Kinetic properties of myosin heavy chain isoforms in mouse skeletal muscle: comparison with rat, rabbit, and human and correlation with amino acid sequence

Oleg Andruchov, Olena Andruchova, Yishu Wang, and Stefan Galler

Department of Cell Biology, University of Salzburg, A-5020 Salzburg, Austria

Submitted 25 May 2004 ; accepted in final form 7 August 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Stretch activation kinetics were investigated in skinned mouse skeletal muscle fibers of known myosin heavy chain (MHC) isoform content to assess kinetic properties of different myosin heads while generating force. The time to peak of stretch-induced delayed force increase (t3) was strongly correlated with MHC isoforms [t3 given in ms for fiber types containing specified isoforms; means ± SD with n in parentheses: MHCI 680 ± 108 (13), MHCIIa 110.5 ± 10.7 (23), MHCIIx(d) 46.2 ± 5.2 (20), MHCIIb 23.5 ± 3.3 (76)]. This strong correlation suggests different kinetics of force generation of different MHC isoforms in the following order:MHCIIb > MHCIIx(d) > MHCIIa >> MHCI. For rat, rabbit, and human skeletal muscles the same type of correlation was found previously. The kinetics decreases slightly with increasing body mass. Available amino acid sequences were aligned to quantify the structural variability of MHC isoforms of different animal species. The variation in t3 showed a correlation with the structural variability of specific actin-binding loops (so-called loop 2 and loop 3) of myosin heads (r = 0.74). This suggests that alterations of amino acids in these loops contribute to the different kinetics of myosin heads of various MHC isoforms.

isoform structure-function relationship; stretch activation; muscle mechanics


SKELETAL MUSCLES ARE COMPOSED of different fiber types to fulfill various functional needs. This diversity relates to the existence of specific myofibrillar protein isoforms in different fiber types (for review, see Refs. 41 and 46). Muscle fiber types are generally categorized according to their specific myosin heavy chain (MHC) isoforms. The head portion of this protein is the essential component of the force-generating mechanism of muscle (for review, see Ref. 30). Three fast fiber types (type II) have been identified in limb muscles of adult rodents: types IIB, IIX (or IID), and IIA, containing the isoforms MHCIIb, MHCIIx(d), and MHCIIa, respectively. Conversely, only one slow fiber type (type I) has to date been characterized by its MHC isoform, called MHCI, but there is increasing evidence that the type I fibers do not represent a homogeneous population (2, 11, 15).

MHC isoform content determines the contractile properties of muscle fibers (for review see Ref. 4). The relation between MHC content, stretch activation kinetics, and unloaded shortening velocity (Vu) has been clearly established for rat, rabbit, and human skeletal muscle fibers (4, 7, 9, 17, 35). Although the mouse is the most important mammalian model for genetic manipulations, the skeletal muscle fibers of this species have been investigated to a much lesser degree. The relationship between shortening velocity and load has been determined in different mouse muscle fiber types. Furthermore, Vu was compared with the actin filament sliding propelled by the different myosin isoforms (28, 39). The kinetics of stretch activation has never been investigated in mouse skeletal muscle fibers, although a strong correlation of this parameter with the MHC isoforms has been found in other species (3, 12, 16, 17, 19, 24, 26, 33). This correlation most likely points to different kinetic properties of myosin heads of various MHC isoforms while generating force (1, 31, 34). Therefore, in the present study stretch activation experiments have been performed on mouse skeletal muscle fibers with known MHC isoform content. To investigate any dependence of stretch activation kinetics on body mass, we compared the results in mice with those of corresponding rat, rabbit, and human fiber types.

Comparisons between different animal species were also used to establish structure-function relationships on the molecular level. We tried to answer the question of which specific structural alterations of the myosin molecule are responsible for the functional differences of mammalian MHC isoforms. So far this problem has been addressed particularly in other animal phyla. In particular, two flexible loops of the myosin head were discussed as possible candidates for modulation of kinetic activities of myosin (22, 37): loop 1, which is the connection of the 25-kDa and 50-kDa domains of the myosin head, and loop 2, which is the connection between the 50-kDa and 20-kDa domains (20). Comparisons of MHC isoforms of striated and smooth muscles of two molluscan species (Argopecten, Placopecten) suggested that the differences in their ATPase activity are likely due to sequence variation of loop 1 (40). Further information about the influence of various myosin head regions on differences in function were provided by studies on chimeric MHC isoforms. In a study of Uyeda et al. (52), loop 2 of Dictyostelium was genetically modified by substitution of nine amino acids with those of myosins of other animal species. Significant changes of ATPase activity after the substitutions in loop 2 were observed. The ATPase activity of the chimeric myosins correlated well with that of myosin from which the substitution was derived. Similarly, in a study of Sweeney et al. (50), loop 1 of different myosin II isoforms was inserted into a smooth myosin backbone. Here a variation of functional properties depending on the substitutions of loop 1 was found. However, the properties of chimeric myosins did not correlate with those of myosins from which the loop was derived. In Drosophila, dramatic changes of kinetic properties were observed after genetic manipulations of the converter region (48).

In the present study, we correlated alterations of the amino acid sequence of the MHC isoforms with the stretch activation kinetics of corresponding fiber types. The correlations were carried out both within and between different animal species. Either whole amino acid sequences or fragments of amino acid sequences of various MHC isoforms were available. In a previous study (39), a similar approach was applied for identification of the structural correlate underlying the differences in filament sliding of mammalian muscle fiber types.


    MATERIALS AND METHODS
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Muscle preparations. Adult C3H mice (~32 wk old) were used in this study. Mice were anesthetized with pentobarbital sodium (55 mg/kg ip) and exsanguinated. The tibialis anterior, gastrocnemius, vastus lateralis, and soleus muscles were quickly excised and split into tissue strips of ~1- to 2-mm diameter.

Solutions. The following solutions (0–5°C; Ref. 53) were used for sequential incubations: 1) skinning solution (in mM): 132 sodium propionate, 5 EGTA, 7 Na2H2ATP, 2 MgCl2, 10 MOPS, 1 dithioerythritol (DTE) adjusted to pH 6.9 with KOH; 2) skinning solution in which sodium propionate was replaced by potassium propionate; 3) solution 2 with 10% (vol/vol) glycerol; 4) solution 2 with 25% (vol/vol) glycerol; and 5) relaxation solution (for composition see Mechanical measurements; pH 6.9) with 50% (vol/vol) glycerol. The preparations were stored in this solution at about –25°C for up to 2 mo. Before the experiments, single fibers were dissected in this solution (around –5°C) and glued to the tips of the two needles of the apparatus.

Mechanical measurements. The experimental apparatus and the method for mechanical measurements have been described previously (14). The attachment points for the muscle fibers (active length ~0.8–2.5 mm, mean diameter 25–55 µm) on the mechanical apparatus were two ~2-mm-long vertically oriented epoxy carbon fiber needles of ~100-µm tip diameter. The needles were connected to the apparatus by silicon plates from force transducer elements (AE 801; SensoNor). One element, the force sensor (resonance frequency ~7.5 kHz), was connected mechanically to a micrometer screw and electrically to a force bridge amplifier. The other element was glued on the lever arm of a stepping motor. Rapid changes of the fiber length (≤1 ms) were achieved by a feedback-controlled stepping motor based on a Ling vibrator. The ability to make rapid solution changes was provided by a cuvette transporting system. Laser diffractometry (He-Ne laser; 632.8 nm, 4 mW) was used for measuring the sarcomere length.

After attachment of the skinned fiber with the tissue glue Vetseal (Braun Melsungen) the fiber ends were fixed by superfusion with a fine, rapidly flowing, downward-directed stream of stained glutaraldehyde solutions (8% glutaraldehyde, 5% toluidine blue, fixative) for 2–3 s. For this purpose the fiber was bathed in a low-ionic-strength rigor solution (in mM: 10 imidazole, 2.5 EGTA, and 7.5 EDTA, 134 potassium propionate, pH 6.9) with a lower specific mass than the fixative. This procedure created a sharp boundary between the functional part of the fiber and the fixed ends. This method considerably improves the maintenance of sarcomere homogeneity and the stability of mechanical properties during prolonged activations (25).

The solutions used during the mechanical measurements had an ionic strength of 0.25 M and contained (in mM) 60 HEPES, 8 Na2H2ATP, 10 sodium creatine phosphate, 10 NaN3, 1 DTE, 40 g/l dextran T-500, 30 U/ml creatine phosphokinase, and 1 mM free Mg2+. The dextran was included in the solutions to prohibit swelling of the skinned muscle fibers (21). In addition, the relaxation solution [pCa > 9, pCa = –log free Ca2+ concentration ([Ca2+])] contained 50 mM EGTA. The maximal activation solution (pCa 4.5) contained 50 mM Ca-EGTA, and the preactivation solution (low Ca2+-buffering capacity, pCa 7) contained 50 mM hexamethylenediamine-N,N,N',N'-tetraacetate (HDTA). pH was adjusted to 7.10 with KOH at 22°C. Free [Ca2+] and free [Mg2+] of the solutions were determined with ion-selective electrodes (13).

Before the experiment, the length of the fibers was adjusted to exactly the slack position (resting length) in relaxation solution, and both fiber dimensions and sarcomere length were recorded. The measurements were carried out at resting length to make them comparable with previous work on other species. Fiber cross-sectional area was calculated, using the largest and the smallest diameters and assuming an elliptical shape. After transfer of the fiber to the preactivating solution (~1 min) and subsequently to the maximal activating solution, a steady force was reached. The maximal isometric tension was determined by dividing this force by the cross-sectional area. A number of quick (≤1 ms) stretches of 0.1–0.2% of fiber length, lasting 1–4 s, were applied to induce force transients. The interval between individual stretches was 0.5–1 min. All experiments were carried out at 22°C. Results are expressed as means ± SD and were analyzed with the two-tailed Student's t-test. Data that did not exhibit a Gaussian distribution were tested with ANOVA.

Biochemical analysis. After completion of the mechanical measurements, the muscle preparations were removed from the apparatus for biochemical analysis, which was described previously (19). The muscle fragments were dissolved in SDS lysis buffer [62 mM Tris·HCl, pH 6.8, 10% (vol/vol) glycerol, 5% (wt/vol) SDS, 5% (vol/vol) 2-mercaptoethanol] and heated at 65°C for 15 min. Subsequently, a part of this extract (~0.1 µg protein) was applied to a SDS polyacrylamide gel system (4% polyacrylamide in stacking gel and 8% in separation gel). For electrophoresis (19), a SE 600 chamber (Hoefer, San Francisco, CA) with 16-cm-long glass plates was used. A constant voltage of 180 V was applied for 28 h. After electrophoresis, gels were silver stained and the relative amount of MHC isoforms was estimated using densitometry.

Comparison of amino acid sequence. For establishing the relationship between the measured kinetic parameters and the amino acid sequence of the MHC isoforms National Center for Biotechnological Information (NCBI) data were used. Sequences with the following GI numbers were compared: mouse: 16508127 (MHCI), 14250231 (MHCIIa), 20883346 (MHCIIx(d)), 38091417 (MHCIIb); rabbit: 2144825 (MHCI), 940233 (MHCIIx(d)); rat: 56657 (MHCI), 543418 (MHCIIa), 34870884 MHCIIx(d), 34870888 and 543419 (MHCIIb); human: 107137 (MHCI), 4808813 (MHCIIa), 4808815 (MHCIIx(d)), 4808811 (MHCIIb). Sequences were aligned with the CLUSTAL W program (Ref. 51; available at http://www.ebi.ac.uk). With this program, the sequence identity score index was determined for pairs of corresponding amino acid sequences.


    RESULTS AND DISCUSSION
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 MATERIALS AND METHODS
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MHC isoforms, morphological data, and maximal tensions of mouse muscle fibers. Figure 1 shows an electrophoresis pattern of MHC isoforms of mouse skeletal muscle fibers for which mechanical properties were previously measured. Four distinct MHC isoforms were separated, namely, MHCIIa, MHCIIx(d), MHCIIb, and MHCI (in order of increasing electrophoretic mobility). These isoforms were assigned to fiber type IIA, type IIX(d), type IIB, and type I, respectively. Either one single MHC isoform (pure fibers, 70% of all fibers investigated) or two MHC isoforms (hybrid fibers, 30% of fibers) were present in individual fibers. The following combinations of MHC isoforms were found: MHCIIb and MHCIIx(d) (type IIBX), MHC IIa and MHCIIx(d) (type IIAX), and MHCIIa and MHCI (type C). Sarcomere lengths measured under resting conditions were similar in different fiber types (Table 1), which is in accordance with other mammalian species (3, 7, 19). The cross-sectional area (measured in relaxation solution containing 40 g/l dextran T-500) differed in different pure fiber types (see Table 1). As in other studies (23, 29) the following order was observed: IIB > IIX(D) > I > IIA (P < 0.05). Soleus muscle fibers contained only MHCI and/or MHCIIa. These fibers were slightly wider (type I: 798 ± 296 µm2, n = 16; type IIA: 663 ± 117 µm2, n = 18) than corresponding fibers in another muscle that contained these fiber types (gastrocnemius; type I: 650 ± 56 µm2, n = 2; type IIA: 562 ± 133 µm2, n = 5). In general, the cross-sectional areas of mouse fibers were smaller than those of rat (coarse range: 1,200–1,900 µm2; Ref. 7), rabbit (700–5,000 µm2; Ref. 3), and human (1,000–5,000 µm2; Ref. 26) fibers. Pure type I fibers of mouse exhibited a smaller maximal isometric tension than pure type II fibers (P < 0.001; Table 1). Within type II fibers there was no significant difference in maximal tension among different fiber types (P > 0.1). This type of distribution of maximal tension values was also found in other mammalian species (7, 39).



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Fig. 1. Silver-stained polyacrylamide gel electrophoresis pattern of myosin heavy chain (MHC) isoforms from mouse skeletal muscle fibers. Lane 1 represents an extract of mouse gastrocnemius muscle and serves as marker for MHCIIb and MHCIIx(d). Lane 2 represents an extract of mouse soleus muscle and serves as marker for MHCI and MHCIIa. The other lanes represent single fibers for which mechanical properties were measured: lane 3, type I (soleus); lane 4, hybrid type C (soleus); lane 5, type IIB (tibialis anterior); lane 6, type IIX(D) (gastrocnemius); lane 7, hybrid type IIAX (vastus lateralis); lane 8, hybrid type IIBX (gastrocnemius); lane 9, type IIA (soleus).

 

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Table 1. Dimensions and stretch activation parameters of mouse skeletal muscle fiber types

 
Stretch activation kinetics of mouse muscle fibers. Stepwise stretches (≤1 ms) applied during maximal Ca2+ activation caused a simultaneous rise in force, followed by a decrease and, subsequently, a delayed rise in force called stretch activation (Fig. 2A). The kinetics of this force transient differed markedly in different fiber types. The order of velocity was IIB > IIX(D) >IIA >> I. The following parameters of the force transient were evaluated: the time from the beginning of the stretch to the lowest force before the onset of delayed rise in force (t2) and the time from the beginning of the stretch to the peak of the delayed rise in force (t3). Within a series of stretches with different amplitudes (0.1–0.2% of fiber length) on an individual fiber, t2 and t3 varied with SD of <10% (t2) or <5% (t3) of the mean. A strong correlation between t2 and t3 values of individual fibers was found (r = 0.99). Both t2 and t3 values were significantly different (P < 0.01) among all (pure and hybrid) fiber types (Table 1). Figure 2B shows the distribution of the t3 values of pure fiber types in a histogram. No overlap of the t3 values existed between the pure fiber types. The ranges of t3 were 18–32 ms for type IIB, 37–62 ms for type IIX(D), 91–150 ms for type IIA, and 520–980 ms for type I fibers. No significant differences (P > 0.1) in t3 values were found in fibers of the same type originating from different skeletal muscles. Hybrid fibers displayed time characteristics intermediate between the corresponding pure fiber types, such that a continuum in a regular order was formed by pure and hybrid fiber types (IIB > IIBX > IIX(D) > IIAX > IIA > C > I; Fig. 2C). The results in mouse fibers are in accordance with previous findings in rat, rabbit, and human skeletal muscle fibers (17, 19, 26).



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Fig. 2. Stretch activation kinetics of skinned fiber preparations of mouse skeletal muscle fiber types at 22°C. A: original recordings of quick stretch experiments on maximally Ca2+-activated skinned fibers of various skeletal muscle fiber types. The top trace shows the fiber length of the type IIB fiber. The traces below represent the force transients of various fiber types induced by quick stretches of similar amplitude (~0.15% of fiber length). The vertical bar is 0.05 mN for type IIB, 0.03 mN for types IIX(D) and IIA, and 0.07 mN for type I. t2, Time from the onset of the stretch to the lowest force before the delayed force increase; t3, time to peak of the delayed force increase. B: t3 for pure skeletal muscle fiber types of mouse. Statistical analysis shows Gaussian distribution for all fiber types: Kolmogorov-Smirnov distance (K-S Dist) = 0.084 (P > 0.2) for type IIB; K-S Dist = 0.147 (P > 0.2) for type IIX(D); K-S Dist = 0.111 (P > 0.2) for type IIA; K-S Dist = 0.117 (P > 0.2) for type I. C: relationship between fiber type and kinetics of stretch activation. Because of the broad range, the reciprocals of the t3 values (means ± SD) are plotted. The diagram includes 195 fibers. The number of the fibers tested is given in parentheses for each type.

 
In mouse fibers, a Gaussian distribution was found for t3 of all pure fiber types. For type II fibers, this is in accord with findings in other mammalian species. However, in contrast to mouse fibers, the t3 values of rat, rabbit, and human type I fibers did not show a Gaussian distribution (2, 3, 17, 26). A Gaussian distribution generally indicates that the data originate from one single identity. Therefore, it can be assumed that, in contrast to rat, rabbit, and human skeletal muscle, the type I fibers of mouse skeletal muscle represent a homogeneous population containing one specific MHC isoform. In rat, rabbit, and human skeletal muscle, MHCI seems to represent either a mixture of more than one isoform or a mixture of a specific isoform that is posttranslationally modified, e.g., by phosphorylation, glucation, or deamination (10, 36, 38, 42).

In hybrid muscle fibers, a relationship between the t3 values and the densitometrically determined ratio of MHC isoform concentrations was established (Fig. 3). For type IIBX hybrids (Fig. 3A), it can be seen that the t3 values depend on the percent distribution of the two isoforms (r = 0.88). Conversely, in mouse type C hybrid fibers, the distribution of t3 was mainly dependent on the predominant MHC isoform (Fig. 3B). Fibers in which MHCIIa was predominant (MHCI-to-MHCIIa+MHCI concentration ratio: 0.10–0.45) exhibited a range of t3 (96–190 ms) close to that of type IIA (91–150 ms). Fibers in which MHCI was predominant (MHCI-to-MHCIIa+MHCI concentration ratio: 0.65–0.95) exhibited a range (568–770 ms) close to that of type I (520–980 ms). In IIBX rabbit fibers (17) and hybrid fibers of rabbit masseter muscle containing combinations of MHCIIa and {alpha}-cardiac MHC (3), the t3 values were distributed in the same way as in mouse type IIBX fibers. Because stretch activation data of type C fibers of other mammalian species are scarce, no comparison is possible.



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Fig. 3. Relationship between the stretch activation parameter t3 and the MHC isoform concentration ratio in hybrid fiber type IIBX (A) and hybrid fiber type C (B) of mouse skeletal muscle: t3 is plotted against the densitometrically determined ratio of MHC isoforms. The values 0 and 1 on the x-axis represent the pure fiber types IIB and IIX(D) in A and the pure fiber types IIA and I in B, respectively. The straight line in A represents the linear regression of the type IIBX hybrid fibers (r = 0.88).

 
Molecular background of stretch activation. The molecular events underlying stretch activation are still unknown. However, a convincing model was deduced on the basis of experiments where maximally Ca2+-activated fibers were exposed to sinusoidal length changes of different frequency (32, 34, 45). According to these studies, t2 (process C of Ref. 32) approximates the step of myosin head detachment from actin after MgATP binding and t3 (process B of Ref. 32) approximates the force generation step before phosphate release (34). If we apply this model to our finding of a close correlation between stretch activation kinetics (t2, t3) and MHC isoforms, we can conclude that both the detachment step and the force generation step of different mouse muscle fiber types (as well as those of rat, rabbit, and human) exhibit the following order of kinetics: MHCIIb > MHCIIx(d) > MHCIIa >> MHCI. The ATPase activity of mammalian skeletal muscle fiber types follows the same order (6). However, the ATPase activity differs maximally by a factor of 5, whereas the t2 and t3 values of mouse fibers differ by factors of ~27 and ~29, respectively. If a direct coupling between the ATPase cycle and the cross-bridge cycle is assumed, it appears that neither t2 nor t3 represents the time-limiting step of the whole ATPase cycle.

Although the correlation between the kinetics of stretch activation and the MHC isoforms is strong, the influence of other myofibrillar protein isoforms is possible in principle. However, the following observations make this unlikely. No correlation was found between t3 and the essential myosin light chain (MLC) isoform content of rat and rabbit fibers (unpublished results). Furthermore, in rabbit and rat fast-twitch muscle fibers, essential MLC isoforms are correlated with Vu (5, 49), but stretch activation kinetics are not correlated with Vu (19). Regarding thin filament proteins, an earlier study (18) suggests that troponin T isoforms do not affect the stretch activation kinetics in rat fast-twitch skeletal muscle fibers.

Comparison of stretch activation kinetics in mouse, rat, rabbit, and human. Although the same relationship between stretch activation kinetics and MHC isoforms was found for mouse, rat, rabbit, and human fibers (3, 17, 19, 26), the t3 values of corresponding fiber types within different animal species are not identical in these mammalian species. Figure 4 shows a double logarithmic plot of the velocity parameter 1/t3 (mean ± SD) of mouse, rat, rabbit, and human fiber types against the corresponding body mass. A 1/t3 value for human type IIB fibers is missing because these fibers do not exist. The values originate from different studies executed under identical conditions (human, Ref. 26; rabbit, Ref. 3). For rat fibers, 1/t3 values of measurements more recent than those in Ref. 19 were taken to have absolutely identical conditions for all species. Although not significant in all cases (see Fig. 4), the 1/t3 values show a trend to decrease with increasing body mass. This trend is expressed by the (negative) slopes of the regression lines (scaling coefficients; Ref. 27). The scaling coefficients were similar for all fiber types (–0.073 for type I, –0.079 for type IIA, –0.102 for type IIX(D), and –0.086 for type IIB). The same type of dependence on body mass was also observed for Vu and the actin filament sliding propelled by different myosins in vitro (8, 28, 39, 44, 47, 55). In type II fibers, the scaling coefficients of stretch activation kinetics were in the range of those of shortening velocity reported in the literature (39); however, in type I fibers, a much smaller scaling coefficient was found for stretch activation (–0.073) than for Vu [–0.175 (39), –0.180 (44)]. Vu is a measure that includes changes of the fiber length, whereas stretch activation kinetics (0.1–0.2% fiber length change) is a measure taken under almost isometric conditions. Thus, for type I fibers, it appears that the cross-bridge cycle steps limiting fiber shortening are more dependent on body mass than specific steps within the cross-bridge cycle during isometric force production.



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Fig. 4. Relation between 1/t3 (mean ± SD) of mouse, rat, rabbit, and human pure muscle fiber types and averaged body mass of these species on logarithmic scales. Significant differences (P < 0.01) were found between type I fibers (except mouse vs. rat and rabbit vs. human), type IIA fibers (except mouse vs. rat, mouse vs. rabbit, and rat vs. rabbit), type IIX(D) fibers (except rat vs. mouse) and type IIB fibers. Lines represent the results obtained by fitting the equation y = axb to the data points (where y is 1/t3, x is body mass, b is the scaling coefficient, i.e., slope of the straight line, and a is y-axis intercept). The scaling coefficients are (means ± SE) –0.073 ± 0.043 (r = 0.94; P = 0.06) for type I, –0.079 ± 0.020 (r = 0.88, P = 0.12) for type IIA, –0.102 ± 0.019 (r = 0.97, P = 0.03) for type IIX(D), and –0.086 ± 0.039 (r = 0.998, P = 0.03) for type IIB. P represents the probability of the slope not deviating from zero.

 
Relationship between stretch activation kinetics and amino acid sequence variation. To find out which region of MHC determines the differences in kinetics, we correlated the variation of stretch activation kinetics with the variation of amino acid sequence of specific regions of the MHC isoforms. For identification of the various regions of MHC we used the amino acid numeration given by Geeves and Holmes (20), which is related to the atomic structure of the chicken skeletal myosin head discovered by Rayment et al. (43). We carried out correlations both within and between different animal species. The method of our correlations consisted of two steps. In the first step the structural variability was quantified by comparing corresponding amino acid sequences of different MHC isoforms. In the second step the quantitative measure of the structural variability of the MHC isoforms was correlated with the ratio of t3 values of corresponding fiber types.

To obtain a quantitative measure of the structural variability the following procedure was undertaken. Pairs of amino acid sequences were aligned in the CLUSTAL W program (51). In short, this program detects identical, nonidentical, and missing amino acids of the two sequences and calculates a sequence identity score index. An index of 100 means 100% identity. Sequence identity score indexes of the following MHC regions were determined: whole MHC, myosin head, loop 1 (amino acids 199–225 of chicken myosin), loop 2 (621–647), loop 3 (567–578) (considered in Refs. 54 and 39 for structure-functional comparisons), whole actin-binding region (487–600 + loop 2), and converter region (712–779). Not all sequences were available in the NCBI database. The GI numbers of the available sequences (Table 2) are mentioned in MATERIALS AND METHODS. For both intra- and interspecies comparisons all possible pairs of available MHC sequences were analyzed. Each isoform was compared with each other MHC isoform of the same species (mouse, rat, rabbit, and human). Likewise, each isoform was compared with each corresponding MHC isoform of the other species (i.e., isoforms with the same name). Thus a considerable number of sequence identity score indexes were obtained.


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Table 2. Correlation of stretch activation kinetics with variation of amino acid sequence of whole or specific parts of MHC isoforms

 
Each score index was related to its functional correlate, namely, the ratio of t3 values of corresponding fiber types (higher value divided by lower value). Correlation between the t3 ratios and the corresponding score indexes was carried out for each MHC region and quantified by linear regression analysis. For all MHC regions, the range of sequence identity score indexes was larger in the intraspecies comparisons than in the interspecies comparisons (Table 2). Thus the structural variability is larger between different MHC isoforms of the same animal species than between corresponding isoforms of different animal species. This pattern is in line with the variation pattern of t3 values (and Vu; Ref. 39), which also showed larger variability within different fiber types of one animal species than within corresponding isoforms of different animal species.

In the intraspecies comparisons, the structure-function correlation revealed correlation coefficients near 0.7 for all MHC regions (Table 2). Because all correlation coefficients were similar, these comparisons were not useful to differentiate the importance of variability in different MHC regions. Therefore, the intraspecies comparisons were not considered for further analysis or reflections. In contrast, the interspecies comparisons revealed a range of correlation coefficients from 0.21 to 0.74. The highest correlation coefficients were found for the whole actin-binding region (r = 0.68; Fig. 5A) and for the combination of actin-binding loops 2 and 3 (r = 0.74; Fig. 5B). This suggests a contribution of these loops to the variation in kinetics (at least for corresponding MHC isoforms of different animal species). It is interesting that the structure-function correlation is less pronounced when sequences of loop 2 and loop 3 are individually correlated to the corresponding t3 ratios. It appears that simultaneous variations in both loops are necessary for contributing to differences in function. This requires a cooperative action of the two loops. The actin-binding loop 2 and loop 3 have also been mentioned as possible origins of the differences in the kinetics of unloaded filament sliding of different mammalian skeletal muscle fiber types (8, 39).



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Fig. 5. Relation between t3 ratios of corresponding fiber types of different animal species and the identity score index of corresponding amino acid sequences of the whole actin-binding region (A) and of loop 2 + loop 3 (B). Mean t3 values of corresponding mouse, rat, rabbit, and human fiber types were related to each other to obtain the ratios shown on the y-axis. Higher values were always divided by lower values. The x-axis represents the identity score index of the amino acid sequence of the considered region of corresponding MHC isoforms. The GI numbers of the sequences used (Table 2) are given in MATERIALS AND METHODS. The correlation coefficients of the linear regression lines are 0.68 for A and 0.74 for B.

 

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This work was supported by grants from the Austrian Fonds zur Förderung der Wissenschaftlicher Forschung (FWF-P14573-MOB) and the South Tyrolean Sparkasse.


    ACKNOWLEDGMENTS
 
We are thankful to E. Puchert (Sydney) and H. Grassberger (Salzburg) for critically reading the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Galler, Dept. of Cell Biology, Univ. of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria (E-mail: stefan.galler{at}sbg.ac.at)

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.


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