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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
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials.
Calyculin A and calphostin C were obtained from Calbiochem. Okadaic
acid, chelerythrine, 4
-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 -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 ml1
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
4-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 [
-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 ml1 [-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 
-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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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 4-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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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 [-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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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We thank Linda Alaniz-Avila for assistance with gel photographs.
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FOOTNOTES |
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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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METHODS
RESULTS
DISCUSSION
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), compared with control
(
). C: data were normalized and fitted to
the Hill equation to obtain the 
Significantly different from TPA treated.
