©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca Affinity of Cardiac Troponin C (*)

(Received for publication, July 21, 1995; and in revised form, October 7, 1995)

Ren Zhang JiaJu Zhao James D. Potter (§)

From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca affinity of the single Ca-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J.(1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D.(1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca dissociation with a reduced Ca sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.


INTRODUCTION

Several lines of evidence have led to a general understanding of how beta-agonist stimulation leads to positive inotropic and chronotropic effects by phosphorylation of cellular substrates through cAMP-dependent protein kinase (PKA)(^1)(1, 2) . It is generally agreed that phosphorylation of sarcolemmal Ca channels (3) and phospholamban(4, 5) , a sarcoplasmic reticular protein regulating the Ca pump, are responsible for the changes seen in the intracellular Ca transient. The resulting increases in intracellular Ca and the rate of Ca resequestration contribute to an increase in cardiac muscle contractility and to faster rates of force development and relaxation. PKA can also phosphorylate contractile machinery proteins, such as C-protein and cardiac troponin I (CTnI) (6, 7, 8, 9) . Many laboratories, including ours, have focused on the mechanism by which phosphorylation of CTnI may be involved in the inotropic effect brought about by beta-agonist stimulation.

Troponin I (TnI), a subunit of the troponin complex, inhibits the actomyosin ATPase activity when muscle is in the resting state. Binding of Ca to the low affinity site(s) of troponin C (TnC) releases the TnI inhibition on actomyosin ATPase through protein-protein interactions among the troponin complex, tropomyosin and actin, and leads to muscle contraction(10) . In comparison with skeletal TnI, CTnI has an additional 32-33 amino acids at its N terminus, and this segment also contains two adjacent serine residues at positions 22 and 23 or 23 and 24, depending on the species(11, 12, 13) . By converting the phosphoserine into the stable S-ethylcysteine derivative on bovine CTnI, Swiderek et al.(14, 15) demonstrated that these two serines were able to be fully phosphorylated by PKA. Biochemical studies have shown that CTnI can be phosphorylated to the level of 2 mol of phosphate/mol of protein (16) , consistent with the sequence prediction. Considerable effort has been made with either perfused hearts (17, 18) or isolated proteins (14, 15, 16) to determine the mechanism by which CTnI phosphorylation may affect cardiac muscle contraction. The decreased Ca sensitivity upon phosphorylation of CTnI has been observed in isolated cardiac myofibrils(8, 19) , in hyperpermeable cardiac fibers(20) , and in skinned cardiac muscle preparations and myocytes(21, 22) . Moreover, it has been shown that CTnI phosphorylation increases the rate of Ca dissociation from a reconstituted troponin complex(23) , suggesting the possible importance of this phosphorylation in modulating the rate of relaxation of cardiac muscle. Some reports, however, have shown that CTnI remains phosphorylated even after removal of beta-agonists and the return of muscle contraction to prestimulated basal levels(4, 19) . These results have suggested that phosphorylation of CTnI may not be as important as the phosphorylation of other proteins, such as phospholamban(4) . In a recent study we have clearly shown that CTnI phosphorylation is directly correlated with the faster muscle relaxation seen after PKA phosphorylation and is probably due to the decreased Ca affinity of CTnC and the consequent faster dissociation of Ca(22) . Our calculations also showed that although PKA phosphorylation of phospholamban is the predominant effector in the inotropic response, the rate of relaxation is significantly influenced by CTnI phosphorylation.

After many years of study there are still numerous questions regarding the mechanism of action of CTnI phosphorylation and of the physiological significance of this process. In the present study we have focussed on the role of the phosphorylation of the two CTnI serines in this process. In order to study the functional and physiological significance of phosphorylation of these two serine residues, we have used our cloned mouse CTnI cDNA to create three mutants of CTnI in which either serine 22 or 23, or both, are mutated into alanine(9) . Our results demonstrate that both serine residues in CTnI are able to be phosphorylated by PKA in vitro but that the rates of phosphorylation for these two serine residues are different. Using a reconstituted skinned cardiac muscle preparation (CSM), we have studied the effect of phosphorylation of each serine residue on the Ca dependence of force development. We show that phosphorylation of both serine residues by PKA is required for the decreased Ca-sensitivity of force development. Since the rate of phosphorylation is different, it is possible that the rate of dephosphorylation is different and may account for the slower dephosphorylation seen in intact systems (4) and suggests that one of the serines may be more or less constitutively phosphorylated with the other serine being functionally more important.


MATERIALS AND METHODS

In Situ Mutagenesis

Mouse CTnI cDNA was obtained by screening a mouse cardiac cDNA library (Clontech) using an oligonucleotide synthesized according to the published rat CTnI sequence(13, 24) . In situ mutagenesis of CTnI was performed using a T7-Gen(TM) in vitro mutagenesis kit (U. S. Biochemical Corp.) according to the protocol provided by the manufacturer. An oligonucleotide (5`-ATC AGC CAT GGT GAT CTC CAG -3`) was used to create an NcoI restriction site 5` to the translation start site, so that the NcoI and BamHI fragment containing the whole translational cDNA sequence could be subcloned into the pET expression vector (Novagen). The following three oligonucleotides were used to create three mutants: 1) 5`-TTG GCA GAG GCG CGG CGT CGG-3` for CTnI mutant, in which Ser-22 was changed to Ala-22; 2) 5`-TAG TTG GCA GCG GAG CGG CGT-3` for the CTnI mutant, where Ser 23 was changed to Ala 23; and 3) 5`-TAG TTG GCA GCG GCG CGG CGT CGG-3` was used to create the CTnI mutant, where both Ser-22 and Ser-23 were changed to Ala-22 and Ala-23.

Affinity Chromatography

Bovine CTnC was isolated as reported previously (25) and was used to prepare a CTnC affinity column. In brief, the protein (100 mg) was dissolved in coupling buffer (0.1 M NaHCO(3), pH 8.3, 0.5 M NaCl) and then mixed with 5 g of CNBr-activated Sepharose 4B (Sigma) equilibrated with 1 mM HCl. After 2 h of incubation at room temperature, the gel was transferred to a blocking solution (0.2 M glycine, pH 8.0) and incubated for 2 h at room temperature. The gel was then washed with coupling buffer and then a solution containing 0.1 M sodium acetate, pH 4.0, and 0.5 M NaCl, and finally by coupling buffer again. The Sepharose was then packed into a column and equilibrated with desired starting buffer.

Expression and Purification of Wild Type CTnI and the CTnI Mutants

The cDNAs from wild type CTnI and the mutants described above were excised from double-stranded M13mp18 DNA using the restriction enzymes NcoI and BamHI and subcloned into the expression vectors pET11d or pET3d (Novagen). The proteins were expressed in Escherichia coli BL21(DE3) (Novagen) using the protocol provided by the manufacturer. The expression was checked by 15% SDS-PAGE and Western blotting using a monoclonal anti-rabbit skeletal TnI antibody (made in Dr. Potter's laboratory). The culture for bacterial expression was collected and centrifuged at 7,000 rpm (JA-10, Beckman). The bacterial pellet was dissolved in a solution containing 6 M urea, 10 mM sodium citrate, pH 7.0, 1 mM DTT, 2 mM EDTA and sonicated (Sonicator, Heat Systems, Inc., Farmingdale, NY) twice at setting 8 for 2 min at 4 °C. After the sonication, the pH of the solution was adjusted to 5.0, and the mixture was centrifuged again at 18,000 rpm (JA-20, Beckman) at 4 °C for 30 min. The supernatant, containing the expressed TnI, was loaded onto a CM-52 ion-exchange column equilibrated with the same buffer used to dissolve the bacterial pellet, except that the pH was 5.0. The CTnI was eluted with a linear KCl gradient of 0-0.4 M in the equilibrating buffer. The fractions containing CTnI (identified by SDS-PAGE and Western blotting) were pooled together and dialyzed against 1 M NaCl, 50 mM Tris, pH 7.0, 2 mM CaCl(2), 1 mM DTT and loaded onto a TnC affinity column equilibrated with the same buffer. Pure TnI was eluted with a double gradient of urea (0-6 M) and EDTA (0-3 mM) in a solution containing 50 mM Tris, pH 7.0, 1 M NaCl, and 1 mM DTT. The resulting yield was 2-4 mg of pure CTnI/liter culture.

Time Course of Phosphorylation of Wild Type CTnI and the CTnI Mutants

The protocol for TnI phosphorylation was modified based on the protocol kindly provided by Dr. Evangelia Kranias, University of Cincinnati. Briefly, wild type CTnI or its mutants (1 µg of each) were phosphorylated in a solution containing 100 mM NaCl, 50 mM MgCl(2), 50 mM sodium phosphate, pH 6.8, 10 µM ATP, [-P]ATP (specific activity, 250-600 cpm/pmol) and the catalytic subunit of PKA (Sigma) 70 units/ml at 30 °C. For measuring maximal phosphate incorporation into the wild type or mutated TnIs, phosphorylation was carried out for 2 h and was terminated by adding 20 µg of bovine serum albumin as carrier for precipitation followed by trichloroacetic acid to 10% to precipitate the proteins. The precipitated proteins were washed again with a 10% trichloroacetic acid solution. The precipitated proteins were mixed with 20 ml of Cytoscint (ICN) and subjected to scintillation counting (LS 1801, Beckman). The phosphate incorporation was calculated according to the specific activity. In a parallel experiment to determine the time course of phosphorylation of CTnI, the phosphorylation reactions were terminated after 30 s, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, or 60 min with the addition of 1 volume of SDS sample buffer (2.0% SDS, 5 mM Tris-glycine, pH 6.8, 2% beta-mercaptethanol, 20% sucrose, 0.05% bromphenol blue) to each sample. Portions of these phosphorylation samples were electrophoresed on SDS-PAGE. The gels were dried and exposed to x-ray film, or the phosphorylated CTnI was cut from the gel, mixed with 20 ml of Cytoscint (ICN) and subjected to scintillation counting. The relative phosphate incorporation for the reactions stopped at different reaction times were calculated by comparing the radioactivity of these gel slices with a gel slice containing a fully phosphorylated TnI whose phosphate incorporation had been determined simultaneously by trichloroacetic acid precipitation as mentioned above.

TnCbulletTnI Complex Formation

A complex of TnC and TnI was formed by combining TnC and TnI in a molar ratio of 1:1.2 in a solution containing 6 M urea, 20 mM MOPS, pH 7.0, 0.5 mM CaCl(2), 1 M KCl, and 1 mM DTT. After dialyzing the protein mixtures against this solution for 2 h, the sample was then successively dialyzed against 1, 0.7, 0.4, 0.2, and 0.05 M KCl in a solution containing 20 mM MOPS, pH 7.0, 0.5 mM MgCl(2), and 1 mM DTT. Any excess TnI that precipitated was removed by centrifugation. The complex formation was confirmed by running the supernatant samples on SDS-PAGE.

Preparation of the Skinned Cardiac and Skeletal Muscle

Cardiac muscle was isolated from the left ventricle of porcine hearts and chemically skinned by incubation with 1% Triton X-100 in the pCa 8.0 relaxing solution (see below) at 4 °C for 1 h. The skinned cardiac muscle (CSM) was then incubated in the same solution plus 50% glycerol for 24 h at -20 °C and stored in 50% glycerol in the pCa 8.0 solution without Triton X-100 at -20 °C. For experiments, CSM was dissected into small fiber bundles (0.5 cm long and 0.1-0.15 cm in diameter).

Skeletal muscle was isolated from rabbit psoas muscle and immersed for 30 min in pCa 8.0 relaxation solution at 4 °C. Then the muscle bundles was excised into small bundles (2-4 mm in diameter, 8-10 cm in length) and tied to wooden sticks. The bundles were incubated in pCa 8.0 solution containing 50% glycerol for overnight at 4 °C. The bundles were changed to a fresh pCa 8.0 solution containing 50% glycerol and then stored at -20 °C until use.

Determination of the CaDependence of Force Development

The CSM or skeletal fibers were mounted using stainless steel clips to a force transducer (26) and immersed in a relaxation solution (pCa 8.0) containing 10M [Ca], 5 mM [Mg], 7 mM EGTA, 20 mM imidazole, 5 mM [Mg-ATP], 20 mM creatine phosphate, and 15 units/ml creatine phosphokinase, pH 7.0, ionic strength = 150 mM. The contraction solution (pCa 4.0) had the same composition as the pCa 8.0 solution except the Ca concentration was 10M and was used to measure the initial force. To determine the Ca dependence of force development, the contraction of CSM was tested in solutions containing intermediate concentrations of Ca.

Phosphorylation of the Skinned Cardiac or Skeletal Muscle

The CSM or skeletal fibers was phosphorylated with PKA in a freshly prepared solution containing 50 mM phosphate, 50 mM MgCl(2), 50 mM NaCl, 10 mM Mg-ATP, and 0.5 unit/µl of the catalytic subunit of PKA, for 1 h at room temperature. The CSM was then washed with the pCa 8.0 relaxing solution. To monitor the level of phosphorylation of CSM, [P-]ATP was added to the phosphorylation mixture. The radiolabeled CSM was solubilized in SDS sample buffer and analyzed by SDS-PAGE (15%) and autoradiography (22) . The Ca dependence of the same CSM or skeletal fibers was measured before and after phosphorylation.

CTnI Extraction and Reconstitution of the CSM

The CSM was first tested for its initial force in the pCa 4.0 solution. Extraction of the endogenous CTnI was performed by incubation of the CSM in a 10 mM orthovanadate solution, pH 6.7, for 10 min(27, 28) . The vanadate was subsequently removed by washing with the pCa 8.0 relaxing solution. The extent of CTnI extraction was estimated from the resultant Ca-independent force seen in the pCa 8.0 relaxing solution. After the Ca-independent contraction had reached the maximum, the CSM was incubated with 10 µM CTnCbulletCTnI complex for 3 h at room temperature. Incubation with the CTnCbulletCTnI complex with the CSM was necessary, since the vanadate treatment extracts both endogenous CTnI and CTnC, and since it has been shown (28) that the reconstitution of Ca regulation is maximal using this protocol. The CTnCbulletCTnI complex was prepared from bovine CTnC and either the wild type or mutated mouse CTnI as described under ``Materials and Methods.'' The extent of reconstitution was estimated from the amount of Ca-dependent force regained after incubation with the CTnCbulletCTnI complex. Force measurements were initiated when the reconstituted force reached more than 50% of the original force. The Ca dependence of force development was determined and compared before and after phosphorylation of the CSM(22) . Phosphorylation of the reconstituted CSM was performed under the same conditions as described for the nonextracted CSM. [-P]ATP was included in the phosphorylation solution to monitor the levels of phosphorylation of wild type CTnI and its mutants after they were reconstituted into the CTnI-depleted CSM.


RESULTS

Expression and Purification of Wild Type CTnI and CTnI Mutants

The expression of wild type CTnI and its mutants was carried out in BL21(DE3) host cells in which the expression was controlled by the expression of the T7 RNA polymerase promoter. The purification of CTnI was reported previously and essentially the same as that used in purifying skeletal TnI(29) . An improvement was made by applying an EDTA (0-3 mM) and urea (0-6 M) double gradient to the TnC affinity column to remove other protein contaminants prior to eluting CTnI from the CTnC affinity column. Since the affinity of CTnI for CTnC is quite high, the bound CTnI can only be eluted from the TnC affinity column in the presence of 6 M urea, 2 mM EDTA, and 1 M NaCl, or near the end of the described gradient, with other contaminating proteins eluted during the first half of the gradient. Fig. 1A shows the SDS-PAGE patterns of the bacterial lysate, and Fig. 1B shows the purified wild type CTnI and the indicated mutants. The levels of expression of CTnI (2-4 mg/liter of culture) was not as high as that seen previously for skeletal TnI in the same system(29) ; however, this method proved to be very efficient and effective to purify CTnI from bacteria expression system.


Figure 1: SDS-PAGE of bacterially synthesized wild type CTnI and its mutants. A: lane 1, bovine cardiac CTnI; lane 2, bacterial lysate with CTnI expression; lane 3, bacteria lysate without CTnI expression. B, purified wild type and mutant CTnIs. Lane 1, purified wild type CTnI; lane 2, purified CTnI; lane 3, purified CTnI; lane 4, purified CTnI; lane 5, bovine CTnI.



Rate of PKA Phosphorylation of Wild Type and Mutated CTnIs

The different levels of CTnI phosphorylation detected in cardiac tissues (16) suggest the possible co-existence of two monophosphate and/or biphosphate forms of CTnI. In this experiment we used wild type CTnI and its mutants to study the time course of CTnI phosphorylation of each of the two serine residues (22 and 23), and thus we could carefully control the experimental variables and we could compare our data with other phosphorylation studies using either CTnI isolated from cardiac muscle or synthetic CTnI peptides(14, 15, 16) . Phosphorylation reactions were stopped at different reaction times (Fig. 2). In order to observe possible differences in the rates of phosphorylation of these two serine residues, the concentration of PKA used for the reaction was limited to 70 units/ml. As expected, we did not observe any phosphorylation of the CTnI mutant (data not shown). We also confirmed previous observations (14) that both serines 22 and 23 are able to be phosphorylated by PKA. Fig. 2, A-C, illustrates the different rates of TnI phosphorylation observed on autoradiograms. Fig. 2C shows that phosphorylation of CTnI was not detected in these autoradiograms before 1 min of phosphorylation, whereas phosphorylation of wild type CTnI (Fig. 2A) and the CTnI mutant (Fig. 2B) were able to be observed during this time. The level of phosphorylation of wild type and the mutated TnIs were calculated and are plotted in Fig. 2D. As can be seen from Fig. 2D, the rate of CTnI phosphorylation is faster than that of CTnI. After 30 min of phosphorylation, CTnI was almost fully phosphorylated, while CTnI was only half-phosphorylated. Wild type CTnI, CTnI and CTnI could be phosphorylated up to 1.61 ± 0.18, 0.92 ± 0.04, and 0.85 ± 0.03 mol of phosphate/mol of protein, respectively. Interestingly, the phosphorylation of the wild type protein is essentially equal to the sum of the phosphorylation of the two mutants, implying that the phosphorylation of Ser-22 and Ser-23 are independent. Under our reaction conditions, there was no additional phosphorylation after 60 min of incubation with PKA, indicating that the proteins were fully phosphorylated.


Figure 2: The time courses of phosphorylation of wild type CTnI and its mutants. Phosphorylation of wild type CTnI and its mutants was performed as described under ``Materials and Methods.'' A-C, autoradiograms showing the phosphorylation of wild type and mutant CTnIs. 1 µg of either wild type CTnI or mutants were phosphorylated by PKA under conditions described under ``Materials and Methods.'' The phosphorylation reactions were stopped with the SDS sample buffer added to the reaction mixtures at different reaction times. Portions of these samples were electrophoresed on 15% SDS-PAGE, and the dried gels were exposed to x-ray films. A: lanes 1-8, phosphorylation of wild type CTnI; B: lanes 1-8, phosphorylation of CTnI; C: lanes 1-8, phosphorylation of CTnI. Lanes 1-8 in the figures represent the different times of phosphorylation. They are 30 s, 1 min, 2 min, 5 min, 10 min, 30 min, and 60 min, respectively. D, time course of phosphate incorporation into wild type and mutant CTnIs by PKA. The amount of phosphorylation of the wild type and mutant CTnIs was calculated as described under ``Materials and Methods'' and was plotted as an average of three to four experiments (X = mean ± S.E.).



Effect of PKA on the CaSensitivity of Muscle Contraction

To determine the effect of PKA on the cardiac skinned muscle, the CSM was phosphorylated as described under ``Materials and Methods,'' solubilized in SDS sample buffer, and analyzed on SDS-PAGE as shown in Fig. 3. The autoradiogram shown in Fig. 3indicates that the primary site of phosphorylation of CSM by PKA is CTnI. Under our experimental conditions the amount of C-protein phosphorylation varied from lightly to barely phosphorylated, while the CTnI phosphorylation was always high, and this result is consistent with a report by Venema and Kuo (7) . Except for CTnI and C-terminal protein, there were no other proteins phosphorylated by PKA under our experimental conditions. The effect of PKA on the Ca sensitivity of muscle contraction was examined and demonstrated in Fig. 3. The Ca dependence of force development of phosphorylated and dephosphorylated CSM was plotted and fit using the Hill equation. We found that phosphorylation of CSM did not change the maximal force at pCa 4.0; however, the pCa decreased from 5.51 ± 0.02 to 5.25 ± 0.04 (Table 2), demonstrating a decreased Ca sensitivity of force development of the CSM after phosphorylation. Moreover, incubation of CSM in the phosphorylation solution without PKA for up to 2 h did not change the Ca dependence of muscle contraction as compared to unphosphorylated CSM (Fig. 3). Therefore, the decrease in Ca sensitivity of the force development of CSM could only be brought about by PKA treatment.


Figure 3: Effect of PKA on the Ca sensitivity of muscle contraction. The Ca sensitivity of muscle contraction was determined as described under ``Materials and Methods.'' Then the CSM was incubated in the phosphorylation solution with PKA for 1 h or without PKA for 1-2 h. The Ca sensitivity of muscle contraction after incubation was determined again. Solid circle with solid line, Ca sensitivity of muscle contraction before incubation; solid inverted triangle with long dashed line, Ca sensitivity of muscle contraction after incubation with PKA; solid triangle with short dashed line, Ca sensitivity of muscle contraction after incubation with phosphorylation solution without PKA. Inset: left, autoradiogram of the SDS gel (on right) of phosphorylated CSM.





CTnI Extraction and Reconstitution

Based on the CTnI extraction method reported by Strauss et al.(27) , a 10 mM orthovanadate solution was used to extract endogenous CTnI from the CSM. The modification we made here was to use the CTnIbulletCTnC complex for the reconstitution, since both endogenous proteins, CTnI and CTnC, are extracted from CSM during the vanadate treatment, and previous results have shown that reconstitution is much greater using this protocol(28) . As shown in Fig. 4A, the force developed by the CSM after 10-min incubation with 10 mM vanadate, gradually became independent of Ca after incubation in the pCa 8 solution for about 30 min. This occurs when the vanadate is washed out by the pCa 8.0 solution, and the CSM contracts even in the absence of Ca. After three washes with the pCa 8.0 solution, there was no further increase in force when the CSM was immersed in the pCa 4.0 contraction solution, suggesting that the extraction was nearly complete. The CTnIbulletCTnC complex in the pCa 8.0 solution was used to reconstitute the extracted CSM. We found that the restoration of the Ca-dependent force was proportional to the time of CSM incubation in the CTnIbulletCTnC solution and was complete within a 3-h time period. The restored force varied between 40 and 70% of the original Ca-dependent force before extraction regardless of which CTnI (wild type or mutants) was used, suggesting that mutations in the N terminus of CTnI had no effect on the efficiency of reconstitution. This extent of reconstitution is consistent with previously reported results(27, 28) . In Fig. 4B we demonstrate a parallel experiment on CSM isolated from the same muscle preparation as in Fig. 4A. The purpose of this experiment is to show the effect of incubating the extracted fiber in the absence of the CTnIbulletCTnC complex for the same time as in Fig. 4A. As can be seen, there is a drop in the Ca-independent force with time, with no change in Ca dependence. It also demonstrates that the CTnIbulletCTnC complex is essential in order to regain Ca-dependent force.


Figure 4: CTnI extraction and reconstitution. CSM was tested for its initial force and was then extracted by treatment with the 10 mM orthovanadate solution described under ``Materials and Methods'' for 10 min. After wash out of the vanadate with the pCa 8.0 solution, the CSM lost its Ca dependence due to the loss of CTnI and CTnC. When maximal force was obtained, the muscle was incubated with 10 µM CTnIbulletCTnC complex in pCa 8.0 solution. The force restoration was tested after a 3-h incubation in CTnIbulletCTnC complex. The scales of different time blocks during the experiment are indicated in the figures. The total time of time block II in A and B is 10 min. A, the CTnIbulletCTnC complex was used for reconstitution; B, control experiment. No CTnIbulletCTnC was used for the muscle reconstitution. C, Coommasie Blue-stained SDS-PAGE of solubilized CSM which has been extracted by vanadate treatment and reconstituted with CTnIbulletCTnC complex. Lane 1, bovine CTnI; lane 2, nonextracted CSM; lane 3, extracted CSM; lane 4, same as lane 3, but the load as twice as much; lane 5, reconstituted CSM.



The amount of CTnI extraction and reconstitution was further checked by SDS-PAGE as demonstrated in Fig. 4C. After vanadate treatment, more than 90% of the endogenous CTnI was extracted. The relative content of other proteins after vanadate treatment was analyzed using the Gelbase/Gelblot program (Ultra Violet Product, version 1.96, 1995). Although the quantities of the proteins loaded onto the gel shown in Fig. 4C, before and after extraction and after reconstitution, were different, the relative amount of protein before and after extraction remained the same (Table 1), suggesting that vanadate specifically extracted CTnI and CTnC without significantly affecting other proteins. The incubation of the extracted CSM with the CTnIbulletCTnC complex resulted in a relatively complete CTnI reconstitution (Table 1). These results are consistent with the findings described in Fig. 4, A and B, i.e. the loss of Ca-dependent force after the vanadate treatment and the restoration of Ca-dependent force of the CSM following its reconstitution with the CTnIbulletCTnC complex. Unfortunately, it was not possible to quantify the extraction and reconstitution of CTnC in CSM due to its poor staining properties (28) .



Effect of PKA on the CaDependence of Force Development in CSM Reconstituted with Either Wild Type or Mutated CTnIs

As shown in Fig. 3and Table 2, phosphorylation of CSM resulted in a decrease of pCa by 0.26 pCa units. This change appears to be specifically brought about by CTnI phosphorylation, since the Ca dependence of force development in skeletal muscle (whose TnI does not have the phosphorylation site for PKA) did not change after the skeletal fibers were phosphorylated by PKA (Table 2). In addition, no change in the Ca dependence of muscle contraction was observed when CSM was incubated in the presence of PKA and PKA inhibitor(22) . The Ca dependence of force development of the extracted CSM, subsequently reconstituted with either wild type or mutated CTnIs, was measured before and after phosphorylation. The CSM extraction and reconstitution with the CTnIbulletCTnC complex, described in detail under ``Materials and Methods,'' was the same as in Fig. 4, A and B. There was no difference in the recovery of Ca-dependent force between wild type CTnI and its mutants, suggesting that reconstitution of the wild type and mutant CTnIs was the same. Fig. 5shows the phosphorylation of wild type CTnI and its mutants when reconstituted back into the CTnI-depleted CSM. As expected, wild type CTnI was phosphorylated to the greatest extent. The levels of phosphorylation of CTnI, and CTnI were essentially the same in the reconstituted CSM, but less than that of wild type CTnI, whereas CTnI was not phosphorylated at all. These data are consistent with the results shown in Fig. 2. The maximal Ca-dependent force of the reconstituted CSM in pCa4.0 after phosphorylation remained the same as that of before phosphorylation, suggesting that there was no effect of the phosphorylation conditions on the dissociation of CTnI or CTnC from the reconstituted muscle preparation.


Figure 5: Phosphorylation of wild type CTnI and its mutants after they were reconstituted back into CTnI-depleted CSM. The photographs represent a Coomassie Blue-stained gel and autoradiogram of the same gel of the solubilized CSM phosphorylated by PKA. The extraction of endogenous CTnI from CSM and the reconstitution of wild type CTnI and its mutants were the same as described in the legend to Fig. 4. Phosphorylation of the reconstituted CSM was described under ``Materials and Methods.'' Lanes 1-4 are an SDS-PAGE representing CSM reconstituted with wild type CTnI, CTnI, CTnI, and CTnI, respectively. Lanes 5-8 are the autoradiography of lanes 1-4.



Fig. 6illustrates the changes in the Ca dependence of force development brought about by PKA phosphorylation. Interestingly, the only PKA-induced change in the Ca dependence of force development was seen in the CSM reconstituted with wild type CTnI. Phosphorylation of this preparation resulted in a decrease in the pCa by 0.18 pCa units. No change in the Ca dependence of force development was observed after phosphorylation of the CSM reconstituted with any of the three mutated CTnIs, independent of the location of the mutation (Table 2). These results show that phosphorylation of both Ser-22 and Ser-23 are required to observe the change in the Ca dependence of force development and confirm that PKA phosphorylation of CTnI, and not other proteins, is responsible for the reduction in the Ca sensitivity of force development. There is no correlation between the extent of recovery of force and the observed pCa of force development. However, the pCa of the reconstituted CSM was 0.17-0.28 pCa units higher than the unextracted CSM (Table 2). There are several possibilities that may explain this. 1) The CTnI in the unextracted CSM was already partially phosphorylated, producing a lower Ca sensitivity. This does not seem possible, since the pCa of the phosphorylated reconstituted CSM was higher than that of phosphorylated unextracted CSM. Also contradictory was the data demonstrating that partially phosphorylated CTnI had no effect on the Ca sensitivity of muscle contraction. 2) Even after 3 h incubation with CTnIbulletCTnC in the pCa 8.0 solution, there was still some Ca-independent force, presumably caused by cross-bridge attachment due to incomplete CTnIbulletCTnC reconstitution. Therefore, the attached cross-bridges may be responsible for the increased Ca sensitivity seen in the reconstituted CSM(26) .


Figure 6: Effect of PKA on the Ca dependence of force development of extracted CSM subsequently reconstituted with the complex of CTnC and either wild type or mutated CTnIs. The extraction and reconstitution procedures and the force measurements were the same as described in the legend to Fig. 4. The results are all based on comparison of the same CSM before and after treatment with PKA. Each CSM was its own control. The results are an average of five to seven independent determinations, and the statistical analysis is shown in Table 2. Phosphorylation was performed as described under ``Materials and Methods.'' The CTnCbulletCTnI complexes used for reconstitution were formed utilizing bovine CTnC and wild type CTnI: A, CTnI; B, CTnI; C, CTnI; D, CTnI.




DISCUSSION

As we have previously reported(9) , mouse CTnI can be expressed in a bacterial expression system. The purification method presented here has proven to be an efficient way to obtain large quantities of CTnI (30) . However, the final yield of CTnI purified from the pET expression system is not as high as for skeletal TnI(29) . This probably results from the codon usages for the first 32 amino acids in the mouse CTnI cDNA sequence, since the expression of a deletion mutant of CTnI devoid of the first 32 amino acids was much higher than that of the intact CTnI(30) .

PKA phosphorylation of CTnI causes a decrease in the Ca dependence of force development or of the myofibrillar ATPase of cardiac muscle and has been observed by many investigators(8, 9, 20, 21, 36) . We have recently found that CTnI phosphorylation increases the rate of Ca dissociation from CTnC, thus contributing to the observed faster relaxation(22, 23) . CTnI contains two adjacent serine residues immediately adjacent to three arginine residues, i.e. R-R-R-S-S, which meets the minimal sequence requirement (R-R-X-S/T) for PKA phosphorylation of both serines(31) . Various studies on CTnI phosphorylation have suggested that CTnI can be phosphorylated up to 2 mol of phosphate/mol of protein(15, 16, 32) , which is consistent with the amino acid sequence of the CTnIs. Our phosphorylation studies have confirmed the prediction that these two serine residues are able to be phosphorylated by PKA. Moreover we have shown that these two serines are the only phosphorylation sites in CTnI, since the mutant, CTnI was not phosphorylated by PKA. In agreement with the study by Mittmann et al.(32) , we found that the rate constants for the phosphorylation of the two serines are different, with Ser-22 being phosphorylated at a slower rate than Ser-23. However, in contrast to the peptide studies (32) , the difference between the rates of phosphorylation of the two serines was not 13-fold but more like 2-fold. The reason of the observed discrepancy is probably due to the different conformation of the two substrates used for phosphorylation. In contrast to our studies on whole CTnI, the peptide used by Mittmann et al. (11) was only ten amino acids in length. The other interesting finding is that the phosphorylation of the wild type CTnI was equal to the sum of the two mutant CTnI phosphorylation. This implies that the phosphorylation of Ser-22 and Ser-23 are independent of each other.

We have demonstrated that only the phosphorylated wild type CTnI was effective in decreasing the Ca sensitivity of force development. The three other mutants, when substituted into CSM, showed no effect of PKA on the Ca dependence of force development. The in vitro experiments ( Fig. 2and Fig. 5) have demonstrated that both CTnI and CTnI can be fully phosphorylated. Our results suggest then that both serines need to be phosphorylated to lower the Ca sensitivity of muscle contraction. The monophosphate forms of CTnI do not appear to be effective in decreasing the Ca sensitivity of muscle contraction.

It is of course possible that, due to the limitation of the methodology used, we were unable to observe small effects when only Ser-22 or Ser-23 are phosphorylated. The limitation of using the vanadate extraction and reconstitution method is that it is difficult to restore the Ca-dependent force close to the initial force before extraction. This is in part due to the fall off in force that occurs during the incubation of the extracted fiber with the CTnIbulletCTnC complex (Fig. 4, A and B). It may also explain why the change in the Ca dependence of force development in CSM reconstituted with wild type CTnI brought about by PKA is only 0.18 pCa units versus a change of 0.26 pCa observed in nonextracted CSM (Table 1). Although we cannot rule out the possibility that these intrinsic methodological weaknesses might contribute to our failure to see any effect of the single serine phosphorylation on the Ca dependence, our data are consistent with other many experimental results. In any event, our results clearly show that PKA phosphorylation of CTnI, and not some other protein(s), is responsible for the PKA induced change in Ca sensitivity.

It has been reported that the majority of the endogenous phosphate in CTnI isolated from cardiac tissue is contained in the second serine (14) . It is also possible to isolate the monophosphate form of native CTnI, in which only the second serine is phosphorylated, from cardiac tissue(33) . Isolation of the monophosphate form of CTnI in which only the first serine is phosphorylated has not been reported, although it appears to exist(33) . Therefore, even in the resting state when beta-agonist levels are low, CTnI is probably partially phosphorylated, mostly at the second serine. These data suggest that not only are the kinetics of phosphorylation of these two serine residues different, but more importantly, phosphorylation of serine 23 alone probably does not seem to affect cardiac muscle contractility. Second, CTnI isolated from cardiac muscle always contains different levels of endogenous phosphate, from 0.5 to 1.5 mol of phosphate/mol of protein(16) . These differences, as pointed out by Swidirek(14) , are probably due to the different methods used to measure the absolute content of phosphate. The interesting point here is that upon beta-agonist stimulation, the net increase in the phosphate of CTnI is 1 mol of phosphate/mol of protein(16) . Concerning the fact that Ser-23 is phosphorylated even in the resting state, it is reasonable to assume that the net increase of phosphate after beta-agonist stimulation is mostly added on the first serine or Ser-22 in the mouse. Thus Ser-23 appears to be constitutively phosphorylated, with the phosphate of Ser-22 being functionally more important. This also suggests that the rate of dephosphorylation of these two serines is probably different and may account for some of the observations in the in vivo studies(4, 19) . Third, using P NMR spectroscopy, Jaquet et al.,(33) found that a new P NMR signal appeared only when both Ser-23 and Ser-24 of bovine CTnI (equivalent to Ser-22 and Ser-23 in mouse CTnI) were phosphorylated in a complex formed with TnT and TnC. Their result suggests that these two phosphorylated serine residues produce a specific interaction within the troponin complex, possibly causing Ca to dissociate faster from CTnC. The two monophospho-forms of CTnI did not show any changes in this specific subunit interaction.

All of these results suggest that partially phosphorylated CTnI has little or no effect on the Ca dependence of force development and therefore on cardiac contractility. Only the doubly phosphorylated form of CTnI appears to contribute to these processes. In combining these results with our phosphorylation data on wild type CTnI and its mutants, we hypothesize that this type of ordered phosphorylation may indeed occur in native CTnI with the major change in phosphorylation occurring on serine 22.

These results raise the question as to what the physiological significance of the existence of the two adjacent serine residues in CTnI is and how the phosphorylation of these two serines affects Ca binding to CTnC. At this point, we do not have a satisfactory answer. The existing sequence data shows that the existence of the two adjacent serine residues on CTnI is shared by avian, and many other mammalian species, implying the possible physiological necessity of the existence of these two serine residues. The effect of CTnI phosphorylation on the Ca affinity of TnC is achieved most likely through a change in the interaction between CTnI and CTnC, which probably requires phosphorylation of both serine residues. Sheng et al. (29) have demonstrated that the N-terminal domain of skeletal TnI interacts with TnC in a Ca-Mg site-dependent manner, and this interaction serves to maintain the structure of troponin complex. Recent NMR studies on the spatial relationships within the CTnIbulletCTnC complex demonstrated that CTnI and CTnC also form an antiparallel arrangement similar to skeletal TnI(34, 35) . Since the two adjacent serines are close to the TnC binding domain in the N-terminal portion CTnI, the phosphate introduced by PKA possibly influences the interaction between the N terminus of CTnI and the C terminus of CTnC. This structural change may also affect the Ca-dependent interaction between the N-terminal region of CTnC and CTnI. The monophosphate form of CTnI may play only a transitory role. Further studies on the interaction between CTnI and CTnC using molecular modeling and their structural determination will help to define these interactions.

In summary, our current view of the mechanism by which CTnI phosphorylation controls the increased rate of muscle relaxation is that: 1) in the resting state, CTnI is partially phosphorylated, mostly at the second serine; 2) when PKA is activated by the beta-agonist pathway, PKA further phosphorylates TnI, mostly at the first serine; 3) the Ca sensitivity of muscle contraction decreases when both serines on CTnI are phosphorylated by PKA, allowing Ca to dissociate faster from the single Ca-specific regulatory site of CTnC; 4) after removal of beta-agonists, CTnI is dephosphorylated, primarily at the first serine.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R37 HL42325. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology, University of Miami School of Medicine, P. O. Box 016189, Miami, FL 33101.

(^1)
The abbreviations used are: PKA, cAMP-dependent protein kinase; TnI, troponin I; CTnI, cardiac muscle troponin I; TnC, troponin C; CSM, skinned cardiac muscle; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Lois Rosenzweig for her expert assistance in the preparation of this manuscript and Dr. Danuta Szczesna for her helpful editorial comments during the preparation of this manuscript.


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