altered ca2' dependence of tension development in skinned

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 13, Issue of May 5, pp. 6096-6099,1986 Printed in U.S.A.

Altered Ca2’ Dependence of Tension Development in Skinned Skeletal Muscle Fibers following Modification of Troponin by Partial Substitution with Cardiac Troponin C*

(Received for publication, August 2, 1985)

Richard L. Moss$, Michael R. Lauer, Gary G. Giulian, and Marion L. Greaser5 From the Department of Physiology, University of Wisconsin School of Medicine and the §Department of Meat and Animal Science and the Muscle Biology Laboratory, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca”’] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by re- extraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.

The regulation of tension development in striated muscles involves the binding of Caz+ t o low-affinity sites on the TnCl subunit of the regulatory protein troponin, which is localized at regular intervals along the thin filament (see Ref. 1 for a review). Troponin in turn is bound to a second regulatory protein, tropomyosin, which in relaxed muscle acts either to sterically block the myosin-binding site on actin (2) or to block a kinetic step in the cross-bridge interaction cycle, possibly the ejection of Pi from the cross-bridge head (3). Once Ca2+ has bound to TnC, tropomyosin undergoes a change in position relative to the thin filament such that the myosin cross-bridges can then cyclically interact with actin to produce tension and mechanical work. The amount of tension developed by a muscle fiber can be varied by changing the amount of free Ca2+ i n the myoplasm, which determines the number of available cross-bridge binding sites on the thin filament. The relationship between isometric tension development and free Ca2+ is much steeper in fast-twitch skeletal than in cardiac (4) or slow-twitch skeletal muscles (5). Such

* This work was supported by Grants HL25861 and AM31806 from the National Institutes of Health and by the School of Medicine and the College of Agricultural and Life Sciences of the University of Wisconsin. A preliminary report of this work has been published elsewhere (Lauer, M. R., Moss, R. L., and Greaser, M. L. (1984) Circulation 70, 11-277). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

j: Performed this work during the tenure of an Established Inves- tigatorship from the American Heart Association and with funds contributed in part by the Wisconsin Affiliate.

The abbreviations used are: Tn, troponin; S-TnC, skeletal troponin C; C-TnC, cardiac troponin C; SDS, sodium dodecyl sulfate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

a difference in steepness, in terms of current models of contractile activation (6) , might be explained on the basis of greater cooperativity in fast-twitch muscle, perhaps resulting from end-to-end interactions of adjacent tropomyosin molecules (7). These interactions are thought to enhance Ca2+ binding in regions of the thin filament adjacent to an already activated troponin-tropomyosin complex. In addition, fast- twitch skeletal TnC (S-TnC) has two low-affinity Ca2+-binding sites (S), whereas cardiac TnC (C-TnC) (9-11) and slow- twitch skeletal TnC have only one (12). However, it is not clear whether this difference in the number of Ca2+-binding sites translates into a difference in the Ca2+ sensitivity of tension development. To examine this possibility, experiments were performed to determine whether partial substitution of S-TnC by C-TnC alters the relationship between tension andpCa (ie. -log[Ca2+]) in single skinned fibers from rabbit psoas muscles.

MATERIALS AND METHODS Psoas muscles were dissected from adult male New Zealand rabbits.

Bundles of fibers were stripped free while in relaxing solution, tied to glass capillary tubes, and then stored at -22 “C in relaxing solution containing 50% (v/v) glycerol for several days before use (13). Indi- vidual skinned fibers were then pulled free and mounted in the experimental chamber (14) between a force transducer (Model 403, Cambridge Technology, Cambridge, MA) and a DC torque motor (Model 300s, Cambridge Technology). Sarcomere length in the relaxed fiber segments was adjusted to 2.5 pm by changing overall segment length, so that sarcomere length during contraction was approximately 2.4 pm. The fiber segments were activated in solutions containing various concentrations of free calcium between 0.1 and 10 p ~ , which are expressed aspCa values (i.e. -log[Ca*+]) in the present report. The relaxing and activating solutions were identical to those of Julian (15). At any given pCa, a steady tension was allowed to develop, a t which time the segment was rapidly (i.e. within 1 ms) slackened and was subsequently relaxed. The difference between the steady developed tension and the tension base line obtained imme- diately following the slack step was measured as total tension. Active tension was calculated as the difference between total tension and the resting tension (usually less than 1 mg in weight) measured in the same segment while in relaxing solution. Tensions (P) at sub- maximally activating levels of calcium were expressed as a fraction of Po, the tension obtained during maximal activation at pCa 5.49. Every third or fourth contraction was performed at pCa 5.49 in order to assess any decline in fiber performance (12). Tension-pCa relationships were obtained (a) in the untreated fiber segment and (b) following partial extraction of endogenous S-TnC by bathing the fiber for 90-120 min at 15 “C in a solution containing 20 mM Tris and 5 mM EDTA, pH 7.8 (16, 17), and subsequent reconstitution with bovine C-TnC. Reconstitution was accomplished by bathing the fiber segment for 10 min in relaxing solution containing 0.5-1.0 mg/ml C- TnC and was followed by two 5-min washes in relaxing solution in order to remove excess C-TnC. In some experiments, a final tension- pCa relationship was determined after re-extraction of the C-TnC and reconstitution with S-TnC. The results of previous control experiments (18) demonstrated that fibers from which S-TnC was first

6096

Contraction in Skeletal Muscle Substituted with Cardiac TnC 6097

extracted and then recombined had contractile properties that were virtually identical to control.

The TnC content of the fibers a t each step of the experimental protocol was determined by performing SDS-polyacrylamide gel electrophoresis on short segments of the same skinned fiber (19). Each fiber was divided into two or three segments of approximately equal lengths. The first of these was dissolved in SDS-containing sample buffer (19). One of the remaining segments was tied between the force transducer and motor for use in the physiological measurements. In those experiments in which the experimental fiber segment was finally reconstituted with C-TnC, an additional segment was tied at both ends to the motor arm so that it was exposed to the Same solutions as the experimental segment. Following the treatments to partially extract S-TnC and recombine C-TnC, the segment tied only to the motor arm was removed and then dissolved in sample buffer. Finally, following the extraction of C-TnC and recombination of s- TnC and the subsequent determination of the relative tension-pca relationship, the experimental segment was dissolved in sample buffer.

Once the staining and drying procedures were completed, the gels were scanned using a Bio-Med Instruments (Fullerton, CAI laser- light scanning densitometer (19). The relative amount of S-TnC or C-TnC present in any given fiber segment was taken as the ratio of the integrated area of the appropriate peak to the sum of areas of the S-TnC and C-TnC peaks. Total TnC content ( i e . C plus s) of the fiber segments was also referenced to the myosin light chain 1 content of the same fiber segment. If total TnC determined in this way varied by more than 10% between control and reconstituted segments, that fiber was excluded from the analysis of the proportions of S-TnC and C-TnC present in the fiber segments at each stage of the protocol.

RESULTS AND DISCUSSION

Fig. 1 represents tension records from a single fiber segment at various stages of the experimental protocol. Following 90 min of exposure to the EDTA-containing solution, maximum developed tension decreased by approximately 70% ( b versus a). The mean S-TnC content of the extracted fibers, determined by SDS-polyacrylamide gel electrophoresis (Fig. 2), was 40% of control levels. Bathing this same fiber in relaxing solution containing bovine C-TnC (0.8 mg/ml) for 10 min resulted in a recovery of Po to over 90% of its control value (Fig. IC). For reasons that are not yet clear, still longer soaks in the solution containing C-TnC or increasing the concen- tration of C-TnC did not result in further recovery of tension but in many cases actually resulted in a decline from the maximum observed at approximately 10 min. On average, maximum Ca2+-activated tension following C-TnC recombination was 92% of the control values measured in the same fibers. At very low Ca2+ concentrations (pCa 6.70 in Fig. 1, d and e) , the developed tension was found to be greater with C- TnC present when compared to control; however, at relatively high Ca2+ concentrations (pCa 6.09 in Fig. 1, f and g), steady isometric tension was less with C-TnC present.

Tension-pCa relationships are shown in Fig. 3 for one fiber segment under control conditions (0), after partial extraction of S-TnC and reconstitution with C-TnC (O), and finally, after another period of extraction followed by reconstitution of the fiber with S-TnC (x). C-TnC substitution into the fiber resulted in an increase in tension relative to control for pCa > 6.51 and a decrease in relative tensions in the range of pCa values between 6.51 and 5.0. Thus, in the presence of C-TnC, there was a rightward shift of the upper part of the tension- pCa relationship and a leftward shift of the lower part. Also apparent from Fig. 3 is that subsequent recombination of S- TnC into the fiber segment resulted in recovery of the tension-pCa relationship to near its original form; however, in most cases, recovery to the form of the control relationship was incomplete, a phenomenon which was correlated with incomplete removal of C-TnC (determined by SDS-polyacrylamide gel electrophoresis) prior to S-TnC recombination. Thus, for many fibers, the tension-pCa relationship following

i

5.00 I A S A

I

5.00 s R 5.00 R

A S

6.70 A S

n 6.70 A s

R

6.09 R A S

6.09 R A S

FIG. 1. Tension records obtained from a single muscle fiber prior to manipulation of TnC content (a, d, and 0, following partial extraction of TnC (b), and in the same fiber following recombination with C-TnC (c, e, and g). Once the fiber has been mounted in the experimental chamber, sarcomere length was adjusted to about 2.4 pm, as determined by light microscopy (14). The fiber was initially placed in relaxing solution containing 100 mM KCI, 2 mM EGTA, 4 mM ATP, 1 mM MgCl,, 10 mM imidazole, pH 7.00 (15). In each tension record, the fiber was transferred from relaxing solution to the indicated pCa at the time point designated A. When a steady active tension was developed, the fiber was rapidly slackened (S) by imposing a length step (complete within 1 ms) at the motor end. This was done to obtain an accurate zero-force base line. Active tension was then measured as the difference between this base line and the peak tension prior to the step from an oscilloscope record at a fast sweep speed (note: the tension trace did not drop to base line on the slow chart records shown). Following the length step, the fiber was returned to relaxing solution (R). The tensions obtained from these records, relative to the tensions obtained at pCa 5.00 in the control condition. were as follows.

pca' Control S-TnC C-TnC extracted recombined

5.00 1.00 (a) 0.30 ( b ) 0.95 ( e ) 6.70 0.05 (d ) 0.10 (e ) 6.09 0.82 (f) 0.65 (g)

S-TnC recombination likely represented a composite relationship, with contributions from both C- and S-TnC as was also the case for the initial C-TnC substitution (Fig. 2, lane 2).

A convenient way to examine the changes in the tension- pCa relationship due to partial substitution with C-TnC is to use the Hill plot transformation of the raw data (20-22). Data from 12 fibers were used to construct the Hill plot in Fig. 4. These data are best fit by two straight lines, one line for P/Po > 0.5 and another for P/Po < 0.5. For P/Po < 0.5, the Hill coefficient, n, was 3.73 under control conditions and 2.44 after partial substitution with C-TnC. For P/Po > 0.5, n was calculated as 1.71 under control conditions and 0.86 after reconstitution with C-TnC. In addition, there were distinct shifts of the two portions of the Hill plot following substitution with C-TnC, and these were consistent with the shifts in the tension-pCa relationship noted previously (Fig. 3). Cer- tainly, the apparent decrease in Ca2+ sensitivity observed for

6098

b

a.

A

C-TnC LC,- - LC,-

1 LANE I

Contraction in Skeletal Muscle Substituted with Cardiac TnC

1 2 3 4 5

LANE 3

FIG. 2. a, sodium dodecyl sulfate-polyacrylamide gel of segmenta of the same single fiber obtained at various stages of the experimental protocol. Lane 1, control segment; lane 2, fiber segment obtained following partial extraction of S-TnC and recombination with C- TnC; lane 3, fiber segment obtained following re-extraction of C-TnC

1.0-

- 0.e

h - ao

-

.- 8

- - 0.6 - ul - e

0.4 - .- - e o u" -

0.2 -

'7.0 L 6.5 6.0 5.5 5.0

PC0

FIG. 3. Relative tension-pCa relationship obtained from a single psoas fiber. Data were first obtained from an untreated control segment (0). then from the same segment following a 90-min extraction of S-TnC and subsequent recombination with C-TnC (O), and finally following a second 90-min extraction period followed by recombination with S-TnC (X). For each of these three conditions, submaximal tensions have been expressed as a fraction of the tension developed at pCa 5.0 under the same condition.

pCa < 6.51 is in keeping with the lower affinity for Ca2+ at the Ca"-specific site on C-Tnc ( K = 2 X lo' M") uersus the sites on S-TnC ( K = 2 X 10' M-') (9).

The biphasic form of the control Hill plot can be interpreted as reflecting cooperativity in cross-bridge binding (6). Coop- erative mechanisms for which there is experimental evidence include effects of bound cross-bridges to increase the affinity of Ca'+ binding by TnC (23) and end-to-end interactions involving tropomyosin to affect cross-bridge binding along the thin filament (7). In this regard, Walsh et al. (24) have recently reported that the cooperative response of the regu- lated actomyosin ATPase to increasing concentrations of Ca'+ was unaffected when the overlapping regions of adjacent tropomyosins were removed. Whereas this result certainly suggests that the cooperative response of the ATPase does not involve end-to-end interactions of tropomyosin, Tawada et al. (25) came to the opposite conclusion based on otherwise similar experiments in which superprecipitation of actomyosin was observed. Given these contradictory findings and

and recombination with S-TnC; lone 4. C-TnC standard; lane 5, S- TnC standard. Lanes 1 3 each contain the equivalent of a 0.5-mm length of fiber. The total acrylamide content in the stacking gel was 3.5% (w/v) and 12% in the separating gel. Otherwise, the details of sample preparation, electrophoresis, silver staining, and densitometry were identical to the methods of Ciulian et of. (19). 6. densitometric scans of lanes 1 3 shown in a. The actual areas (in arbitrary units) of the peaks corresponding to the myosin light chains (LC), TnI, and TnC were as follows (note that the load in lane 2 was approximately 20% less than in l anes I and 3 as judged from the areas under the mvosin lieht chain Deaks).

Lune I Lone 2 (C-TnC

Lune 3 (S-TnC

(contm') recombined) recombined)

LC1 492 390 496 TnI 315 235 295 S-TnC 137 58 154 C-TnC 78 LC, 591 43 1 510 LC8 125 107 124

Contraction in Skeletal Muscle Substituted with Cardiac TnC 6099

1 . q -

1.0 I-

v' '

I 2

Log [Ca"]

FIG. 4. Hill plot of tension-pCa data before (0) and after (0) partial substitution with C-TnC. The straight lines were tit to the data using least-squares regression analysis. The error bars indicate one standard deviation ( n = 12). P, represents relative tension (P/Po).

the apparently lower degree of cooperativity in the ATPase response to Ca2+ compared to tension and Ca2+, it remains to be shown whether nearest neighbor interactions of tropomyosin can account for the cooperativity apparent in the Ca" activation of tension development. In addition, evidence has been presented suggesting that there may be direct interactions between the troponin-?' of one functional group and the tropomyosin of the adjacent functional group (26).

The idea of cooperativity within the thin filament has been incorporated by Hill and co-workers (6, 27) into models of Ca2+ activation of contraction. Asymmetry in simulated tension-pCa relationships of fast-twitch muscle, corresponding to the biphasic nature of our Hill plots, was achieved in Hill's (6) model by imposing the constraint that no movement of tropomyosin could occur unless both low-affinity sites on TnC were occupied with Ca2+. With this is mind, changes in the form of the Hill plot following C-TnC reconstitution lead to relatively straightforward interpretations. For P/Po > 0.5, the value of n following C-TnC substitution (n = 0.86) is approximately one-half that obtained under control conditions (n = 1.71). This result is consistent with the finding that C-TnC contains only one low-affinity Ca2+-binding site as compared to two on S-TnC (9-11). For P/Po < 0.5, the Hill coefficient following C-TnC substitution (n = 2.44) was smaller than that measured under control conditions (n = 3.73). Since the overall steepness of the Hill plot may reflect cooperativity among Ca2+-binding sites along the thin filament (28), as well as between cross-bridge binding and Ca2+ binding (23), the present result suggests that partial substitution of C-TnC into fast-twitch fibers produces a decrease in such cooperativity. The decrease in steepness with C-TnC may, at least in part, simply reflect the need to bind only one Ca2+ (versus two in fast-twitch muscle) in order to induce movement of the asso- ciated tropomyosin.

In summary, the present results have shown that partial substitution of C-TnC for S-TnC in skinned fast-twitch muscle fibers from the rabbit causes a reversible change in the form of the tension-pCa relationship to one which is more characteristic of skinned cardiac or slow-twitch skeletal muscle. In relation to Ca2+ regulation of contraction, there are at least two important implications of this finding. First, the

form of the tension-pCa relationship is affected by the TnC that is present within the thin filament and appears to depend strongly on the number of low-affinity Ca2+-binding sites on TnC and their relative affinities for Ca2+. Second, the degree of cooperativity within the thin filament differs depending upon the type of TnC bound. Possible explanations for this effect may involve a decrease in interactions between bound cross-bridges and the low-affinity Ca2+-binding site on C-TnC or a reduction in end-to-end interactions among tropomyosin molecules. Further experiments will be required to determine the relative importance of these two mechanisms in the Ca2+ regulation of tension development in striated muscles.

Acknowledgment-We are grateful to James Graham for his as- sistance and to Susan Krey for preparation of the manuscript.

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altered ca2' dependence of tension development in skinned

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