Identification of a Functionally Critical Protein Kinase C Phosphorylation Residue of Cardiac Troponin T *

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr 197 , Ser 201 , Thr 206 , and Thr 287 ) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC- (cid:1) or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca 2 (cid:2) concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca 2 (cid:2) sensitivity of the myofilaments. An important and novel finding of our study was that Thr 206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC- (cid:1) or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin smaller, (cid:2) 20%, decrease. A recent study in our laboratory has indicated that pan-activation of PKC results in phosphorylation of cTnT and cTnI and induces a 30% reduction of maximum force of mouse cardiac myofilaments (18). When cTnT was partially replaced with a fast skeletal (fs) isoform, which is not phosphorylated by PKC, the effect of PKC activation was no longer evident. These results indicated that cTnT phosphorylation might be pivotal for the PKC-induced depression of tension in the myofilament. Tension and Actomyosin Mg-ATPase Activity Measurements — The method of de Tombe and Stienen used in these experiments was described in detail elsewhere (21). The fiber bundles (control or exchanged) were attached to a displacement generator at one end and a force transducer at the other end using aluminum T-clips. The sarcomere length was adjusted to 2.3 (cid:3) m using a laser diffraction pattern and the cross-sectional area was determined based on an elliptical model. Fiber bundles were equilibrated for 5 min in Relaxing Buffer (100 m M BES, pH 7.1, 8.37 m M MgCl 2 , 5.80 m M Na 2 ATP, 20 m M EGTA, 42.5 m M potassium propionate), followed by 2 min in Pre-Activating Buffer (100 m M BES pH 7.1, 7.78 m M MgCl 2 , 5.80 m M Na 2 ATP, 0.50 m M EGTA, 19.5 m M HDTA, 43.6 m M potassium propionate) and then im- mersed into a bath containing a Maximal Activating Solution (100 m M BES, pH 7.1, 7.63 m M MgCl 2 , 5.87 m M Na 2 ATP, 20 m M Ca 2 (cid:1) -EGTA, 43.6 m M potassium propionate). Relaxing, pre-activating, and maximum activating solutions also contained 900 (cid:3) M NADH, 5 m M sodium azide, 10 m M phospho(enol)pyruvate, 1 mg/ml pyruvate kinase (500 units/mg), 0.12 mg/ml lactate dehydrogenase (870 units/mg), 10 (cid:3) M oligomycin B, 20 (cid:3) M P 1 P 5 -di(adenosine-5 (cid:3) )pentaphosphate, and 10 (cid:3) M leupeptin, 1 (cid:3) M pepstatin, 1 m M DTT, and 10 (cid:3) M phenylmethylsulfonyl fluoride. After each contraction, fiber bundles were incubated for 1

charges of sarcomeric proteins are critical to these proteinprotein interactions. Charge-charge interactions are involved in the reaction of the molecular motor myosin with actin and in the control of the actin-myosin interaction by Tm and the troponin complex (Tn). 1 Troponin is a heterotrimeric protein consisting of a Ca 2ϩ -binding protein, TnC; an actomyosin Mg-ATPase-inhibiting protein, TnI, and a Tm-binding protein, TnT (for reviews see Refs. [1][2][3][4]. Excellent examples of the major significance of the charged amino acids in these proteins are the functional effects of missense mutations and deletions that are causal in familial hypertrophic cardiomyopathies (FHC) (5,6) or dilated cardiomyopathies (DCM) (7). Moreover, charge modifications induced by phosphorylation of cTnI at tissuespecific PKA sites and at PKC sites modifies the Ca 2ϩ sensitivity, tension, and velocity of shortening of the myofilaments (8 -10).
In experiments reported here we have focused on the functional significance of PKC phosphorylation and specific charge modifications, mimicking phosphorylation, of cardiac cTnT. cTnT is the "lever" that transmits the signal generated by Ca 2ϩ -induced conformational changes in cTnC-cTnI structures to the filamentous protein, Tm. Upon Ca 2ϩ -activation Tm undergoes both a movement and a rotation on the actin filament to facilitate the binding of myosin heads to actin and to promote the process of contraction (11)(12)(13). As a critically positioned molecule in the thin filament, it is clear that modifications of cTnT have the potential for versatile interactions with adjacent proteins and for producing significant functional effects (14). Even so, the specific effects of cTnT phosphorylation on tension and ATP hydrolysis have not been well studied in the myofilament lattice. There is no evidence for phosphorylation of cTnT by PKA. However, in vitro studies demonstrated that there are four main sites for PKC-dependent phosphorylation on cTnT (15), which are at Thr 197 , Ser 201 , Thr 206 , and Thr 287 (mouse sequence). These sites are located in the functionally significant C-terminal half of the molecule. It is this region of cTnT that is likely to interact with cTnI and cTnC and possibly with Tm, and is essential in the transmission of the Ca 2ϩ -binding signal to Tm-actin (16). Noland and Kuo (17) reported that exclusive phosphorylation of cTnT results in a ϳ50% decrease in maximum actomyosin Mg-ATPase activity using in vitro fully reconstituted systems. When cTnI was exclusively phosphorylated under the same conditions, there was a much smaller, ϳ20%, decrease. A recent study in our laboratory has indicated that pan-activation of PKC results in phosphorylation of cTnT and cTnI and induces a 30% reduction of maximum force of mouse cardiac myofilaments (18). When cTnT was partially replaced with a fast skeletal (fs) isoform, which is not phosphorylated by PKC, the effect of PKC activation was no longer evident. These results indicated that cTnT phosphorylation might be pivotal for the PKC-induced depression of tension in the myofilament.
However given the large number of PKC isoforms, multiple phosphorylation sites, the lack of homogeneously phosphorylated samples and inability of performing site-directed phosphorylation, the role of each cTnT phosphorylation site remains unknown. In the present study we generated cTnT mutants in which Glu (mimic of phosphorylation) or Ala residues were placed at the PKC phosphorylation sites. cTnT was selectively mutated to generate single, double, triple, and quadruple mutants (see Fig. 3). The wild-type and mutated cTnTs were used to reconstitute a recombinant cTn complex, which was incorporated into detergent extracted left ventricular papillary muscle fiber bundles by replacing the endogenous troponin complex (19,20). We simultaneously determined tension development and actomyosin Mg-ATPase activity in the reconstituted preparations as a function of Ca 2ϩ concentration (21). Our data provide the first evidence that Thr 206 is the functionally critical phosphorylation residue. Its exclusive phosphorylation by PKC-␣ or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca 2ϩ -sensitivity and cooperativity. On the other hand the charge modification of the other 3 residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing cTnT-wt-P (but not T197/S201/T206/ T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt implicating cTnT phosphorylation in regulation of cross-bridge detachment rate.

MATERIALS AND METHODS
Preparation of Site-directed cTnT Mutants-cDNA of adult mouse cardiac TnT (a generous gift of Dr. Jil Tardiff) was previously cloned into a pSBETa vector (22) and selectively mutated using the Quick-Change Site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Sense primers (from Operon) shown in Table I were used for the respective mutations. Mutated residues are underlined. The identity of constructs was verified by DNA sequencing.
Contractile Protein Expression and Purification-Human cardiac TnC and mouse cardiac TnI were expressed and purified as previously described (23). Bovine cardiac Tm for the cosedimentation assay was purified as previously described by Tobacman and Adelstein (24). Recombinant adult mouse cTnT-wt and mutants were expressed and purified by a modified method of Chandra et al. (25). cTnT was expressed in BL21(DE3) cells using the pSBETa expression plasmid. BL21(DE3) cells grown over night in Luria Broth supplemented with 30 g/ml kanamycin, were collected by centrifugation at 6000 ϫ g for 10 min. at 4°C. The cell pellet was then resuspended well in a suitable volume (about 200 ml) of TnT-Buffer A (20 mM Tris, pH 8.0, 6 M urea, 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM DTT) containing 0.5% Triton X-100. The cells were lysed by sonication on ice, followed by 60 min of centrifugation at 48,000 ϫ g, at 4°C. The supernatant fraction was subjected to ammonium sulfate fractionation as previously reported (25). The final ammonium sulfate pellet was solubilized in TnT-Buffer A, and then dialyzed at 4°C against TnT-Buffer A. The dialyzed sample was applied on a DEAE-Fast Flow Sepharose column (Amersham Biosciences) connected to a FPLC System (Amersham Biosciences). cTnT was eluted with a 0.0 -0.4 M KCl gradient in TnT-Buffer A. The fractions containing cTnT were analyzed by 12% SDS-PAGE and those containing Ͼ90% pure cTnT were pooled, extensively dialyzed against 0.1% formic acid, 1 mM DTT in H 2 O, lyophilized, and stored in powder form at Ϫ80°C.
PKC-␣ Expression and Purification-Recombinant human PKC-␣ (26) was purchased from ATCC (American Type Culture Collection, ATCC 80045). The following sense primers (from Operon) were used for the creation of a 5Ј-BglII and 3Ј-EcoRI restriction sites by PCR, for the subsequent subcloning into pVL1392 vector (BD Pharmingen): PKCAB-glII, 5Ј-ata ata aga tct atg gct gac gtt ttc ccg ggc aac-3Ј; PKCAEcoRI 5Ј-att aat gaa ttc tca tac tgc act ctg taa gat ggg-3Ј. Mutated residues are underlined. The identity of constructs was verified by DNA sequencing.
Sf-9 cells (Invitrogen) transfected with PKC-␣ using the BaculoGold Kit (BD Pharmingen) were used to produce high-titer baculovirus stock. For protein expression, Sf-9 cells were grown at 27°C in suspension culture to 2 ϫ 10 6 cells/ml and infected with a MOI of 10. After 3 days cells were harvested and resuspended in 40 ml of Lysis Buffer containing 25 mM Tris-HCl, pH 7.5, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 50 g/ml leupeptin, 1% Triton X-100, and 0.2 mM phenylmethylsulfonyl fluoride. The cell suspension was lysed in a 50-ml hand-held homogenizer chilled on ice. The lysate was centrifuged at 50,000 ϫ g and 4°C for 60 min. The supernatant was loaded onto a HP Q-Sepharose Fast Flow column (Amersham Biosciences). PKC-␣ was eluted with a 0.0 -0.4 M KCl gradient in PKC-Buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, and 1 mM DTT). Active PKC fractions were pooled, adjusted to 2 M KCl, and loaded onto a POROS 20 HP2 column (Per-Septive Biosystems) and eluted with linear salt gradient from 2 to 0 M KCl in Buffer A. Active PKC fractions were concentrated in an Ultrafree-15 centrifugal filter device (Millipore). Activity of PKC-␣ was assayed by measuring the initial rate of [ 32 P]phosphate incorporation into 5 M cTn (modified from Ref. 27).
Recombinant cTn Complex Reconstitution-The recombinant heterotrimeric cTn complex was reconstituted by mixing equimolar amounts of cTnT, cTnI and cTnC in a solubilization buffer containing 6 M urea, 50 mM Tris pH 8.0, 1 M KCl, 5 mM MgCl 2 , 1 mM CaCl 2 , 1 mM DTT (28). The solution was subjected to sequential dialysis using 0.7 M KCl, 0.4 M KCl, and finally 0.2 M KCl in the above buffer minus urea. Next the cTn complex was further separated from the monomeric troponins by anion exchange on a RESOURCE-Q column (Amersham Biosciences) connected to a FPLC system. The cTn complex identity and purity was verified by 12% SDS-PAGE and then concentrated to about 2 ml using a Centriprep 10K (Millipore) and dialyzed against the Exchange Buffer containing 20 mM MOPS pH 6.5, 200 mM KCl, 5 mM EGTA, 5 mM MgCl 2 , 1 mM DTT (19,20). Next, the cTn complex was aliquoted and stored at Ϫ80°C until it was used for exchange experiments.

Exchange of Recombinant cTn in Left Ventricular
Fiber Bundles-Fiber bundles were prepared from hearts of 5-6 months old, FVBN mice purchased from Harlan or Charles Rivers laboratories. The mice were anesthetized with ether and hearts were quickly removed and rinsed free of blood in ice-cold saline (0.9% NaCl). Muscle strips (ϳ150 -200 m wide and 3-4 mm long) were dissected from left ventricular papillary muscle. Then the fiber bundles were detergent-treated at 4°C, in a High Relax Buffer containing 20 mM MOPS pH 7.0, 50 mM potassium propionate, 6.8 mM MgCl 2 , 10 mM EGTA, 25 M CaCl 2 , 12 mM phosphocreatine, 5 mM Na 2 ATP, 10 UI/ml creatine kinase, 0.5 mM DTT, a mixture of protease inhibitors and 1% Triton-X100 (21). Following the detergent treatment the fiber bundles were transferred to a bath containing a recombinant cTn complex in Exchange Buffer (see above) and incubated overnight at 4°C. The extent of recombinant cTn exchange was determined after isometric tension and actomyosin Mg-ATPase activity measurements (see below) by immunoblot analysis of control versus exchanged fiber bundles using anti-Myc antibody (clone 9E10, Santa Cruz Biotechnology) followed by treatment of the same membrane (after c-Myc removal) with anti-TnT antibody (clone JLT-12, Sigma). The presence of the 9-amino acid Myc tag at the N terminus of recombinant cTnT (29) allowed us to separate it from the endogenous mouse cTnT on a 15% SDS-PAGE (acrylamide:bis-acrylamide ratio 200:1).
Isometric Tension and Actomyosin Mg-ATPase Activity Measurements-The method of de Tombe and Stienen used in these experiments was described in detail elsewhere (21). The fiber bundles (control or exchanged) were attached to a displacement generator at one end and a force transducer at the other end using aluminum T-clips. The sarcomere length was adjusted to 2.3 m using a laser diffraction pattern and the cross-sectional area was determined based on an elliptical model. Fiber bundles were equilibrated for 5 min in Relaxing Buffer (100 mM BES, pH 7.1, 8.37 mM MgCl 2 , 5.80 mM Na 2 ATP, 20 mM EGTA, 42.5 mM potassium propionate), followed by 2 min in Pre-Activating Buffer (100 mM BES pH 7.1, 7.78 mM MgCl 2 , 5.80 mM Na 2 ATP, 0.50 mM EGTA, 19.5 mM HDTA, 43.6 mM potassium propionate) and then immersed into a bath containing a Maximal Activating Solution (100 mM BES, pH 7.1, 7.63 mM MgCl 2 , 5.87 mM Na 2 ATP, 20 mM Ca 2ϩ -EGTA, 43.6 mM potassium propionate). Relaxing, pre-activating, and maximum activating solutions also contained 900 M NADH, 5 mM sodium azide, 10 mM phospho(enol)pyruvate, 1 mg/ml pyruvate kinase (500 units/mg), 0.12 mg/ml lactate dehydrogenase (870 units/mg), 10 M oligomycin B, 20 M P 1 P 5 -di(adenosine-5Ј)pentaphosphate, and 10 M leupeptin, 1 M pepstatin, 1 mM DTT, and 10 M phenylmethylsulfonyl fluoride. After each contraction, fiber bundles were incubated for 1 min in Relaxing Buffer, followed by 2 min in Pre-Activating Buffer. The final contraction was induced in Activating solution containing maximally activating [Ca 2ϩ ]. Only those fibers able to generate greater than 80% of initial tension in their final contraction were kept for analysis. The isometric tension and actomyosin Mg-ATPase activity were determined simultaneously at 20°C in the presence of variable Ca 2ϩ concentrations as described (30). Data were analyzed using Labview (National Instruments, Austin, Texas). Tension-, actomyosin Mg-ATPase activity-[Ca 2ϩ ] relations were fit by a nonlinear fit procedure to a modified Hill equation shown in Equation 1, where P is the parameter of interest (isometric tension, actomyosin Mg-ATPase activity); Max is the maximum value at saturating [Ca 2ϩ ]; EC 50 is the [Ca 2ϩ ] at which 50% of Max is reached; and H represents the slope of the relationship (Hill coefficient). Cosedimentation Assay-The binding affinity of cTn complexes containing cTnT-wt or mutants to bovine cardiac tropomyosin (Tm) was assessed using a cosedimentation assay (31). Variable amounts of cTn complex (wt or mutant) in the presence of 1 M Tm were incubated in 10 mM MOPS pH 7.0, 5 mM MgCl 2 , 100 mM KCl and of either 1 mM EGTA or 100 M CaCl 2 , at 4°C (conditions shown to promote sedimentation of cTn-Tm complex, (31)). The Tm-bound cTn was determined by cosedimentation at 74,000 rpm for 45 min using a TLA rotor. Sedimentation supernatants and pellets were analyzed by 15% SDS-PAGE. Densitometric analysis of the gels was carried out using NIH Image software.
Animal Care-All animals were handled in accordance with the guidelines of the Animal Care Committee at the University of Illinois, Chicago.
Statistical Analysis-All values are presented as mean Ϯ S.E., and values of p Ͻ 0.05 were the criteria for statistical significance. Data was analyzed using a one-way ANOVA and post-hoc Dunnett's t test.

Recombinant cTn Incorporation into the Myofilament
Lattice-To assess the functional role of each cTnT phosphorylation site we used a modified version (see "Materials and Methods") of the technique of whole troponin exchange introduced by Brenner et al. (19). This method allows the gentle replacement of the endogenous cTn with a recombinant cTn in muscle fiber bundles. The exchange procedure introduces no major alteration in the structure and properties of the fiber bundles, since the myofilament is never depleted of cTn. Fig. 1 shows the Western blot analysis of representative fibers used in our measurements (1 fiber bundle per lane). The samples were separated on 15% SDS-PAGE, and after transfer to a nitrocellulose membrane probed with an anti-c-Myc antibody (data not shown) and then with anti-cTnT antibody. Lanes 1, 5, 6, and 10 show representative native (no exchange) fibers bundles that have been used in control measurements, whereas the other lanes contain fibers that had undergone exchange with recombinant cTn-wt or Tn-T3SE. The middle lanes 3 and 8 serve as standards for recombinant cTn. The endogenous mouse cTnT and recombinant mouse cTnT have different mobilities on the SDS-PAGE due to the presence of a c-Myc tag at the N terminus of recombinant cTnT. The c-Myc tag retards the recombinant cTnT migration on the gel allowing us to assess the exchange efficiency. The Western blot analysis demonstrated that exchange of endogenous cTn with recombinant cTn was basically 100% since the fibers bundles treated with the recombinant cTn showed no band corresponding to endogenous cTnT.
Mechanoenergetic Characteristics of Native and Recombinant cTn-wt Exchanged Fibers-In a first set of experiments, FIG. 1. Complete substitution of endogenous cTn by recombinant cTn complex. The Western blot demonstrates the efficiency of recombinant cTn complex in exchanging the whole endogenous cTn complex in detergent-treated left ventricular fiber bundles. Detection of cTnT using an anti-cTnT antibody serves as a marker of exchange effectiveness. Lanes 1, 5, 6, and 10 are representative fiber bundles that were used in mechanoenergetic measurements for the native control group. The adult mouse cTnT in native fiber bundles had a higher mobility on the SDS-PAGE, whereas the recombinant cTnT was retarded due to presence of N terminus c-Myc tag. Lanes 2 and 4 are representative fiber bundles from the cTn-wt exchange group, and lanes 7 and 9 are representative fiber bundles of the T3SE (see Fig. 3) exchange group. Lanes 3 and 8 are standard cTnT-wt and cTnT-T3SE, respectively from the cTn complex used in exchange reaction.
we determined the functional effect of introducing recombinant cTn-wt into the myofilament lattice of left ventricular fiber bundles. To determine the effect of cTn-wt incorporation we compared the mechanoenergetic characteristics of native (no exchange) and recombinant cTn-wt exchanged fiber bundles. The data (Fig. 2 and Table II) indicate that the basal mechanics and energetics of native versus cTn-wt were not significantly altered. Native and cTn-wt exchange fibers demonstrated no significant differences in myofilament Ca 2ϩ sensitivity or Hill coefficients for any parameter (Table II).
Effects of Glu Substitution at the cTnT PKC Phosphorylation Sites-In a second set of experiments, we tested whether glutamate incorporation, mimicking phosphorylation, at the PKC phosphorylation sites of cTnT has a functional effect on myofilament mechanics and energetics. This pseudo-phosphorylation method was recently used in our laboratory to study the effect of PKC phosphorylation on cTnI residues Ser 43 , Ser 45 , and Thr 144 (8) by selectively mutating them to glutamic acid. The same approach was employed by Hoshijima et.al. (32) in constructing a S16E phospholamban (PLN) recombinant adeno-associated vector (rAAV) to imitate the conformational changes induced by PKA phosphorylation. This method proved to be useful in mimicking the effects of PKA phosphorylation of PLN. In a similar fashion, Wick et al. (33) showed that substitution of the Thr 516 with glutamic acid, mimicking phosphorylation, confers constitutive activity to mouse PDK-1 in cells. There is an increase utilization of glutamate substituted Ser/ Thr residues as a useful model for studying the functional consequences of protein phosphorylation (33)(34)(35)(36)(37).
Our approach was to selectively mutate the main PKC phosphorylation sites on cTnT (Thr 197 , Ser 201 , Thr 206 , and Thr 287 ) to Glu, express and purify the recombinant proteins, reconstitute the cTn complex using wild type (see above) or mutated cTnT with cTnI-wt and cTnC-wt, exchange this complex into left ventricular fiber bundles, and then measure simultaneously the effect on myofilament activation and ATP consumption at various [Ca 2ϩ ]. Our experimental rationale was to have different permutations of the mutated residues to Glu resulting in single, double, triple, and quadruple mutants. Fig. 3 schematically depicts the amino acid substitution and the location in the adult mouse cTnT. Table II summarizes the mechanical and energetic profiles of left ventricular fiber bundles harboring the various forms of cTnT as part of the exchanged cTn complex.
The cTn complex containing all four PKC sites mutated to Glu (T3SE) compared with cTn-wt had a severe inhibiting effect (see Fig. 4) on the myofilament tension development (reduced by 62%), actomyosin Mg-ATPase activity (reduced by 51%), Ca 2ϩ sensitivity and cooperativity. EC 50 Table II demonstrate a 51% depression in tension development and a 42% decrease in actomyosin Mg-ATPase rate. Myofilament Ca 2ϩ sensitivity and cooperativity were also affected. The EC 50 of tension changed from 1.7 to 18.6 M and the Hill coefficient from 4.7 to 2.8; also EC 50 , and Hill coefficient of actomyosin Mg-ATPase activity changed from 1.5 to 15.7 M and from 4.8 to 2.4, respectively.
These results indicate that T206E is sufficient for the functional effect seen in the mutants containing this charge modification. To verify this result we generated a set of 3 constructs T2SE, STE, and T197E in which the Thr 206 site was left unchanged. Fig. 5 shows the results of experiments in which left ventricular fiber bundles underwent whole cTn exchange with recombinant complexes that were "pseudo-phosphorylated" at residues other than Thr 206 . Compared with cTn-wt exchange T2SE, STE, T197E demonstrated no significant differences for any parameter (see Table II). T197E demonstrated a slight increase in isometric tension and actomyosin Mg-ATPase rate, displaying features that resemble the native fiber profile even closer than the wild-type exchange. These data thus support our conclusion that Thr 206 is the functionally critical cTnT PKC phosphorylation residue.
Effects of Ala Substitution at the cTnT PKC Phosphorylation Sites-We investigated the effect of Ala substitution at the cTnT PKC sites on myofilament mechanics and energetics. The Ala mutants were originally designed to serve as controls in our exchange experiments and we expected no major changes of mechanoenergetic parameters. However, the results obtained with these mutants were quite intriguing. As shown by the Tension and actomyosin Mg-ATPase activity were slightly decreased in cTnT-wt exchange fibers compared with native. However there were no significant differences between any parameter. Average values Ϯ S.E. are presented in Table II. data summarized in Table II and Fig. 6, the substitution of Thr 206 with Ala, had an effect comparable in severity with T206E (depressed tension and actomyosin Mg-ATPase, and a notable change in Ca 2ϩ sensitivity and cooperativity). We also observed an increase in tension cost, but somewhat smaller than with T206E. T3SA and T3A also contain T206A, but its effect seems to be buffered by the presence of Ala at the other PKC sites. Overall we conclude that Thr 206 is positioned in a hypersensitive region of the protein and any changes (such as phosphorylation by PKC) have the potential of depressing myofilament functional properties and energetics.
Analysis of Glu Substitutions on cTn-Tm Binding-To test whether cTnT phosphorylation affects the binding between cTnT and Tm we used a modified cosedimentation assay of Hill and Tobacman (31). For these experiments the recombinant cTn complexes were selected from the same batch used in fiber exchange measurements. Fig. 7 shows the binding curves of cTn-wt and mutants T3SE and T2SE to Tm. Our results indicated that there is no significant difference between the binding of cTn-wt and cTn containing the mutants cTnT to Tm. This result fits with previous findings (31) that identified the cTnT fragment spanning amino acids 95-153 (bovine sequence) as responsible for most of the binding affinity of cTnT to Tm. This area of the molecule remained unchanged by our charged mutations.
Effects of Thr 206 and cTnT-wt Phosphorylation by PKC-The above "pseudo-phosphorylation" study proved to be a useful tool in identification of Thr 206 as the functionally critical phosphorylation residue. The next logical step was to phosphorylate Thr 206 by PKC and see if we get the same functional effect. However, we found out that the PKC-⑀ used in the previous study (8) to phosphorylate cTnI performed poorly in phosphorylating cTnT. In order to find which PKC isozyme prefers cTnT and especially Thr 206 we compared recombinant PKC-␣, -␤II, and -⑀. Our data demonstrate that PKC-␣ preferentially phosphorylates Thr 206 , followed by PKC-␤II and -⑀. 2 The PKC-␣ phosphorylated cTnT-wt-P and cTnT-T2SA-P (used for exclusive Thr 206 phosphorylation and from now on referred to as cTnT-T206-P), were reconstituted in the cTn and incorporated in fiber bundles. Fig. 8 and Table II show the mechanic and energetic profile of these proteins. The phosphorylation of both Thr 206 and cTnT-wt induced a significant reduction in maximal tension of 22-24% and maximal ATPase of 28 -36%, as well as a significant Ca 2ϩ desensitization and change in cooperativity. It is interesting that both cTnT-T3SE and cTnTwt-P gave a more pronounced Ca 2ϩ desensitization than the cTnT-T206E and the corresponding cTnT-T206-P, implying that the effect of Thr 206 phosphorylation may be enhanced by the phosphorylation of the other sites (1). Here we show that phosphorylation and Glu substitution at Thr 206 differentially affect isometric tension and actomyosin Mg-ATPase rate. Fiber bundles containing cTnT-wt-P (but not T3SE) exhibited a significant decrease of tension cost (see Table II) as compared with cTnT-wt, implicating cTnT phosphorylation in regulation of cross-bridge detachment rate.

DISCUSSION
Data presented here are the first to report the specific effect of phosphorylation and charge change at the PKC sites on cTnT in the cardiac myofilament lattice generating tension and hydrolyzing ATP. Moreover, our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity and Ca 2ϩ sensitivity of the myofilaments. An important and novel finding of our study was that Thr 206 is a functionally critical cTnT PKC phosphorylation residue. PKC-␣ phosphorylation and Glu incorporation at this site, as a mimic of phosphorylation, resulted in a significant reduction of isometric tension and actomyosin Mg-ATPase activity, Ca 2ϩ desensitization, change in cooperativity. Glu modification of the other three phosphorylation sites Thr 197 , Ser 201 , and Thr 287 had no significant effect on any of the parameters studied. Interestingly the incorporation of cTnT-wt-P in papillary muscle fiber bundles resulted in a decrease of tension cost that was not observed with T3SE.
The only other investigations of the specific role of cTnT phosphorylation were reported by Noland and Kuo (38,39), who measured Ca 2ϩ -dependent Mg-ATPase activity of a fully reconstituted thin filament preparation reacting with myosin or myosin heads. These studies reported that PKC-dependent phosphorylation of cTnT induced about a 50% depression in maximum actomyosin Ca 2ϩ -dependent Mg-ATPase activity. They showed that specific PKC-dependent phosphorylation of cTnI was also able to inhibit the Mg-ATPase activity, but to a lesser extent than cTnT phosphorylation. Moreover, whereas increasing the ratio of myosin to actin was able to greatly overcome the inhibition by the phosphorylated cTnI, the inhibition of Ca 2ϩ -dependent Mg-ATPase activity by phosphorylated cTnT remained about the same regardless of the myosin to actin ratio. These data not only indicated a difference between cTnI and cTnT in the mechanism by which phosphorylation affects myofilament activation, but also that the state of cTnT has more significant control over the activation state of the thin filament. Indirect evidence from our previous work (18) also indicated that specific phosphorylation of cTnT may modulate myofilament activity. We showed that pan-activation of endog-  Table II. FIG. 5. Mechanoenergetic characteristics of cTn-wt versus mutants containing unaltered Thr 206 . Steady-state isometric tension (A) and actomyosin Mg-ATPase activity (B) were simultaneously measured in fiber bundles at various [Ca 2ϩ ]. There were no significant changes of any parameter in fiber bundles exchanged with cTn complexes in which the native Thr 206 residue remained unchanged. Average values Ϯ S.E. are presented in Table II. enous PKC in mouse heart muscle preparations resulted in a phosphorylation of cTnT and cTnI that induced a 30% reduction of maximum tension generated by the skinned fiber bundles. This effect of PKC activation was no longer evident, when cTnT was partially replaced by transgenesis with a fast skeletal isoform that it is not phosphorylated by PKC.
The depression of myofilament function by the PKC-dependent phosphorylation of cTnT may be of significance in the decompensation seen at end-stage heart failure and associated with a history of hypertrophic signaling. Animal and human studies strongly support the idea that PKC activation is a key signal in the hypertrophic process and that PKC-dependent phosphorylation of myofilament proteins may be a critical fac-tor in hypertrophy/failure (40,41). However there are no detailed studies showing precisely how PKC phosphorylation of contractile proteins is linked to alterations in contractile function.
Several lines of evidence indicate that signaling through the PKC pathway, which involves phosphorylation of sites on cTnI and cTnT, is important in regulation of cardiac myofilament function. In the case of cTnI, this role has been demonstrated in a transgenic mouse model (TG-S43A/S45A) expressing a mutant cTnI in which the PKC sites, Ser 43 and Ser 45 , were mutated to Ala (18,42). Compared with wild-type controls the TG-S43A/S45A hearts, demonstrated an enhanced response to intracellular Ca 2ϩ with activation of PKC, and an enhanced  Table II. induction of contracture with ischemia. These results fit with the hypothesis that cTnI in wild-type hearts was partially phosphorylated at Ser 43 and Ser 45 , and that this phosphorylation inhibits contraction. Previous indirect support for this hypothesis came from the studies of Strang and Moss (43), who reported a significant depression in unloaded shortening velocity of cardiac myocytes skinned after pretreatment with the ␣-1 adrenergic agonist, phenylephrine. In support of this hypothesis, studies comparing intact and detergent extracted papillary muscle from controls and TG hearts demonstrated a diminished response to activation of the PKC pathway by phenylephrine in papillary muscles from the TG-S43/S45-A. Maximum tension developed by skinned fiber bundles from TG-S43A/ S45A hearts was depressed by activation of the PKC pathway prior to skinning, but to a significantly lower extent than the controls. Interestingly, determination of levels of protein phosphorylation demonstrated that phosphorylation of cTnT in the TG-S43/S45-A heart preparations was increased relative to controls following activation of the PKC pathway. These studies thus indicated the potential for thin filament PKC-dependent protein phosphorylation to regulate contractility, and indirectly demonstrate the potential for an in vivo effect of cTnT phosphorylation by PKC. Further evidence for this hypothesis must await the generation of heart muscle preparations either by transgenesis or by viral transfer of cDNA that contain variations in the PKC sites of cTnT.
There is evidence that Thr 206 phosphorylation or Glu substitution may have short and long range effects on regulation of the thin filament by cTnT. cTnT can be separated into 2 domains: T1 (residues 1-183, adult mouse sequence) and T2 (residues 184 -291). T1 is considered as essential in cTnT binding to Tm, whereas T2 is the region of interaction with cTnI-cTnC. Recent studies reported that the T1 fragment, bovine cardiac residues 1-153 (44) and rabbit fast skeletal residues 1-158 (45), inhibits myosin binding to actin, potentially by stabilizing the Tm binding to actin. Maytum et al. (45) showed that T1 causes a much greater inhibitory effect on actomyosin Mg-ATPase activity than the whole cTnT. This suggests that the T2 region of cTnT might have an important role in the thin filament activation by modulating the inhibitory effect of the T1. This fits well with the evidence presented by Potter (46) that cTnT is not just a scaffold molecule anchoring cTnI and cTnC to Tm, but is directly involved in the Ca 2ϩ regulation of actomyosin S1-ATPase activity. Therefore alterations of the T2 half of cTnT, either by phosphorylation or by charge mutations, could prevent the release of inhibitory effect of T1 resulting in the decrease of actomyosin Mg-ATPase activity, isometric tension generation and desensitization to calcium as reported in our study. Malnic et al. (47) demonstrated that a cTnT fragment  encompassing the T1 plus a small fragment of the T2 region can actually activate actomyosin Mg-ATPase activity rather than inhibit it. Furthermore Oliveira et al. (48) showed that a region of chicken fsTnT (158 -191), corresponding to mouse cardiac cTnT region (190 -229), has actin binding properties and stimulates ATPase activity. Taken as a whole these results indicate that the cTnT region (190 -229) is important in controlling the thin filament activation. This could explain why an alteration of this region by phosphorylation could have such an inhibitory effect on tension development and actomyosin ATPase activity.
It is noteworthy that charge alterations in this region of cTnT (190 -229) by the deletion of the charged amino acid Lys 210 (⌬Lys 210 ) have been shown to cause DCM, characterized by cardiac dilation and reduced systolic function leading to heart failure with high mortality (7). In vitro motility studies showed that ⌬Lys 210 resulted in a decrease in the maximum ATPase activity, sliding filament velocity and an alteration in the Ca 2ϩ activation of the thin filament (49). When the TnT containing the ⌬Lys 210 was exchanged into fiber bundles it caused a significant Ca 2ϩ desensitization of force generation (50). Lys 210 is part of the region 190 -229 and is also part of the ␣-helix structure (as Lys 213 in mouse sequence) that has Thr 206 as its starting (N-cap) residue (51). These results also show the importance of this cTnT region in thin filament regulation and homeostasis.
The decrease in tension cost observed with fiber bundles containing cTnT-wt-P (but not with T3SE) supports the hypothesis that alterations in the thin filament proteins affect the reaction of cross-bridges with the thin filament by an allosteric mechanism (8). This result implicates that cross-bridge detachment rate (which is a function of ADP release and ATP binding to the cross-bridge) could be regulated by cTnT phosphorylation.
Computational analysis of cTnT fragment (184 -227) containing the PKC phosphorylation cluster Thr 197 , Ser 201 , and Thr 206 was performed using the facility at EMBL-Heidelberg (www.embl-heidelberg.de/Services/serrano/agadir). The prediction algorithm AGADIR (52) is based on the helix/coil transi-  Table II. tion theory. AGADIR allows prediction of the helical content and the transition from random coil to helix of peptides. Fig. 9 depicts the graphic representation of the computational data. The critical residue Thr 206 was identified as the N terminus capping residue of an ␣-helix that extends between residues 207 and 225. The existence of this helix with Thr 206 N-cap residue was verified by x-ray crystallography (51). Modifications at this critical site either by Glu or Ala substitutions have an effect in extending the ␣-helix content of the peptide leftward to the new N-cap residue Gly 200 . Alterations of Thr 197 and/or Ser 201 either by Glu or Ala seem to have no major impact on the helix structure prediction. When the chicken fsTnT underwent the same computational analysis it showed little propensity for an ␣-helix between residues 207 and 225. Also, although Thr 206 is present in fsTnT it is likely to be involved in a position other than the N-cap of an ␣-helix. This may make the Thr 206 inaccessible to PKC and may be the reason why fsTnT is not phosphorylated by PKC (18).
Our results are validated by studies of Smart and McCammon (53) who used computer simulations of model peptides to examine the role of phosphorylation events in ␣-helices. Their study revealed a clear stabilization of the helix after N terminus phosphorylation. This was attributed to favorable electrostatic interactions between the phosphate group and the helix backbone. Likewise Andrew et al. (54) investigated the effect of phosphorylation event occurring at the N-cap or the middle of an alanine-based ␣-helical peptide. They demonstrated that positioning a phosphoserine/phosphothreonine residue at the N terminus of a helix it stabilizes the helix through favorable electrostatic interactions with the helix dipole by as much as 2.3 kcal/mol, while the same residue when located in the interior of the helix destabilizes it by as much as 1.2 kcal/mol. Taken together these observations suggest a possible mechanism by which cTnT PKC phosphorylation modulates protein structure and function. On the basis of current concepts of thin filament structure and function (55-58), we consider cTnT as a lever in transmitting the Ca 2ϩ -induced conformational changes in cTnC-cTnI structures to Tm. We hypothesize that the fulcrum of the lever is located in the cTnT region (190 -229). Simple lever mechanics suggest that the closer the fulcrum is to the load the less effort is needed, and the farther the fulcrum is to the load the greater the effort needed to move it. We propose that PKC phosphorylation of cTnT might induce its effect on the mechanics and energetics of the myofilament through repositioning the "fulcrum" through ␣-helix (206 -225) stabilization and extension.