305

Journal of Physiology (1990), 441, pp. 305-324 With 12 figures Printed in Great Britain

THE ROLE OF TROPONIN C IN THE LENGTH DEPENDENCE OF Ca2+SENSITIVE FORCE OF MAMMALIAN SKELETAL AND CARDIAC MUSCLES By JAGDISH GULATI*t, EDMUND SONNENBLICK* AND ARVIND BABU* From the Departments of *Medicine and tPhysiology/Biophysics, Molecular Physiology Laboratory, Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

(Received 11 September 1990) SUMMARY

1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2' and the length dependencies of their pCa (-log [Ca2+])-force relationships were compared. 2. Ca2+ sensitivity of the myocardium was higher at 22-24,tm than that at 1V7-19 ,um. The length dependence was at least twofold greater in cardiac muscle than in fast skeletal fibres at identical temperatures and salt concentrations. Slowtwitch fibres gave a response similar to that in the myocardium. 3. The effect of the troponin C (TnC) phenotype on the length dependence of Ca2+ sensitivity was measured on both fast skeletal fibres and cardiac muscle with TnC exchange in situ. The length-induced increase in Ca21 sensitivity was found to be greater in the presence of cardiac TnC than with fast skeletal TnC. Thus the results indicate that a certain domain of TnC is specialized in this length function, and that this domain is different in the two phenotypes. 4. The possibility that the enhanced length dependence of Ca2+ sensitivity after cardiac TnC reconstitution was attributable to reduced TnC binding was excluded when the length dependence of partially extracted fast fibres was reduced to one-half the normal value after a 50% deletion of the native TnC. 5. Two recombinant forms of cardiac TnC (kindly provided by Dr John Putkey, Houston, TX, USA) were used next, to investigate the roles of two specific domains in TnC in the control of length dependence of Ca2+ sensitivity and in the contraction-relaxation switching of cardiac muscle: 6. Using mutant CMB 1, in which site 1 was modified such as to bind the 4th Ca21 ion, as in skeletal TnC, the length-induced Ca2+ sensitivity in cardiac muscle was suppressed. The effect was intermediate between cardiac and skeletal TnCs under the same conditions. The pSr (-log [Sr2+])-force relationship of cardiac muscle was also measured. In the presence of the mutant, skinned trabeculae manifest pSr-activation curves identical to those of fast fibres. This indicates that the metal ion binding properties of site 1 in TnC modulate the regulatory action of site 2. 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected N1S 8789

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result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle. Also, when this mutant was loaded into a moderately extracted fibre, the length dependence remained at the reduced level observed after partial TnC extraction. This shows that the modified state of the thin filament following such partial extraction occurs in response to the loss of active TnC rather than the vacancy per se in the thin filament. 8. The results of this study firmly indicate a direct role of TnC in the modified length dependence of cbardiac function when compared with that in skeletal muscle, and further, provide direct evidence that site 1 of the N-terminus of TnC is a key component of the length sensing instrument in the myocardium. This novel function of cardiac TnC in the length-sensing mechanism is additional to its classical role as the Ca21 switch. INTRODUCTION

Troponin C (TnC) plays a key role in the contraction-relaxation switching mechanism in cardiac and skeletal muscles where Ca2" initiates a series of events that trigger contraction (Ebashi & Endo, 1968; Zot & Potter, 1986). Recently, studies with TnC exchange in cardiac muscle have shown that TnC also critically participates in the length-sensing mechanism of the sarcomere in the myocardium and that, in this function, cardiac TnC is more efficient than skeletal TnC (Gulati, 1990a). An efficient length-sensing mechanism in the myocardium is of paramount importance since the heart needs to make instantaneous adjustments during its beat-to-beat performance, as described by Starling's law of the heart (Jewell, 1977; Allen & Kentish, 1985; Lakatta, 1986). As a first step in the investigation of the mechanisms by which TnC contributes to the length-sensing process, therefore, we have studied the efficacies of cardiac and skeletal TnCs in relation to their Ca2' binding properties and primary structures, in both skeletal and cardiac milieus. Cardiac and fast-twitch skeletal TnC are proteins whose sequences manifest 70 % homology (Van Eerd & Takahashi, 1975). Both isoforms have four putative helix-loop-helix (EF-hand) motifs (Kretsinger, 1980), which are potential Ca21 binding sites, and are expected to form dumbbell-shaped global structures (Herzberg & James, 1985). The 30% sequence dissimilarity observed constitutes the basis of distinct functional properties of the two TnC forms. One major difference between the two TnCs is in their actual Ca2' binding capability: cardiac TnC binds only three Ca21 ions (Leavis & Kraft, 1978) compared with skeletal TnC which binds four (Potter & Gergely, 1975). This observation is explained by the misplacement of two aspartic acid residues needed to co-ordinate the Ca2` ion in the first loop (site 1) in cardiac TnC. Such remarkable distinction in the Ca2+ binding property of site 1 is thus the first natural candidate in the length-sensing mechanism, a possibility investigated in the present study. Two types of experiments are described. In one, cardiac TnC-skeletal TnC exchange is performed in skinned fast-twitch fibres to measure any modifications of the length dependencies of Ca2" sensitivity specific to the TnC type present. The objective is to compare the results derived from fast fibres with results of similar earlier experiments using cardiac muscle (Babu, Sonnenblick & Gulati, 1988b), to establish whether the higher efficacy of cardiac TnC in the myocardium was a special

LENGTH-DEPENDENT Ca2+ SENSITIVITY IN MUSCLE

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effect in this system, mediated by the interactions of cardiac TnC with the cardiac milieu or, conversely, whether the length-sensitive domains in TnC produced their characteristic effects irrespective of the resident differences between skeletal and cardiac milieus. The latter possibility would both confirm and firmly establish a direct role of TnC in the length-sensing instrument within the sarcomere. In the second type of experiments, a mutated form of cardiac TnC is substituted in skinned cardiac trabeculae to study specifically the effects of changing the Ca2+ binding property of site 1. Aspartic acid residues were inserted in strategic positions of cardiac TnC (CMB1) to promote Ca2+ co-ordination in site 1 similar to that observed in skeletal TnC (Putkey, Sweeney & Campbell, 1989). CBM1 increased the slope of the pCa (- log [Ca2+])-force relation of slow fibres, a behaviour indeed expected of fast-type TnC. The CMB1 mutant, however, was surprisingly unable to regulate contraction in fast fibres. Here we investigate the effects of the improved Ca2+ binding property of CBM1 on contractility in cardiac muscle. The effect of this mutant on regulation and co-operativity has previously been tested both in slowtwitch fibres (Putkey et al. 1989) and in fast-twitch fibres (Gulati, Babu & Putkey, 1989). METHODS

The methods for the isolation and permeabilization of fibres were generally similar to those described earlier (Babu, Pemrick & Gulati, 1986; Babu, Scordilis. Sonnenblick & Gulati, 1987). Trabeculae from the right ventricle of hamsters were used as the cardiac muscle preparation (2 mm segment length, 50-60 ,tm wide). These manifest slack sarcomere lengths of about 1-9 ,tm in the relaxing solution (see below for solution composition). Hamster soleus muscle was used as the source of slow-twitch fibres. Rabbit and hamster psoas muscles were used as the fast-twitch fibre preparation (2-5 mm segment length, 50-150 ,tm wide). Fibre phenotype was checked in each experiment by characterizing Sr2+-activation curves (Babu et al. 1986). The identification of TnC and myosin light chain (LC) isoforms, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at the end of experiments, was used as confirmatory evidence of phenotype. Gels were silver stained with a modified BioRad procedure (Silver Stain Kit, BioRad, CA, USA), where a brief incubation with 5% glutaraldehyde (Oakley, Kirsch & Morris, 1980) was used for fixation (30 min). This modification of the kit protocol was found to be critical in the quantitative reproducibility of staining of the TnC bands (Babu et al.

1987). TnC extraction and reconstitution Native TnC was extracted from fibres by initially transferring briefly from the relaxing solution (see below) to the rigor solution (see below). A steady level of rigor force developed in this solution, following which the fibre was manually shortened by about 1 % of its length. The temperature was then raised to 28-30 °C, and the fibre was transferred to the extraction solution (see below, 28-30 °C) (Babu et al. 1987). Extraction times used varied from 5-12 min for fast-twitch fibres to 45-60 min for cardiac muscle. These times were considered critical with respect to the retention of other important factors within the fibre (Gulati & Babu, 1988). Higher salt solutions with longer extractions have been used successfully by others on fast fibres (e.g. Moss, Lauer, Giulian & Greaser, 1986), but the recovery of the myocardium has been somewhat problematic with this treatment (Hoar, Potter & Kerrick, 1988; Harrison & Bers, 1990). TnC reconstitution in cardiac and skeletal fibres was achieved in relaxing solution (see below) containing TnC, the requisite mutant, or purified LC2, at a concentration of 1 mg/ml, over 30 min intervals, at 20 °C (hamster), or 5 °C (rabbit). Once fast-twitch fibres were reconstituted they were generally maintained in a bathing solution containing a total ionic strength of 200 mm. When fast-twitch fibres were reconstituted with cardiac TnC, however, fibre tension development was enhanced in the activating solution containing

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100 mm salt (as discussed below). In these experiments rabbit fibres were used at 5 °C, rather than hamster fibres, because of their greater retention of contractility under these conditions. At 20 'C in a solution containing a salt concentration of 100 mm, fibres were not stable. As the temperature dependence of Ca2` sensitivity of skinned fibres varies with TnC phenotype (Harrison & Bers, 1990), control experiments were performed under each of the test conditions described. 100 _

a

0

u

75

-

50

000O0O

25

1

2

3

4

O0Oo5

5 6 7 8 Segment no.

9

10 11

12 13

Fig. 1. Uniformity of TnC extraction. Consecutive segments from the same fibre were used (7-8 mm long, 81 ,um wide). The TnC contents as indicated were normalized to the LC1 band in the same lane.

Experimental solutions Relaxing solution. This contained 20 mM-imidazole, 5 mM-ATP, 5 mM-EGTA, 20 mM-phosphocreatine. Potassium propionate was added to adjust the salt concentration to 200 mm, and enough MgCl2 to yield 1 mm free [Mg2+]. Activating solutions contained a total EGTA concentration of 5 mM. Ca2' additions required to establish the requisite pCa were calculated by standard procedures (Martell & Smith, 1974). [MgATP] and free [Mg2+] were maintained constant by the addition of the requisite amounts of ATP; pH was adjusted to 7-0 at the desired temperature of activation (see below). Rigor solution. This contained 165 mM-potassium propionate, 20 mM-imidazole, 2-5 mM-EGTA, 2-5 mM-EDTA, pH 7-2. Extraction solution. The solution used was a modified version of that described by Cox, Comte & Stein (1981) and Brandt, Diamond, Rutchik & Schachat (1987) such that very low salt concentrations were used, and EDTA was added. The final concentrations used were 10 mMimidazole and 5 mM-K3EDTA, pH 7-2. TnC extraction and reconstitution controls Extraction controls Sarcomere length determinations. Sarcomere length was monitored by first-order laser diffraction at various times throughout the experiment, under both activated and relaxed conditions. Experiments were continued only if sarcomere length was maintained during steady activations to within 01 ,um of the adjusted value. The sarcomere length range was 1-7-2-2 um in the initial experiments on psoas fibres, which covered a good deal of the ascending limb of the length-tension relation (see p. 44 of Woledge, Curtin & Homsher, 1985). Subsequently, when comparison was being made of the results between cardiac and skeletal muscles, the range employed was 1-9-2-4 ,um. The lower limit was set at 19 ,um using isolated trabeculae, because smaller segment lengths made visualization of sarcomere length below 1-9 ,tm difficult. Uniformity of TnC extraction along the fibre length. An unusually long fibre segment (7-8 mm) isolated from the rabbit psoas bundle was extracted to levels of 01 P. (resting fibre tension, see Results section). Subsequently the fibre was divided into twelve small segments about 0 5-0{7 mm long which then were individually processed using SDS-PAGE to reveal the uniformity of the residual TnC in all segments (Fig. 1) Differential silver staining of cardiac and skeletal TnCs. Since we have relied heavily on

LENGTH-DEPENDENT Ca2+ SENSITIVITY IN MUSCLE 60

309

/Cardiac TnC

C

40 -Skeletal TnC

20 CL

0

l

10

I

20 30 TnC (ng)

I

40

50

Fig. 2. Comparison of the silver staining of cardiac and skeletal TnCs in solution. Areas under the peaks of skeletal TnC and cardiac TnC bands in gel scans are compared over a 12-fold concentration range. Arrow marks the approximate amount of TnC in a fibre segment of 2 5 mm length, typical for gels. 0, free skeletal TnC; *, free cardiac Tnc; *, CBM2A; A, CBM1. Cardiac TnC (and CBM2A) stain 1-45 times more intensely than skeletal TnC (and CBM1).

SDS-polyacrylamide gels to quantitatively estimate the exchanged amounts of TnC in this study, it was necessary to establish the staining properties of cardiac, skeletal and mutant TnCs. This was done by loading various known amounts of purified TnCs on the same gel in different lanes. The amounts loaded were pre-determined by BioRad protein assay using bovine serum albumin as the standard. Cardiac TnC stained more intensely than skeletal TnC (Fig. 2) by a factor of 1-45 + 0-07 (n = 8 separate gels). This correction factor for cardiac TnC was applied throughout. The staining of mutant CBMI was similar to that of skeletal TnC; that of mutant CBM2A was similar to that of cardiac TnC. Reconstitution controls Uniformity of TnC reconstitution within the sarcomere. Force levels were measured at the sarcomere lengths of 1 7 and 2-2 ,um, and the observed force ratios (1 7/2-2 ,tm) in the native and reconstituted states were compared. On all reconstituted fibres used in the experiments below, the mean value of the force ratio was 0-65 + 0-02 (n = 13). In the native state the corresponding value was 0-68+0-02 (n = 6). These values are in good agreement with the 'theoretical' value on frog fibres (0 72 in Gordon, Huxley & Julian, 1966), and also, notably, with the previously reported value on untreated skinned rabbit fibres (0-65 in Allen & Moss, 1987). If the non-overlap region of the thin filament in the vicinity of the Z-line had remained devoid of restored TnC (the worst case possibility), a force ratio of 0 35 would have been observed. Completeness of cardiac TnC reconstitution in fast-twitch fibres. When fast-twitch fibres were reconstituted with cardiac TnC, fibre tension development (P.) was dependent both on the salt content of the bathing solution and on the extraction level prior to reconstitution (Fig. 3). Residual TnC, in this case, was monitored by the force response immediately following extraction in the experiments indicated. Fibres reconstituted with homologous skeletal TnC generated P. values of 0851-00. As indicated in Fig. 3 tension generated in these reconstituted fibres was markedly higher in a solution containing 100 mm salt than that in a 200 mm salt solution, at low levels of residual native TnC. Thus cardiac TnC in reconstituted fast fibres could be fully functional in 100 mM salt, but down-regulated at physiological salt concentrations under otherwise the same conditions (Babu et al. 1987; Gulati et al. 1989). As the tension developed at each salt concentration was tested in each fibre, in random order, and as the reconstitution technique is known to fully load extracted fibres (Fig. 4, see below), neither the possibility of (a) different degrees of loading nor (b) some interaction of salt in a partially loaded fibre can contribute to this observed salt dependence. Full occupancy of the vacated sites by cardiac TnC, and binding with affinity similar to that

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200 mM salt

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.

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0 Cl)

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0-1

0-2

0.3

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Tension with residual native TnC (PO)

Fig. 3. Effect of residual skeletal TnC and of salt on the effectiveness of cardiac TnC in fast-twitch fibre. Triangles, rabbit fibres; circles, hamster. Tension is expressed as the fraction of maximal tension in a fibre at the indicated ionic strengths. Vertical dashed line indicates the average residual TnC of fibres used in the present study of length dependence. manifest by skeletal TnC, is indicated also in Fig. 4. In addition, in 100 mm salt, reconstituted fibres manifest Sr2" sensitivity curves characteristic of cardiac TnC (Babu, Lehman & Gulati, 1989). If cardiac TnC reconstitution had been partial, the force level with maximal activation (with Sr2+ or Ca2+) would have remained submaximal in both low and high salt concentrations. The increase in Sr2+ sensitivity too would be incomplete, remaining at a point intermediate between the values characteristic of skeletal and cardiac TnCs. Finally, the addition of skeletal TnC to the medium also had no further effect on the TnC content or force generation of the cardiac TnC-loaded fibre, indicating that no vacant sites remained. In contrast to the results described above, however, Potter and co-workers in a recent study (Sheng, Strauss, Francois & Potter, 1990) find close to full force generation in fast-twitch fibres with CTnC reconstitution following apparent extraction to P. of 0-2, at ionic strengths of 200 mm. But caution is necessary in interpreting the Potter study. A fibre bundle (two to four fibres) was

TABLE 1. Relative intensities of TnCs in fibres with STnC-CTnC exchange estimated from runs on polyacrilamide gels (normalized to LC1 band in the same run) Fibre treatment STnC/LC1 CTnC/LC1 0-190+0-018 (+)STnC

(n = 5)

(100%)

(+)CTnC

0-043 + 0004

0146 + 0.009*

(n = 5) (23%) (77%) STnC, skeletal TnC; CTnC, cardiac TnC; LC1, myosin light chain 1. * Corrected for intensity ratio of CTnC to STnC (1-45, see Fig. 2). STnC/LC1 for native segments was 0-172+0-010 (n = 6).

LENiSGTH-_DEPENDENT Ca2+ SENiSITIVITY IN MUSCLE 0 C

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50

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Percentage CTnC in reconstitution solution

Fig 4. Cardiac TnC exchange in fast-twitch fibre. A, 15% acrylamide gel, silver stained. (+ )STnC, fibre extracted and reloaded with skeletal TnC; (+ )CTnC, reloaded with cardiac TnC. The densitometric scans for the bottom part of the gel are shown. To evaluate the extent of CTnC exchange, the area under the CTnC band in lane 2 was compared with the STnC band in lane 1 after proper normalization and correction by the factor of 1-45. With these manipulations. the CTnC intensity was found to equal the difference in the STnC bands in lanes 1 and 2. B, competition between the TnC isoforms for binding. Fibres were extracted by the standard procedure described in the text. For reconstitution, different fibres were incubated in solutions with mixtures of CTnC and STnC, and the uptake of each isoform was evaluated from the gels. Note that each data point is from a separate gel lane on a particular fibre segment. Also, the data for cardiac TnC intensity are modified by 1-45. LCI-3, myosin light chains 1-3.

used, and sarcomere length was not directly monitored at any stage of the experiment. Both fibre heterogeneity and lack of control of fibre slippage through attached ends could thus contribute to their observations. Wherever appropriate, data are given as means ± S.E.M. with the number of independent

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J. GULATTI, E. SONNENBLICK AND A. BABU

measurements included within parentheses. Statistical determinations of the significance of the data were made routinely with Student's t test. In a number of experiments activated fast-twitch fibres were also subjected to the release-stretch steps as a further precaution to maintain the sarcomere uniformity (Brenner, 1985; Gulati & Babu, 1986). The experiments with mutants in this study were completed by December 1989. RESULTS

Length dependence of pCa-force relation in fast fibres with TnC exchange In fibres initially extracted to 0-15Po (±002, n = 12), and reconstituted with either skeletal or cardiac forms of TnC, the pCa-force relationships changed markedly when sarcomere length was changed from 2-2 to 1-7 ,um. (Fig. 5A). Both skeletal TnC and cardiac TnC curves were shifted to the left at the longer sarcomere length. The net increase in Ca2+ sensitivity (ApCa50, ApK) observed using an activation solution containing 100 mm salt (5 °C) was 014 (range, 009-X18) with skeletal TnC and 0-26 (range, 0 20-028) with cardiac TnC. The mean values, which include the results derived from all twelve fibres tested, are indicated in the inset bar diagram. Interestingly, when the length-induced increase in sensitivity with cardiac TnC is compared with that in fibres reconstituted with skeletal TnC, the response is nearly doubled. We have previously reported that conversely, in cardiac muscle, such length dependence decreased in the presence of skeletal TnC (Babu et al. 1988b). Hill coefficients (nH) were also evaluated from the slopes of the pCa-force curves by computer-derived least-squares fit of the Hill equation. These coefficients are indicated in Fig. 5B. The values were markedly higher at the shorter length (1 7 ,um) compared with those at 2-4 ,um. Similar results were reported by Martyn & Gordon (1988) in the 2 3-3-4 ,am length range. Finally, the length dependence of Ca2+ sensitivity in partially extracted fibres with no added TnC was studied as an additional control for cardiac TnC loading in fast fibres (Fig. 6). It was possible that, even though the force level generated by cardiac TnC-loaded fibres (after maximal activation) reached that of untreated fibres in a solution of 100 mm ionic strength, loaded TnC might not have contributed fully to the observed length dependence; that is, the agreement with results in cardiac TnCloaded trabeculae (Babu et al. 1988b) may have been fortuitous. Under such circumstances, one would expect the length dependence of the cardiac TnC-loaded fibre to be similar to that of the partially extracted fibre. The results on partially extracted fibres shown in Fig. 6 (first and second hatched bars in the left panel), however, indicate that length dependence was much reduced in the presence of vacant TnC sites, and/or associated structural changes. This result is opposite to that observed in cardiac TnC-loaded fibres (Fig. 5). This observation is, therefore, further indication that increased length dependence of Ca21 sensitivity with cardiac TnC is indeed a cardiac TnC effect.

pCa-force relationships of slow-twitch skeletal fibres The TnC of slow fibres is phenotypically homologous with the cardiac isoform (Wilkinson, 1980), unlike the skeletal fast-twitch isoform. Thus, it was worthwhile to compare the length-dependent Ca2+ sensitivities of slow fibres with cardiac muscle and with fast-twitch fibres. The results are shown in Fig. 7. In the sarcomere length

LENGTH-DEPENDENT O,a2+ SENS-ITIVITY IN MUSCLE

3313

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Fig. 5. A, pCa-force relations at two sarcomere lengths before and after cardiac TnC (CTnC)-skeletal TnC (STnC) exchange. The data points for pCa-force curves were all on the same fibre. The curves are the computer-generated least-square fits of the Hill equation. Long length, 22 ,m; short length. 1 7 ,um. The length-induced shifts in Ca2+ sensitivities are indicated by horizontal bars. These values are plotted in the inset for a number of fibres (n = 9 for STnC and 4 for CTnC). ApK is the difference in pCa50 values for the two lengths. Note that of the nine STnC fibres, six were native and the other three were treated as with CTnC except that STnC was reinstated after extraction. In every case the STnC fibre gave the length dependence one-half that with CTnC, 100 mm salt. 5 'C. B. the effect of length on Hill coefficient. The results are on twelve fibres in 100 mM salt.

of 1V9-2 4 ,um, slow fibres give results comparable to cardiac muscle. This is additional evidence that TnC phenotype is a critical determinant of the effects of length on Ca21 sensitivity in the present experiments.

range

Studies with genetically manipulated cardiac TnC on heart muscle We then began a series of experiments aimed at the identification of length-sensing molecular domains in TnC. This goal was first approached with the use of the mutant of cardiac TnC in which Ca2' binding to site 1 was restored; CBM1 (Putkey et al.

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0.15

1 °r a) C)

0

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O

Native

Partial extraction

Partial extraction

(+)CBM2A

(+)STnC)

0

Fig. 6. Length-induced Ca2+ sensitivity in moderately extracted fibres. Note the reduction in the length effect with partial TnC. The addition of inactive mutant CBM2A had no further effect. ApK (hatched bars) is the change in sensitivity (pCa50) when the length was increased from 1-7 to 2-2 /tm. Force is indicated by filled bars. 0.15

Native tissues

TnC exchange in myocardium

CY)

- 0.10

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Fig. 7. Length-induced Ca2+ sensitivity: a comparison. The data on three slow fibres are compared with other tissues (n = 5 for cardiac muscle; n = 4 each for psoas fibres of hamster and rabbit). The data on cardiac muscle with TnC exchange are also indicated (n = 5 for CTnC; n = 4 for STnC). 200 mm salt. 20 'C.

1989) (Fig. 8). A second mutant, CBM2A (Putkey et al. 1989) in which site 2 was inactivated (Fig. 8) was also used. Presumably, in this mutant therefore both sites 1 and 2 were non-functional. The quantitative substitution of CBM1 for native cardiac TnC and its restoration of Ca2+-activated force in cardiac muscle is indicated in Fig. 9. The length-induced shift in Ca2+ sensitivity after replacement of cardiac TnC with CBM1 in skinned trabeculae is indicated in Fig. 10. The resultant shift was intermediate between those observed after cardiac and skeletal TnC reconstitution. In this set of experiments (180 mm salt), the increase in Ca2+ sensitivity (ApCa50) at

LENGTH-DEPENDENT Ca2 SENSITIVITY IN MUSCLE

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the long length (2-4 ,im) was 0-05 with skeletal TnC and 0-16 with cardiac TnC. With CBM1, the mean shift was 0 09 pCa, which places the pCa-force curve about half-way between skeletal and cardiac TnCs. The difference in the CBM1 effect from the cardiac and skeletal isoforms is statistically significant (P < 0 001). Note also that Anchoring sites

Trigger sites

_

N

N 4I

|

~IJ

LJ

K LJ

_ I-J

LJ

N N

_

f

C

Cardiac TnC

C

Skeletal TnC

C

CBM1

C

CBM2A

Fig. 8. Diagrammatic representation of TnC isoforms and their calcium-binding properties. The four helix-loop-helix structures are indicated. Sites 1 and 2 are the Ca2+specific sites, and 3 and 4 the Ca2+-Mg2+ sites. The underlined regions for skeletal TnC indicate the approximate domains with sequence dissimilarities. Inactive sites that do not bind calcium are left vacant.

the control in Fig. 10 (the first bar) indicates the results with bacterially synthesized cardiac TnC (CTnC3, Putkey et al. 1989); results similar to those observed using cardiac TnC. Thus any bacterial post-translational modifications of the protein did not alter the functions under investigation. The CBM1 derived results (Fig. 10) thus indicate that the Ca2+ binding to site 1 participates, in the determination of length dependence of Ca21 sensitivity: when Ca2+ ion is bound to site 1, the length dependence is suppressed.

Modifications of Sr2+ sensitivity and co-operativity in cardiac muscle with CBMI Because the restored metal ion binding to site 1 in CBM1 produced only half of the length response expected with skeletal TnC, it was of immediate interest to find out whether CBM1 was similarly a partial analogue of skeletal TnC in all its various functions concerning the Ca2+ switching of contraction in the myocardium, or whether each of the functions was separately controlled by a different set of molecular interactions within TnC. We tested this by investigating the effects of CBM1 on two other parameters that are characteristic of the TnC phenotype. (1) Cardiac TnC Sr21 sensitivity has previously been demonstrated to be 5- to 6-fold greater than that of skeletal TnC (Babu et al. 1987, 1989). (2) The skeletal TnC Hill coefficient for the pCa-force relation has previously been demonstrated to be higher than that manifest by cardiac TnC, indicating greater co-operativity in the skeletal Ca2+-activation mechanism (Moss et al. 1986; Gulati, Scordilis & Babu, 1988). The CBM1 results in cardiac muscle are shown in Figs 11 and 12. The maximal force levels generated (pCa 4 or pSr 3 5) were identical with cardiac TnC, skeletal TnC and CBM1.

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A

25 kN/m2 20

(+)CBM 1

(-)TnC

Native

s

B

-

..i

l

LC1

_

LC2 CTnC

-

-

1

CBM1

2 3

Fig. 9. Effect of genetically produced mutant of cardiac TnC on force recovery in skinned myocardium. A, force traces for maximal activation (pCa = 4) on a typical trabecula. Note that the maximal force after CBM1 reconstitution is the same as before extraction (180 mm salt; 20 °C). B, the gel is 12% acrylamide and silver stained on three different trabeculae. Lane 1, native; lane 2, TnC extracted; lane 3, CBM1 loaded. Segment 3 is that in force records; segments 1 and 2 are separate segments from the same heart. Note that CBMI band is indistinguishable from the TnC band.

' 0.15 a)

0.10 0

i-

-z

0.05

U)

Q 0

CTnC3

STnC

CBM 1

Fig. 10. CBM1-induced recovery of length-dependent Ca2l sensitivity in cardiac muscle (n = 7 for CBMt; n = 5 each for the other two bars). CTnC3 is bacterially synthesized cardiac TnC. 180 mm salt. 20 'C. Maximal force with pCa4 was indistinguishable in the three cases.

317

LENGTH-DEPENDENT Ca2+ SENSITIVITY IN MUSCLE 1.0 -4 4 pSr 5 ~~~~~~~~~~~~~~pSr

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Fig. 12. Comparison of Hill coefficients in cardiac muscle. The number of experiments was different in each case (n = 9 for CTnC3; n = 6 for STnC; n = 7 for CBM1). For further comparison, the Hill coefficient for fast fibres was 4-7 under the same conditions of 180 mM salt concentration (Gulati, 1990 b). The data for CBM1 were indistinguishable from STnC.

The effect of CBM1 replacement on the pSr-force relation of cardiac muscle is indicated in Fig. 11. The curve is shifted to the right, indicating a sensitivity lower than that observed with the native or cardiac TnC-loaded myocardial tissue preparation. The CBM1-induced shift, however, was identical to that found with skeletal TnC. This indicates that the control of Sr2+ sensitivity is fully restored on reintroducing the Ca2+ binding property in site 1 (see Fig. 8) in contrast to the length-

sensing mechanism.

318

J. GJLA TI, E. SOANNEANBLICK AND A. BABU

The Hill coefficients (from pCa-force relations) in cardiac muscle reconstituted with CBM1, CTnC3 and skeletal TnC are plotted in Fig. 12. The values of the Hill coefficients observed using CTnC3 and skeletal TnC were 3 5 and 4 7 respectively, in agreement with our previous results (Gulati et al. 1988). The value of the Hill coefficient using CBM1 was also 45, a value not different from the skeletal TnC generated value, but significantly higher than that generated by cardiac TnC. This CBM1 mutant result therefore extends the finding on slow fibres reported originally by Putkey et al. (1989): site 1 Ca2' binding in CBM1 completely explains its effect on co-operativity in cardiac muscle. Thus the difference between the Hill coefficients generated by cardiac and fast-skeletal TnCs (like the Sr2" sensitivity described above, but unlike the length-sensing effect) is fully explained by the additional metal ion binding ability of site t in skeletal TnC and, conversely, by its loss in cardiac TnC. Thus, the effectiveness of the length signal in modifying Ca21 sensitivity requires participation of both the residues which constitute the Ca2' binding site 1, together with those outside the loop region of the EF-hand.

Modification of the cardiac contraction-relaxation switch with inactive sites 1 and 2 When hamster trabeculae were reconstituted using CBM2A, a mutant in which both sites 1 and 2 are inactivated (Putkey et al. 1989), no force development was detectable at any Sr2+ concentration (inset of Fig. 11). This dramatic lack of a force response therefore extends the previous findings of blocked Ca2' activation manifest by this mutant in slow-twitch and fast-twitch fibres (Putkey et al. 1989; Gulati et al. 1989). To test the possibility that CBM2A might bind less firmly than the native protein we challenged trabeculae loaded with CBM2A by the addition of cardiac TnC in the bathing solution (CBM2A free). No further physiological effect was elicited, nor was the bound mutant released, as indicated by SDS-PAGE. This indicates that CBM2A binds at least as firmly as the native protein. Thus, as in skeletal fibres (Gulati et al. 1989; Putkey et al. 1989), sites 3 and 4 are the main anchoring sites for the Ca21 switch. The observations described above of the function of cardiac muscle reconstituted with CBM2A also have an additional significance. The present results show that the inactivity of CBM2A is not associated with previously established differences in Tnls (troponin Is). The cardiac isoform of TnI has previously been demonstrated to manifest an additional 28-residue segment at the N-terminus, absent in skeletal fast and slow fibres (Perry, 1979). Thus the 28-residue segment is probably situated outside the domain critical for the target action between TnCs and cardiac TnI.

Length dependence of Ca21 sensitivity in partially extracted fibres loaded with CBM2A Earlier in this paper, in Fig. 6 (second hatched bar), we showed that the contractile system was less responsive to sarcomere length when 50 % or more of the TnC was extracted. We now consider the alternatives that either the extracted system is modified by site vacancy per se on the thin filament, or that 'partial' activation, and/or Ca2"-bound TnC is/are key factors. The use of the CBM2A mutant addresses these alternatives directly as its use promotes vacant site occupation without affecting the degree of activation. The additional results plotted in Fig. 6 indicate

LENGTH-DEPENVDENT Ca2+ SENSITIVITY IN MU,SCLE

319

that the length-induced shift in Ca21 sensitivity was the same with and without CBM2A. Thus the second alternative must prevail, that is the lack of appropriately Ca2+-bound TnC forms appear instrumental in shifting the activation characteristics of cardiac muscle. DISCUSSION

Two principal findings are reported in the present study. Firstly, we show that substitution of cardiac TnC for fast skeletal TnC increases the length-induced Ca2+ sensitivity of fast-twitch fibres. This complements our previous demonstration that substitution of skeletal TnC in skinned cardiac trabeculae decreased the length effect (Babu et al. 1988b). The results thus provide firm evidence that the characteristic length dependence of cardiac muscle is specified intrinsically by a particular domain of the TnC moiety, and that the expression of this effect by cardiac TnC can occur similarly in both cardiac and skeletal milieus. Secondly, efforts to identify the length-sensitive domains in TnC were initiated with the use of cardiac TnC mutants constructed using site-specific mutagenesis. When the Ca2+ co-ordination ability of site 1 was restored (CBM1, Putkey et al. 1989), the length-induced shift in Ca2+ sensitivity was readjusted midway between the values characteristic of skeletal and cardiac TnCs. These results further support the role of TnC in the length-sensing instrument, previously indicated by skeletal TnC exchange experiments (Babu et al. 1988b), and show that the length signal for Ca2+ sensitivity is linked to the metal ion binding property of site 1 in TnC. Whether the partial recovery in the length-induced shift in Ca2+ sensitivity of trabeculae containing CBM1, with respect to those containing cardiac TnC, reflects the additional contribution of residues outside the Ca2+ binding loop, or whether it is a seconday effect of site-directed mutation, however, remains to be determined. Interestingly, by contrast, such exchange of CBM1 for cardiac TnC fully restored the S2'-activation curve and Hill coefficient of the pCa-force relationship to values characteristic of skeletal TnC, indicating that, unlike the length dependence of Ca2+ sensitivity described above, these properties are precisely controlled by the metal ion binding properties of site 1 (see Note added in proof).

Length-sensing mechanism: additional implications for Starling's law of the heart The instantaneous ability of the heart to adjust its function on a beat-to-beat basis, as predicated by Starling's law (Frank, 1895; Starling, 1918), depends ultimately on a responsive length-sensing element in the cardiac sarcomere (Jewell, 1977; Allen & Kentish, 1985). The definition of the molecular basis of the lengthsensing mechanism, as described above, is now being addressed (Gulati, 1990a). The present results of TnC exchange in fast-twitch fibres, coupled with the characterization of the function of TnC mutants indicate that the metal ion binding properties of the regulatory sites of TnC are critical both in the switching mechanism of the contraction-relaxation process as well as in the modulation of the length dependence of Ca2+ sensitivity in the myocardium such as to promote the Starling mechanism. 45Ca uptake in skinned fibres has also previously been demonstrated to be modified in the 1P8-244tm range (Hofmann & Fuchs, 1988), an additional indication of the

J. G lILA TI, E. SONVNENSBLICK AND A. BABU 320 effects of length on the Ca2+-TnC reaction. The results of the present study extend these results and raise the further question as to whether Ca2+-TnC reactions (at sites 1 and 2) are sensitive to length directly, or whether some length-derived alteration in filament geometry is involved, as an intermediate step (see also Allen & Kentish, 1988). Possible alteration in cross-bridge number with length has often been mentioned as one such intermediate step (Allen & Kentish, 1985). Such variation in cross-bridge population could contribute to the transduction of the length signal to the TnC molecule, since the fall in tension at the shorter sarcomere length probably reflects a decreased number of attachments in the force-producing configuration. Certainly, it has been long known that attached bridges (especially in their rigor form) can cause 'thin-filament potentiation' (Bremel & Weber, 1972). Similarly, there are recent indications that both rigor and active cross-bridges modify the fluorescent properties of skeletal TnC, indicating some functional modification of this moiety (Guth & Potter, 1987; Gordon, Ridgeway, Yates & Allen, 1988). Such a mechanism appears of major importance in explaining the hysteresis loop of contraction (Gordon & Ridgeway, 1987; Harrison, Lamont & Miller, 1988; Babu & Gulati, 1989), as well as the related phenomenon of Ca2' release from binding sites on myofilaments (Allen & Kentish, 1988; Sys & Brutsaert, 1989) in skinned fibres. However, it seems unlikely that variation in the cross-bridge population provides a primary mechanism with which to explain Starling's law because: (1) the decrease in the number of cycling cross-bridges at the short length is relatively small, if any (5-15 %; Kentish, ter Keurs & Allen, 1988; 0% in Stephenson, Stewart & Wilson, 1989), (2) stretching the length beyond 24,um continues to increase Ca2+ sensitivity (Endo, 1972; Fabiato & Fabiato, 1978), even as cross-bridge number decreases with partial filament overlap. This observation might have been explained by invoking separate mechanisms in the two length regions. Data reported by Gulati & Babu (1990), however, argue against this alternative possibility. Sarcomere length also affects interfilament separation (Matsubara & Millman, 1974), which could conceivably alter TnC by modifying, for instance, its charge distribution (Naylor, Bartels, Bridgman & Elliott, 1985). Whether this may be important in Starling's law, however, remains in question, because the change in filament separation with length is diminished in permeabilized cells (Matsubara & Elliott, 1972) but the length-induced effects on force development appear to be similar in both permeabilized and intact cells. Alternatively, however, some third filament spanning the entire sarcomere may gauge sarcomere length change and may communicate the information to TnC (Babu et al. 1988b; see also Winegrad, 1984). Connectin (also known as titin) is the only known filament joining the Z-lines at the opposite ends of the sarcomere (Wang, 1984; Maruyama, 1986). It thus may be considered a contender for the lengthsensing role. Finally, the effect of the intrinsic polarity of the thin filament on TnC, may also be important in the length dependence of Ca2+ sensitivity. The polarity of S-1 decorated F-actin is commonly visualized under the electron microscope in the form of arrow-heads directed towards the Z-line (see p. 12 of Woledge et al. 1985). In addition, motility assays with free floating F-actin filaments and S-1 heads similarly indicate a strictly unidirectional motion (Toyoshima, Toyoshima & Spudich, 1989).

LENGTH-DEPENDENT Ca2 SENSITIVITY IN MUSCLE

321

Thus, if actin filaments are indeed polarized in the cell, the Ca2' affinity of TnC could possibly increase progressively towards the sarcomere centre. If so, the polarization of actin filaments could conceivably make an important contribution to the Starling mechanism. Relation to earlier studies and the role of four sites in TnC The present studies indicate a second role for TnC, one additional to its previously established function as the contraction-relaxation switch in striated muscle, length sensing. Specifically, both sites 1 and 2 contribute to TnC's length-sensing function in addition to satisfying their previously assigned role as the regulatory sites in the switching mechanism. Studies in which the various natural TnC isoforms (Babu et al. 1989) and TnC mutants (Putkey et al. 1989) were used in skinned fibres have definitively resolved these individual roles. The contraction-relaxation trigger role was first assigned to sites 1 and 2 in skeletal TnC by the finding that the low affinity for Ca2+ binding to these sites (in comparison with the higher affinity for sites 3 and 4 at the C-terminus) was comparable to the overall Ca2+ regulation of actomyosin ATPase (Potter & Gergely, 1975). This was true also for cardiac TnC with the exception that metal ion binding to site 1 was blocked (Leavis & Kraft, 1978), presumably rendering cardiac site 1 'inactive'. Thus cardiac TnC site 2 alone appeared to perform the trigger role. The hypothesis that site 1 modulates the action of site 2 in skeletal TnC is now derived from observations of the effects of TnC exchange in cardiac muscle, in which the Sr2+ activation properties of skeletal and cardiac TnCs were compared (Babu et al. 1987, 1989). The studies described above in which the CMB1 mutant was used provide important support for the hypothesis. The results of experiments in which the CBM2A mutant was used (p. 14; Putkey et al. 1989; Gulati, et al. 1989), support the earlier view that sites 3 and 4 are critical for the binding of TnC to Tnl (Potter & Gergely, 1975; Babu, Orr & Gulati, 1988a).

Co-operativity in the Ca2+-force relations and muscle relaxation Length shifted the co-operativity in the Ca2+-TnC reaction, as indicated by the observation that Ca2+-force relations were steeper at 1-7 than at 2-2 gm (Fig. 5B). This point has further implications with respect to muscle relaxation, in so far as the process is influenced by Ca2+ release from the thin filament. Because of the cooperativity between sites within each TnC on the one hand and that between individual TnC molecules on the other, the release of the first few ions would be expected to promote subsequent release and induce higher co-operativity, to result in faster relaxation. Interestingly, this has actually been observed in cardiac muscle when rates of the fall of force following twitch contraction were compared at different lengths (Sys & Brutsaert, 1989). The data generated indicated that the rate of terminal relaxation was significantly higher with only a 10% reduction in length. Note added in proof. The cardiac muscle results on Sr2+-activation with CBM1 (present Fig. 11; also, Babu, Gulati & Putkey, 1989) have now also been reproduced on the slow-twitch fibre (Fig. 4 in Sweeney, Brito, Rosevear & Putkey, 1990). The combined results further indicate that the modulatory role of site 1 is independent of cardiac/skeletal isoforms of TnI and TnT. 11

PHY 441

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J. GULA TI, E. SONNEATBLICK AND A. BAB U

We dedicate this paper to the memory of a good friend Ichiro Matsubara (Tohoku University, Japan) for his important work in Muscle and Cardiac Physiology. We thank John Putkey (University of Texas Medical School) for the mutants, Hong Su for her highly helpful technical assistance, and Barbara Rayson (Cornell University Medical College) for her critical reading of the manuscript. We also thank our colleague John Russell for his generous help throughout. Grant support for J. G. was from NIH (AR33736) and the New York Heart Foundation. REFERENCES

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