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Am J Physiol Heart Circ Physiol 280: H1011-H1018, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 3, H1011-H1018, March 2001

Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force

David E. Montgomery1, Murali Chandra1, Qi-Quan Huang2, Jian-Ping Jin2, and R. John Solaro1

1 Program in Cardiovascular Sciences, Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612; and 2 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca2+ sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 ± 2.5 vs. 33.4 ± 2.6 mN/mm2) reduction in maximum tension in the nontransgenic (NTG) preparations (n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations (n = 11). TPA induced a small but significant (P < 0.02) increase in Ca2+ sensitivity (untreated pCa50 = 5.63 ± 0.01 vs. treated pCa50 = 5.72 ± 0.01) only in TG preparations. In TG preparations, 32P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.

troponin; phosphorylation sites; myofilament activation; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF THE CARDIAC MYOFILAMENTS involves Ca2+-dependent alterations in regulatory protein interactions that lead to increased actomyosin activity. In the physiological state, scaling of myofilament activation occurs partly through reversible covalent modification of protein-protein interactions induced by phosphorylation (21). However, excessive protein phosphorylation, because of upregulation of protein kinase C (PKC) isoforms, may ultimately lead to impaired myofilament activation (1, 2, 23). Although the presence of multiple subcellular substrates and isoforms of PKC confounds our understanding of the mechanism of the impairment, specific myofilament substrates have been identified (15, 23). PKC-mediated phosphorylation of cardiac troponin I (cTnI) was shown to decrease actomyosin MgATPase activity in myofibrillar and fully reconstituted preparations (14, 25). Studies aimed at understanding the role of the phosphorylation sites on cTnI showed that phosphorylation at Ser43 and Ser45 is important for PKC-mediated regulation of reconstituted actomyosin S-1 MgATPase (13). However, little is known about how phosphorylation of cardiac troponin T (cTnT) impacts the activation of the myofilaments. Noland and Kuo (14) showed that exclusive phosphorylation of cTnT results in a ~60% decrease in maximum actomyosin MgATPase activity in fully reconstituted systems. When cTnI was exclusively phosphorylated under the same conditions, there was a much smaller, ~20%, decrease. However, under conditions where both cTnI and cTnT are phosphorylated, an intermediate decrease in activity resulted (14).

These observations suggest an important role for cTnT in mediating functional effects of PKC activation and in modulating the functional effects of PKC-mediated phosphorylation of cTnI. Three putative PKC-specific phosphorylation sites (Thr195, Thr204, and Thr285 in the bovine sequence) are located at the carboxyl terminus of cTnT (15) in the region that interacts with the carboxy terminus of cardiac troponin C (cTnC) and the amino terminus of cTnI (9, 24). Given that the functionally significant sites for cTnI phosphorylation (Ser43 and Ser45) are also located in this same region of the thin filament, it is conceivable that interactions among the PKC phosphorylation sites on cTnI and on cTnT may amplify or repress Ca2+ activation (19, 22, 24). Although specific functional roles have not been assigned to PKC sites on cTnT, it is apparent that Thr285 may be especially important. Thr285 is found only in cardiac variants of TnT (8, 9), and this site was shown to be preferentially phosphorylated in vitro (8, 15). Yet, the specific contribution of phosphorylated cTnT to the PKC-mediated effects on myofilament activation has not been tested in the intact myofilament lattice.

In experiments reported here, we tested the hypothesis that PKC-mediated phosphorylation of myofilaments lacking PKC-specific sites on TnT alters activation in the intact lattice. We employed a line of transgenic mice that expresses ~50% fast skeletal TnT (fsTnT), which naturally lacks phosphorylation sites homologous with cardiac Thr285 and Thr195, in a cardiac-specific manner (6). Our results suggest that the phosphorylation state of TnT may be critical for the PKC-induced depression of tension in the intact myofilament lattice. Furthermore, we provide evidence that changes in the primary structure of troponin components may alter the level and, ultimately, the functional consequences of phosphorylation of the troponin complex.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Calyculin A and calphostin C were obtained from Calbiochem. Okadaic acid, chelerythrine, 4alpha -phorbol, and 12-O-tetradecanoylphorbol 13-acetate (TPA) were all obtained from Sigma. The anti-chicken fsTnT monoclonal antibody (mAb) 6B8, rabbit anti-TnT (RATnT) polyclonal antibody and anti-cardiac TnT mAb CT3 were described previously (26, 7).

Transgenic mice. The transgenic (TG) mice were produced as previously described (9). A mouse alpha -myosin heavy chain promoter in a C57Bl/6 background drove the expression of the full-length chicken fsTnT cDNA. The heart-specific expression of the transgene was confirmed by Western blot analysis using anti-chicken fsTnT mAb. Nontransgenic (NTG) littermates or age- and sex-matched C57Bl/6 mice (Charles River) served as controls. There were no statistical differences between measurements made in either control preparations, and, therefore, the data were pooled for comparison with data from TG preparations.

Western blot analysis. Ventricular muscle samples from the TG and NTG mice were homogenized in SDS-PAGE buffer containing 1% SDS and heated at 80°C for 5 min to solubilize the proteins. As described previously (7, 9) the muscle protein extracts were resolved by SDS-PAGE (14%, acrylamide-to-bisacrylamide ratio = 180:1) for best separation of TnT isoforms and transferred to nitrocellulose membranes using a semidry apparatus (Bio-Rad). Western blots were probed by using the 6B8 and CT3 mAbs together with RATnT, recognizing all TnT isoforms (26) via alkaline phosphatase-labeled anti-mouse or anti-rabbit immunoglobulin second antibodies. Densitometric analysis was carried out on the Western blots with the use of National Institutes of Health Image version 1.61 software to quantify the relative amounts of the endogenous cTnT and the exogenous fsTnT expressed in the TG mouse cardiac muscle

Steady-state tension measurements. Mice were anesthetized by injection with pentobarbital sodium (50 mg/kg body wt) into the peritoneal cavity. The hearts were quickly removed and left ventricular papillary muscle fiber bundles were freshly isolated and dissected into strips ~150-200 µm in width and 3-4 mm in length on a cold plate (4°C). These fiber bundles were then placed in a high relaxing (HR) solution containing (in mM) 20 MOPS (pH 7.0), 10 EGTA, 1 free Mg2+, 5 Mg ATP2-, 12 creatine phosphate, 0.5 1,4-dithiothreitol, and 10 U ml-1 creatine kinase (bovine heart, Sigma). Fiber bundles were immediately treated in a stirring bath that included either 100 nM TPA or an equal volume of vehicle, DMSO, control, for 10 min in the presence of a cocktail of phosphatase inhibitors (0.1 µg/ml calyculin A and 0.2 µg/ml okadaic acid). Some preparations were pretreated with a PKC-specific inhibitor chelerythrine (5 µM) for 5 min. A smaller (3 µM) concentration of chelerythrine only partially prevented the effect of PKC on the myofilaments (data not shown). Furthermore, at effective concentrations (3-4 µM), calphostin C pretreatment resulted in a shift in the pCa50 (increased Ca2+ sensitivity; data not shown). As a result, we discontinued the use of calphostin C in these experiments. In some experiments, fiber bundles were treated with the inactive phorbol ester 4alpha -phorbol (100 nM) for 10 min. After the drug treatment, detergent extraction of the myofilaments was performed in HR containing 1% Triton X-100 and the phosphatase inhibitors for 30 min. The initial sarcomere length (2.3 µm) was determined by laser diffraction onto a calibrated screen. The Ca2+ dependence of tension development was determined, as described in Chandra et al. (3).

Labeling of myofilament proteins with [gamma -32P] ATP. In a method adapted from Gupta et al. (5), fiber bundles were incubated in HR that contained 0.1 mM cold ATP and 75 µCi ml-1 [gamma -32P] ATP for 1 h on ice. At least four fiber bundles were used per condition. Fiber bundles were incubated at room temperature with 100 nM TPA or an equal volume of DMSO (vehicle control) and shaken for 10 min in the presence or absence of the phosphatase inhibitor cocktail. Some preparations were treated with 5 µM chelerythrine for 5 min before TPA or DMSO treatment. Triton X-100 (1% final) was then added, followed by shaking for 30 min. This buffer was subsequently aspirated off, and the bundles were rinsed twice with fresh HR. We then added ~40 µl of 1% SDS (10 µl/fiber bundle), containing 0.5 mM EDTA, to the bundles. This was followed by sonication for 10 min and then boiled for 5 min. After the bundles were completely solubilized by using the method of Chandra et al. (3), the protein concentration was measured by the Lowry method. An equal volume of gel loading buffer (125 mM Tris · HCl, pH 6.8, 20% glycerol, 2% SDS, 0.01% bromophenol blue, and 50 mM beta -mercaptoethanol) was then added.

Polyacrylamide gel electrophoresis and autoradiography. Gels were run on the same day as treatments. A 12.5% polyacrylamide resolving gel, 4% stacking gel with a ratio of 25:0.4 (acrylamide-to-bisacrylamide) gave optimum separation of myofilament proteins. Samples were boiling for 5 min before loading 20 µg protein/lane. Samples were diluted with 1:1 mixture (1% SDS: gel loading buffer) to load equal volumes to each well. Proteins were visualized by Coomassie brilliant blue staining. Destained gels were exposed to the phoshor screen overnight for quantification of labeled proteins. The phosphorimager (Molecular Dynamics) was used to quantify the amount of 32P incorporation into protein bands. The comparison of 32P incorporation across lanes was determined after background correction with the use of ImageQuant software. Phosphorylation into TnI and TnT in treatment groups from the same day was expressed as percent increase in 32P incorporation with DMSO treatment alone taken as 100%.

Statistical analysis. Data from the normalized pCa-tension relations were fitted to the Hill equation, as previously described (3), by using a nonlinear least-squares regression procedure to obtain the pCa50 (-log of free Ca2+ concentration required for half-maximum activation) and the Hill coefficient (nH). Statistical differences were analyzed by an unpaired Student's t-test or one-way ANOVA and Student-Newman-Keuls post hoc for multiple comparisons with significance set at P < 0.05. All data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TG expression and incorporation of fsTnT into the cardiac myofilament lattice. Figure 1 shows Western blot analysis of total protein extracts from TG mouse hearts probed with three different anti-TnT antibodies. The blots using the chicken breast muscle-specific antibody 6B8 revealed a significant overexpression of the fsTnT in the transgenic but not control mouse hearts together with the endogenous cTnT. The blots using polyclonal antibody RATnT and anti-cardiac mAb CT3 indicated the presence of both isoforms with distinct gel mobility in the TG hearts. When the myocardial samples were probed with both 6B8 and CT3 mAbs, the Western blots demonstrated that the exogenous fsTnT makes up 48 ± 3% of the total TnT in the TG mouse heart, whereas the endogenous cTnT was 51 ± 3%, corresponding to a ratio of 0.93:1.00 for the fsTnT and cTnT. We previously (6) showed that the similar ratio was present in the extensively washed cardiac myofibrils. By using phase-contrast microscopy and immunofluorescence microscopy, we also showed that although normal amounts of endogenous cTnT were expressed in this TG mouse, the overexpression of fsTnT significantly increased the amount of exogenous protein incorporated into the thin filament.


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  		Fig. 1.   Western blots demonstrating expression of the fast skeletal TnT (fsTnT) isoform in transgenic (TG) mouse hearts. Expression of TnT isoforms in TG and nontransgenic (NTG) wild-type (WT) hearts was determined with the use of the specific anti-chicken fsTnT monoclonal antibody (mAb) 6B8, anti-cardiac TnT mAb CT3, and the anti-rabbit polyclonal antiserum (RATnT) that recognizes all TnT isoforms. Western blots using mAb 6B8 demonstrated the expression of fsTnT in all of the TG mouse hearts but not the NTG mouse hearts. Western blots probed with CT3 and RATnT demonstrated expression of cardiac TnT (cTnT) isoforms and showed coexpression of cTnT and fsTnT in TG hearts. When the myocardial samples were probed with both 6B8 and CT3 mAbs, the Western blots demonstrate a near 1:1 ratio of the exogenous fsTnT and the endogenous cTnT.

Effect of TPA on maximum steady-state tension development and pCa tension relation in fiber bundles. Figure 2A shows that treatment of NTG cardiac fiber bundles with 100 nM TPA for 10 min decreased the maximum developed tension by 29% (46.1 ± 2.5 mN/mm for DMSO-treated control vs. 33.4 ± 2.7 mN/mm for TPA-treated preparations). Figure 2B shows that the tension remained significantly depressed over the range of physiological Ca2+ concentrations. There was no effect of TPA treatment on the Ca2+ sensitivity in NTG myofilaments, as measured by pCa50 of the normalized pCa tension relation. The pCa50 was 5.66 ± 0.01 in the control group and 5.63 ± 0.01 in the TPA-treated group (Fig. 2C). TPA treatment did not significantly affect the cooperativity (nH) in these preparations (control n = 2.86 ± 0.15 vs. TPA n = 2.55 ± 0.11). To test whether the TPA-induced effect on maximum tension was caused by PKC-mediated phosphorylation, we pretreated NTG fiber bundles with the specific PKC inhibitor, chelerythrine (5 µM), for 5 min before adding TPA. Figure 2A shows that the inhibitor abolished the TPA-induced decrease in maximum isometric tension development (control, 46.1 ± 2.5 vs. chelerythrine, 44.3 ± 2.7 mN/mm2) There were no significant changes in the Ca2+ sensitivity after pretreatment with the inhibitor, and chelerythrine alone had no significant effect on maximum tension development or cooperativity (data not shown). To establish that indirect effects of TPA did not cause our results, we did experiments using an inactive phorbol ester. Figure 2A shows that the inactive 4alpha -phorbol (100 nM) had no significant effect on the maximum tension development (42.0 ± 1.6 mN/mm2) and no significant effect on the Ca2+ sensitivity or cooperativity (data not shown).


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  		Fig. 2.   Effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the maximum steady-state tension development and the pCa-tension relation. A: fiber bundles from NTG mice were treated under conditions described in METHODS with TPA (100 nM, n = 7), DMSO (vehicle in equal volume; n = 7), or the inactive phorbol ester 4alpha -phorbol (100 nM; n = 4) for 10 min in the presence of a phosphatase inhibitor cocktail (0.1 µg/ml calyculin A and 0.2 µg/ml okadaic acid). Pretreatment with the PKC-specific inhibitor chelerythrine (Chel, 5 µM) for 5 min was followed by a 10-min TPA treatment (n = 5). TPA treatment resulted in a 29% decrease (46.1 ± 2.5 vs. 33.4 ± 2.6 mN/mm2) in the maximum tension developed. B: maximum tension over a range of physiological [Ca2+] was also significantly depressed in TPA-treated preparations (), compared with control (). C: data were normalized and fitted to the Hill equation to obtain the -log of free [Ca2+] required for half-maximum activation (pCa50) in control 5.66 ± 0.01 and after TPA 5.63 ± 0.01. Data are means ± SE. *Significantly different from control, P < 0.02. dagger Significantly different from TPA treated.

Figure 3 shows results of experiments in which we measured tension in TG fiber bundles expressing fsTnT treated with either DMSO or TPA in the presence of phosphatase inhibitors. In preparations treated with DMSO alone, there was a consistent 18% decrease in the maximum tension developed by myofilaments containing fsTnT (Fig. 3A) compared with NTG myofilaments (Fig. 2A). This difference agrees with previous findings (3, 17, 24), indicating that variations in the NH2-terminus of TnT, as seen between cTnT and fsTnT, result in a reduction in maximum activation of the myofilaments. Furthermore, the pCa tension relation was significantly steeper (nH) in the DMSO-treated preparations containing fsTnT (nH = 3.36 ± 0.06) compared with the DMSO-treated NTG (nH = 2.86 ± 0.15). These data are in agreement with our previous findings (6) that incorporation of fsTnT in the myofilament lattice increases the cooperative activation, as reflected in nH. Interestingly, Fig. 3A shows that the maximum tension generated by TG fiber bundles was unchanged after TPA treatment (37.8 ± 1.4 mN/mm for controls and 36.4 ± 1.2 mN/mm for TPA-treated preparations). However, TPA treatment resulted in a small but significant (P < 0.05) increase in the Ca2+ sensitivity (pCa50) from 5.63 ± 0.01 in controls to 5.72 ± 0.01 in TPA-treated preparations (Fig. 3B).


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  		Fig. 3.   Effect of TPA on the maximum steady-state tension development and the pCa-tension relation in TG preparations. Fiber bundles (expressing fsTnT) were treated with DMSO (n = 12) or TPA (n = 11) in the presence of phosphatase inhibitors under the same conditions as NTG preparations (see Fig. 2). A: TPA treatment did not affect the maximum tension development in TG preparations (37.8 ± 1.4 for controls vs. 36.4 ± 1.2 mN/mm2 TPA treated). Values are means ± SE. B: data were normalized and fitted to the Hill equation to obtain the pCa50 and the Hill coefficient (nH). TPA treatment resulted in a small but significant (P < 0.05) increase in the Ca2+ sensitivity (pCa50) from 5.63 ± 0.01 in controls to 5.72 ± 0.01 TPA treated. The nH was slightly decreased after TPA treatment (n = 3.56 ± 0.07 vs. 3.05 ± 0.13) and is significantly different compared with NTG fiber bundles with or without TPA.

Effect of fsTnT incorporation on TPA-induced phosphorylation of fiber bundles. Figure 4 shows a representative gel from three experiments aimed at identification of proteins phosphorylated under the conditions of our experiments. The Coomassie blue-stained gel in Fig. 4 shows the mobility of the myofilament proteins that had been incubated with [gamma -32P]ATP before PKC activation, as described in METHODS. Autoradiography (Figs. 4 and 5) demonstrated that under our experimental conditions TPA treatment alone induced consistent increases in 32P incorporation only into the bands corresponding to cTnI (123% of control) and cTnT (124% of control). We also phosphorylated the myofilaments (Fig. 4) under the same conditions in the presence of a cocktail of phosphatase inhibitors (0.1 µg/ml calyculin A, 0.2 µg/ml okadaic acid). As shown in Figs. 4 and 5, phosphatase inhibition led to an increase in the 32P incorporation into cTnT (150% of control without inhibitors) and an increase into cTnI (300% of control without inhibitors). Pretreatment of fiber bundles with the specific PKC inhibitor, chelerythrine, decreased the TPA-induced 32P incorporation to control levels (Fig. 5). Figure 6 shows the 32P incorporation into TG fiber bundles. The Coomassie blue-stained gel shows the migration pattern of fsTnT compared with cTnT (Fig. 6). In the autoradiogram there were no visible bands corresponding to TnT in any of the lanes. However, bands corresponding to cTnI were clearly phosphorylated. Quantification of the area of the autoradiogram where cTnT and fsTnT migrate showed no difference in 32P incorporation between lanes loaded with DMSO, TPA, and chelerythrine-treated preparations in the presence of phosphatase inhibitors (Fig. 7). However, there was a small but significant 19.5 ± 4.5% increase in the 32P incorporation into cTnI in the TG autoradiogram.


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  		Fig. 4.   Effect of TPA on 32P incorporation into NTG myofilament proteins. Lane 1 shows analysis of NTG myofilaments by SDS-PAGE (12.5%). Autoradiograms after treatment with [gamma -32P]ATP as described in METHODS are shown after treatment with DMSO alone (lane 2), or with phosphatase inhibitors (lane 3), TPA alone (lane 4), TPA with phosphatase inhibitors (lane 5), and TPA treatment after chelerythrine pretreatment (lane 6). 32P incorporation was significant in the TnI and TnT bands only.



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  		Fig. 5.   Summary of the effect of TPA on 32P incorporation into NTG myofilaments. Histogram summarizing % change in 32P incorporation into TnI (A) and TnT (B). DMSO treatment alone (control) preparations were taken as 100%. +/-, Presence or absence of the phosphatase inhibitor cocktail, and Chel pretreatment, followed by TPA treatment. *Significantly different from DMSO control (P < 0.05). dagger Significantly different from DMSO plus phosphatase inhibitors (P < 0.05). Data are means ± SE from determinations on 3 separate NTG mouse heart preparations.



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  		Fig. 6.   Effect of TPA on the 32P incorporation into TG myofilament proteins. Lane 1, analysis of myofilaments from TG mouse hearts by SDS-PAGE (12.5%). The TG fsTnT migrates just above cTnT. The autoradiogram shows 32P incorporation into myofilaments treated with DMSO (lane 2), TPA (lane 3), or Chel pretreatment, followed by TPA treatment (lane 4). No 32P incorporation into TnT was apparent, suggesting that the lack of phosphorylatable residues (corresponding to cardiac Thr285 and Thr195) on fsTnT completely precludes phosphorylation of TnT.



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  		Fig. 7.   Summary of the effect of TPA on 32P incorporation in TG myofilaments. Histogram showing change expressed as % increases in 32P incorporation over control (DMSO) into TnI (A) and TnT (B). Chel pretreatment was followed by TPA treatment. Determinations were on the basis of 3 mouse heart preparations. TPA caused a significant (*P < 0.05) 19.5 ± 4.5% increase in the 32P incorporation into the band that corresponds to cTnI, compared with the 200% increase in NTG myofilaments under the same conditions (Fig. 5). Data are means ± SE from determinations on 3 separate NTG mouse heart preparations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our experiments provide evidence for a potentially important role of TnT phosphorylation in inducing depression of myofilament force. The putative PKC-specific sites on cTnT have been identified (15). However, the regulatory role that phosphorylated TnT has on activation or whether it acts synergistically with phosphorylated TnI remains unclear. Although scant, the evidence that exists concerning the role of phosphorylated TnT has come from experiments where TnT was exclusively phosphorylated in vitro with exogenous PKC isoforms and, subsequently, incorporated into fully reconstituted preparations. However, whether PKC activation leads to phosphorylation of TnT independently of TnI or vice versa has not been established in situ. Our results suggest that the degree of TnT phosphorylation, induced by endogenous PKC, affects myofilament activation, perhaps by altering structural interactions with TnI, which in turn affects the state of TnI phosphorylation in the intact lattice.

TG incorporation of fsTnT into the myofilament lattice. The TG approach was advantageous in this study for two reasons. First, it allowed us to determine the effect of endogenous mouse PKC activation. Second, we could test the effect of PKC activation on myofilaments incorporated with fsTnT in a native manner. Mochly-Rosen et al. (10-12) have shown that activated PKC does not simply diffuse to its substrate. Instead, PKC isoforms are ushered to their substrates via binding proteins (receptors for activated C-kinase or RACKs), which play a crucial role in substrate specificity in the cardiomyocyte. Although useful information can be obtained, it is unclear whether phosphorylation of the myofilaments using exogenous kinases results in the same signaling and, therefore, the ultimate response as occurs in the intact cardiomyocyte. Our experiments used a phorbol ester to induce endogenous PKC signaling and activity.

Our Western blot analysis showed that 48% of the total population of TnT incorporated into the myofilament lattice is fsTnT. This potentially means that every other functional unit has the fast skeletal isoform, assuming uniform distribution. Using immunofluorescent microscopy, our previous data (6) showed that fsTnT was incorporated normally into the cardiac myofilament lattice. In our experiments, we used the same line of mice, which was found to be free of developmental and systemic side effects.

Effect of PKC-mediated phosphorylation on Ca2+-dependent myofilament activation. Activation of the actin-myosin interaction requires the Ca2+-TnC-mediated reversal of a prevailing inhibition imposed by TnI, and a possible activation by an interaction of Ca2+-TnC with TnT. Our findings indicate that the ability of Ca2+-TnC to fully turn on the actin-myosin reaction is blunted with PKC-mediated phosphorylation of cTnT and cTnI. On the basis of studies with a deletion mutant of fsTnI missing residues 1-57 that normally bind fsTnT, Potter et al. (18) concluded that direct interactions between Ca2+-fsTnC and fsTnT may activate actin-myosin interactions. Therefore, a straightforward mechanism for the action of TnT phosphorylation on maximum tension could be a decrease in the affinity of TnT for Ca2+-TnC. This, in turn, would result in a tighter association of TnT and tropomyosin (Tm) during Ca2+ activation (21). Ultimately, a population of myosin binding sites on actin would remain blocked, and the thin filament would be submaximally activated at saturating Ca2+ levels. This is by no means a modest decrease, given that only a fraction of cross-bridges operate during resting conditions in the organism. Interestingly, when TnI was phosphorylated without TnT phosphorylation in the TG preparations, no change in the tension development occurred. In the absence of phosphorylation, it is possible that TnT binding to Tm is unaltered on Ca2+ activation and that full activation results. The issue of whether differential in vivo phosphorylation levels could affect our results does not apply here because there was no phosphorylation of TnT in the TG mice even in the presence of phosphatase inhibitors. Together, these results indicate that the functional consequence of PKC-mediated phosphorylation is dependent on the phosphorylation state of both TnI and TnT in the intact lattice.

PKC activation in the TG myofilaments resulted in a modest but significant increase in the Ca2+ sensitivity. Although it is not clear why this occurs, a more complex mechanism involving altered interactions with cTnI may come into play here. It is apparent that TnT not only mediates the activation of neighboring functional units in a cooperative manner, but may also link TnI phosphorylation to changes in actomyosin interactions. We hypothesize that the 1:1 relation of fsTnT:cTnT in the myofilament lattice, which considerably alters the cooperative activation, is also responsible for structural changes between functional units (20).

Effect of fsTnT incorporation on troponin phosphorylation. The clustering of phosphorylation sites at regions of interaction between TnT and TnI suggests that PKC-mediated phosphorylation of these components and its functional consequences may not be exclusive of each other. cTnT and cTnC interact in a region composed of cTnI amino acid residues 33-80. cTnC and cTnI interact with cTnT in a region composed of amino acids 190-289 (16). These regions on both cTnI and cTnT contain the sites for PKC-mediated phosphorylation. It is, therefore, apparent that substitution of fsTnT, which lacks two of the three sites homologous to those on cTnT, may significantly alter the level of cTnI phosphorylation. Structural changes induced by the absence of these TnT sites could alter the accessibility of TnI sites, Ser43, Ser45, and Thr144, to PKC or could alter the substrate specificity of the TnI sites for PKC. It is also possible that increased activity or altered localization of protein phosphatases in the TG hearts could affect levels of TnI phosphorylation. Because of the relatively high activity of phosphatases, we do not believe the lack of incorporation of 32P into TnI is caused by an increased basal state of TnI phosphorylation in the TG preparations versus controls.

An important finding of our experiments was the demonstration that replacement of about one-half of the endogenous cTnT with fsTnT completely inhibited PKC-induced phosphorylation of TnT. These results provide further evidence that alterations of troponin components in one structural unit, consisting of actin-Tm-Tn complex in a 7:1:1 ratio, influence the properties of neighboring units (20). A change in steepness of the pCa force relation induced by incorporation of fsTnT into the cardiac myofilaments (6) also provides evidence that interactions among these molecules is altered. What is new here is the finding that the remaining cTnT molecules become poor substrates for PKC. This could occur because of conformational changes that result in decreased accessibility of the sites to PKC, which could alter the Michaelis constant (Km) of maximal velocity (Vmax) activity. These conformational changes could also alter the binding signal for RACKs (12) and thereby account for a loss of PKC-induced phosphorylation. Whatever the case, our data suggest that incorporation of modified TnT molecules into the lattice may not only alters the response of the myofilaments to Ca2+, but also alters the substrate specificity of myofilament proteins to kinases.

We have isolated functional effects of Tn phosphorylation by treating the preparations with phorbol esters in low Ca2+. Therefore, the functional consequence of PKC activation is the result of changes in the degree of troponin phosphorylation. Because we did not induce phosphorylation of myosin binding protein C (MyBPC) and myosin light chain-2 (MLC2), proteins shown to change Ca2+ responsiveness (27), the effect of the phosphorylated state of these proteins on myofilament activity has been excluded. There has been some controversy regarding the effect of PKC-mediated phosphorylation on myofilament Ca2+ sensitivity. PKC-mediated phosphorylation MLC2 has been shown by some investigators to increase the Ca2+ sensitivity and maximum ATPase rate (4), whereas others find no change in Ca2+ sensitivity (14, 25). We observed no changes in Ca2+ sensitivity in the NTG preparations (i.e., all phosphorylation sites present). These data suggest that the discrepancies in the literature concerning the effect of PKC on Ca2+ sensitivity may be because of differential phosphorylation of MLC2 or MyBPC.

In conclusion, the precise role of PKC activation in the cardiac myocyte is not entirely understood. It is apparent that sustained activation and upregulation of PKC isoforms, as occurs in response to hypertrophic signals, may prove counterproductive in the long term. In fact, a proposed mechanism for the maladjustment of the heart in the transition from compensated hypertrophy to decompensated failure is unchecked cTnI phosphorylation (21, 23). Our data add a new dimension to this hypothesis and suggest an important role for cTnT. Furthermore, we report the interesting possibility that a synergy between cTnT and cTnI is required for PKC-mediated phosphorylation to exert its inhibitory effect on the actin-myosin interaction. Thus our data provide insight into a potential physiological role for TnT phosphorylation.


    ACKNOWLEDGEMENTS

We thank Linda Alaniz-Avila for assistance with gel photographs.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant R37-HL-22231 (to R. J. Solaro) and an American Heart Association grant (to J.-P. Jin).

Address for reprint requests and other correspondence: R. J. Solaro, Dept. of Physiology and Biophysics, College of Medicine, Univ. of Illinois at Chicago, 835 S. Wolcott, M/C 901, Chicago, IL 60612 (E-mail: SolaroRJ{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 August 2000; accepted in final form 5 October 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(3):H1011-H1018
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