unknown.

The alkali myosin light chains (MLC; essential) play a role in determining unloaded shortening velocity in fibers with MHC type II isoforms. Sweeney et al. (25) and Bottinelli et al. (4) report that velocity is elevated in type II fibers that contain larger relative amounts of MLC 3f isoform (MLC3f). Moreover, velocity is proportional to the relative content of MLC3f in a group of single fast fibers (MHC type II) (4, 11). These results suggest that the proportion of MLC3f is an important determinant of unloaded shortening velocity among fast fibers. Thus it is possible that the decline in shortening velocity in MHC type IIB fibers following unloading is due to changes in the relative content of MLC3f. To date, the effect of unloading on the relative content of MLC3f in skeletal muscle fibers that contain MHC type IIB has not been thoroughly investigated.

Consequently, the purpose of this study is to define the roles of myosin ATPase activity and the relative content of MLC3f in the muscle unloading-induced decline in shortening velocity in MHC type IIB and MHC type IIB-IIX fibers. We hypothesize that the decrease in velocity following muscle unloading is related to reduced ATPase activity and/or a reduction in the relative content of MLC3f. To study the effect of unloading on contractile properties the single-permeabilized fiber preparation is used. This cellular preparation allows investigation of contractile protein function in a cell with an intact filament lattice. Next, a sensitive SDS-PAGE is used to separate and quantify the relative content of the MLC isoforms and determine the MHC isoforms.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animals, unloading protocol, and tissue preparation. An animal care protocol was approved by the Institutional Animal Care and Use Committee and was in accordance with guidelines established by the American Physiological Society. Twenty-four Fisher 344 male rats were randomly assigned to a control (weight bearing) group or an unloading (non weight bearing) group (6 rats/group). The non-weight-bearing rats were suspended for either 2, 3, or 4 wk by a tail harness attached to the proximal two-thirds of the tail (1, 9, 22, 30). All rats were obtained from the colony maintained by the Minneapolis Veterans Administration. After 2, 3, or 4 wk of non-weight-bearing or normal cage activity for the control group, rats were anesthetized with pentobarbital sodium (55 mg/kg ip) and weighed. Next, the semimembranosus muscles were dissected and weighed. The semimembranous muscle was selected because it is predominantly composed of MHC type IIB fibers and MHC type IIB-IIX fibers and shows hindlimb unweighting-induced changes (36). Although the study focused on the semimembranosus muscle, the soleus muscles from control rats, composed predominantly of MHC type I fibers, were dissected and weighed, too. The soleus and semimembranosus muscles were used in the ATPase experiments to demonstrate the difference in ATPase activities between muscles composed of different fiber types in non-weight-bearing rats.

Permeabilized single fiber preparation. The semimembranosus muscles were placed in relaxing buffer composed of (in mM) 7 EGTA, 0.016 CaCl2, 5.6 MgCl2, 80 KCl, 20 imidazole, pH 7.0, 14.5 creatine phosphate, and 4.8 ATP on ice. Bundles of fibers ~8 mm long and ~1 mm in diameter were formed for single fiber contractile analyses. These bundles were tied to pieces of capillary tubes and stored in 50% glycerol: 50% relaxing buffer for up to 4 wk at –20°C (29).

Fiber bundle preparation for ATPase experiments. Additional bundles of fibers (from the semimembranosus muscles and soleus muscles), ~25 mm long and ~2 mm in diameter, were permeabilized and stored at –20°C in fresh 50% glycerol: 50% relaxing buffer composed of (in mM) 60 KPr, 25 MOPS, 2 MgCl2, 1 EGTA, and 1 NaN3, containing 0.1 DTT for ATPase analyses.

ATPase analyses. Glycerinated muscle bundles (from the semimembranosus muscles and soleus muscles) were homogenized in 20 mM Tris·HCl and protein concentration was determined using spectrophotometrical protein assay. BSA was used as protein standard. In general, myosin ATPase activity of myofibrils was determined spectrophotometrically at 25°C by measuring the release of phosphate resulting from the hydrolysis of ATP (10, 12). Myosin ATPase activities were measured under resting condition (i.e., low Ca2+ buffer, which contained 248 mM K-propionate, 50 mM MOPS, pH 7.0, 4 mM MgCl2, and 2 mM EGTA) and Ca2+-activated conditions (i.e., high Ca2+ buffer, which contained 242 mM K-propionate, 50 mM MOPS, pH 7.0, 4 mM MgCl2 and 3 mM CaCl2). Reactions were started by the addition of 4 mM ATP and stopped by 34% citrate. The concentration of phosphate was determined by comparing the sample absorbance at 660 nm with the absorbance of phosphate standards. Specifically, the ATPase enzyme activity is activated with the addition of ATP, and at five defined time intervals (1, 2, 3, 4, and 5 min), 50 µl of the reaction solution is removed. Following removal of the 50-µl solution, the ATPase reaction of each specific time interval is stopped with citrate (15-min incubation). The phosphate content of each specific time interval is then determined spectrophotometrically by comparing it to phosphate standards. To determine specific activity, the data from the five time points along with an experimental blank are regressed (umoles phosphate against time) and normalized to protein content.

Single permeabilized fiber contractile properties analyses. On the day of the experiment, individual fiber segments (~2 mm long) from permeabilized bundles were randomly isolated and studied at 15°C as described in detail previously (28, 29). Fiber segments were mounted in relaxing buffer, the segment length was adjusted a sarcomere spacing of 2.5 µm. Maximal Vo was determined using the slack test methods. In brief, the fiber was maximally activated (7 mM EGTA, 7 mM CaCl2, 5.6 mM MgCl2, 80 mM KCl, 20 mM imidazole, pH 7.0, 14.5 mM creatine phosphate, 4.8 mM ATP) and then rapidly shortened (slacked) by 7–18% of fiber length such that force dropped to zero. The time between zero force and force redevelopment was measured by computer analysis. This procedure was done 4 to 5 times at different slack distances. The slack distances were then regressed against the corresponding times of force redevelopment and the slope of that line (mm/s) is reported as Vo after normalizing to fiber length (fl/s). Six fibers from each animal were studied and their values averaged to represent fiber contractility for that rat.

MHC analysis for ATPase experiments. After ATPase determination, a portion of the glycerinated muscle bundle homogenate was solubilized in reducing buffer. Subsequently, MHC composition of the homogenates was determined by SDS-PAGE using a 4% (wt/vol) stacking gel, an 8% separating gel, followed by silver staining (12, 13). Stained gels were imaged using a Fluor-S MultiImaging System (Bio-Rad) and the relative expression of the MHC isoforms (type I, type IIB, type IIX/A) were determined from the optical density (densitometric analysis) using image analysis software as previously described (SigmaScan, Jandel Scientific) (12, 13).

MHC and MLC analysis for single skeletal fiber experiments. After single fiber velocity measurements, the fiber was solubilized in 50 µl of sample buffer (24 mM EDTA, 60 mM Tris, pH 6.8, 1% SDS, 5% beta-mercaptoethanol, 15% glycerol, 2 mg/ml bromophenol blue) and stored at –80°C (12, 30). In general, MHC isoform expression and MLC compositions of each fiber were determined by gel electrophoresis and silver staining. MHC and MLC compositions were determined by SDS-PAGE with the use of a 4% (wt/vol) stacking gel and either an 8% separating gel for MHC or 12% separating gel for the MLC. Stained gels were imaged using a Fluor-S MultiImaging System (Bio-Rad) (12). The relative expression of the essential MLC isoforms (MLC1f, MLC2f, MLC3f) were determined from the optical density (densitometric analysis) using image analysis software as previously described (SigmaScan, Jandel Scientific) (12). Specifically, the optical density of MLC3f was compared with the optical density of MLC2f (ratio of MLC3f /MLC2f). The ratio of MLC3f/MLC2f is a good index of the ratio between the two essential light chains in MHC type II muscle fibers (4). Although the ratio of MLC3f/MLC2f is preferable, we also determined the MLC3f/(MLC1f + MLC3f) ratio and the MLC1f/(MLC1f + MLC3f) ratio. The MLC3f/MLC2f ratios, MLC3f/(MLC1f + MLC3f) ratios, and MLC1f/(MLC1f + MLC3f) ratios of 90 fibers of known Vo value were successfully determined.

Because of the nature of determining MHC isoform and MLC isoforms, it was necessary to repeat SDS-PAGE analysis for clarity of the individual fiber samples. Sometimes the protein expression of the individual fibers on the gels was not clear enough to give reliable measurements of the MLC3f/MLC2f ratio, the MLC3f/(MLC1f + MLC3f) ratio, and MLC1f/(MLC1f + MLC3f) ratio along with MHC isoform clarity. Thus ~50% of the fast fibers of known Vo and containing the fast MHC could be characterized for MLC content with confidence. Figure 1 shows an example of two individual fibers, the unloaded shortening velocity determined by the slack test, the MLC region of the SDS-PAGE and analysis of MLC3f/MLC2f ratio.


Figure 1
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  		Fig. 1. Representative results of slack tests from two semimembranosus fibers each containing myosin heavy chain (MHC) type II isoform and their respective SDS-PAGE of myosin light chain (MLC) isoforms. The duration of unloaded shortening (milliseconds) after several imposed slacks is plotted vs. slack distances (µm) for a single semimembranosus fiber from a control rat ({blacksquare}), and a single semimembranosus fiber from a hindlimb unweighted rat ({blacktriangleup}). Unloaded shortening velocity (Vo) was determined from the slope of the fitted line and normalized by fiber length (9.95 and 3.54 fl/s). Subsequently, the MLC isoforms of the two fibers were separated on 12% SDS-PAGE and stained with silver (insets). The ratio of the relative content of MLC3f isoform to MLC2f isoform (MLC3f/MLC2f) was calculated by determining the relative content of each isoform densitometrically. The arrows identify the three MLC isoforms. The fiber with a fast shortening velocity (9.95 fl/s) had an MLC3f/MLC2f ratio of 0.89, whereas the fiber with a slower Vo (3.54 fl/s) had a MLC3f/MLC2f ratio of 0.26.

 
In summary, in this study, we are reporting ATPase activities along with MHC compositions of the glycerinated bundle homogenate (whole muscle) and individual skeletal fiber shortening velocities along with MHC and essential MLC composition (single fiber).

Statistical analyses. Values in the results are means ± SE. A one-way ANOVA was used to evaluate ATPase activity and MHC composition in glycerinated muscle bundles, and single fiber Vo, MLC3f/MLC2f ratio, MLC3f/(MLC1f + MLC3f) ratio, and MLC1f/(MLC1f + MLC3f) ratio. The Dunnett post hoc test was used to estimate the differences among means. Differences were considered significant when P < 0.05. Student's t-tests were used to compare the ATPase activity between the soleus and semimembranosus muscles from control rats, P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of unloading on muscle mass. Significant muscle atrophy of the semimembranosus muscle was noted following unloading. Two, three, and four weeks of unloading resulted in a 35, 32, and 40% decline, respectively, in the semimembranosus muscle wet weight. However, when semimembranosus muscle mass was normalized to body mass, no significant effect of unweighting was detected.

Myosin ATPase activity and MHC composition in glycerinated fiber bundles. Resting myosin ATPase rates were not different between myofibrils prepared from corresponding muscles of control and hindlimb unloading rats (P = 0.86; Fig. 2A). Ca2+-activated myosin ATPase activities also were not different between myofibrils prepared from corresponding muscles of control and hindlimb unloading rats (P = 0.13; Fig. 2A). In Fig. 2B, resting myosin ATPases rate for soleus samples (muscles composed of predominately MHC type I fibers) was 0.131 and increased to 0.302 µmol Pi·min–1·mg protein–1 with Ca2+ activation. In contrast, resting myosin ATPases rate for semimembranosus samples (muscles composed of predominately MHC type II fibers) was 0.110 and increased to 0.630 µmol Pi·min–1·mg protein–1 with Ca2+ activation. The Ca2+-activated myosin ATPase of the semimembranosus samples is significantly greater than the Ca2+-activated myosin ATPase of the soleus samples, demonstrating activity differences between muscles of different fiber types. The MHC composition of the semimembranosus muscle (bundles) was predominantly fast MHC isoforms, 56 ± 2% type IIB, 35 ± 2% type IIX/IIA, and 11 ± 2% type I. The MHC fiber type composition of the semimembranosus muscle bundles did not change with unloading.


Figure 2
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  		Fig. 2. Resting myosin ATPase activity (i.e., low Ca2+ condition) and Ca2+-activated myosin ATPase activity of freely shortening myofibrils. A: ATPase activity values (means ± SE) of the semimembranosus muscles from rats that were cage control or hindlimb unloaded (HU) for 2 wk (HU2), 3 wk (HU3), and 4 wk (HU4). B: ATPase activity values (means ± SE) of the soleus muscles (composed of MHC type I fibers) compared with the semimembranosus muscles (composed of MHC type II fibers) from control rats. *Significantly different from soleus muscles.

 
Unloading-induced alterations in individual MHC type II fibers. The Vo of type II fibers decreased by 28% with 2 wk of unloading, 40% with 3 wk of unloading, and 36% with 4 wk of unloading (Table 1). The decrease in Vo with unloading is also observed when the individual fibers are analyzed by specific fiber type group, fibers expressing only MHC type IIB (Table 2) or fibers co-expressing MHC type IIB-IIX only (Table 3).


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  		Table 1. Vo and ratio of MLC3f/MLC2f, MLC3f/(MLC1f + MLC3f), and MLC1f/(MLC1f + MLC3f) of fast type II fibers (MHC type IIB, MHC type IIB-IIX) from SM of control and HU animals

 

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  		Table 2. Vo and ratio of MLC3f/MLC2f and MLC3f/(MLC1f + MLC3f) of fast MHC type IIB fibers from SM of control and HU animals

 

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  		Table 3. Vo and ratio of MLC3f/MLC2f and MLC3f/(MLC1f + MLC3f) of fast MHC type IIB-IIX fibers from SM of control and HU animals

 
Effect of unloading on Vo and MLC content of individual fibers expressing MHC type IIB and coexpressing type IIB-IIX fibers. To evaluate the role of MLC and Vo, we focused our analysis on the individual fibers expressing MHC type IIB and MHC type IIB-IIX (grouped together). Table 1 shows the analysis of MLC compositions in the same fibers that were analyzed for unloaded shortening velocity [changes of Vo and the ratios of MLC3f/MLC2f, MLC3f/(MLC1f + MLC3f), and MLC1f/(MLC1f + MLC3f)]. Two, three, and four weeks of unloading resulted in single fiber Vo decreasing from control values (Table 1). The ratio of the MLC3f and MLC2f of single fibers decreased by 37, 32, and 48% with unloading, respectively (Table 1). The ratio of the MLC3f/(MLC1f + MLC3f) of the same single fibers followed the same hindlimb unweighting-induced pattern (decrease). Moreover, there was an increase in the ratio of MLC1f/(MLC1f + MLC3f) (Table 1).

In contrast to Table 1, Tables 2 and 3 summarize Vo and MLC isoform compositions by specific fiber type (fibers with only MHC type IIB or hybrid fibers that express both MHC type IIB/IIX). The hindlimb unweighting-induced decreases in Vo and in the ratios MLC3f/MLC2f and MLC3f/(MLC1f + MLC3f) are observed when the fibers are grouped by specific fiber type.

Relationship between Vo and MLC composition. Figure 3 shows the relationship between the MLC3f/MLC2f ratio and Vo for each individual fiber containing MHC type IIB and MHC type IIB-IIX. A significant correlation between the relative amount of MLC3f and Vo was found. The slope of the linear regression was 5.18 for all fibers (r = 0.48, P ≤ 0.05). In other words, as the relative amount of MLC3f decreased among these fibers, so did Vo. Moreover, the relationship between MLC3f/MLC2f ratio and Vo is significant when the data was analyzed by specific fiber type. The slope of the linear regression was 5.43 for the MHC type IIB fibers (r = 0.52, P ≤ 0.05) and 5.11 for the MHC type IIB-IIX fibers (r = 0.50, P ≤ 0.05).


Figure 3
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  		Fig. 3. Relationship between Vo (fl/s) and the proportion of the MLC3f isoform to the MLC2f in single fibers, MLC3f/MLC2f. The Vo was determined by the slack test, followed by MLC3f and MLC2f isoforms ratio determination by SDS-PAGE separation, silver staining, and densitometric analysis for MLC3f/MLC2f ratio. Single fibers from rats that were cage control or hindlimb unloaded for 2 wk (HU2), 3 wk (HU3), and 4 wk (HU4). The regression line is 5.18x + 3.89, R2 = 0.27, r = 0.48, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of the present study was to compare individual skeletal muscle fibers that contain identical MHC but express different ratios of myosin light chain (MLC3f). Thus addressing the question of whether or not alterations in MLC3f relative content can explain the non-weight-bearing-induced decline in contractile velocity. The hindlimb unweighting model was used to promote the non-weight-bearing condition, which alters muscle contractility (30, 31). The strength of the current study was using the same skeletal muscle fiber for velocity measurements and subsequent MHC and MLC analyses, which permitted an unambiguous analysis of the correlation between contractile function and relative MLC3f isoform content.

The primary results of the present study are (1) muscle unloading induces a decline in maximal unloaded shortening velocities among individual rat semimembranosus fibers (MHC type IIB and MHC type IIB-IIX), (2) variations in the MLC3f relative content of single fibers from rat semimembranosus were quantitatively correlated with Vo, suggesting the non-weight-bearing-induced decline in Vo may be, in part, due to a loss in MLC3f relative content (and a subsequent increase in MLC1f relative content).

Effects of unloading on ATPase. The shortening velocity of an individual muscle is thought to be primarily related to the ATPase activity of myosin within the muscle. Myosin ATPase activity is, in turn, related to distribution of MHC isoforms expressed in the fiber. For example, fast-twitch muscles (MHC type II) have faster ATPase activity compared with slow-twitch muscle (MHC type I) (26). So a logical explanation for the decrease of Vo in the semimembranosus fibers with unloading would be slower myosin ATPase activity in samples from non-weight-bearing rats. However, our Ca2+-activated myosin ATPase measurements on myofibrils from the semimembransosus muscle do not substantiate this explanation. Although there is a significant difference in the ATPase activity between slow-twitch and fast-twitch muscles, fourfold (33), the difference between the fast myosins (MHC IIA, IIX, IIB) is not as great (33). Thus, it is not surprising we did not detect a change in ATPase activity in the semimembranosus muscles following unloading, since the fiber-type composition of the muscle is predominantly MHC type IIB and MHC type IIB-IIX. Furthermore, this general fiber type composition of the muscle does not change with hindlimb unweighting.

In a previous study, we showed a shift in MHC isoform expression in individual fibers with 3 wk of unloading, such that the percentage of fibers that coexpressed MHC type IIB/IIX in non-weight-bearing rats was twofold greater than that in control rats, yet this shift did not explain the decrement in contractile velocity (36). It is important to point out, in the current study, the analysis by specific fiber type (MHC type IIB, MHC type IIB-IIX) and analysis by pooled fiber types (MHC type IIB and MHC type IIB-IIX) report the same conclusions. Therefore we conclude that other factors, such as changes in the essential MLC composition, must contribute to unloading-related reductions in contractile velocities.

Vo and MLCs. Although the MHC isoform composition of an individual muscle fiber is the primary determinant of its maximal shortening velocity, the MLC isoform complement has a modulatory influence on regulating this property, especially in fast fibers. Variations in the essential MLC isoform relative composition of single fibers, whether naturally occurring (8, 24), in transgenic animals (5, 18), or in experiments involving myofilament protein substitutions (18), are associated with differences in fiber contractile properties. The results of an in vitro motility assay utilizing myosin reconstituted with different MLC subunits (14, 15) further contribute to this theory. Collectively, these studies show that the unloaded velocity of shortening in fast fibers increases with greater relative levels of MLC3f.

In the present study, the decrease in MLC3f relative content may be the molecular mechanism for the decreases in contractile velocities with unloading because Vo was proportional to MLC3f relative content in fast fibers of same MHC composition. A change of 32–48% in MLC3f/MLC2f [25–38% in MLC3f/(MLC1f + MLC3f)] or MLC3f proportion induces a change in Vo of 28–40%. The slope of the relationship of Vo to MLC3f/MLC2f ratio (5:18) is similar to a previous study investigating MLC3f/MLC2f ratio and contractile velocities (8).

Muscle contraction and the role of MLCs. Muscle contraction is due to cyclic interactions of myosin and actin. Myosin is composed of two MHCs and two pairs of MLCs. MHCs are described to have a rod part that forms the backbone whereas the myosin heads form the globular ends. An {alpha}-helix (8.5 nm long) connects the globular end with the rod part of each MHC. One "alkali" or "essential" MLC and one "regulatory" MLC wrap around this helix, and form the myosin neck (20). The myosin neck is thought to act as a lever arm, which amplifies small conformational changes occurring in the catalytic domain of the MHC during ATP hydrolysis cycles into larger movements (20). The MLCs appear to be necessary for increasing the rigidity of the lever arm while transmitting force during the myosin head power stroke.

It is possible that any modulatory role of MLC may vary with its size, as well as with differences in primary sequence, which may, in turn, contribute to differences in contractile properties of fast fibers. For instance, a higher proportion of MLC1f is associated with slower unloaded shortening velocity (8, 25). This slowing of velocity by MLC1f is likely due to interaction of its NH2-terminal extension with actin (2, 8, 14, 25, 32). MLC1f appears to interact directly with actin through a ~40 amino acid NH2-terminal extension that is lacking in MLC3f (32). This extension is rich in proline and has four lysines at the distal end. The size and the positively charged residues enable this extension to interact with the negatively charged COOH-terminus of actin (2). It appears that the binding of the NH2-terminal extension of MLC1f to actin slows the speed of filament sliding but not the cross-bridge kinetics underlying the force transients and the rate of the whole cross-bridge cycle (ATPase activity) (19, 21). These contractile characteristics can be explained by assuming that MLC1f binding to actin reduces the distance of filament displacement during one cross-bridge cycle, but does not affect the kinetics. The results of the present study are consistent with this theory that a decrease in velocity can occur without a change in ATPase (kinetics). Furthermore, a decrease in the relative content of MLC3f would result in an increase in the relative content of MLC1f. Although this study reports a decrease in the relative content of MLC3f, most likely there is a concomitant increase in the relative content of MLC1f content, which is observed in the increase in the ratio MLC1f/(MLC1f + MLC3f). The change in essential MLC isoforms (from MLC3f to MLC1f) is based on the assumption that one alkali light chain and one regulatory light chain wrap around the {alpha}-helix of the myosin molecule described in the proceeding paragraph.

Summary. In conclusion, the non-weight-bearing-induced changes in Vo, in the ratio of MLC3f/MLC2f and in the ratio of MLC3f/(MLC1f + MLC3f) of single semimembranous fibers have been characterized. Non-weight-bearing changes induced a decline in fiber Vo that was likely caused, at least in part, by a decrease in the relative MLC3f content (and subsequent increase in MLC1f content). However, this decline in Vo is independent of ATPase activity. Future studies are needed to identify the cellular processes responsible for the preferential decline in MLC3f content, such as changes in gene expression and upregulation of proteins involved in degradation processes.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute on Aging Grants AG-17768 and AG-21626 (to L. V. Thompson).


   ACKNOWLEDGMENTS
 
We thank Janice Shoeman for valuable technical skills.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. V. Thompson, Dept. of Physical Medicine and Rehabilitation, Univ. of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455 (e-mail: thomp067{at}umn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
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