Evidence That the Sr2+ Activation Properties of Cardiac Troponin C ...

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Troponin C (TnC) was extracted from skinned skel- etal muscle fibers by a method similar to that used previously on myofibrils (Zot, H. G., and Potter, J. D..
THEJOURNAL OF B~OLOGICAL CHEMISTXY 0 1985 hy The American Suciity of Biological Chemists, IRC.

VoL 260, No. 29,Issue of December 15, pp. 15687-15693.1985 Printed in U S A .

Evidence That the Sr2+Activation Properties of Cardiac TroponinC Are Altered When Substituted into Skinned Skeletal Muscle Fibers* (Received for publication, November 5,1984, and inrevised form, July 12,1985)

W. Glenn L. Kerrick$§, HenryG . Zotg, Phyllis E. Hoar$, and James D. Potter8 From the ~ e p a ~ m e noft s~ P h y s i o ~ and a g ~Siophysics and $Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

Troponin C (TnC) was extractedfrom skinnedskel- the troponin subunits (Potter and Gergely, 1975). However, etal muscle fibers by a method similar to that used preliminary results suggest that Ca2+ binding to the Ca2+previously on myofibrils (Zot,H. G., and Potter, J. D. specific sites in the Tn.Tm.actin complex has similar prop(1982) J. BioL Chem. 257, 7678-7683) and replaced erties as in TnC alone, suggestingfurther modification of the with eitherskeletal (fast-twitch)or cardiac TnC. The Ca2+binding properties by actin andfor tropomyosin (Zot et relationship between isometric tension and Srz+con- al,, 1983). The above results are supported by the fact that centration remained essentially the same before re- the Ca2+ sensitivity of tension activation in skinned fastmoval and after replacement with skeletal or cardiac twitch muscle fibers is the same as the Caz+ dependenceof TnC. Therefore, the origin of the TnC made differno ence in the Sr2+activation properties of the skinned Ca2+binding to theTnC rather thanto whole Tn (Kerrick et al., 1977).Recentevidencesuggests that Ca2+binding to fiber. In contrast, the activation of skinned cardiac troponin affects a kinetic step in the hydrolysis of ATP by fibers is approximately an order of magnitude more myosin subfragment 1 (Chalovich et al., 1981),which in turn sensitive to Sr2+ than skinned skeletal fibers. These results show that theaffinity of cardiac TnC for Srz+ would suggest that subfragment 1 binding to actin may affect is altered when substituted into skinned skeletal mus-binding of Ca2+to troponin. Two types of evidence exist suggesting myosin interactions with actin can affect Ca2+ cle fibers through protein-protein interactions. binding to troponin. Rigor complexes in myofibrils increase the Ca2+affinity for troponin (Bremel and Weber, 1972),and in the presence of rigor, glycerinated skeletal muscle fibers The protein primarily responsible for the Ca" activation are reported to bind more Ca" than in its absence (Fuchs of striated muscle contraction is troponin (Ebashi et al., 1968) and Fox, 1982). The question we wished to answer in thisstudy is whether which is made up of three subunits (Greaser and Gergely, substitution of cardiac TnC for fast-twitch skeletal TnC in 1971).The Ca2+-bindingsubunit TnCl binds 4 mol of Ca" in fast-twitch skeletal muscle (Potter and Gergely, 1975) and 3 skinned skeletal muscle fibers couldaffect the S?+ activation mol of Ca2+in cardiac (Potter et al.,1977; Leavis and Kraft, properties of cardiac TnC. Cardiac troponin has been shown 1978; Holroyde, et al., 1980).The amino acid sequencesof the to have a binding constant for Sr2+approximately 30 times two TnCs show a high degree of homology (Van Eerd and that of skeletal troponin (Ebashi et al., 1968). Other studies Takahashi, 1976).The loss of one Ca2+-bindingsite in cardiac have shown that cardiac skinned fibers are activated by 10TnC probably occurs in region I due to critical amino acid fold lower S P concentrations than skinned skeletal muscle fibers (Kerrick et al., 1980). It was also shown that when the substitutions in this site. Both proteins contain twohigh affinity Ca2+-Mg2+sites, the occupancy of which is probably native Tn. Tmcomplex from cardiac or skeletal muscle was responsible for the structural integrity of the troponin com- used to regulate a purified skeletal muscle acto-HMM system, plex (Zot and Potter,1982). Ca2+ binding to thelower affinity the S?+ dependence of ATPase activation was the same for it for the skeletal Tn Tm complex Ca2+-specificsites is believed to be responsible for the regu- the cardiac Tn .Tm as was lation of muscle contraction (Potter and Gergely, 1975; Hol- (Kerrick et al., 1980). We showin this paper that substitution royde et al., 1980). The Ca2+affinity of TnC in the whole of cardiac for skeletal TnC in skinned fast-twitch muscle troponin complex is greatly increased by the interaction of fibers causes the cardiac TnC to mimic the activation properties of skeletal muscle TnC. These results emphasize the * This work was supported by grantsfrom the Volusia-Flagler Area importance of myofibrillar protein-protein interactions in and Palm Beach County Florida Chapters of the American Heart determining the divalent binding properties of TnC.

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Association (P. E. H.) and from the American Heart Association and Muscular Dystrophy Association (W. G. L. K.) and by National Institutes of Health GrantHL22619-3A (J.D.P.) and National Institutes of Health training grant HL07188 (H. G. Z.). A preliminary report of this work was presented at the 27th Annual Meeting of the Biophysical Society (Kerrick, W. G. L., Zot, H. G., Hoar, P. E., and Potter, J. D. (1983) Biophys. J. 41,148a). 'The abbreviations used are: TnC, troponin-C; STnC, skeletal TnC;CTnC, cardiac TnC; Tn, troponin; Tm, tropomyosin; TnI, troponin-I; STnI, skeletal TnI; TnT, troponin-? STnT, skeletal TnT EGTA, ethylene glycol his(@-aminoethyl ether)-N,N,N',N'tetraacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; LC,, 18,000-Da lightchain of myosin; pSr, -log[Sr*+]; HMM, heavy meromyosin; Mops, 4-morpholinepropanesulfonic acid.

MATERIALS AND METHODS

M e ~ ~ r e m e ofn t Sr2+ and Caz+-aetiuated Tension-Functionally skinned fibers from cardiac and fast-skeletal (adductor magnus) muscles were prepared by the method of Kerrick and Krasner (1975). The functionally skinned fibers were mounted in stainlesssteel clamps and attached to a tension transducer similar to thatused by Hellam and Podolsky (1969). The fibers were then immersed into the various test relaxing and contracting solutions and the steady state tensions were measured. The tension data were collected as described by Kerrick et ak. (1980) for various Sf' or Ca2+concentrations and are expressed as a percentage of the maximum tension developed at the highest Sf+ or Ca2' concentrations in Figs. 5 and 6. The sarcoplasmic reticulum in the fibers was rendered nonfunctional by im-

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Alteration of Sr2' Activation Properties

of Cardiac Troponin C

mersing the fibers in a relaxing solution containing 1%Lubrol WX (Sigma) solution for 10 min. All tension measurements were carried out atroom temperature (21 "C) except as noted below. Solutions-AI1 activating (S?+ or Caz+)and relaxing solutions used for tension measure men^ contained 70 mM K* + Na+, 1.0 mM Mg+, 2.0mM MgATP2-, 7 mM EGTA, 15 mM creatine phosphate, and 15 units of creatine phosphokinase/ml. The ionic strength was set at EDTA 0.15 and the pH set at7.00 & 0.02 with imidazole propionate. The total concentrations of magnesium propionate, potassium propionate, I I , strontium or calcium propionate, disodium creatine phosphate, diso5 9 pCa 9 5 9 9 dium adenosine triphosphate, and imidazole required to make the TNC solutions were determined by a computer program with binding FIG. 2. Tension versus time records for single rabbit adducconstants for the various ionic species taken from the literature tor fibers showing responses after overnight treatment under (Donaldson and Kerrick, 1975). The calculated apparent binding EDTA (top)and control (bottom)conditions. These fibers were constants for Ca/EGTA and Sr/EGTA are 106.07and respec- chosen from groups of fibers treated under these conditions. Recovery tively. The Na+ added with phosphocreatine and adenosine triphos- of tension in theEDTA-treated fiber with skeletal TnC treatmentis phate was treated asKCby the computer program, since Na+ neither shown in the lower trace. Horizontal calibration bar, 1 min; vertical binds significantly to anyof the ligands nor affects tension measure- calibration bars, 9.5 mg (top) and 3.4 mg (bottom). ments differently than Kt (Kerrick and Krasner, 1975). The relaxing solution contained no added Ca" or Sr2+. The wash solution contained 40 mM Tris HC1, pH 7.0, and 25 mM relations hi^ between Sr2+concentration and tension was also deterKC1. The EDTA extraction solution contained 20 mM Tris, and 5 mined before and after the extraction process and after s u b ~ q u e n t mM EDTA, pH 7.8. The control incubationsolution contained 20 mM treatment with exogenous TnC. Groups of fibers at various stages of TnC extraction and replacement were dissolved in boiling SDS samTris HC1, pH 7.8. ple buffer (Kerrick et at., 1980) to be run on SDS-PAGE gels. Removal and ReDlacement of TroDonin C inSkinned FibersSDS-PAGE-The protein content of the skinned fibers was anaRemoval of TnC frdm skinned kbers was accomplished by a series of washes in low ionic strength EDTA solutionsby a method previously lyzed by SDS-polyacrylamide gel electrophoresis using 20-cm glass described (Cox et al. 1981) for the purification of TnC and subse- tubes (Zot and Potter, 1981). Densitometry scans of the SDS gels quently used for the removal and replacement of TnC in myofibrils were done using a Soft Laser Scanning Densitometer (Biomed In(Zot and Potter, 1982).Following the preparation of the skinned struments) (Zot and Potter, 1982). Purified Proteins-Skeletal TnC, Tm,and actin were purified from fibers, TnC was extracted either from fibers mounted in tension transducers or from groups of fibers suspended in the extraction an etherpowder made from rabbit back and leg muscle (Potter, 1982). solutions. Control fibers were prepared in a parallel manner in solu- Cardiac TnC was purified by the same method from bovine left tions not containing EDTA. All TnC extractions were done at 4 "C ventricle. Skeletal HMM was prepared from rabbit back muscle by since at higher temperatures the LC, light chainof myosin is removed chymotrypsin digestion of myosin in 0.6 M Kc1 and 1 mM CaCb and Pope, 1977). (Moss et al., 1982). For fibers extracted in a tension transducer, a (Weeds P r e ~ r a t i o nof ~ e g u ~ Actin-~oponin ~ed was ~ c o n s t i ~ t from ed control contraction was obtained and the fiber transferred to the purified subunits by the method of Potter (1982). Two types of extraction solutionsfor the indicated times. Following the extraction troponin were prepared. The firstconsisted of skeletai the fibers were transferred to relaxing and contracting solutionsand reconstituted TnT, TnI, and TnC, and the second consisted of skeletal TnT, the fibertestedfor Ca2+ or Sr2+ sensitivity. Following this test skeletal TnI, and cardiac TnC. The subunits were combined in an contraction, the fiber was then transferred to a relaxing solution equimolar ratio. The reconstituted troponin complexes were sepacontaining eithercardiac or skeletalTnC (I mg/ml) for the indicated rated from uncomplexed subunits by chromatography on G-150. times at 21 "C. The fibers were again contracted in Ca" or S?+ to Regulated actin was prepared from purified skeletal actin, skeletal determine their sensitivity. Fig. 1shows Caz+-activatedcontractions Tm, and either troponin complex. The components were combined, for fibers which undergo control or EDTA extraction protocols fol- respectively, in a 7:l:l molar ratio. The unbound soluble proteins lowed by treatment with STnC. For fibers extracted inbulk suspen- were separated by centrifugation at 100,000 X g for 2 h. The pellets sions, the fibers were extracted in the low ionic strength EDTA were resuspended and constituted the regulated actin. All steps were solutions overnightat 4 "C. Control fibers were extracted in a similar carried out at 4 "C. solution not containing EDTA. Fibers were then mounted in tension Measurement of Acto-HMM ATPase-One unit of regulated actin transducers and tested for Ca2+or Sr2+sensitivity. Fibers were next is defined as seven actin monomers, one Tm, and one T n in a complex transferred to relaxing solutions containingthe appropriate TnCs for of Mr 432,000. Both types of regulated actin, that regulated with each varying lengths of time andagain t e s M for Caz+or S?+ ~ n s i t i v i ~ .type of Tn complex, were tested for their ability to activate HMM Fig. 2 shows the protocol for Ca'+-activated contractions of EDTA- ATPase with Srz+. treated andcontrol fibers before and after treatment with STnC. The An assay tube was prepared for each Srz" addition. Each tube contained 1.0 nmol of regulated actin in 120 mM Mops, pH 7.0, 68 mMK", 5 mM Na+, 15 mM Cl-, 0.7 mM N3, 2 mM MgATP, 1 mM Mg2" (free), 2 mM EDTA, the ionic strength estimated to be 0.08 M. Addit,ion of SrCI2 was made to eachtube in order to achieve a predetermined free Sr2+ concentration. (These solutions are called low ionic strength solutions elsewhere in this manuscript.) The reaction was started with the addition of 2 nmol of HMM to theassay mixture and incubated at 25 "C. At 20 min after the HMMaddition, the reaction was stopped by adding an equal volume of the reagent used for the determination of inorganic phosphate (Pi). KC1 was used to bring ionic strength to 0.15 M for high ionic strength measurements. The Pi released by the hydrolysis of ATP was determined by r 1 r + I t z t 94 9 9 M Q 9 PC0 93*9 Wash control method of Piper and Lovell(1981). From the Pi released, the rateof TNC ATP hydrolysis and specifk activity were calculated. In the low ionic 21 nsn .si Wfn strength solutions, the minimum and maximum specific activities, in FIG. 1. Tension versus time recordshowing removal of en- pmol of Pi/pmol of actinlmin, for STnT .STnI. STnC-regulated actin dogenous TnC and its replacement with purified skeletal TnC averaged 6.6 and 13.8, respectively, and for STnT.STnI-CTnCregulated actin were 7.2 and 11.0, respectively. In the high ionic (top) in a single rabbit adductor fiber mounted in a tension transducerand acontrolfiber (bottom). See Materials and strength solutions, the minimum and maximum specific activities, in Methods" for wash, EDTA, control, and TnCsolutions. All solutions pmol of Pilpmol of actin/min, for STnT .STnI STnC-regulated actin were standard relaxing or contracting solutions except the above- averaged 5.1 and 7.0, respectively. The per cent maximum ATPase mentioned solutions. Horizontal calibration bar, 1 min; vertical cali- was calculated from the ratio of the increase in activity over basal to the totalchange in activity. bration bars, 9.5 mg (top) and 6.8 mg (bottom). I

I

.

.

. *

I

9

,

Alteration of Sr2+Activation Properties RESULTS

SDS-PAGE gels were run with samples taken from groups of control and EDTA-treated fibers and from the extracts comprising the supernatantsfrom these extracted fibers. The supernatants of control and EDTA-treated fibers show several prominent bands on SDS gels (Fig. 3). A major band that comigrates with purified rabbit skeletal TnC is visible in the EDTA extract (Fig. 3, lane B ) ;a weak corresponding band in the control supernatant (Fig. 3, lane A ) may be the result of a small amount of TnC or whole troponin solubilized under control conditions. Although several other major proteins are present in both supernatants, as shown in Fig. 3, lanes A and B, the major difference resulting from the two treatments is in the presence of the prominant TnC band of the EDTA extract and the near absence of this band in the control supernatant, which confirms that solubilization of TnC is the primary action of EDTA as previously reported for myofibrils prepared from rabbit back muscle (Zot and Potter, 1982). A faint band which co-migrates with LC2 standard was found in the EDTA extract andnot in the control supernatant (Fig. 3). This is consistent with the myofibril results (Zot and Potter, 1982) and a report showing that LC2 is extensively extracted at higher temperatures (25 "C) (Moss et al., 1982) than used here. The band corresponding to TnC in the gel of the fibers after extraction with EDTA appears to be much reduced in comparison to fibers treated under control conditions (Fig. 4, lanes F and I ) . A densitometric scan of the control and EDTAtreated fibers in Fig. 4 shows that 26-3296 of the control level of TnC remains in the fiber after extraction with EDTA (Table I). Groups of EDTA-treated (extracted) and control fibers were incubated with purified skeletal or cardiac TnC and extensively washed to remove any unbound TnC. The added skeletal TnC appears to restore the intensity to this band in fibers previously extracted with EDTA (Fig. 4, lane E). The densitometric scan shows that added skeletal TnC fully restores the amountof TnC in extracted fibers to control levels while no change in the contentof TnC in control fibers incubated with added skeletal TnC is observed (Table I). EDTA-treated fibers that were incubated with cardiac TnC demonstrated an additional band between the endogenous TnC and LC2(Fig. 4, lane D). The standard containing skeletal TnC, cardiac TnC, and total myosin light chains (Fig. 4) shows that cardiac TnC does migrate between skeletal TnC and LC2, demonstrating that the extra band in lane D is cardiac TnC and the band that co-migrateswith skeletal TnC is the endogenous TnC remaining after treatmentof the fibers with EDTA (Fig. 4). A similar band for cardiac TnC is not observed in control fibers that were incubated with added cardiac TnC (Fig. 4, lane C). The sum of the peak areas for skeletal and cardiac TnCs brings the estimated total TnC content of EDTA-treated fibers that were incubated with cardiac TnC back to control levels (Table I). Single fibers that were selected from groups of EDTAtreated fibers initially exhibited as low as 0% (Fig. 2, bottom) of the final tension after subsequent incubation with skeletal or cardiac TnC. This is in contrast to control fibers that produced approximatelythe same or somewhat highertension before incubation with TnC. No difference was noticed in the maximum tension restored by the cardiac uersus skeletal TnCs. The tension restored by the two types of TnC is consistent with the incorporation of exogenous TnC in the fibers treated with EDTA. Fig. 5 shows the relationship between the percentage of maximal S13"activated tension and pSr for a typical skinned fiber of skeletal muscle (pre-EDTA) and abundle of skinned

of Cardiac Troponin C ~

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.-

I@

-LC, -sTnC IcTnC

- LC,

A B C D E FIG.3. EDTA extract from skinned fibers. Groups of chemically skinned fibers were incubatedat 4 "C for12-14 h in the presence or absence of EDTA. The solvent conditions for control fibers were 20 mM Tris-HCI, pH 7.8. The other group of fibers were incubated in 2 mM EDTA. Tris, pH 7.8. The supernatants from each group were collected after a brief spin in a clinical centrifuge. The particulates that carried over in the Supernatant were removed by centrifugation at 20,000 rpm for 30 min ina Beckman JA 20 rotor. To each supernatant, 50% trichloroacetic acid was added to achieve a final concentration of 6% and allowed to incubate at 4 "C overnight. The precipitate was collected by centrifugation, resuspended in a small volume of water, and dialyzed exhaustively against water prior to a final dialysis in 1% SDS and 1% 8-mercaptoethanol. The samples were then placed into a sample buffer and run on SDS tube gels:lane A, the control-extracted supernatant; lane B, the EDTA-extracted supernatant; lane C, standards ofrabbit fast-twitch myosin light chains and STnC; lane D,lane C plus CTnC; lane E, lane C plus CTnC without STnC. Although the standards were run at the same time, the bands do not line up exactly with those of the supernatant samples. The identity of the bands in the supernatant samples were inferred by their location relative to each other and in comparison with the relative mobilities of the standards.

cardiac cells (control). As can be seen, cardiac skinned fibers require approximately 8.5 times less Sr2' than fast-twitch skinned skeletal muscle fibers. This suggests that the S?' affinity of the endogenous cardiac TnC in the cardiac fiber is much higherthan it is forthe endogenousTnC in the skinned skeletal fiber.

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TABLE I Effect of EDTA on the TnC content of skinned skeletal muscle fibers TnCb/TnI

Treatment'

Area

Control 96

TnCb/(LCI + LC*) Area

Control %

0.25 100 Control 0.50 100 0.46 92 0.26 104 Control STnC 96 0.24 Control + CTnC 0.50 100 EDTA 0.13 26 0.08 32 0.49 98 0.25 100 EDTA STnC 96 0.24' EDTA CTnC 84 0.42' Groups of fibers were incubated overnight at 4 'C in either 20 mM Tris, pH 7.8 (control), or 2 mM EDTA adjusted to pH 7.8 with Tris base (EDTA). Following several changes of relaxing solution (Fig. 4), the fibers were tested for tension development and prepared for SDS-PAGE. * The ratios of the peak areas for TnC were normalized to both TnI and myosin light chains in order to detect variations in the protein content of the thin and thick filaments due to the control and EDTA treatments. Includes peak area of residual endogenous TnC.

+

+ +

LC,

-

sTnC. cTnC LC,

RabbitAdductor 0

0

Pre-EDTA EDTA then C-TNC

R a b b i tH e a r t v Control

L%'

A B C D E

F

G

H

I

FIG. 4. Reconstitution of EDTA-extracted fibers with TnC. The fibers that were recovered from the treatmentsdescribed for Fig. 3 were washed five times with 10 pellet volumes of relaxing solution in order to remove unbound TnC. Aliquots of these fibers were likewise run on SDS tubegels: lane A, standards of rabbit fast-twitch myosin light chains, STnC, and CTnC; lane B, same as in lane A without STnC; lane C, same as in lane A, without CTnC; lane D, EDTA-extracted fibers incubated with CTnC; lane E, EDTA-extracted fibers incubated with STnC; lane F,EDTA-extracted fibers; lane G, control extractedfibers incubated with CTnC; lane H , control extracted fibers incubated with STnC; lane I , control extractedfibers. The standardswere run at thesame time with the fiber samples. The standards show the relative positions of STnC and CTnC to the myosin light chains and indicate that despite the similarity in molecular weight for STnC and CTnC, they can be separated by this gel system.

P*

FIG.5. The relationship between percentage of maximum tension and pSr for a single rabbit adductor magnusfiber (0, 0)and acontrol bundle of rabbit cardiaccells (V).The adductor fiber was first tested before EDTA treatment (0).After overnight treatment with EDTA (see "Materials and Methods"), the fiber did not contract inCa2+or Sr2+ activating solutions. Subsequent addition of cardiac TnC caused recovery of tension generating capacity (0). The slope ( n )and midpoint @K) were determined from a least squares fit of a linearized version of the Hill equation: T%/100 = [Ca]"/ V, n = 2.39,pK = 5.39; 0, n = 2.42,pK = 4.46; 0, ([Ca]" + n = 2.15, pK = 4.43.

related to the type of TnC (cardiac or skeletal). These data strongly suggest that theS I +binding or activation properties After removal of the endogenous skeletal TnC from a skinned aciductor fiber and replacement with fast-twitch skel- of TnC are not solely determined by the protein itself, but etal TnC, the relationship between the percentage of maxi- also by the protein-protein interactions in the cardiac and mum SI+-activated tension and pSr was restored approxi- skeletal muscle fibers. In order to check further our conclusions, reconstitution mately to itspre-EDTA values (Fig. 6). The maximum tension was also restored to at least 55% of its pre-EDTA level (Fig. experiments using skeletal acto-HMM, Tm, TnI, TnT, and 1). The incorporation of cardiac TnC into the skinned skeletal either skeletal or cardiac TnC were carried out. Fig. 7 shows fibers also restored the relationship between SI+-activated the results from these experiments. The relationship between tension and pSr to its pre-EDTA values (Fig. 5). Likewise, pSr and acto-HMM ATPase in the reconstituted system is maximum tension is restored to levels comparable to that similar regardless of whether skeletal or cardiac TnC is used obtained with skeletal muscle TnC, suggesting that the de- (Fig. 7). In contrast, the pSr tension relationships obtained crease in tension observed after reincorporation of TnC is not from control cardiac and skeletal muscle fibers werevery

Alteration of Sr2+Activation Properties of Cardiac Troponin C RabbitAdductor Pre-EDTA

0

pSr

FIG. 6 . The relationship between percentage of maximum tension and pSr for a single rabbit adductor magnus fiber. This fiber was tested before (0)and after overnight treatment with EDTA (see "Materials and Methods") followed by addition of exogenous skeletal TnC (W). After the EDTA treatment, the fiber was incapable of contraction in either Ca2+or Sr2+solutions. The slope (n)and midpoint (PK) were determined as described for Fig. 5 . 0 , n = 3.27,pK = 4.41; W, n = 2.15,pK = 4.55. S k e l e t a l AM-Tm-Tnl-TnT 0

E

S k e l e t a l TnC Cardiac TnC

looi

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(superprecipitation) to Ca2+and Sr2+mimicked the affinity ratios for isolated skeletal and cardiac Tn. For instance, a skeletal myosin B preparation (isolated actomyosin) regulated by skeletal T n - T m had a Sr2+/Ca2+sensitivity ratio of 1:23 while skeletal myosin B regulated by cardiac Tn.Tm had a Sr2+/Ca2+sensitivity ratio of1:3. These data suggested that the S?+/Ca2+ affinity ratio of Tn in the thinfilament is the same as in isolated Tn and regulation is purely a function of metal binding to Tn. More recently, Kerrick et al. (1980) have reported values for the Ca2+- and S?+-sensitive tension development of skinned fibers and theATPase of acto-HMM and myofibrils from rabbit fast-skeletaland cardiac muscles. While the Ca2+ sensitivities of cardiac and skeletal muscles were nearly the same, the cardiac muscle wasfound to be about 6.3-fold more sensitive to activation by S?+ than skeletal muscle. Skeletal acto-HMM had the lower S?+ sensitivity, typical of the Sr2+ activation of an entirely skeletalsystem, regardless of whether it was regulated by skeletal or cardiac TmaTn complexes. A recalculation of the datafrom Kerrick et al. (1980) shows that the Sr2+/Ca2+sensitivity ratios for regulation by skeletal Tm . Tn andcardiac Tm. Tncomplexes were 1:lO and 1:8, respectively. These data suggest that either the Sr2+/Ca2+affinity ratio or the process of activation isdifferent in cardiac muscle than when cardiac native Tn .Tmis reconstituted with skeletal acto-HMM. The cause for the discrepancy between the reports by Ebashi et al. (1968) and either our earlier work (Kerrick et aL, 1980) or our data in Fig. 7 is not immediately apparent. We show that skinned skeletal fibers treated with EDTA lose their ability to be activated by divalent cation, that the loss in activationis directly related to thedissociation of TnC from the fiber, and that either cardiac or skeletal TnCs are capable of restoring Ca2+-or Sr2+-dependent tension development to theextracted fibers to atleast 55% of control value. Thus, the loss in tension upon reconstitution does not have anything to do with the type of TnC substituted but rather with some irreversible effects of the extraction process. The sole difference between the reconstituted fibers is the tissue type of the metal-binding subunit (TnC) of the Tncomplex. In both cases, the activation by Sr2+after readdition of TnC occurred at approximately the same [S?'] as before EDTA treatment, indicating that theregulation of skeletal fibers by cardiac or skeletal TnC is the same. The Sr2+sensitivity of this hybrid fiber is consistent with the earlierreport by Kerrick et al. (1980) and supports the concept that the activation process itself plays a part in the affinity of TnC for divalent metals. This conclusion is strengthened by the data in Fig. 7 which show that the Sr2+concentration required for the activation of acto-HMM ATPase in a reconstituted skeletal acto-HMM/ Tm/TnI/TnT system is similar regardless of whether cardiac or skeletalTnC isused to confer S?+ activation. The concentration of S?+ required for the activation of acto-HMM ATPase by cardiac TnC (Fig. 7) is higher than theconcentration of Sr2+ required for the activation of cardiac skinned fibers (Fig. 5) but less than the Sr2+concentration required for the activation of contrblJ and cardiac TnC substituted skeletal muscle skinned fibers (Fig. 5). Also, the Sr2+concentration required for activation of acto-HMM ATPaseby skeletal TnC is lower than that required for control or reconstituted skeletal fibers. Thus, the protein environment affects the binding or activation properties of both cardiac and skeletalTnCs differently inthe reconstitutedskeletalactoHMM/Tm/TnI/TnT system than in the control cardiac or skeletal skinned fibers. Although the ATPase measurements

2oL 0

7

5

6

3

pSr

FIG. 7. The relationship between the percentage of maximum acto-€€" ATPase and pSr for a skeletal acto-HMM system containingskeletal Tm, TnI, TnT, and either skeletal TnC (0)or cardiac TnC (W). The slope (n)and midpoint (pK) were determined as described for Fig. 5 . 0 , n = 1.94, pK = 4.96; W, n = 1.92, pK = 5.19.

different, with cardiac fibers being 8.5 times more sensitive to S?+ than skeletal (Fig. 5). DISCUSSION

The various factors responsible for regulating the affinity of TnC for divalent cations arevery important to our understanding of the regulation of muscle contraction. It was previously shown that theaffinity of TnC for Ca" was approximately 10-fold higher in a TnC .TnI complex or in isolated Tn than in theisolated protein (Potter and Gergely, 1975). This difference in affinity holds true for both cardiac and skeletal TnC. Ebashi et al. (1968) reported that the S?+/Ca2+ affinity ratio of isolated skeletal T n is 1:27 while the Sr2+/Ca2+affinity ratio of isolated cardiac T n is only 1:3. Using hybrid preparations of skeletal and cardiac actomyosin, Ebashi et al. (1968) also reported that the sensitivity of the activation process

Alteration of Sr2+Activation Properties

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in Fig. 7 were done at a lower ionic strength than thetension measurements, it is not a problem because ionic strength has no noticeable effect upon the concentration of S1' required to activate the ATPase (Fig. 8). The ATPase measurements in Fig. 7 were done at low ionic strength because acto-HMM ATPase is very low at high ionic strength and toodifficult to measure accurately in the skeletal system in which cardiac TnC was substituted. There is evidence that theinteraction of the Tn-Tmcomplex with actin decreases the affinity of T n for Ca2+.Wnuk and co-workers (Wnuk and Stein, 1980; Wnuk et al., 1984) have reported that the binding constant for the single Ca2+binding site of crayfish isolated T n is an order of magnitude greater than that in thecrayfish actin. Tm Tncomplex. Another report for rabbit skeletal T n supports in principle the results with the Frayfish system (Zot et al., 1983). However, rabbit TnC is complicated by having four sites that are available for binding Ca2+.The preliminary results of Zot et al. (1983) suggest that only the lower affinity Ca2+-specific sites of T n are affected when T n is reconstituted into a rabbit skeletal actin.T n .Tm complex, and compared to isolated Tn, the actin. Tn-Tm complex exhibits a 8-10-fold decrease in affinity. The affinity at all four sites of the binary Tn. Tm complex from the rabbit skeletal system is indistinguishable from that in isolated T n (Zot et al., 1983), suggesting that the interaction with actin formed the basis for the decrease in affinity. Also, the Ca2+activation of skinned fibers parallels Caz+ binding to the Ca2+-specificsites of TnC rather than Ca2+ binding to whole troponin (Kerrick et al., 1977). A comparison of Sr2+-activated skeletal acto-HMMATPase regulated by either skeletalor cardiac native Tm Tn (Kerrick et al., 1980) with similar data inFig. 7 where cardiac TnC was substituted into an otherwise completely skeletal acto-HMM system suggests that the Sr2+binding to TnC in the muscle fiber is most likely altered by actin and/or myosin. Recent work (El-Saleh et al., 1984) shows that the modification of a single amino acid residue of actin can drastically affect the ability of the Tn. Tm complex to regulate acto-HMM ATPase. Thus, the known difference of four amino acids between cardiac and skeletal actin (Vandekerchove and Weber 1979) could affect the divalent cation sensitivity. Reports have appeared which suggest that myosin interacS k e l e t a l AM-Tm-Tnl-TnT-sTnC 0 Law I.S. A H l g h I.S.

100

5

80

E (1

P

..

60

0

al

-

40

c

e 0

a"

20

0

7

5

6

4

3

pSr

FIG. 8. The relationship between the percentage of maximum acto-HMM ATPase and pSr for a skeletal acto-€€" system (containing skeletal Tm, TnI, TnT, and TnC) at high (0.15 M) (A) and low (0.08 M) (0)ionic strengths. The curue is drawn through the low ionic strength data. The slope (n)and midpoint ( p K )were determined as described for Fig. 5 . A, n = 1.04, pK = 4.89; 0, n = 1.94, p K = 4.96.

of Cardiac Troponin

C

tion with the thin filament may affect the Ca2+sensitivity of an actomyosin system. Myofibrils in rigor or low ATP conditions have been shown to bind Ca2+with a greater affinity than in the presence of ATP (Bremel and Weber, 1972). Also, measurements of Ca2+binding to glycerinated fibers suggest an increase in total Ca2+binding in rigor as opposed to in the presence of ATP (Fuchs andFox, 1982). More recent studies, where myosin is hypothesized to be weaklybound to the thin filament in relaxed muscle rather than to be sterically blocked from interacting with actin by tropomyosin, suggest that Ca2+ binding to troponin may bring about a change in a kinetic step of the hydrolysis of ATP (Chalovich et al., 1981). Consequently, myosin bound to the thin filament could affect the affinity of troponin for divalent cations. It has been shown both for skeletal (Godt, 1974) and cardiac (Best et al., 1977) muscle that decreases in MgATP2- concentration in skinned fibers increase the sensitivity of the activation process for Ca2+.Recently, it was reported that adding MgADP to contracting solutions will cause an increase in both maximal Ca2+-activatedtension and the Ca2+sensitivity of tension in skinned skeletal fibers (Kerrick and Hoar, 1985). Therefore, the interaction of actin and myosin may also have an affect on the affinity of cardiac TnC for Sr2' and explain the differences in affinity observed when cardiac TnC is in an intact cardiac fiber versus when it issubstituted into skeletal a fiber. In summary, the data presented in this paper suggest that when cardiac TnC is substituted for the endogenous TnC of skinned skeletal muscle fibers, the Sr2+activation properties of cardiac muscle TnC arechanged to those of skeletal muscle TnC. The most likely explanation for this is that the kinetic interaction of the contractile and regulatory proteins of a particular fiber type strongly affect the divalent cation binding properties of TnC. REFERENCES Best, P. M., Donaldson, S. K. B., and Kerrick, W.G. L. (1977) J. Physiol. (Lond.) 265,l-17 Bremel, R. D., and Weber, A. (1972) Nature New Biol. 238,97-101 Chalovich, J. M., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256,575-578 Cox, J. A., Comte, M., and Stein, E. A. (1981) Biochem. J. 195,205211 Donaldson, S. K. B., and Kerrick, W.G. L. (1975) J. Gen. Physiol. 66,427-444 Ebashi, S., and Endo, M. (1968) Prog. Biophys. Mol. Bid. 18, 123183 Ebashi, S., Kodama, A., and Ebashi, F. (1968) J. Biochem. (Tokyo) 64,465-477 El-Saleh, S. C., Thieret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259,11014-11021 Fuchs, F., and Fox, C. (1982) Biochim. Biophys. Acta 679, 110-115 Godt, R. (1974) J. Gen. Physiol. 63, 722-739 Greaser, M. L., and Gergely, J. (1971) J. Bid.Chem. 246,4226-4233 Hellam, D. C., and Podolsky, R. J. (1969) J . Physiol. (Lond.) 200, 807-819 Holroyde, M. J., Robertson, S. P., Johnson, J. D., Solaro, R. J., and Potter, J. D. (1980) J. Biol. Chern. 255,11688-11693 Kerrick, W. G. L., and Krasner, B. (1975) J. Appl. Physiol. 39,10521055 Kerrick, W. G. L., and Hoar, P. E. (1985) Bwphys. J. 47,296a Kerrick, W. G. L., Hoar, P. E., Malencik, D. A., Pocinwong, W., Coby, R.L., and Fischer, E. H. (1977) Proceedings Third US-USSR Joint Symposium on Myocardial Metabolism, pp. 195-209 United States Department of Health, Education, and Welfare Publication NO(NIH) 78-1457 Kerrick, W. G. L., Malencik, D. A., Hoar, P. E., Potter, J. D., Coby, R. L., Pocinwong, S., and Fischer, E. H. (1980) Pfluegers Archiv. Eur. J. Physiol. 386,207-213 Leavis, P. C., and Kraft, E. L. (1978) Arch. Biochem. Biophys. 186, 411-415

Alteration of Sr2+Activation Pivperties of Cardiac Troponin C

15693

Moss, R L., Giulian, G. G., and Greaser, M.L. (1982) J. Bwl. Chem. Van Eerd, J. P., and Takahashi, K. (1976) Biochemistry 15, 11711180 257,8588-8591 Vandekerchove, J., and Weber, K. (1979) ~ i ~ e r e n t 14, ~ ~123i o ~ Piper, J. M., and Lovell, S. J. (1981) Anal; Bioclaem. 117,70-75 133 Potter, J. D. (19823 M e t ~ s E n z 85,241-263 ~mo~ Weeds, A. G., and Pope, B. (1977) J. MOLBbl. 111,129-157 Potter, J. D., and Gerggly, J. (1975) J. Biol. Chem. 250,46284633 Wnuk, W., and Stein, A. E. (1980) Deu. Biochem. 14,343-344 Potter, J. D.,Johnson, J. D., Dedran, J. R., Schreiber, W. E., Mandel, Wnuk, W., Schoechlin, M., and Stein, E. A. (1984) J. Biol. Chem. F., Jackson, R. L., and Means, A. R. (1977) in C a ~ i u m - B i n d i ~ 259,9017-9023 Proteins a d Calcium Function (Wasserman, R. H., Caradino, R. Zot, H. G., and Potter, J. D.(1981) Prep. Biochem. 11, 381-395 A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and Siegel, Zot, H. G., and Potter, J. D. (1982) J. BioL Chem. 257,7678-7683 F. L., eds) pp. 239-250, Elsevier/North-Holland, New York Zot, H. G., Iida, S., and Potter, J. D.(1983) Chem. Scr. 21,133-136