From the University of Illinois at Chicago,
Department of Physiology and Biophysics, Program in Cardiovascular
Sciences, College of Medicine, Chicago, Illinois 60612 and the
¶ University of California at Los Angeles, Department of
Physiology, School of Medicine, Center for Health Sciences, Los
Angeles, California 90025
Received for publication, October 18, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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There is evidence that multi-site phosphorylation
of cardiac troponin I (cTnI) by protein kinase C is important in
both long- and short-term regulation of cardiac function. To determine
the specific functional effects of these phosphorylation sites
(Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI
was replaced with cTnI phosphorylated by protein kinase C- Specific modifications of charge in thin filament proteins are
known to have significant effects on myofilament regulation and heart
function. Single point mutations linked to familial hypertrophic
cardiomyopathies involve charged amino acids on a number of thin
filament proteins that induce alterations in myofilament regulation by
Ca2+, as well as alterations in modulation of
Ca2+ regulation by pH and sarcomere length (1, 2).
Moreover, these charge modifications of the thin filament are well
correlated with altered myocardial dynamics that may be significant in
the evolution of hypertrophy and sudden death (3). An important question is whether charge changes introduced into the thin filament proteins by protein phosphorylation may also be causal in cardiac dysfunction (4). In experiments reported here we introduced charge
changes into specific protein kinase C
(PKC)1 sites of cardiac
troponin I (cTnI) by either protein phosphorylation or mutation of
these sites to glutamic acid. We tested the effect of these changes on
Ca2+ regulation of steady-state tension generated by the
myofilaments as well as the speed of thin filament sliding on myosin
heads in the in vitro motility assay.
cTnI is a key regulatory and inhibitory protein of the thin filament,
which together with the tropomyosin binding unit of troponin (Tn),
cardiac troponin T (cTnT), the Ca2+ binding unit, cardiac
troponin C (cTnC), and tropomyosin (Tm) confers Ca2+
sensitivity to the actin-myosin reaction. The phosphorylation of cTnI
at specific sites (Ser-23 and Ser-24) that are substrates for protein
kinase A and specific sites (Ser-43, Ser-45, and Thr-144) that
are substrates for PKC appears to be of special significance with
regard to modulation of myofilament function and control of cardiac
dynamics (5-8). Our hypothesis has been that covalent modification of
cTnI not only plays a role in the homeostasis of cardiac function, but
also in the decline of cardiac function that occurs in the transition
from compensated hypertrophy to heart failure. Ample evidence indicates
that phosphorylation of cTnI at Ser-23 and Ser-24 results in a
reduction in myofilament Ca2+ sensitivity (9) and an
increase in cross-bridge cycling (6, 10). These effects of
protein kinase A-mediated phosphorylation of cTnI are important
elements in the enhanced relaxation that is critical to the response of
the heart to adrenergic stimulation (6, 11-13). The role of the PKC
sites on cTnI in the regulation of myofilament response to
Ca2+ and regulation of cross-bridge cycling is less clear.
An understanding of the role of these sites is particularly important
in view of evidence from studies on failing human hearts (14, 15) and on hearts from transgenic mouse models (16, 17). These studies indicate
that increased activity and expression of PKC may lead to excessive
and/or persistent phosphorylation of cTnI by PKC with an associated
depression in tension-generating capability. Myofilaments from hearts
of transgenic mice expressing the mutant cTnI-S43A/S45A (substitution
of alanine residues, A, for serine residues, S) demonstrate a
blunting of the effect of PKC-mediated phosphorylation to depress
maximum tension (17). Moreover, these transgenic mouse hearts contract
and relax much faster than the controls and demonstrate significant
alterations in the Ca2+-pressure relationship of isolated,
perfused hearts (18).
Exactly how phosphorylation of the PKC sites on cTnI affects
cross-bridge function in the myofilament lattice remains unknown. Studies with transgenic hearts are revealing but difficult to interpret. Apart from the existence of multiple sites for PKC-mediated phosphorylation, substitution of alanine for serine in the transgenesis is not benign. This substitution itself causes a depression in both
maximum tension and maximum Ca2+-stimulated actomyosin
MgATPase activity in fully reconstituted preparations (7). In addition,
modification of the PKC sites on cTnI influences the ability of PKC to
phosphorylate cTnT (19). The reverse is true as well (17).
We report here new evidence on the role of the specific PKC sites of
cTnI in regulation of the actin-myosin interaction. We phosphorylated
cTnI directly by PKC- Protein Purification and Preparation--
Expression and
purification of recombinant human cTnC, mouse cTnI (wild-type (WT) and
mutant), and mouse cTnT was previously described (21). The preparation
of actin, myosin, heavy meromyosin, and rhodamine phalloidin-labeled
actin for use in the motility assay was previously described by Homsher
et al. (22). Bovine ventricular tropomyosin was prepared
according to the method described by Tobacman and Adelstein (23).
Troponin reconstitution for in vitro motility studies was
accomplished by mixing cTnI, cTnC, and cTnT in final concentrations of
10 µM each, in the presence of 6 M urea. The
mixture was sequentially dialyzed at 4 °C, each time for at least
12 h, against 10 mM MOPS (pH 7.0), 1 mM
DTT, 0.01% NaN3, 50 µM CaCl2,
and 1) 1 M KCl, 2 M urea; 2) 1 M
KCl; 3) 0.1 M KCl; 4) 0 M KCl. After the last
dialysis, proteins were centrifuged and purified using fast protein
liquid chromatography. Purified whole Tn was collected and a mixture of
protease inhibitors (5 mg/ml each of TPCK, TLCK, and 0.3 mM
phenylmethylsulfonyl fluoride) was added before proteins were separated
into aliquots and stored at Phosphorylation of cTnI by PKC- Preparation of Detergent-skinned Fiber Bundles and Treatment with
cTnT-cTnI--
Left ventricular papillary muscle fiber bundles were
dissected from male CD-1 mice (age 3 months) and detergent-skinned
overnight as previously described (25). The detergent-skinned fiber
bundle was mounted between a force transducer and a micro-manipulator, and the sarcomere length was adjusted to 2.3 µM. Initial
maximum isometric force was measured in activating solution
(pCa 4.5) containing 20 mM MOPS (pH 7.0), 33.8 mM KCl, 10 mM EGTA, 9.96 mM
CaCl2, 5.39 mM ATP, 6.47 mM
MgCl2, 12 mM creatine phosphate, 10 IU/ml
creatine kinase, and 1 mM DTT. The fiber bundle was then placed in high relaxing solution containing 20 mM MOPS (pH
7.0), 53.5 mM KCl, 10 mM EGTA, 0.025 mM CaCl2, 1 mM free
Mg2+, 5 mM MgATP2 In Vitro Motility Assay--
Rhodamine phalloidin-labeled actin
movement over heavy meromyosin-covered nitrocellulose surfaces was
monitored in motility chambers by video epifluorescence microscopy and
analyzed as previously described (22). Forty µl of 270-300 µg/ml
heavy meromyosin was injected into the motility chamber and allowed to
incubate for 2 min. This was followed by 40 µl of 1 mg/ml bovine
serum albumin (Sigma), which incubated for 1 min. The slide chamber was
then washed with two 40 µl aliquots of low salt assay (AB) buffer (20 mM KCl, 25 mM MOPS (pH 7.4), 0.5 mM
EGTA, 2 mM MgCl2, 1 mM DTT). Next,
40 µl of rhodamine phalloidin-labeled actin was injected into the
motility chamber and incubated for 1 min in the chamber. Again, the
slide chamber was washed with two 40-µl aliquots of AB buffer. This
was followed by injection of 40 µl of a 50-mM ionic
strength reconstitution solution containing regulatory proteins (Tn and
Tm, ranging in concentrations from 0.1-0.4 µM). The
reconstitution solution consisted of 25 mM MOPS (pH 7.4),
25 mM KCl, 2 mM MgCl2, 2 mM EGTA (with varying ratios of CaKEGTA to
K2EGTA), and 10 mM DTT. The chamber was
incubated in this solution for 5 min, and the solution replaced by two
40-µl washes of motility solution, which was a reconstitution
solution to which 1 mM Na2-ATP and a
glucose/oxidase/catalase mixture had been added to slow photobleaching (3 mg/ml glucose, 100 µg/ml glucose oxidase, 10 µg/ml catalase).
Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Cardiac myofilament proteins from skinned fiber bundles
were analyzed on 12.5% SDS-polyacrylamide gels as well as 8% alkaline urea polyacrylamide gels as previously described (25). The exchange of
endogenous Tn with recombinant Tn components was demonstrated by
Western blot analysis using a primary anti-cTnT antibody (Sigma) and
secondary goat anti-mouse IgG antibody conjugated to
horseradish-peroxidase (Sigma).
Statistical Analysis--
Data from the pCa-force
measurements in skinned fibers were normalized and fitted to the Hill
equation by using a nonlinear least-square regression procedure to
obtain the pCa50 ( Tension Measurements in Skinned Fiber Bundles--
Our strategy to
analyze the functional role of charge modification at the PKC sites on
cTnI involved exchange of native Tn components in detergent extracted
(skinned) fiber bundles with Tn containing phosphorylated or mutant
forms of cTnI. We used a procedure illustrated in a chart recording
depicted in Fig. 1A, in which
the tension of the skinned fiber bundle was first measured over a range
of pCa values. The fiber bundle was then exposed to a large
excess of cTnT-cTnI under conditions described under "Experimental
Procedures." The advantage of this approach, which was
developed in our lab by Chandra et al. (25), over the use of
cTnT alone (27), is that the fiber bundle is more relaxed during the
exchange. The recording in Fig. 1A shows that after the
incubation in cTnT-cTnI solution, tension generated by the fiber bundle
at pCa 4.5 was a very small fraction of the control value
(varied from 3-30% depending on cTnI mutant used). The ratio of this
tension to the control maximum tension serves as an index of the extent
of replacement of Tn with cTnT-cTnI and the associated removal of cTnC.
Fig. 1B shows Coomassie-stained SDS-PAGE analysis of
myofilament proteins from skinned fiber bundles with endogenous Tn
complex (lane 1), after removal of endogenous Tn complex by
extraction with cTnT-cTnI complex (lane 2), and after
subsequent reconstitution of Tn complex with cTnC treatment (lane
3). A pure cTnC standard is shown in lane 4. The
exogenous, recombinant mouse cTnT was modified with a myc tag, which
slowed its mobility so it runs closer with actin. In lanes 2 and 3, the band for actin appears larger, due to the
presence of recombinant cTnT running closer to actin. The myc tag has
no effect on cTnT function but served to alter the mobility of cTnT,
permitting demonstration of exchange of Tn. Also, the band for cTnC is
usually hard to detect in SDS gels, as it runs closely with myosin
light chain-2 and does not normally stain well with Coomassie blue. However, in lane 3 of Fig. 1B, the band for
myosin light chain-2 appears darker, probably due to the presence of
recombinant cTnC due to incubation of the skinned fiber with the cTnC
reconstitution, which has a high concentration of cTnC (4 mg/ml). Fig.
1C shows Western blot analysis of cTnT from skinned fiber
bundles with endogenous Tn complex (lane 1), after removal
of endogenous Tn complex by extraction with cTnT-cTnI complex
(lane 2), and after subsequent reconstitution of Tn complex
with cTnC treatment (lane 3). The endogenous cTnT shown in
lane 1 demonstrates a faster mobility, compared with
recombinant cTnT shown in lanes 2 and 3, and this
band essentially disappears after extraction and reconstitution (lane 3), indicating exchange of Tn occurred. A pure cTnT
standard is shown in lane 4. Further evidence of Tn exchange
is illustrated in Fig. 1D, which visualizes the cTnC band
more clearly by non-SDS alkaline urea PAGE analysis. The presence of
cTnC in the untreated skinned fiber bundles (i.e. still
containing endogenous Tn complex) is shown in lane 1. Subsequent removal of the endogenous Tn complex by extraction with
cTnT-cTnI complex results in loss of cTnC from the fiber bundle
(lane 2), which accounts for the decrease in tension
generated by the fiber bundle at pCa 4.5 (Fig.
1A). After reconstitution of Tn complex with cTnC treatment
the cTnC band reappears (lane 3). This incubation of the
fiber bundle in cTnC solution restored maximum tension to a value close
(~75%) to that of the control, and was maintained during a series of
measurements over a range of pCa values. A pure cTnC
standard is shown in lane 4.
The pCa-tension curves obtained after the exposure of the
fiber bundle to the same series of solutions, which did not contain cTnT-cTnI or cTnC, were essentially the same as those obtained after
replacement of the endogenous Tn with exogenous native Tn (data not
shown). This result indicated that the fall-off of maximum tension was
time-dependent and that the exchange protocol using native
Tn components did not result in changes in the myofilament activity due
to nonspecific interactions of the cTnT-cTnI complex to other regions
of the thin filament. The pCa-tension relation of fiber
bundles incubated with exchange solutions without proteins (cTnT-cTnI
or cTnC) gave pCa50 values of 5.70 ± 0.01, whereas fiber bundles incubated with exchange solutions containing
native Tn components gave pCa50 values of
5.72 ± 0.01.
Fig. 2 compares pCa-tension
relations for fiber bundles in which the endogenous cTnI was exchanged
with various forms of exogenous cTnI. Data in Fig. 2A show
that compared with controls, exchanged with unphosphorylated WT cTnI,
which had a pCa50 = 5.55 ± 0.01, fiber
bundles containing cTnI exhaustively phosphorylated by PKC- In Vitro Motility Assay Measurements--
Our studies employing
the in vitro motility assay also demonstrated that specific
charge changes at the PKC sites on cTnI depress thin filament sliding
over myosin heads and its sensitivity to Ca2+. Compared
with control filaments regulated by WT Tn, the speed of sliding of thin
filaments regulated by Tn complexes containing cTnI phosphorylated at
cTnI-PKC sites decreased by as much as 60% at various pCa
values. Thin filaments containing cTnI-P demonstrated a decrease in
maximum sliding speed (Vmax) of 43% and a
decrease in the pCa50 of 1.3 pCa
units (Fig. 4A and Table I)
compared with controls. Sliding speed of actin filaments regulated by
Tn complexes containing cTnI-S43E/S45E/T144E decreased by as much as
85% at various free Ca2+ concentrations;
Vmax decreased by 55% and the
pCa50 decreased by 0.7 pCa units
(Fig. 4B and Table I) compared with controls. Sliding speed
of actin filaments regulated by Tn complexes containing cTnI-S43E/S45E
decreased by as much as 30% at various pCa values; Vmax decreased by 15% compared with controls,
whereas the change in pCa50 was not
statistically different compared with controls (Fig. 4C and
Table I). Lastly, actin filaments regulated by Tn complexes containing
cTnI-T144E demonstrated a decrease in the pCa50
of 0.5 pCa units, with no significant change in maximum filament sliding speed (Fig. 4D and Table I) compared with
controls. It is apparent, therefore, that all three PKC sites must be
phosphorylated to elicit a maximum inhibition of sliding speed.
Data presented here provide new evidence on the scope of
functional effects of cTnI phosphorylation on myofilament function. Previous data indicated that phosphorylation of the N-terminal protein
kinase A sites is able to decrease Ca2+ sensitivity with no
effect on maximum tension and to increase shortening velocity and
cross-bridge cycling rate (6, 11-13). We report here that
phosphorylation of Ser-43 and Ser-45 (not Thr-144) dominates regulation
of the level of maximum tension. However, phosphorylation of Thr-144,
in addition to phosphorylation of Ser-43 and Ser-45, appears to be
required for regulation of thin filament sliding speed. This result may
not be surprising in that the cTnI domain containing Ser-43 and Ser-45
is located at sites distinct and apparently distant from the domain
containing Thr-144. Ser-43 and Ser-45 are located in the near
N-terminal region of cTnI that binds to the C terminus of cTnT and the
C-lobe of cTnC, whereas Thr-144 is located in the highly basic
inhibitory region. Together with a C-terminal region of cTnI (28), the cTnI inhibitory region is the molecular switch that turns on
contraction when this region of cTnI moves from actin-Tm to the
N-terminal lobe of cTnC upon Ca2+ binding to the regulatory
site. It is also apparent from our data that Thr-144, which is a
cardiac-specific residue (Pro in ssTnI and fsTnI), may represent a
structural specialization in cTnI related to the regulation of cardiac
muscle, which lacks the ability to regulate function through the
recruitment of motor units.
The depression of maximum tension and Ca2+ sensitivity
induced by phosphorylation or glutamic acid substitution at Ser-43 and Ser-45 may involve altered interactions of the near N-terminal region
of cTnI with the C-terminal lobe of cTnC. Data on the crystal structure
of a complex containing cTnC, a large proportion of cTnI, and a large
peptide comprised of most of the C-terminal region of cTnT demonstrate
an interaction between the C-lobe of cTnC and the cTnI region
containing Ser-43 and Ser-45 (29). Although the C-lobe of cTnC is
generally considered to be important in the structural stability of the
thin filament, evidence on the binding sites and functional effects of
the Ca2+ sensitizing agent EMD 57033 indicates a role for
the C-lobe of cTnC and its interaction with the near N terminus of cTnI
in tension regulation. EMD 57033, which increases sub-maximal and
maximal tension developed by skinned fiber preparations (30), docks in
a stereospecific manner to the C-lobe of cTnC (31). Moreover, a cTnI
peptide comprised of the near N-terminal region of cTnI (cTnI34-71) and containing Ser-43 and Ser-45 was
demonstrated to displace EMD 57033 from its binding site (31).
Interestingly, EMD 57033 was not displaced by the presence of a cTnI
peptide containing the inhibitory region
(cTnI128-147).
The effect of a charge change at Ser-43 and Ser-45 of cTnI on
steady-state tension may involve effects on the affinity of cross-bridges for the thin filament as well as
cross-bridge-dependent activation. In earlier studies of
ATPase rate of reconstituted preparations, we reported a decrease of
the affinity (Kapp) of myosin S-1 for regulated
thin filaments containing cTnI phosphorylated at Ser-43 and Ser-45
residues when compared with those containing unphosphorylated residues
(7). However, data reported by Morimoto et al. (32) also
indicate that phosphorylation-induced modulation of interactions of
cTnI with the C-lobe of cTnC may alter
cross-bridge-dependent activation of cardiac myofilaments.
In these studies, activation of myofilaments with and without cTnC was
accomplished at pCa 9 by varying MgATP concentration and
thus the number of strongly bound rigor cross-bridges. The ability of
rigor cross-bridges to activate the thin filament depended on the
presence of cTnC and was also dependent on the isoform population of
TnI. Inasmuch as the C-lobe of cTnC forms the major interaction with
cTnI at low Ca2+, Morimoto et al. (32) concluded
that activation of the myofilaments by the strongly bound cross-bridges
is dependent on an interaction between the C-lobe of cTnC and cTnI. The
activation of the thin filament by myosin is generally thought to
involve a movement of Tm induced by strongly bound cross-bridges (33).
Yet, the data of Morimoto et al. (32) indicate that the
ability of strong cross-bridges to move Tm may also involve cTnI and
its interaction with the C-lobe of cTnC. It seems reasonable to
speculate that this role of cTnI in cross-bridge-dependent
activation may be modified by phosphorylation at Ser-43 and Ser-45.
The mechanism by which phosphorylation at Ser-43 and Ser-45 together
with phosphorylation at Thr-144 might alter the unloaded velocity of
thin filament sliding is not likely to involve altered binding of
cross-bridges to the thin filament. Previous studies (34, 35) have
demonstrated that unloaded shortening velocity is independent of the
filament overlap and thus cross-bridge availability. Velocity of thin
filament sliding is more likely to be modified by the step-size of the
cross-bridge (i.e. how far the cross-bridge pulls the thin
filament in its cycle) or the rate of cross-bridge detachment (which is
a function of ADP release and ATP binding to the cross-bridge).
Our results support the hypothesis that alterations in the thin
filament proteins affect the reaction of cross-bridges with the thin
filament by an allosteric mechanism. The depression in speed of thin
filament sliding especially at the saturating levels of
Ca2+ suggests that the charge change at the PKC sites of
cTnI either decreases the step-size of the cross-bridge or decreases
the rate of cross-bridge detachment at the end of the power stroke. As argued previously (36) it seems unlikely that phosphorylation of cTnI
would alter the step-size. However, there is evidence that
phosphorylation of cTnI at Ser-43 and Ser-45 may affect cross-bridge detachment. In studies comparing force and ATPase rate of myofilaments from non-transgenic (NTG) mice and transgenic (TG) mice expressing a
mutant cTnI, cTnI-S43A/S45A, Pyle et al. (19) reported that compared with the skinned fiber bundles from NTG mice, skinned fiber
bundles from the TG cTnI-S43A/S45A mice demonstrated an increase in
tension cost. That is, the ATPase rate at a given level of tension was
higher in the TG than the NTG preparations. As expected, levels of
phosphorylation at PKC sites were higher in the NTG preparations than
the TG preparations. These data indicate that the cross-bridge
detachment is indeed affected by PKC-mediated phosphorylation of
the myofilaments.
Although the precise physiological significance of signaling through
the PKC pathway and phosphorylation of myofilament proteins in the
intact heart remains unknown, there is indirect evidence that
PKC-mediated phosphorylation of cTnI may have important effects on
short-term, beat-to-beat regulation of the heart. PKC-mediated phosphorylation of the myofilaments has been shown to be associated with reduced myofibrillar ATPase rate (7, 37-39) and also with decreased unloaded shortening velocity (40, 41). Studies on transgenic
mice missing PKC phosphorylation sites on cTnI indicate that these
changes in myofilament activity are translated into altered cardiac
function (17, 42). Montgomery et al. (17) reported that the
depression of maximum tension of papillary muscles from NTG controls by
the Myofilament protein phosphorylation through the PKC pathway may also be
of significance in long-term regulation of heart function in adaptation
to hemodynamic stressors such as hypertension and myocardial
infarction. Up-regulation of the PKC pathway is well known to promote
transcription and growth of heart cells (15, 43, 44). Our hypothesis is
that this increased activity of PKC is associated with myofilament
phosphorylation and decreased force and shortening. Thus, at a time
when the heart has engaged pathways to induce cellular hypertrophy, the
power of muscle contraction may be declining and leading to a vicious
cycle resulting in decompensation and heart failure. Evidence in
support of this idea comes from the data of Bowling et al.
(14), who reported an increase in the expression level of PKC- Yet, it is a challenge to understand the exact role of PKC in the
myocardium. PKC has multiple and varied effects on cardiac function
that could be attributed to the multiple PKC phosphorylation sites on
cTnI, as well as on its other substrates, which include cTnT, myosin
binding protein C, and myosin light chain-2 (49). Furthermore, PKC can
also phosphorylate ion pumps, channels, and exchangers. We think data
presented here, which show the specificity of functional effects of
phosphorylation of a specific substrate, provide an important tool for
understanding the complex integration of PKC-mediated signaling in both
short- and long-term regulation of cardiac function.
or
mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used
detergent-skinned mouse cardiac fiber bundles to measure changes in
Ca2+-dependence of force. Compared with controls,
fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or
cTnI-S43E/S45E were desensitized to Ca2+, and maximum
tension was as much as 27% lower, whereas fibers reconstituted with
cTnI-T144E showed no change. In the in vitro motility assay
actin filaments regulated by troponin complexes containing
phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in
Ca2+ sensitivity and maximum sliding speed compared with
controls, whereas filaments regulated by cTnI-S43E/S45E showed only
decreased maximum sliding speed and filaments regulated by cTnI-T144E
demonstrated only desensitization to Ca2+. Our
results demonstrate novel site specificity of effects of PKC
phosphorylation on cTnI function and emphasize the complexity of
modulation of the actin-myosin interaction by specific changes in the
thin filament.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TnI-P), one of the predominant isoforms of PKC found in the adult cardiac myocyte (20), and incorporated either the unphosphorylated or the phosphorylated cTnI
into thin filaments. To determine the specific role of the phosphorylation induced change in charge, we also generated the following mutant forms of cTnI: cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, and cTnI-T144E in which serine (S) and/or threonine (T) residues were
replaced by glutamic acid (E). Our experiments provide direct evidence
that charge change at PKC sites of phosphorylation inhibits the
actin-myosin interaction by causing decreases in maximum tension, Ca2+ sensitivity, and thin filament sliding speed. Our data
also indicate that phosphorylation of one region of cTnI may be more
significant in regulating tension than filament sliding speed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C for up to 6 months.
--
Recombinant
PKC-
was prepared in our laboratory using a recombinant baculovirus
system as previously described (24). Phosphorylation of cTnI for
in vitro motility assay studies was accomplished by first
forming a 10 µM cTnI-cTnC complex in 50 mM
Tris-HCl (pH 8.0), 1 M KCl, 10 mM DTT, and 0.2 M phenylmethylsulfonyl fluoride. This complex was then
phosphorylated in the presence of 0.4 mM [
-35S]ATP (Sigma), 0.1% Triton X-100 (Sigma),
0.3 mM phosphatidylserine (Avanti Polar-Lipids), and 0.2 mM diacylglycerol (Avanti Polar-Lipids) in a reaction
initiated by ~0.5 µM PKC-
for 1 h at 30 °C.
The reaction was stopped by adding 6 M urea to the
solution. cTnT (10 µM) was then added to the mixture to
reconstitute the whole Tn complex. The mixture was sequentially
dialyzed at 4 °C, each time for at least 12 h, against 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 100 µM CaCl2, 1 mM DTT and 1) 1 M KCl, 2) 0.7 M
KCl, and 3) 0.3 M KCl, before purification by fast phase
liquid chromatography. Purified whole Tn was collected, and a mixture of protease inhibitors was added. The protein kinase A sites present in
cTnI-S43E/S45E/T144E were not phosphorylated by PKC-
under our
conditions. Phosphorylation of cTnI for skinned fiber studies was
similar, except cTnI was not complexed to cTnC for phosphorylation. The
phosphorylation reaction was stopped by adding 6 M urea to the solution. cTnT (10 µM) was then added to the mixture
to form a cTnT-cTnI complex used for the extraction procedure in
skinned fiber studies. The mixture was dialyzed overnight against
extraction buffer containing 20 mM MOPS (pH 6.5), 250 mM KCl, 5 mM EGTA, 5 mM
MgCl2, and 1 mM DTT at 4 °C. The next day,
insoluble protein was removed by centrifugation at 5,000 rpm for 30 min. After centrifugation, protease inhibitors were added to the
supernatant fraction containing the cTnT-cTnI complex.
, 12 mM creatine phosphate, and protease inhibitors (1 µg/ml
pepstatin, 5 µg/ml leupeptin, and 0.2 mM
phenylmethylsulfonyl fluoride. Force was measured while the fibers were
bathed in sequentially increasing Ca2+ concentrations
ranging from pCa 8 to 4.5. pCa values were
calculated as previously described using binding constants reported by
Godt and Lindley (26). The Tn exchange was carried out by treating the
fiber for 90 min with 2 ml of extraction solution containing cTnT-cTnI,
prepared as previously described (25), then 10 min with 2 ml of
extraction buffer (without cTnT-cTnI) and 10 min with 2 ml of high
relaxing. Maximum Ca2+-activated force was then measured in
pCa 4.5 solution to determine the extent of endogenous
troponin removed. Next, the cTnT-cTnI-treated fiber was treated for 90 min with a TnC reconstitution solution, prepared as previously
described (25), followed by measurement of maximum
Ca2+-activated force in pCa 4.5 solution and
subsequent force measurements ranging from pCa 8 to
pCa 4.5.
log of free
Ca2+-concentration required for half-maximal activation)
and the Hill coefficient (n). Statistical differences were
analyzed by an unpaired t test and ANOVA with the criteria
for significance set at p < 0.05. Data are expressed
as means ± S.E. The in vitro motility assay data were
acquired and analyzed as previously described in detail (22). Typically
the speed at a given pCa is reported as a mean for
~150-300 filaments. Data are reported as means ± S.E.
Plots of the mean filament sliding speed versus
pCa were fitted to the Hill equation. Fits to the Hill
equation were made from a regression using all of the observed speeds
so that a fit was generated from over 1200 filaments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Procedure for Tn exchange in Triton X-100
skinned mouse cardiac fiber bundles. A, original chart
recording of the force tracings generated by skinned fibers during the
extraction-reconstitution procedure (see "Experimental
Procedures"). The force is measured and recorded while the fibers are
bathed in solutions of varying Ca2+ concentrations,
expressed as pCa or the log [Ca2+].
Fibers are mounted and sarcomere length is set to 2.3 µM.
Fibers are first maximally contracted in a solution of pCa
4.5. Next, the fiber is bathed in solutions of sequentially decreasing
pCa values, ranging from pCa 8 to 4.5. Removal of
the native Tn complex is accomplished by incubating the fiber in a
cTnT-cTnI extraction solution. B, Coomassie-stained SDS-PAGE
analysis of myofilament proteins from skinned fiber bundles with
endogenous Tn complex (lane 1), after removal of endogenous
Tn complex by extraction with recombinant cTnT-cTnI complex (lane
2), and after subsequent reconstitution of Tn complex with cTnC
treatment (lane 3). A pure cTnC standard is shown in
lane 4. C, Western blot analysis of cTnT presence
(using anti-cTnT antibody) from skinned fiber bundles with endogenous
(e) Tn complex (lane 1), after removal of
endogenous Tn complex by extraction with recombinant (r)
cTnT-cTnI complex (lane 2), and after subsequent
reconstitution of Tn complex with cTnC treatment (lane 3).
Pure cTnT standard is shown in lane 4. D, non-SDS
alkaline urea PAGE analysis of TnC presence from skinned fiber bundles
with endogenous Tn complex (lane 1), after removal of
endogenous Tn complex by extraction with cTnT-cTnI complex (lane
2), and after subsequent reconstitution of Tn complex with cTnC
treatment (lane 3). Pure cTnC standard is shown in
lane 4.
(cTnI-P)
had a reduced sensitivity to Ca2+
(pCa50 = 5.38 ± 0.01). As
summarized in Fig. 3B and
Table I, there was also a significant
26% decrease in maximum tension of the myofilaments induced by cTnI
phosphorylation at the PKC sites. Replacement of endogenous Tn with Tn
complex containing cTnI-S43E/S45E/T144E, in which charged glutamic acid
residues replaced the two serines and one threonine that are substrates
for PKC (Fig. 2B), resulted in a desensitization of the
myofilaments to Ca2+ with a decrease in the
pCa50 to 5.31 ± 0.01 and a 26% depression in maximum tension (Fig. 3B and Table I). Thus, the presence of cTnI-S43E/S45E/T144E in place of WT cTnI decreased the
pCa50 by 0.24 units and decreased
Ca2+ sensitivity to a slightly greater extent than that of
cTnI-P, which gave a decrease in the pCa50 of
0.17 pCa units. When unphosphorylated cTnI was replaced with
cTnI-S43E/S45E (Fig. 2C), the pCa50
decreased by 0.21 units and tension decreased by 27% (Fig.
3B and Table I). Replacement of cTnI with cTnI-S43D/S45D
(serine residues replaced by aspartic acid residues), however, induced
only a relatively small change in myofilament Ca2+
sensitivity (a decrease in the pCa50 of 0.11 units) and a 20% decrease in maximum tension (data not shown) compared
with the effects of replacement of cTnI with cTnI-S43E/S45E. Lastly,
when unphosphorylated cTnI was replaced with cTnI-T144E (Fig.
2D), there was neither a decrease in myofilament
Ca2+ sensitivity nor a decrease in maximum tension (Fig.
3B and Table I).
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Fig. 2.
Force-pCa relationships of
skinned fibers in which the endogenous Tn complex was exchanged for Tn
containing either cTnI WT or cTnI with charge changes at PKC sites of
phosphorylation. A, force-pCa curve for
skinned fibers containing cTnI WT (filled squares) or cTnI
that had been phosphorylated by PKC- , cTnI-P (open
circles). Skinned fibers reconstituted with cTnI-P
(pCa50 = 5.38 ± 0.01) were significantly
desensitized to Ca2+ compared with skinned fibers
containing cTnI WT (pCa50 = 5.55 ± 0.01).
cTnI WT, n = 10; cTnI-P, n = 4. B, force-pCa curve for skinned fibers containing
cTnI WT (filled squares) or cTnI-S43E/S45E/T144E (open
circles). Skinned fibers reconstituted with cTnI-S43E/S45E/T144E
(pCa50 = 5.31 ± 0.01) were significantly
desensitized to Ca2+ compared with skinned fibers
containing cTnI WT (pCa50 = 5.55 ± 0.01).
cTnI WT, n = 10; cTnI-S43E/S45E/T144E,
n = 11. C, force-pCa curve for
skinned fibers containing cTnI WT (filled squares) or
cTnI-S43E/S45E (open circles). Skinned fibers reconstituted
with cTnI-S43E/S45E (pCa50 = 5.34 ± 0.01)
were significantly desensitized to Ca2+ compared with
skinned fibers containing TnI WT (pCa50 = 5.55 ± 0.01). cTnI WT, n = 10; cTnI-S43E/S45E,
n = 13. D, force-pCa curve
for skinned fibers containing cTnI WT (filled squares) or
cTnI-T144E (open circles). Skinned fibers reconstituted with
cTnI-T144E (pCa50 = 5.55 ± 0.02) were not
desensitized to Ca2+ compared with skinned fibers
containing TnI WT (pCa50 = 5.55 ± 0.01).
cTnI WT, n = 10; cTnI-T144E, n = 14.
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Fig. 3.
Summary of skinned fibers results.
A, summary of pCa50 values obtained
in skinned fibers after removal of endogenous Tn complex and
substitution with Tn containing cTnI WT, cTnI-P, cTnI-S43E/S45E/T144E
cTnI-S43E/S45E, or cTnI-T144E. The decrease in
pCa50 was significantly lower in skinned fibers
containing cTnI-P, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E
(p < 0.05). B, summary of maximum tension
measurements in skinned fibers after removal of endogenous Tn complex
and substitution with Tn containing TnI WT, cTnI-P,
cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. Maximum
Ca2+-dependent tension was determined after the
fibers were bathed in a solution of pCa 4.5 and normalized
to the maximum tension determined in fibers reconstituted with cTnI WT.
The decrease in maximum tension was significantly lower in skinned
fibers containing cTnI-P, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E
(p < 0.05).
Summary of results from skinned fibers and in vitro motility assay
studies
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Fig. 4.
Thin filament sliding speed measured for
actin filaments regulated by Tn containing cTnI WT or cTnI containing
charge changes at PKC sites of phosphorylation. A,
filament sliding speed measured for actin filaments regulated by Tn
containing cTnI WT (filled squares) or cTnI that had been
phosphorylated by PKC- , cTnI-P (open circles). Actin
filaments regulated by cTnI-P showed decreased maximum filament sliding
speed (Vmax = 3.49 ± 0.2 µM/s) and decreased Ca2+ sensitivity
(pCa50 = 5.64 ± 0.1) compared with actin
filaments regulated by cTnI WT (Vmax = 6.13 ± 0.4 µM/s, pCa50 = 6.97 ± 0.2). B, filament sliding speed measured for actin filaments
regulated by Tn containing cTnI WT (filled squares) or
cTnI-S43E/S45E/T144E (open circles). Actin filaments
regulated by cTnI-S43E/S45E/T144E showed decreased maximum filament
sliding speed (Vmax = 2.16 ± 0.2 µM/s) and decreased Ca2+ sensitivity
(pCa50 = 5.72 ± 0.1) compared with actin
filaments regulated by cTnI WT (Vmax = 4.86 ± 0.2 µM/s, pCa50 = 6.42 ± 0.1). C, filament sliding speed measured for actin filaments
regulated by Tn containing cTnI WT (filled squares) or
cTnI-S43E/S45E (open circles). Actin filaments regulated by
cTnI-S43E/S45E showed decreased maximum filament sliding speed
(Vmax = 4.46 ± 0.3 µM/s) and
no significant change in Ca2+ sensitivity
(pCa50 = 7.13 ± 0.2) compared with actin
filaments regulated by cTnI WT (Vmax = 5.30 ± 0.4 µM/s, pCa50 = 7.11 ± 0.2). D, filament sliding speed measured for actin filaments
regulated by Tn containing cTnI WT (filled squares) or
cTnI-T144E (open circles). Actin filaments regulated by
cTnI-T144E showed no significant decrease in maximum filament sliding
speed (Vmax = 5.35 ± 0.1 µM/s), but decreased Ca2+ sensitivity
(pCa50 = 5.66 ± 0.1) compared with actin
filaments regulated by cTnI WT (Vmax = 5.61 ± 0.3 µM/s, pCa50 = 6.17 ± 0.1). (n = 150-300 filaments for each pCa
value).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic agonist, phenylephrine, was substantially blunted in
papillary muscles from TG cTnI-S43A/S45A hearts. Montgomery et
al. (17) reported no differences in twitch dynamics between NTG
and TG preparations before or after treatment with phenylephrine. In
contrast, the studies of Pi et al. (42), in which all three
PKC sites of cTnI were mutated to alanine, implicated altered
cross-bridge cycling kinetics as a possible result of phosphorylation
of cTnI. Pi et al. (42) investigated the effect of the PKC
agonist endothelin 1 on ventricular myocytes from wild-type mice or
mouse lines that expressed non-phosphorylatable cTnI in which protein
kinase A and PKC sites of phosphorylation (Ser-23, Ser-24, Ser-43,
Ser-45, and Thr-144) were substituted with alanine
(cTnI-Ala5) on a cTnI-null background. In response to
endothelin 1, twitches were prolonged by 24-41% in WT mice, but only
by 5-8% in cTnI-Ala5 mice. The data of Pi et
al. (42) indicate that the depression of cross-bridge cycling has
the more predominant effect on twitch duration than desensitization of the myofilaments to Ca2+, which we found to be the case in
the present investigation. Therefore, PKC-mediated phosphorylation of
cTnI could play an important role in prolonging the cardiac twitch,
which may be a direct result of altered cross-bridge cycling kinetics.
,
-
1, and -
2 in failed heart cells. More recent data indicate that
phosphorylation of cTnI is responsible for decreases in sliding
velocity of thin filaments isolated from explanted hearts of patients
with end-state heart failure (45). Moreover, expression of PKC-
2 in
the hearts of transgenic mice induces hypertrophy as well as reduced
myofilament response to Ca2+ (16, 46). It thus seems
reasonable to speculate that increases in the activity of PKC-
2,
induced by some hemodynamic stress, may initially promote compensatory
hypertrophic mechanisms that with sustained stress lead to
decompensation and heart failure. This is a particularly important
mechanism to understand inasmuch as agents that specifically inhibit
PKC are being developed (47, 48).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 HL 64035 (Project 1, to R. J. S.), P01 HL 62426 (to R. J. S. and A. F. M.), RO1 AR-30988 (to E. H.), and T3207692 (to M. P. S. and E. M. B.), Individual National Research Service Award F32 HL 10409 (to M. P. S.), and American Heart Association Scientist Development Grant 0230038N (to T. K.).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.
§ Supported by an American Heart Association Pre-Doctoral Fellowship (Midwest Affiliate).
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, (M/C 901), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Tel.: 312-996-7620; Fax: 312-996-1414; E-mail: SolaroRJ@uic.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210712200
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ABBREVIATIONS |
---|
The abbreviations used are:
PKC, protein kinase
C;
Tn, troponin;
cTn, cardiac troponin;
TnI-P, PKC phosphorylated TnI;
Tm, tropomyosin;
AB, low salt assay buffer;
DTT, dithiothreitol;
MOPS, 3-(N-morpholino)propanesulfonic acid;
TPCK, L-1-tosylamide 2-phenylethyl chloromethyl ketone;
TLCK, N-p-tosyl-L-lyrine chloromethyl
ketone;
pCa,
log[Ca2+];
ANOVA, analysis of
variance;
WT, wild-type;
TG, transgenic;
NTG, non-transgenic.
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